Placental DNA methylation analysis of selective fetal growth restriction in monochorionic twins reveals aberrant methylated CYP11A1 gene for fetal growth restriction.

Fetal growth restriction (FGR) is associated with increased susceptibility to perinatal morbidity and mortality. Evidence suggests that epigenetic changes play critical roles in the regulation of fetal growth. We sought to present a comprehensive analysis of the associations between placental DNA methylation and selective fetal growth restriction (sFGR), which is a severe complication of monochorionic twin pregnancies, characterized by one fetus experiencing restricted growth. Genome-wide methylation analysis was performed on 24 placental samples obtained from 12 monochorionic twins with sFGR (Cohort 1) using Illumina Infinium MethylationEPIC BeadChip. Integrative analysis of our EPIC data and two previous placental methylation studies of sFGR (a total of 30 placental samples from 15 sFGR twins) was used to identify convincing differential promoter methylation. Validation analysis was performed on the placentas from 15 sFGR twins (30 placental samples), 15 FGR singletons, and 14 control singletons (Cohort 2) using pyrosequencing, quantitative real-time polymerase chain reaction, western blot, and immunohistochemistry (IHC). A globe shift toward hypomethylation was identified in the placentas of growth-restricted fetuses compared with the placentas of normal fetuses in monochorionic twins, including 5625 hypomethylated CpGs and 452 hypermethylated CpGs, especially in the regions of CpG islands, gene-body and promoters. The analysis of pathways revealed dysregulation primarily in steroid hormone biosynthesis, metabolism, cell adhesion, signaling transduction, and immune response. Integrative analysis revealed a differentially methylated promoter region in the CYP11A1 gene, encoding a rate-limiting enzyme of steroidogenesis converting cholesterol to pregnenolone. The CYP11A1 gene was validated to have hypomethylation and higher mRNA expression in sFGR twins and FGR singletons. In conclusion, our findings suggested that the changes in placental DNA methylation pattern in sFGR may have functional implications for differentially methylated genes and regulatory regions. The study provides reliable evidence for identifying abnormally methylated CYP11A1 gene in the placenta of sFGR.

Congenital heart defects in monochorionic twin pregnancy complicated by selective fetal growth restriction.

OBJECTIVES: To evaluate the prevalence, subtypes and postnatal outcomes of congenital heart defects (CHD) in a cohort of monochorionic diamniotic (MCDA) twin pregnancies complicated by selective fetal growth restriction (sFGR), and to compare this population with a cohort of uncomplicated MCDA pregnancies evaluated during the same period. METHODS: This was a retrospective analysis of all consecutive MCDA pregnancies referred between 2009 and 2018, including those complicated by sFGR (Group A) and those without complications (Group B). All neonates delivered in our center were screened for CHD before discharge. Discharge letters for all those delivered elsewhere were retrieved. Pregnancies with complications other than sFGR and those without perinatal follow-up were excluded. Pregnancies in Group A were divided into three types according to the Gratacós system of sFGR classification. RESULTS: A total of 870 MCDA twin pregnancies were included: 296 in Group A and 574 in Group B. In Group A, the prevalence of CHD was 3.7% (22/592 twins), with no significant difference in CHD frequency between the three types of sFGR (Type I, 3.7%; Type II, 3.2%; Type III, 4.2%; P = 0.55). Of four Type-III sFGR pregnancies with CHD, one had pulmonary stenosis (PS) in the larger twin and isolated coarctation of the aorta in the smaller cotwin, and three had PS in the larger twin only. No Type-III sFGR pregnancies in which only the smaller twin was affected by CHD were observed. Of 11 CHD cases in the larger twin, 10 (91%) were right ventricular outflow tract abnormalities (RVOTA), and one (9%) was a ventricular septal defect. In the smaller twins, 11 cases of CHD were observed, covering a broad spectrum of cardiac abnormalities. In Group B, the CHD prevalence was 1.1% (13/1148 twins), which was similar to that in the general population, according to the EUROCAT registry for the same period and geographical area of the study (0.96%; P = 0.579). The CHD prevalence was significantly higher in Group A compared with Group B (3.7% vs 1.1%; P = 0.0002; odds ratio, 3.57 (95% CI, 1.78-7.22)). In all pregnancies with CHD in the study population, the anomaly was discordant. CONCLUSIONS: In MCDA twin pregnancy, sFGR was associated with a three-fold higher prevalence of CHD. Women with such pregnancies should be referred to a tertiary care hospital for pre- and postnatal cardiac evaluation, treatment and long-term follow-up. In larger twins, the only major CHD observed was RVOTA, while a broad spectrum of CHD was noted in smaller twins. The higher risk of CHD in MCDA pregnancies appears to be due to the typical complications of the monochorionic pregnancy, rather than to the monochorionic nature of the pregnancy itself. © 2022 International Society of Ultrasound in Obstetrics and Gynecology.

Growth patterns of monochorionic twin pregnancy complicated by Type-III selective fetal growth restriction.

OBJECTIVES: Little is known regarding fetal growth patterns in monochorionic twin pregnancy complicated by Type-III selective fetal growth restriction (sFGR). We aimed to assess fetal growth and umbilical artery Doppler pattern in Type-III sFGR across gestation and evaluate the effect of changing Doppler flow pattern on growth and intertwin growth discordance. METHODS: This was a retrospective cohort study of all Type-III sFGR pregnancies managed at nine fetal centers over a 12-year time period. Higher-order multiple pregnancy and cases with major fetal anomaly or other monochorionicity-related complications at presentation were excluded. Estimated fetal weight (EFW) was assessed on ultrasound for each twin pair at five timepoints (16-20, 21-24, 25-28, 29-32 and > 32 weeks' gestation) and compared with singleton and uncomplicated monochorionic twin EFW. EFW and intertwin EFW discordance were compared between pregnancies with normalization of umbilical artery Doppler of the smaller twin later in pregnancy and those with persistently abnormal Doppler. RESULTS: Overall, 328 pregnancies (656 fetuses) met the study criteria. In Type-III sFGR, the smaller twin had a lower EFW than an average singleton fetus (EFW Z-score ranging from -1.52 at 16 weeks to -2.69 at 36 weeks) and an average monochorionic twin in uncomplicated pregnancy (Z-score ranging from -1.73 at 16 weeks to -1.49 at 36 weeks) throughout the entire gestation, while the larger twin had a higher EFW than an average singleton fetus until 22 weeks' gestation and was similar in EFW to an average uncomplicated monochorionic twin throughout gestation. As pregnancy advanced, growth velocity of both twins decreased, with the larger twin remaining appropriately grown and the smaller twin becoming more growth restricted. Intertwin EFW discordance remained stable throughout gestation. On multivariable longitudinal modeling, normalization of fetal umbilical artery Doppler was associated with better growth of the smaller twin (P = 0.002) but not the larger twin (P = 0.1), without affecting the intertwin growth discordance (P = 0.09). CONCLUSIONS: Abnormal fetal growth of the smaller twin in Type-III sFGR was evident early in pregnancy, while EFW of the larger twin remained normal throughout gestation. Normalization of umbilical artery Doppler was associated with improved fetal growth of the smaller twin. © 2021 International Society of Ultrasound in Obstetrics and Gynecology.

Outcome of radiofrequency ablation for selective fetal reduction before vs at or after 16 gestational weeks in complicated monochorionic pregnancy.

OBJECTIVE: To investigate whether gestational age at intervention (< or ≥ 16 weeks) and other factors affect the risk of loss of the cotwin after selective fetal reduction using radiofrequency ablation (RFA) in monochorionic (MC) pregnancy. METHODS: This was a single-center retrospective analysis of 63 consecutive RFA procedures performed at our institution from January 2011 to October 2019 for selective fetal reduction in complicated MC pregnancies. Indications for RFA were twin reversed arterial perfusion sequence (13 cases), twin-to-twin transfusion syndrome (12 cases), twin anemia-polycythemia sequence (two cases), selective fetal growth restriction (10 cases), discordant anomalies (17 cases) and multifetal pregnancy reduction in triplets or quadruplets with a MC pair (nine cases). Twenty-six (41.3%) of these procedures were performed before and 37 (58.7%) after 16 weeks. Potential factors that could affect the risk of loss of the cotwin, including gestational age at RFA, order of multiple pregnancy, amnionicity, indication for RFA and number of ablation cycles, were assessed first by univariate analysis and then by multivariate analysis. RESULTS: There were 17 (27.0%) cotwin losses. Ablation cycles numbering four or more was the only factor among those investigated to be associated with loss of the cotwin after RFA (P = 0.035; odds ratio, 5.21), while the indication for RFA, order of multiple pregnancy, amnionicity and gestational age at RFA had no effect. Comparing RFA performed at < 16 vs ≥ 16 weeks, there was no difference in the rate of cotwin loss (23.1% vs 29.7%; P = 0.558) or preterm prelabor rupture of the membranes before 34 weeks (7.7% vs 5.4%; P = 0.853), or in the median gestational age at delivery (36.2 vs 37.3 weeks; P = 0.706). CONCLUSIONS: RFA is a promising tool for early selective fetal reduction in MC pregnancy before 16 weeks. Four or more ablation cycles is a major risk factor for cotwin loss. Careful assessment pre- and post-RFA, together with proficient operative skills to minimize the number of ablation cycles, are the mainstay to ensure that this procedure is effective and safe. © 2020 International Society of Ultrasound in Obstetrics and Gynecology.

Seroprevalence of Spotted Fever Group Rickettsiae in extensively managed sheep and goats in Nigeria, West Africa.

Sheep and goats raised extensively are frequently infested by Ixodid ticks that may act as vectors or reservoirs of Spotted Fever Group Rickettsiae (SFGR). A study to determine the seroprevalence of SFGR infection in 300 sheep and goats in Plateau State, Nigeria was conducted from September to November, 2018 using the Indirect Fluorescence Antibody Test (IFAT). Overall, 85 out of 300 animals (28.3%) were seropositive to SFGR. Relatively higher seroprevalence was recorded in sheep than goats (28.8% vs 28.0%) but the difference was not statistically significant (p > 0.05). Furthermore, seropositivity was not affected by age, sex or location of the animals screened in this study. This is the first serological study to report the prevalence of SFGR in sheep and goats using IFAT in this study area. The presence of SFGR antibodies in domestic ruminants is of public health concern considering the close association between farmers and their animals occasioned by the management system practiced in the study area. This finding calls for further studies to evaluate the level of human exposure to this group of pathogen.

Early- and late-onset selective fetal growth restriction in monochorionic diamniotic twin pregnancy: natural history and diagnostic criteria.

OBJECTIVES: To evaluate the natural history and outcome of selective fetal growth restriction (sFGR) in monochorionic diamniotic (MCDA) twin pregnancy, according to gestational age at onset and various reported diagnostic criteria, and to quantify the risk of superimposed twin-to-twin transfusion syndrome (TTTS). METHODS: This was a cohort study of MCDA twin pregnancies that had their routine antenatal care from the first trimester at St George's Hospital, London, UK. Pregnancies had ultrasound examinations every 2 weeks at 16-24 weeks and then every 2-3 weeks until delivery. The diagnostic criteria for sFGR were estimated fetal weight (EFW) of one twin < 10th centile and intertwin EFW discordance ≥ 25%. We also applied other diagnostic criteria reported in a recent Delphi consensus. Pregnancies in which the diagnosis of TTTS was made before that of sFGR were not included in the analysis. Pregnancies that underwent fetal intervention for sFGR were excluded. The incidence of sFGR was compared between the different diagnostic criteria, overall and according to gestational age at onset. In all subsequent analyses, cases of sFGR included those diagnosed according to any of the criteria. The Gratacós classification of sFGR was applied (Type I, II or III). Pregnancy outcomes included miscarriage, intrauterine death, neonatal death and admission to the neonatal unit. Comparisons between groups were carried out using the Mann-Whitney U-test for continuous variables and the chi-square or Fisher's exact test for categorical variables. RESULTS: The analysis included 287 MCDA twin pregnancies. According to the International Society of Ultrasound in Obstetrics and Gynecology diagnostic criteria, the incidence of early (< 24 weeks) sFGR was 4.9%, while that of late sFGR was 3.8%. When applying the various diagnostic criteria, the incidence of early sFGR varied from 1.7% to 9.1% and that of late sFGR varied from 1.1% to 5.9%. In early-onset cases, the incidence of Type I sFGR was 80.8%, that of Type II was 15.4% and that of Type III was 3.8%. The corresponding figures in late-onset cases were 94.4%, 5.6% and 0%. The incidence of superimposed TTTS was 26.9% in cases affected by early-onset sFGR and 5.6% in those affected by late-onset sFGR. The incidence of perinatal death was 8.0% in early-onset sFGR and 5.6% in late-onset sFGR (P = 0.661). Admission to the neonatal unit occurred in 61.0% and 52.9% of cases, respectively (P = 0.484). CONCLUSIONS: In MCDA twin pregnancies, early-onset sFGR is slightly more common than is late-onset sFGR, although this difference was not significant, and is associated with worse perinatal outcome. The incidence of Types II and III sFGR is higher in early-onset sFGR. The incidence also varies according to the diagnostic criteria used, which supports the use of standardized international diagnostic criteria. Superimposed TTTS is more common in early- than in late-onset sFGR. Copyright © 2019 ISUOG. Published by John Wiley & Sons Ltd.

Consensus definition and essential reporting parameters of selective fetal growth restriction in twin pregnancy: a Delphi procedure.

OBJECTIVES: Twin pregnancy complicated by selective fetal growth restriction (sFGR) is associated with increased perinatal mortality and morbidity. Inconsistencies in the diagnostic criteria for sFGR employed in existing studies hinder the ability to compare or combine their findings. It is therefore challenging to establish robust evidence-based management or monitoring pathways for these pregnancies. The main aim of this study was to determine, by expert consensus using a Delphi procedure, the key diagnostic features of and the essential reporting parameters in sFGR. METHODS: A Delphi process was conducted among an international panel of experts in sFGR in twin pregnancy. Panel members were provided with a list of literature-based parameters for diagnosing sFGR and were asked to rate their importance on a five-point Likert scale. Parameters were described as solitary (sufficient to diagnose sFGR, even if all other parameters are normal) or contributory (those that require other abnormal parameter(s) to be present for the diagnosis of sFGR). Consensus was sought to determine the cut-off values for accepted parameters, as well as parameters used in the monitoring, management and assessment of outcome of twin pregnancy complicated by sFGR. The questions were presented in two separate categories according to chorionicity. RESULTS: A total of 72 experts were approached, of whom 60 agreed to participate and entered the first round; 48 (80%) completed all four rounds. For the definition of sFGR irrespective of chorionicity, one solitary parameter (estimated fetal weight (EFW) of one twin < 3rd centile) was agreed. For monochorionic twin pregnancy, at least two out of four contributory parameters (EFW of one twin < 10th centile, abdominal circumference of one twin < 10th centile, EFW discordance of ≥ 25%, and umbilical artery pulsatility index of the smaller twin > 95th centile) were agreed. For sFGR in dichorionic twin pregnancy, at least two out of three contributory parameters (EFW of one twin < 10th centile, EFW discordance of ≥ 25%, and umbilical artery pulsatility index of the smaller twin > 95th centile) were agreed. CONCLUSIONS: Consensus-based diagnostic features of sFGR in both monochorionic and dichorionic twin pregnancies, as well as cut-off values for the parameters involved, were agreed upon by a panel of experts. Future studies are needed to validate these diagnostic features before they can be used in clinical trials of interventions. Copyright © 2018 ISUOG. Published by John Wiley & Sons Ltd.

Severe Fever with Thrombocytopenia Syndrome Complicated by Co-infection with Spotted Fever Group Rickettsiae, China.

During 2013-2015 in central China, co-infection with spotted fever group rickettsiae was identified in 77 of 823 patients infected with severe fever with thrombocytopenia syndrome virus. Co-infection resulted in delayed recovery and increased risk for death, prompting clinical practices in the region to consider co-infection in patients with severe fever with thrombocytopenia syndrome.

Amniotic fluid metabolic fingerprinting contributes to shaping the unfavourable intrauterine environment in monochorionic diamniotic twins.

BACKGROUND & AIMS: Amniotic fluid (AF) is the primary intrauterine environment for fetal growth throughout gestation. Selective fetal growth restriction (sFGR) is an adverse complication characterized by unequal growth in twins with nearly identical genetic makeup. However, the influence of AF-mediated intrauterine environment on the development and progression of sFGR remains unexplored. METHODS: High-throughput targeted metabolomics analysis (G350) was performed on AF samples collected from sFGR (n = 18) and MCDA twins with birth weight concordance (MCDA-C, n = 20) cases. Weighted correlation network analysis (WGCNA) was used to identify clinical features that may influence the metabolite composition in AF. Subsequently, partial least-squares discriminant analysis (PLS-DA) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to compare the different types of sFGR and MCDA-C twins. Receiver operating characteristic (ROC) and multivariate ROC curves were utilized to explore potential AF markers in twins with sFGR. RESULTS: In our study, 182 metabolites were quantified in 76 AF samples. WGCNA indicated that the metabolite composition in late AF may not be influenced by gestational age. PLSDA demonstrated distinct variations between the metabolite profiles of AF in the sFGR and MCDA-C twins, with a significant emphasis on amino acids as the primary differential metabolite. The dissimilarities observed in sFGR twins were predominantly attributed to lipid metabolism-related metabolites. In particular, the KEGG enrichment metabolic pathway analysis revealed significant associations of both types of sFGR twins with central carbon metabolism in cancer. The multivariate ROC curves indicated that the combination of carnosine, sarcosine, l-alanine, beta-alanine, and alpha-n-phenylacetylglutamine significantly improved the AUC to 0.928. Notably, the ROC curves highlighted creatine (AUC:0.934) may be a potential biomarker for severe sFGR. CONCLUSION: The data presented in this study offer a comprehensive metabolic map of the AF in cases of sFGR, shedding light on potential biomarkers associated with fetal growth and development in MCDA twins.

Mitochondrial dysfunction and oxidative stress in selective fetal growth restriction.

INTRODUCTION: Placental dysfunction is the primary cause of selective fetal growth restriction (sFGR), and the specific role of mitochondria remains unclear. This study aims to elucidate mitochondrial functional defects in sFGR placentas and explore the roles of mitochondrial genomic and epigenetic alterations in its pathogenesis. METHODS: The placental villi of MCDA twins with sFGR were collected and the morphology and number of mitochondria were observed by transmission electron microscopy. Meanwhile, the levels of reactive oxygen species (ROS), ATP and oxidative damage markers were assessed. Mitochondrial DNA (mtDNA) copy number detection, targeted sequencing and methylation sequencing were performed. The expression of placental cytochrome c oxidase subunit I (COX I) and mitochondrial long non-coding RNAs (lncRNAs) were evaluated by Western blotting and qPCR. RESULTS: Compared with placentae from normal fetuses, pronounced mitochondrial damage within cytotrophoblast was revealed in sFGR placentae, alongside augmented mitochondrial number in syncytiotrophoblast. Enhanced oxidative stress in these placentae was evidenced by elevated markers of oxidative damage, accompanied by increased ROS production and diminished ATP generation. In sFGR placentae, a notable rise in mitochondrial copy number and one heterozygous mutation in the MT-RNR2 gene were observed, along with decreased COX Ⅰ levels, increased lncND5, lncND6, lncCyt b, and MDL1 synthesis, and decreased RMRP synthesis. DISCUSSION: Findings collectively confirmed an exacerbation of oxidative stress within sFGR placentae, coinciding with mitochondrial dysfunction, compromised energy production, and ultimately the failure of compensatory mechanisms to restore energy balance, which may result from mutations in the mitochondrial genome and abnormal expression of epigenetic regulatory genes.

Is There a Cumulative Effect for Congenital Heart Defects in Monochorionic Twins after Assisted Reproduction? - A Retrospective Analysis at a Tertiary Referral Center.

Introduction: The aim of our study was to compare maternal, chorionicity and neonatal complications in monochorionic (MC) twins between spontaneously conceived (SC) and assisted reproductive technologies (ART) pregnancies. Material and Methods: This was a retrospective cohort study between January 2010 to December 2019 at a tertiary referral University center. All consecutive pregnancies with MC twins that delivered at our University hospital were included. Maternal, chorionicity and neonatal complications were recorded and compared between SC and ART pregnancies. Results: 393 MC pregnancies were included for final analysis, including 353 (89.8%) SC and 40 (10.2%) pregnancies conceived after ART. Hypothyroidism was the only maternal condition seen significantly more often in ART pregnancies (35.0% vs 12.5%, p = 0.001). There were no significant differences in chorionicity complications, such as twin-twin transfusion syndrome, selective fetal growth restriction and twin anemia-polycythemia sequence (40.0% in ART pregnancies vs 31.6% in SC pregnancies, p = 0.291). At least one congenital anomaly in one twin was seen significantly more often in ART pregnancies (18.8% vs 8.1%, p = 0.004), especially congenital heart defects (16.3% vs 6.2%, p = 0.005). There were no other significant differences in neonatal outcomes between both groups, however, there were non-significant trends in gestational age at delivery (34 weeks in ART pregnancies vs 35 weeks, p = 0.078) and birthweight (1951 g ± 747 in ART pregnancies vs 2143 g ± 579, p = 0.066). Conclusion: This is the largest cohort study to date comparing maternal, chorionicity and neonatal complications between MC twin pregnancies after ART and after SC. Hypothyroidism was the only maternal condition occurring more frequently in pregnancies conceived after ART. There were no significant differences in chorionicity complications, in contrast to previously reported studies. While MC twins and ART pregnancies per se are known to be at risk for congenital heart defects, there seems to be a cumulative effect in MC pregnancies conceived after ART.

Molecular detection of Rickettsiales and a potential novel Ehrlichia species closely related to Ehrlichia chaffeensis in ticks (Acari: Ixodidae) from Shaanxi Province, China, in 2022 to 2023.

Important tick-borne diseases include spotted fever group Rickettsia (SFGR), Anaplasma, and Ehrlichia, which cause harm to animal and human health. Ixodidae are the primary vectors of these pathogens. We aimed to analyze the prevalence and genetic diversity of SFGR, Anaplasma, and Ehrlichia species in the Ixodidae in Shaanxi Province, China. Herein, 1,113 adult Ixodidae ticks were collected from domestic cattle and goats, and detected using nested PCR. A total of four Ixodidae species were collected and Ca. R. jingxinensis (20.58%, 229/1113), A. bovis (3.05%, 34/1113), A. capra (3.32%, 37/1113), A. marginale (0.18%, 2/1113), E. sp. Yonaguni138 (0.18%, 2/1113), and a potent novel Ehrlichia species named E. sp. Baoji96 (0.09%, 1/1113) were detected. A. marginale was detected for the first time in Rhipicephalus microplus. E. sp. Baoji96 was closely related to E. chaffeensis and was first identified in Haemaphysalis longicornis. In addition, co-infection with two Rickettsiales pathogens within an individual tick was detected in 10 (1.54%) ticks. This study provides a reference for the formulation of biological control strategies for ticks and tick-borne diseases in Shaanxi Province, and could lead to an improved control effect.

Knowledge, attitudes, and practices of Illinois medical professionals related to ticks and tick-borne disease.

Background: The rising incidence of tick-borne disease (TBD) underscores the importance of proficiency in TBD diagnosis. Clinicians' knowledge about vector ticks and TBDs in their area may influence whether patients are questioned about potential tick exposure and the consideration of diagnostic testing for TBDs. Objective: Our objective was to assess the knowledge, attitudes, and practices of Illinois clinicians towards ticks and TBDs. The study aimed to 1) identify predictors associated with knowledge, 2) identify knowledge gaps, and 3) evaluate attitudes and practices related to TBDs. Methods: A web-based knowledge, attitudes, and practices survey about Illinois ticks and TBDs was disseminated to physicians, mid-level practitioners, and nurses between August 2020 and February 2022. Poisson regression analysis was conducted to identify predictors of higher scores. Results: Of 346 respondents, 80% correctly identified Lyme disease as endemic to Illinois, and 95% were familiar with diagnostic testing for Lyme. Knowledge of other TBDs present in the state was highest among physicians, yet only 26% of physicians believed Rocky Mountain spotted fever (RMSF) to be present in Illinois, and only 17% believed ehrlichiosis to be endemic. Only 32% of physicians knew the cause of Alpha-gal syndrome and fewer than 18% were aware of available diagnostic testing. Tick or TBD-related education within the past two years was the most significant predictor of higher scores, increasing overall knowledge scores by 26% (RR 1.26, 95% CI 1.13-1.41) and increasing scores specific to TBDs by 42% (RR 1.42, 95% CI 1.19-1.69). Conclusion: Illinois clinicians were informed about Lyme disease but lacked knowledge of other TBDs endemic to the state, including RMSF, ehrlichiosis, and Alpha-gal syndrome. The strongest predictor of knowledge was tick/TBD training in the previous two years, highlighting the importance of frequent region-specific training on ticks and TBDs.

Phase-1 clinical study of tadalafil administered for selective fetal growth restriction in twin pregnancy.

BACKGROUND: Selective fetal growth restriction (sFGR) is a condition of twin pregnancy in which the development of one fetus is restricted, despite normal growth of the other fetus. A method of intrauterine therapy for sFGR does not currently exist. The only treatment for sFGR is to terminate the pregnancy before the FGR worsens. In twin pregnancies, maternal and intrauterine environments are common in both fetuses, thus a placental factor is considered the cause of FGR in fetuses. Tadalafil is a phosphodiesterase (PDE)-5 inhibitor that induces an increase in uterine blood flow by dilatation of blood vessels in cases of FGR with placental dysfunction, which improves FGR. PURPOSE: The aim of this study was to investigate the safety and maximum tolerated dose (MTD) of tadalafil administered for twin pregnancy (diamniotic-monochorionic twin or diamniotic-dichorionic twin). METHODS: In this phase I, open-label, dose-escalation trial, sequential patient cohorts (3 + 3 dose-escalation design) for twin pregnancy received tadalafil (20 or 40 mg/d) as a single dose by oral administration from the day they were diagnosed with sFGR, defined as estimated fetal weight (EFW) < 3% tiles, that is, -1.8 SD the mean EFW for gestational age (GA) to unacceptable toxicity or the day of delivery. This study evaluated the safety of maternally administered tadalafil for sFGR, examining maternal, fetal, and neonatal adverse events. Maternal adverse events were graded on the basis of the Common Terminology Criteria for Adverse Events v4.0. RESULTS: Six women with sFGR who were pregnant with twins were treated with tadalafil. There were no severe adverse events in either cohort, although the most common (≥3 patients) drug-related adverse events were headache and heart failure. The MTD of tadalafil among Japanese patients was 40 mg. CONCLUSIONS: Tadalafil has a manageable safety profile up to an MTD of 40 mg/d.

Whole genome miRNA profiling revealed miR-199a as potential placental pathogenesis of selective fetal growth restriction in monochorionic twin pregnancies.

INTRODUCTION: Placental-related mechanism of fetal growth restriction (FGR) is still unknown. Here we aimed to profile whole-genome miRNA between selective FGR twin (sFGR-T) and normally larger co-twin (sL-T) in monochorionic (MC) twin pregnancies and to further investigate effect of the miRNA on placental pathogenesis, including angiogenesis and mitochondrial functions. METHODS: MC twin pregnancies with or without sFGR were recruited, and their placental miRNAs were profiled (n = 3 vs 5). Ratio of placental miRNAs in the sFGR twin pairs (sFGR-T/sL-T) were calculated and compared to that in the control twin pairs (cS-T/cL-T). Differentially expressed miRNAs and associated markers were validated qRT-PCR, immunohistochemistry staining (n = 8 vs 13) and electron microscopy (n = 3 vs 3). RESULTS: Placental miR-199a-5p was significantly upregulated in sFGR-T (p = 0.004), which was validated by qRT-PCR (1.03 vs 0.56; p = 0.020). Compared to control twin pairs, ratio of CD31-positive vessels and volume density of vessels in sFGR twin pairs was lower (0.65 vs 0.92 and 18.7% vs 36.3%; both p < 0.001), while that of cyclooxygenase 2 (COX2)-positive trophoblast cells was higher (3.50 vs 2.22; p = 0.001), indicating an impaired angiogenesis and oxidative stress in the sFGR placenta. In addition, ratio of mitochondrial DNA (mtDNA) mitochondrial encoded NADH dehydrogenase 1 (MTND1) copy numbers (2.10 vs 0.90; p = 0.013), H-score ratios of mitochondrial markers citrate synthase (CS) and cytochrome c oxidase subunit 4 isoform 1 (COX4, 0.53 vs 0.95, p < 0.001; 0.29 vs 1.06, p < 0.001) in trophoblast cells of sFGR twin pairs were also altered significantly and correlated with angiogenesis. Furthermore, ratio of mitochondrial numbers per trophoblasts (8.67 vs 18.67; p = 0.006) and percentage of swollen mitochondria (84.33 vs 11.33; p = 0.003) were converted significantly, indicating mitochondrial damage. DISCUSSION: Our results suggested miR-199a-5p may play a role in the placental angiogenesis, oxidative stress and mitochondrial damage and dysfunction as an underlying pathogenesis of sFGR.

Seroprevalence of spotted fever group rickettsiae in canines along the United States-Mexico border.

Portions of northern Mexico are experiencing a re-emergence of Rocky Mountain spotted fever (RMSF), a tickborne disease caused by Rickettsia rickettsii, a member of the spotted fever group of rickettsiae (SFGR). Infection with R. rickettsii can result in serious and life-threatening illness in people and dogs. Canine seroprevalence has been used as a sentinel for human RMSF in previous studies. This study aims to quantify SFGR seroprevalence in canines in three northern Mexican states and identify risk factors associated with seropositivity. A total of 1,136 serum samples and 942 ticks were obtained from dogs participating in government sterilization campaigns and from animal control facilities in 14 Mexican cities in three states. SFGR antibodies were detected using indirect immunofluorescence antibody assays at titre values ≥1/64. Six per cent (69 dogs) showed antibodies to SFGR, with the highest seroprevalence reported in Baja California (12%), Coahuila (4%) and Sonora (4%). Dogs from Baja California had three times higher odds of having SFGR antibodies compared to dogs from Sonora (OR = 3.38, 95% CI, 1.81-6.37). Roughly one quarter (25%) of surveyed dogs were parasitized by ticks (Rhipicephalus sanguineus sensu lato) at the time of sample collection. A portion of collected ticks were tested for rickettsial DNA using polymerase chain reaction. Positive samples were then sequenced, showing evidence of SFGR including R. massiliae, R. parkeri and R. rickettsii. Dogs that spent the majority of time on the street, such as free-roaming or community-owned dogs, showed a greater risk of tick infestation, seropositivity, bearing seropositive ticks, and may play a pivotal role in the spread of SFGR among communities. Estimating the seroprevalence of SFGR in the canine population can help public health campaigns target high-risk communities for interventions to reduce human RMSF cases.

Serological and molecular evidence of Bartonella henselae in cats from Luanda city, Angola.

A total of 100 domestic cats from Luanda (Angola) were tested for the presence of antibodies against Bartonella henselae and spotted fever group of Rickettsia (SFGR) using indirect immunofluorescence assay (IFA). Molecular screening targeting the riboflavin synthase (ribC) gene for Bartonella and outer membrane protein B (ompB) gene for Rickettsia, using conventional PCR and sequencing was also performed in cat´s blood samples. Sixty-six percent of the cats from Luanda had IgG antibodies against Bartonella species but none of them had antibodies against SFGR. Of the total seroreactive cats for Bartonella henselae, 4.5% had an IgG titre of 64 (cut-off), 60.6% a titre of 128, 28.8% a titre of 256 and 6.1% a titre of 512. A statistically significant association was observed between seropositivity for Bartonella henselae and the lack of access to prophylaxis against ectoparasites (p = 0.018). Molecular detection and further sequence analysis of the positive amplicons allowed identification of Bartonella henselae in a 2-year old male cat. To the best of our knowledge this study confirms for the first time, the presence of Bartonela henselae circulating in domestic cats from Luanda. This fact call the attention for the possible cases of cat-scratch disease in humans.

Importation of exotic ticks and tick-borne spotted fever group rickettsiae into the United States by migrating songbirds.

Birds are capable of carrying ticks and, consequently, tick-transmitted microorganisms over long distances and across geographical barriers such as oceans and deserts. Ticks are hosts for several species of spotted fever group rickettsiae (SFGR), which can be transmitted to vertebrates during blood meals. In this study, the prevalence of this group of rickettsiae was examined in ticks infesting migratory songbirds by using polymerase chain reaction (PCR). During the 2009 and 2010 spring migration season, 2064 northward-migrating passerine songbirds were examined for ticks at Johnson Bayou, Louisiana. A total of 91 ticks was removed from 35 individual songbirds for tick species identification and spotted fever group rickettsia detection. Ticks were identified as Haemaphysalis juxtakochi (n=38, 42%), Amblyomma longirostre (n=22, 24%), Amblyomma nodosum (n=17, 19%), Amblyomma calcaratum (n=11, 12%), Amblyomma maculatum (n=2, 2%), and Haemaphysalis leporispalustris (n=1, 1%) by comparing their 12S rDNA gene sequence to homologous sequences in GenBank. Most of the identified ticks were exotic species originating outside of the United States. The phylogenetic analysis of the 71 ompA gene sequences of the rickettsial strains detected in the ticks revealed the occurrence of 6 distinct rickettsial genotypes. Two genotypes (corresponding to a total of 28 samples) were included in the Candidatus Rickettsia amblyommii clade (less than 1% divergence), 2 of them (corresponding to a total of 14 samples) clustered with Rickettsia sp. "Argentina" with less than 0.2% sequence divergence, and 2 of them (corresponding to a total of 27 samples), although closely related to the R. parkeri-R. africae lineage (2.50-3.41% divergence), exhibited sufficient genetic divergence from its members to possibly constitute a new rickettsial genotype. Overall, there does not seem to be a specific relationship between exotic tick species, the rickettsiae they harbor, or the reservoir competence of the corresponding bird species.

Multispacer Typing (MST) of Spotted Fever Group Rickettsiae Isolated from Humans and Rats in Chengmai County, Hainan Province, China.

Spotted fever caused by spotted fever group rickettsiae (SFGR) is found throughout China. During 2007-2008, 28 human SFGR isolates and 34 rat SFGR isolates including 15 isolates from Rattus fulvescens, 5 isolates from R. edwardsi, 7 isolates from Callosciurus erythraeus roberti and 7 isolates from Dremomys rufigenis) were obtained from L929 cell culture. Previous research indicated that the 62 strains of SFGR mentioned above shared not only the same serophenotype but also 100% of identity sequences of 16S rRNA, gltA, ompA, groEL and 17KD, which enabled us to apply multispacer typing (MST) to the 62 SFGR isolates in the study. Six primer pairs, which were used for typing of Rickettsia rickettsii and Rickettsia conorii, were chosen, and the results exhibited greater nucleotide polymorphisms among the 62 isolates tested. A total of 48 distinct genotypes were identified. The dominant genotype, represented by h3 isolates, accounted for 21.7% (13/60) of the isolates tested, and the remaining 47 genotypes were all unique. Phylogenetic analysis showed that all the 48 genotypes could be classified in the same clade, while the genetically related strain, R. heilongjiangensis, was close but not the same as the cluster. We concluded that the genetically diverse of spotted fever group rickettsiae strains are endemic in Chengmai County, Hainan Province, China.