Identification and validation of DNA methylation changes in pre-eclampsia.

INTRODUCTION: Pre-eclampsia (PE) is a dangerous placental condition that can lead to premature labour, seizures and death of mother and infant. Several studies have identified altered placental DNA methylation in PE; however, there is widespread inconsistency between studies and most findings have not been replicated. This study aimed to identify and validate consistent differences in methylation across multiple PE cohorts. METHODS: Seven publicly available 450K methylation array datasets were analysed to identify consistent differentially methylated positions (DMPs) in PE. DMPs were identified based on methylation difference (≥10%) and significance (p-value ≤ 1 × 10-7). Targeted deep bisulfite sequencing was then performed to validate a subset of DMPs in an additional independent PE cohort. RESULTS: Stringent analysis of the seven 450K datasets identified 25 DMPs (associated with 11 genes) in only one dataset. Using more relaxed criteria confirmed 19 of the stringent 25 DMPs in at least four of the remaining six datasets. Targeted deep bisulfite sequencing of eight DMPs (associated with three genes; CMIP, ST3GAL1 and DAPK3) in an independent PE cohort validated two DMPs in the CMIP gene. Seven additional CpG sites in CMIP were found to be significantly differentially methylated in PE. DISCUSSION: The identification and validation of significant differential methylation in CMIP suggests that the altered DNA methylation of this gene may be associated with the pathogenesis of PE, and may have the potential to serve as diagnostic biomarkers for this dangerous condition of pregnancy.

Human Papillomavirus E6/E7 Expression in Preeclampsia-Affected Placentae.

Whether HPV is causative of pregnancy complications is uncertain. E6 and E7 affect functions underling preeclampsia (PET) in cultured trophoblasts, but whether E6 and E7 is produced in the placenta is uncertain. Here, we investigated whether E6/E7 was expressed in the placentae from pregnancies with PET, other pregnancy complications (fetal growth restriction (FGR) and diabetes mellitus), and uncomplicated pregnancies. Placental tissues collected from two geographical locations were subjected to RNAscope analyses of high- and low- risk E6/E7, and individual HPV types identified using an HPV array. High-risk E6/E7 expression was increased in both PET cohorts, (81% and 86% of patients positive for high-risk HPV DNA compared to 13% of control patients). Various HPV types were identified. Although HPV 18 was the most frequent in all cohorts, the majority of individuals had multiple HPV types (55% of the PET compared to 25% of the control cohort). Further evidence that E6 and E7 is present early when placental pathology underlying preeclampsia is established, is provided with the finding of high-risk E6/E7 in the first-trimester placenta anchoring trophoblast. In conclusion, E6/E7 expression and multiple HPV types were frequent in placentae from preeclampsia-complicated pregnancies.

RETRACTED: Association of prior HPV vaccination with reduced preterm birth: A population based study.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This paper is being retracted at the request of the authors. The authors report that there was an incorrect interpretation of the odds ratio meaning that instead of HPV vaccination potentially being protective, there may be an associated increased risk of preterm delivery. The authors believe that an increased risk of preterm delivery is unlikely and not consistent with the evidence to date. Further, the authors have not been able to access the original source data as per protocol to check the data validity. The authors wish to repeat the study to reassure themselves that there were no data processing or other errors in the databases in order to reach definitive conclusions. Lastly, it is of serious concern to the Editor-in-Chief that the Conflict of Interest statement was only added to the paper by the authors after acceptance and was not made visible to the editor or reviewers prior to acceptance. The authors state that there was no input to the methodology, implementation and results of this study by any commercial entity. The pharma distribution company CSL mentioned in the conflict of interest statement only knew about the study after publication.

Placental Hypomethylation Is More Pronounced in Genomic Loci Devoid of Retroelements.

The human placenta is hypomethylated compared to somatic tissues. However, the degree and specificity of placental hypomethylation across the genome is unclear. We assessed genome-wide methylation of the human placenta and compared it to that of the neutrophil, a representative homogeneous somatic cell. We observed global hypomethylation in placenta (relative reduction of 22%) compared to neutrophils. Placental hypomethylation was pronounced in intergenic regions and gene bodies, while the unmethylated state of the promoter remained conserved in both tissues. For every class of repeat elements, the placenta showed lower methylation but the degree of hypomethylation differed substantially between these classes. However, some retroelements, especially the evolutionarily younger Alu elements, retained high levels of placental methylation. Surprisingly, nonretrotransposon-containing sequences showed a greater degree of placental hypomethylation than retrotransposons in every genomic element (intergenic, introns, and exons) except promoters. The differentially methylated fragments (DMFs) in placenta and neutrophils were enriched in gene-poor and CpG-poor regions. The placentally hypomethylated DMFs were enriched in genomic regions that are usually inactive, whereas hypermethylated DMFs were enriched in active regions. Hypomethylation of the human placenta is not specific to retroelements, indicating that the evolutionary advantages of placental hypomethylation go beyond those provided by expression of retrotransposons and retrogenes.

A clinicopathological study of episomal papillomavirus infection of the human placenta and pregnancy complications.

Viral infections are known to adversely affect pregnancy, but scant attention has been given to human papilloma virus (HPV) infection. We aimed to determine the molecular and histopathological features of placental HPV infection, in association with pregnancy complications including fetal growth restriction, pre-maturity, pre-eclampsia, and diabetes. Three hundred and thirty-nine placentae were selected based on the presence or absence of pregnancy complications. Five independent methods were used to identify HPV in the placenta, namely, immunohistochemistry for L1 viral capsid, in situ hybridization to high-risk HPV DNA, PCR, western blotting, and transmission electron microscopy. Pregnancy complications and uterine cervical smear screening results were correlated with placental HPV histopathology. In this study, which was deliberately biased towards complications, HPV was found in the decidua of 75% of placentae (253/339) and was statistically associated with histological acute chorioamnionitis (P<0.05). In 14% (35/253) of the HPV positive cases, HPV L1 immunoreactivity also occurred in the villous trophoblast where it was associated with a lymphohistiocytic villitis (HPV-LHV), and was exclusively of high-risk HPV type. HPV-LHV significantly associated with fetal growth restriction, preterm delivery, and pre-eclampsia (all P<0.05). All cases of pre-eclampsia (20/20) in our cohort had high-risk placental HPV. A further 55 cases (22%, 55/253) of HPV positive placentae had minimal villous trophoblast HPV L1 immunoreactivity, but a sclerosing pauci-immune villitis, statistically associated with diabetes (49.1%, 27/55, P<0.05). For women with placental HPV, 33% (69/207) had an HPV-related positive smear result before pregnancy compared with (9.4% 8/85) of women with HPV-negative placentae (P=0.0001). Our findings support further investigations to determine if vaccination of women and men will improve pregnancy outcomes.

Loss of ATRX and DAXX expression identifies poor prognosis for smooth muscle tumours of uncertain malignant potential and early stage uterine leiomyosarcoma.

Uterine smooth muscle tumours of uncertain malignant potential (STUMP) are diagnostically and clinically challenging. The alternative lengthening of telomeres (ALT) telomere maintenance mechanism is associated with poor survival in soft tissue leiomyosarcoma. Time to first recurrence and survival were known for 18 STUMP and 43 leiomyosarcomata (LMS). These were screened for ALT telomere maintenance by the presence of ALT-associated PML bodies (APBs) and for changes associated with the ALT phenotype, namely aberrant p53 expression, isocitrate dehydrogenase 1 mutation (R132H substitution) expression, mutant ATRX (αthalassemia/mental retardation syndrome X-linked) expression and mutant DAXX (death-domain-associated protein) expression by immunohistochemistry (IHC). Overexpression of p16(INK4A) was examined immunohistologically in a subset of cases. Many of the tumours associated with death or recurrence demonstrated APBs commensurate with ALT telomere maintenance. However, all uterine STUMP (4/4), and vaginal STUMP (2/2) patients, and almost all LMS patients (88.4%, 23/26, including 90% (9/10) of stage 1 LMS cases), who had died of disease or who had recurrent disease, displayed loss of ATRX or DAXX expression. Loss of ATRX or DAXX expression identified poor prognosis (95% CI 2.1 to 40.8, p < 0.003), in the LMS group. Thus, loss of ATRX or DAXX expression in uterine smooth muscle tumours identifies a clinically aggressive molecular subtype of early stage LMS and when histopathological features are problematic such as in STUMP. As ATRX and DAXX IHC is readily performed in diagnostic laboratories these are potentially useful for routine histopathological classification and management.

Smoking during pregnancy causes double-strand DNA break damage to the placenta.

Despite the adverse effects of smoking, many pregnancies are exposed to tobacco smoke. Recent studies have investigated whether smoking damages placental DNA by measuring DNA adducts. This study investigated whether a more severe lesion, double-strand DNA breaks, was also present in the tobacco smoking-exposed placenta. Term placentae from women who smoked during their entire pregnancies (n = 52), from those who had ceased smoking for at least 4 weeks before delivery (previous smokers, n = 34), and from nonsmoking women (n = 150) were examined using the DNA double-strand break marker phosphorylated γ H2AX. The extent of DNA damage was assessed according to cell type and additional markers were applied for cell fate (apoptosis and DNA repair), and function (human chorionic gonadotropin, human placental lactogen, and glucose transporter 1), to characterize the effect of the DNA damage on placental integrity. Marked phosphorylated γ H2AX-positive cells occurred in the villous syncytiotrophoblast and syncytial knot nuclei in placentae from smokers (P < .001). Phosphorylated γ H2AX foci did not colocalize with the DNA repair protein 53BP1, and damaged nuclei had a marked reduction in expression of human chorionic gonadotropin, human placental lactogen, and glucose transporter 1. Minimal DNA damage, similar to nonsmokers, was present in previous smokers including those that had ceased smoking for just over 4 weeks before delivery. In summary, smoking during pregnancy was associated with marked double-strand DNA break damage to the syncytiotrophoblast. We suggest that smoking cessation is important to prevent additional DNA damage and to facilitate DNA repair.

The alternative lengthening of telomeres pathway may operate in non-neoplastic human cells.

The alternative lengthening of telomeres (ALT) mechanism represents an alternative to the enzyme telomerase in the maintenance of mammalian telomeres in 25-60% of sarcomas and a minority of carcinomas (about 5-15%). ALT-positive cells are distinguished by long and heterogeneous telomere length distributions by terminal restriction fragment (TRF) Southern blotting. Another diagnostic marker of ALT is discrete nuclear co-localized signals of telomeric DNA and the promyelocytic leukaemia protein (PML), referred to as ALT-associated PML bodies (APBs). Recently, we detected smaller sized co-localized PML and telomere DNA (APB-like) bodies in endothelial cells adjacent to astrocytoma tumour cells in situ. In this study, we examined a wide variety of non-neoplastic tissues, and report that co-localized signals of PML and telomere DNA are present in endothelial, stromal, and some epithelial cells. Co-localized signals of PML and telomere DNA showed an increased frequency in non-neoplastic cells with DNA damage. These results suggest that a mechanism similar to that in ALT-positive tumours also operates in non-neoplastic cells, which may be activated by DNA damage.

Is there a correlation between bacterial vaginosis and preterm labour in women in the Otago region of New Zealand?

CONTEXT: While an association between bacterial vaginosis and preterm labour has been established, the relative contribution of this condition remains controversial. OBJECTIVE: To determine whether bacterial vaginosis is likely to be an important contributing factor in preterm births in Otago, New Zealand, a region with a historically high rate of such births. DESIGN AND SETTING: Women receiving antenatal care from Queen Mary Maternity Services were studied prospectively. Cases were women presenting with preterm labour or premature rupture of membranes. Controls had uncomplicated pregnancies and delivered at term. PATIENTS AND METHODS: Vaginal swabs from 44 cases and 72 controls were examined by amplification of bacterial 16S rRNA genes followed by denaturing gel gradient electrophoresis. Atopobium vaginae, a bacterial vaginosis-associated bacterium, was detected in a separate polymerase chain reaction. Nugent Gram stain scoring of vaginal swabs from 44 cases and 69 controls was also carried out. RESULTS: Denaturing gel gradient electrophoresis revealed three major types of band profiles corresponding to normal, intermediate and bacterial vaginosis microflorae. There were significantly more cases with bacterial vaginosis band profiles compared with controls (P = 0.024). More cases had intermediate or bacterial vaginosis Nugent scores compared with controls (P = 0.022). Conversely, controls were more likely to have normal scores than cases (P = 0.022). Atopobium vaginae was equally distributed between the cases and controls. CONCLUSIONS: Women in the Otago region undergoing preterm labour were approximately twice as likely to have a bacterial vaginosis type vaginal microflora as controls. In preterm labour, the incidence of bacterial vaginosis was comparable with that found elsewhere, suggesting that current guidelines for treatment and detection of this condition are appropriate.

A survey of New Zealand RANZCOG Fellows on their use of the levonorgestrel intrauterine device in adolescents.

BACKGROUND: The levonorgestrel intrauterine device (LNG-IUD) is an established treatment for adult women. Although it is being used in adolescents, there is little published research in this age group to date. Recent reviews and editorials have challenged the long-held views that intrauterine devices should not be used in young women. AIMS: This study aimed to identify the patterns of use, including indications and contraindications of the LNG-IUD in adolescents by RANZCOG Fellows practising in New Zealand. METHODS: A postal survey of New Zealand RANZCOG Fellows on their use of the LNG-IUD in females aged 10-19 years. RESULTS: There was a 72% response rate. Half of the respondents had inserted the LNG-IUD in adolescents. Non-inserters identified a significantly greater number of contraindications than inserters (chi2, P < 0.0001). Over half of those respondents who had inserted a device in an adolescent did so fewer than three times per year. Intellectual disability and endometriosis, both unlicensed indications, were the two most commonly identified circumstances for insertion by respondents. CONCLUSIONS: Patterns of insertion of the LNG-IUD in adolescents by RANZCOG Fellows in New Zealand differ and there was equipoise over its use. Further research is required to establish the efficacy, safety and acceptability of the LNG-IUD in adolescents.

Periconceptional folic acid use among women giving birth at Queen Mary Maternity Hospital in Dunedin.

BACKGROUND: The New Zealand Ministry of Health advises that all women planning a pregnancy take a folic acid supplement to reduce the risk of having a neural tube defect (NTD)-affected pregnancy. There is little information available to determine if women are following this advice. OBJECTIVE: The purpose of this study was to determine periconceptional folic acid use among women in the postnatal ward of Queen Mary Maternity Hospital in Dunedin. METHODS: A questionnaire was administered to women in the postnatal ward between 14 November and 22 December 2004. RESULTS: One hundred and six women were interviewed during the study period. Forty women (39%) used folic acid supplements before conception. Sixty-seven women (64%) planned their pregnancy. The proportion of women (P<0.001) who planned their pregnancy (53%) and used folic acid before conception was higher than those who did not (11%). The proportion of women 30 years of age (55%) who took folic acid supplements before conception was higher than women aged 17 to <25 years (10%). CONCLUSION: Despite a lack of a public health campaign in New Zealand, a high proportion of participants, especially those who planned their pregnancy, took folic acid during the periconceptional period. A comprehensive public health campaign is needed to increase folic acid use. Fortification may be required to reach those women who do not plan their pregnancies.

A preliminary survey of Atopobium vaginae in women attending the Dunedin gynaecology out-patients clinic: is the contribution of the hard-to-culture microbiota overlooked in gynaecological disorders?

Preliminary studies have indicated that the recently described bacterium Atopobium vaginae may have an association with bacterial vaginosis (BV). Fifty-five women attending the gynaecology out-patient's clinic were tested for the presence of this micro-organism, Gardnerella vaginalis, Mobiluncus and Bacteroides species by polymerase chain reaction (PCR)-based assays. The frequency of detection was 40%. PCR detection of Gardnerella vaginalis with A. vaginae, occurred in 50% of A. vaginae-positive cases. Due to the high detection rate of A. vaginae we believe that it is important to determine whether this and other hard-to-culture microorganisms have a role in gynaecological disorders.