(Cost-)effectiveness of personalised multimodal physiotherapy compared to surgery in patients with cervical radiculopathy: A systematic review.

RATIONALE: Cervical radiculopathy is initially typically managed conservatively. Surgery is indicated when conservative management fails or with severe/progressive neurological signs. Personalised multimodal physiotherapy could be a promising conservative strategy. However, aggregated evidence on the (cost-)effectiveness of personalised multimodal physiotherapy compared to surgery with/without post-operative physiotherapy is lacking. AIM/OBJECTIVES: To systematically summarise the literature on the (cost-)effectiveness of personalised multimodal physiotherapy compared to surgery with or without post-operative physiotherapy in patients with cervical radiculopathy. METHODS: PubMed, Embase, CINAHL, PsycINFO and Web of Science were searched from inception to 1st of March 2023. Primary outcomes were effectiveness regarding costs, arm pain intensity and disability. Neck pain intensity, perceived recovery, quality of life, neurological symptoms, range-of-motion, return-to-work, medication use, (re)surgeries and adverse events were considered secondary outcomes. Randomised clinical trials comparing personalised multimodal physiotherapy versus surgical approaches with/without post-operative physiotherapy were included. Two independent reviewers performed study selection, data-extraction, and risk of bias assessment using the Cochrane RoB 2 and Consolidated Health Economic Evaluation Reporting Standards statement. Certainty of the evidence was determined using Grading of Recommendations, Assessment, Development and Evaluations. RESULTS: From 2109 records, eight papers from two original trials, with 117 participants in total were included. Low certainty evidence showed there were no significant differences on arm pain intensity and disability, except for the subscale 'heavy work' related disability (12 months) and disability at 5-8 years. Cost-effectiveness was not assessed. There was low certainty evidence that physiotherapy improved significantly less on neck pain intensity, sensory loss and perceived recovery compared to surgery with/without physiotherapy. Low certainty evidence showed there were no significant differences on numbness, range of motion, medication use, and quality of life. No adverse events were reported. CONCLUSION: Considering the clinical importance of accurate management recommendations and the current low level of certainty, high-quality cost-effectiveness studies are needed.

Human PKD1 sequences form R-loop structures in vitro.

Autosomal dominant polycystic kidney disease results from the loss of the PKD1 gene product, polycystin 1. Regulatory mechanisms are unresolved, but an apparent G/C sequence bias in the gene is consistent with co-transcriptional R-loop formation. R-loops regulate gene expression and stability, and they form when newly synthesized RNA extensively pairs with the template DNA to displace the non-template strand. In this study, we tested two human PKD1 sequences for co-transcriptional R-loop formation in vitro. We observed RNase H-sensitive R-loop formation in intron 1 and 22 sequences, but only in one transcriptional orientation. Therefore, R-loops may participate in PKD1 expression or stability.

High-fat diet during pregnancy promotes fetal skeletal muscle fatty acid oxidation and insulin resistance in an ovine model.

Maternal diet during pregnancy is associated with offspring metabolic risk trajectory in humans and animal models, but the prenatal origins of these effects are less clear. We examined the effects of a high-fat diet (HFD) during pregnancy on fetal skeletal muscle metabolism and metabolic risk parameters using an ovine model. White-faced ewes were fed a standardized diet containing 5% fat wt/wt (CON), or the same diet supplemented with 6% rumen-protected fats (11% total fat wt/wt; HFD) beginning 2 wk before mating until midgestation (GD75). Maternal HFD increased maternal weight gain, fetal body weight, and low-density lipoprotein levels in the uterine and umbilical circulation but had no significant effects on circulating glucose, triglycerides, or placental fatty acid transporters. Fatty acid (palmitoylcarnitine) oxidation capacity of permeabilized hindlimb muscle fibers was >50% higher in fetuses from HFD pregnancies, whereas pyruvate and maximal (mixed substrate) oxidation capacities were similar to CON. This corresponded to greater triacylglycerol content and protein expression of fatty acid transport and oxidation enzymes in fetal muscle but no significant effect on respiratory chain complexes or pyruvate dehydrogenase expression. However, serine-308 phosphorylation of insulin receptor substrate-1 was greater in fetal muscle from HFD pregnancies along with c-jun-NH2 terminal kinase activation, consistent with prenatal inhibition of skeletal muscle insulin signaling. These results indicate that maternal high-fat feeding shifts fetal skeletal muscle metabolism toward a greater capacity for fatty acid over glucose utilization and favors prenatal development of insulin resistance, which may predispose offspring to metabolic syndrome later in life.NEW & NOTEWORTHY Maternal diet during pregnancy is associated with offspring metabolic risk trajectory in humans and animal models, but the prenatal origins of these effects are less clear. This study examined the effects of a high-fat diet during pregnancy on metabolic risk parameters using a new sheep model. Results align with findings previously reported in nonhuman primates, demonstrating changes in fetal skeletal muscle metabolism that may predispose offspring to metabolic syndrome later in life.

Corticotropin releasing hormone is present in the feline placenta and maternal serum.

Background: In women, placental corticotropin releasing hormone (CRH) can be detected in maternal blood throughout pregnancy and is important in the regulation of the timing of parturition. However, its role in other mammalian species is unclear. In fact, very little is known about the presence and localization of CRH in placentas other than human. In this study we report for the first time the presence of CRH in feline placenta and maternal serum. Methods: Presence of CRH mRNA and protein was assessed using RT-PCR and Western blot, respectively, in at term domestic cat placentas opportunistically obtained at a local animal shelter and spay clinic. In addition, CRH localization within the placenta was demonstrated via immunohistochemistry. Finally, presence of CRH in maternal blood from early (¾21 days) and mid (25-35 days) stages of pregnancy was investigated by ELISA. Results: CRH mRNA and protein were detected in feline placentas, and localized to larger decidual cells and fetal trophoblast cells, including the binucleate cells. CRH was detectable in maternal blood collected from early-stage pregnancies, and amounts significantly increased in mid-gestation samples. Conclusion: This is the first report on the presence and localization of CRH in the feline placenta, and its increase in maternal serum during the first half of pregnancy. These data lay the foundation for future studies to determine if CRH can be used as potential novel marker for early pregnancy diagnosis, determination, and monitoring in felids, and could greatly increase efficiency and success in zoo breeding programs utilizing artificial reproductive technologies for endangered feline species.

Characterization of trophoblast mitochondrial function and responses to testosterone treatment in ACH-3P cells.

INTRODUCTION: Trophoblast mitochondria play important roles in placental energy metabolism, physiology and pathophysiology. Hyperandrogenism has been associated with mitochondrial abnormalities in pregnancy disorders such as pre-eclampsia, gestational diabetes, and intrauterine growth restriction, but the direct impacts of androgen exposure on placental mitochondrial function are unknown. Given the inherent limitations of studying the human placenta during pregnancy, trophoblast cell lines are routinely used to model placental biology in vitro. The aim of this study was to characterize mitochondrial respiratory function in four commonly used trophoblast cell lines to provide a basis for selecting one well-suited to investigating the impact of androgens on trophoblast mitochondrial function. METHODS: Androgen receptor expression, mitochondrial respiration (JO2) and reactive oxygen species (ROS) release rates were evaluated in three human trophoblast cell lines (ACH-3P, BeWo and Swan-71) and one immortalized ovine trophoblast line (iOTR) under basal and substrate-stimulated conditions using high-resolution fluorespirometry. RESULTS: ACH-3P cells exhibited the greatest mitochondrial respiratory capacity and coupling efficiency of the four trophoblast lines tested, along with robust expression of androgen receptor protein that was found to co-localize with mitochondria by immunoblot and immunofluorescence. Acute testosterone administration (10 nM) tended to decrease ACH-3P mitochondrial JO2 and increase ROS release, while chronic (7 days) testosterone exposure increased expression of mitochondrial proteins, JO2, and ROS release. DISCUSSION: These studies establish ACH-3P as a suitable cell line for investigating trophoblast mitochondrial function, and provide foundational evidence supporting links between hyperandrogenism and placental mitochondrial ROS production with potential relevance to several common pregnancy disorders.

Clinical outcome in decompression alone versus decompression and instrumented fusion in patients with isthmic spondylolisthesis: a prospective cohort study.

OBJECTIVE: In the surgical treatment of isthmic spondylolisthesis, it is debatable whether instrumented fusion is mandatory in addition to decompression. The objective of this prospective cohort study was to assess the long-term effect of decompression alone compared with decompression and instrumented fusion in patients who underwent the intervention of their own preference. The results were compared with those in patients who underwent randomly assigned treatment. METHODS: The authors performed a prospective observational multicenter cohort study, including 91 patients with isthmic spondylolisthesis assigned to undergo either decompression alone (n = 44) or decompression and fusion (n = 47). The main outcomes were the Roland-Morris Disability Questionnaire (RDQ) scores and the patient's perceived recovery at the 2-year follow-up. Secondary outcomes were visual analog scale (VAS) leg pain and back pain scores and the reoperation rate. A meta-analysis was performed for data from this cohort study (n = 91) and from a randomized controlled trial (RCT) previously reported by the authors (n = 84). Subgroup analyses were performed on these combined data for age, sex, weight, smoking, and Meyerding grade. RESULTS: At the 12-week follow-up, improvements of RDQ scores were comparable for the two procedures (decompression alone [D group] 4.4, 95% CI 2.3-6.5; decompression and fusion [DF group] 5.8, 95% CI -4.3 to 1.4; p = 0.31). Likewise, VAS leg pain scores (D group 35.0, 95% CI 24.5-45.6; DF group 47.5, 95% CI 37.4-57.5; p = 0.09) and VAS back pain scores (D group 23.5, 95% CI 13.3-33.7; DF group 34.0, 95% CI 24.1-43.8; p = 0.15) were comparable. At the 2-year follow-up, there were no significant differences between the two groups in terms of scores for RDQ (difference -3.1, 95% CI -6.4 to 0.3, p = 0.07), VAS leg pain (difference -7.4, 95% CI -22.1 to 7.2, p = 0.31), and VAS back pain (difference -11.4, 95% CI -25.7 to 2.9, p = 0.12). In contrast, patient-perceived recovery from leg pain was significantly higher in the DF group (79% vs 51%, p = 0.02). Subgroup analyses did not demonstrate a superior outcome for decompression alone compared with decompression and fusion. Nine patients (20.5%) underwent reoperation in total, all in the D group. The meta-analysis including both the cohort and RCT populations yielded an estimated pooled mean difference in RDQ of -3.7 (95% CI -5.94 to -1.55, p = 0.0008) in favor of decompression and fusion at the 2-year follow-up. CONCLUSIONS: In patients with isthmic spondylolisthesis, at the 2-year follow-up, patients who underwent decompression and fusion showed superior functional outcome and perceived recovery compared with those who underwent decompression alone. No subgroups benefited from decompression alone. Therefore, decompression and fusion is recommended over decompression alone as a primary surgical treatment option in isthmic spondylolisthesis.

Peptidylarginine deiminase enzymes and citrullinated proteins in female reproductive physiology and associated diseases†.

Citrullination, the post-translational modification of arginine residues, is catalyzed by the four catalytically active peptidylarginine deiminase (PAD or PADI) isozymes and alters charge to affect target protein structure and function. PADs were initially characterized in rodent uteri and, since then, have been described in other female tissues including ovaries, breast, and the lactotrope and gonadotrope cells of the anterior pituitary gland. In these tissues and cells, estrogen robustly stimulates PAD expression resulting in changes in levels over the course of the female reproductive cycle. The best-characterized targets for PADs are arginine residues in histone tails, which, when citrullinated, alter chromatin structure and gene expression. Methodological advances have allowed for the identification of tissue-specific citrullinomes, which reveal that PADs citrullinate a wide range of enzymes and structural proteins to alter cell function. In contrast to their important physiological roles, PADs and citrullinated proteins are also involved in several female-specific diseases including autoimmune disorders and reproductive cancers. Herein, we review current knowledge regarding PAD expression and function and highlight the role of protein citrullination in both normal female reproductive tissues and associated diseases.

Trophectoderm Transcriptome Analysis in LIN28 Knockdown Ovine Conceptuses Suggests Diverse Roles of the LIN28-let-7 Axis in Placental and Fetal Development.

The proper conceptus elongation in ruminants is critical for the successful placentation and establishment of pregnancy. We have previously shown that the trophectoderm-specific knockdown of LIN28A/B in day 9 ovine blastocysts resulted in increased let-7 miRNAs and reduced conceptus elongation at day 16 of gestation. In this current study, by transcriptome analysis of LIN28A knockdown (AKD) or LIN28B knockdown (BKD) trophectoderm (TE), we explored the downstream target genes of the LIN28-let-7 axis and their roles in the placental and fetal development. We identified 449 differentially expressed genes (DEGs) in AKD TE and 1214 DEGs in BKD TE compared to non-targeting control (NTC). Our analysis further revealed that 210 downregulated genes in AKD TE and 562 downregulated genes in BKD TE were the potential targets of let-7 miRNAs. Moreover, 16 downregulated genes in AKD TE and 57 downregulated and 7 upregulated genes in BKD TE were transcription factors. The DEGs in AKD and BKD TE showed enrichment in the biological processes and pathways critical for placental development and function, and fetal development and growth. The results of this study suggest the potential roles of the LIN28-let-7 axis in placental and fetal development beyond its involvement in trophoblast proliferation and conceptus elongation.

The Roles of Extracellular Vesicles and Organoid Models in Female Reproductive Physiology.

Culture model systems that can recapitulate the anatomy and physiology of reproductive organs, such as three-dimensional (3D) organoid culture systems, limit the cost and welfare concerns associated with a research animal colony and provide alternative approaches to study specific processes in humans and animals. These 3D models facilitate a greater understanding of the physiological role of individual cell types and their interactions than can be accomplished with traditional monolayer culture systems. Furthermore, 3D culture systems allow for the examination of specific cellular, molecular, or hormonal interactions, without confounding factors that occur with in vivo models, and provide a powerful approach to study physiological and pathological reproductive conditions. The goal of this paper is to review and compare organoid culture systems to other in vitro cell culture models, currently used to study female reproductive physiology, with an emphasis on the role of extracellular vesicle interactions. The critical role of extracellular vesicles for intercellular communication in physiological processes, including reproduction, has been well documented, and an overview of the roles of extracellular vesicles in organoid systems will be provided. Finally, we will propose future directions for understanding the role of extracellular vesicles in normal and pathological conditions of reproductive organs, utilizing 3D organoid culture systems.

Microdiscectomy for sciatica: reality check study of long-term surgical treatment effects of a Lumbosacral radicular syndrome (LSRS).

PURPOSE: It remains unclear whether the long-term results of RCTs regarding the outcome of microdiscectomy for lumbosacral radicular syndrome (LSRS) are generalizable. The purpose of this study was to determine the external validity of the outcome preseneted in RCTs after microdicectomy for LSRS in a patient cohort from a high-volume spine center. METHODS: Between 2007 and 2010, 539 patients had a single level microdiscectomy for MRI disk-related LSRS of whom 246 agreed to participate. Questionnaires included visual analogue scores (VAS) for leg pain, RDQ, OLBD, RAND-36 and Likert scores for recovery, leg and back pain. Lumbar re-operation(s) were registered. RESULTS: Mean age was 51.3, and median time of follow-up was 8.0 years. Re-operation occurred in 64 (26%) patients. Unfavorable perceived recovery was noted in 85 (35%) patients, and they had worse leg and back pain than the 161 (65%) patients with a favorable recovery: median VAS for leg pain 28/100 mm versus 2/100 mm and median VAS for back pain 9/100 mm versus 3/100 mm, respectively. In addition, the median RDQ and OLBD scores differed significantly: 9 vs 3 for RDQ and 26 vs 4 for OLBD, respectively (p < 0.001). CONCLUSION: In this cohort study, the long-term results after microdiscectomy for LSRS were less favorable than those obtained in RCTs, possibly caused by less strict patient selection than in RCTs. Our findings emphasize that patients, who do not meet the same inclusion criteria for surgery as in RCTs, should be informed about the chances of a less favorable result.

Effectiveness of an Annular Closure Device to Prevent Recurrent Lumbar Disc Herniation: A Secondary Analysis With 5 Years of Follow-up.

Importance: Patients with large annular defects following lumbar microdiscectomy for disc herniation are at increased risk for symptomatic recurrence and reoperation. Objective: To determine whether a bone-anchored annular closure device in addition to lumbar microdiscectomy resulted in lower reherniation and reoperation rates vs lumbar microdiscectomy alone. Design, Setting, and Participants: This secondary analysis of a multicenter randomized clinical trial reports 5-year follow-up for enrolled patients between December 2010 and October 2014 at 21 clinical sites. Patients in this study had a large annular defect (6-10 mm width) following lumbar microdiscectomy for treatment of lumbar disc herniation. Statistical analysis was performed from November to December 2020. Interventions: Lumbar microdiscectomy with additional bone-anchored annular closure device (device group) or lumbar microdiscectomy only (control group). Main Outcomes and Measures: The incidence of symptomatic reherniation, reoperation, and adverse events as well as changes in leg pain, Oswestry Disability Index, and health-related quality of life when comparing the device and control groups over 5 years of follow-up. Results: Among 554 randomized participants (mean [SD] age: 43 [11] years; 327 [59%] were men), 550 were included in the modified intent-to-treat efficacy population (device group: n = 272; 270 [99%] were White); control group: n = 278; 273 [98%] were White) and 550 were included in the as-treated safety population (device group: n = 267; control group: n = 283). The risk of symptomatic reherniation (18.8% [SE, 2.5%] vs 31.6% [SE, 2.9%]; P < .001) and reoperation (16.0% [SE, 2.3%] vs 22.6% [SE, 2.6%]; P = .03) was lower in the device group. There were 53 reoperations in 40 patients in the device group and 82 reoperations in 58 patients in the control group. Scores for leg pain severity, Oswestry Disability Index, and health-related quality of life significantly improved over 5 years of follow-up with no clinically relevant differences between groups. The frequency of serious adverse events was comparable between the treatment groups. Serious adverse events associated with the device or procedure were less frequent in the device group (12.0% vs 20.5%; difference, -8.5%; 95% CI, -14.6% to -2.3%; P = .008). Conclusions and Relevance: In patients who are at high risk of recurrent herniation following lumbar microdiscectomy owing to a large defect in the annulus fibrosus, this study's findings suggest that annular closure with a bone-anchored implant lowers the risk of symptomatic recurrence and reoperation over 5 years of follow-up. Trial Registration: ClinicalTrials.gov Identifier: NCT01283438.

Decompression alone versus decompression and instrumented fusion for the treatment of isthmic spondylolisthesis: a randomized controlled trial.

OBJECTIVE: The most advocated surgical technique to treat symptoms of isthmic spondylolisthesis is decompression with instrumented fusion. A less-invasive classical approach has also been reported, which consists of decompression only. In this study the authors compared the clinical outcomes of decompression only with those of decompression with instrumented fusion in patients with isthmic spondylolisthesis. METHODS: Eighty-four patients with lumbar radiculopathy or neurogenic claudication secondary to low-grade isthmic spondylolisthesis were randomly assigned to decompression only (n = 43) or decompression with instrumented fusion (n = 41). Primary outcome parameters were scores on the Roland Disability Questionnaire (RDQ), separate visual analog scales (VASs) for back pain and leg pain, and patient report of perceived recovery at 12-week and 2-year follow-ups. The proportion of reoperations was scored as a secondary outcome measure. Repeated measures ANOVA according to the intention-to-treat principle was performed. RESULTS: Decompression alone did not show superiority in terms of disability scores at 12-week follow-up (p = 0.32, 95% CI -4.02 to 1.34), nor in any other outcome measure. At 2-year follow-up, RDQ disability scores improved more in the fusion group (10.3, 95% CI 3.9-8.2, vs 6.0, 95% CI 8.2-12.4; p = 0.006, 95% CI -7.3 to -1.3). Likewise, back pain decreased more in the fusion group (difference: -18.3 mm, CI -32.1 to -4.4, p = 0.01) on a 100-mm VAS scale, and a higher proportion of patients perceived recovery as showing "good results" (44% vs 74%, p = 0.01). Cumulative probabilities for reoperation were 47% in the decompression and 13% in the fusion group (p < 0.001) at the 2-year follow-up. CONCLUSIONS: In patients with isthmic spondylolisthesis, decompression with instrumented fusion resulted in comparable short-term results, significantly better long-term outcomes, and fewer reoperations than decompression alone. Decompression with instrumented fusion is a superior surgical technique that should in general be offered as a first treatment option for isthmic spondylolisthesis, but not for degenerative spondylolisthesis, which has a different etiology.

A Potential Role and Contribution of Androgens in Placental Development and Pregnancy.

Successful pregnancy requires the establishment of a highly regulated maternal-fetal environment. This is achieved through the harmonious regulation of steroid hormones, which modulate both maternal and fetal physiology, and are critical for pregnancy maintenance. Defects in steroidogenesis and steroid signaling can lead to pregnancy disorders or even fetal loss. The placenta is a multifunctional, transitory organ which develops at the maternal-fetal interface, and supports fetal development through endocrine signaling, the transport of nutrients and gas exchange. The placenta has the ability to adapt to adverse environments, including hormonal variations, trying to support fetal development. However, if placental function is impaired, or its capacity to adapt is exceeded, fetal development will be compromised. The goal of this review is to explore the relevance of androgens and androgen signaling during pregnancy, specifically in placental development and function. Often considered a mere precursor to placental estrogen synthesis, the placenta in fact secretes androgens throughout pregnancy, and not only contains the androgen steroid nuclear receptor, but also non-genomic membrane receptors for androgens, suggesting a role of androgen signaling in placental function. Moreover, a number of pregnancy disorders, including pre-eclampsia, gestational diabetes, intrauterine growth restriction, and polycystic ovarian syndrome, are associated with abnormal androgen levels and androgen signaling. Understanding the role of androgens in the placenta will provide a greater understanding of the pathophysiology of pregnancy disorders associated with androgen elevation and its consequences.

MicroRNA Expression Changes and Integrated Pathways Associated With Poor Outcome in Canine Osteosarcoma.

MicroRNAs (miRNA) are small non-coding RNA molecules involved in post-transcriptional gene regulation. Deregulation of miRNA expression occurs in cancer, and miRNA expression profiles have been associated with diagnosis and prognosis in many cancers. Osteosarcoma (OS), an aggressive primary tumor of bone, affects ~10,000 dogs each year. Though survival has improved with the addition of chemotherapy, up to 80% of canine patients will succumb to metastatic disease. Reliable prognostic markers are lacking for this disease. miRNAs are attractive targets of biomarker discovery efforts due to their increased stability in easily obtained body fluids as well as within fixed tissue. Previous studies in our laboratory demonstrated that dysregulation of genes in aggressive canine OS tumors that participate in miRNA regulatory networks is reportedly disrupted in OS or other cancers. We utilized RT-qPCR in a 384-well-plate system to measure the relative expression of 190 miRNAs in 14 canine tumors from two cohorts of dogs with good or poor outcome (disease-free interval >300 or <100 days, respectively). Differential expression analysis in this subset guided the selection of candidate miRNAs in tumors and serum samples from larger groups of dogs. We ultimately identified a tumor-based three-miR Cox proportional hazards regression model and a serum-based two-miR model, each being able to distinguish patients with good and poor prognosis via Kaplan-Meier analysis with log rank test. Additionally, we integrated miRNA and gene expression data to identify potentially important miRNA-mRNA interactions that are disrupted in canine OS. Integrated analyses of miRNAs in the three-miR predictive model and disrupted genes from previous expression studies suggest the contribution of the primary tumor microenvironment to the metastatic phenotype of aggressive tumors.

MicroRNA-mRNA Networks in Pregnancy Complications: A Comprehensive Downstream Analysis of Potential Biomarkers.

Pregnancy complications are a major cause of fetal and maternal morbidity and mortality in humans. The majority of pregnancy complications initiate due to abnormal placental development and function. During the last decade, the role of microRNAs (miRNAs) in regulating placental and fetal development has become evident. Dysregulation of miRNAs in the placenta not only affects placental development and function, but these miRNAs can also be exported to both maternal and fetal compartments and affect maternal physiology and fetal growth and development. Due to their differential expression in the placenta and maternal circulation during pregnancy complications, miRNAs can be used as diagnostic biomarkers. However, the differential expression of a miRNA in the placenta may not always be reflected in maternal circulation, which makes it difficult to find a reliable biomarker for placental dysfunction. In this review, we provide an overview of differentially expressed miRNAs in the placenta and/or maternal circulation during preeclampsia (PE) and intrauterine growth restriction (IUGR), which can potentially serve as biomarkers for prediction or diagnosis of pregnancy complications. Using different bioinformatics tools, we also identified potential target genes of miRNAs associated with PE and IUGR, and the role of miRNA-mRNA networks in the regulation of important signaling pathways and biological processes.

Maternal Influenza A Virus Infection Restricts Fetal and Placental Growth and Adversely Affects the Fetal Thymic Transcriptome.

Maternal influenza A viral infections in humans are associated with low birth weight, increased risk of pre-term birth, stillbirth and congenital defects. To examine the effect of maternal influenza virus infection on placental and fetal growth, pregnant C57BL/6 mice were inoculated intranasally with influenza A virus A/CA/07/2009 pandemic H1N1 or phosphate-buffered saline (PBS) at E3.5, E7.5 or E12.5, and the placentae and fetuses collected and weighed at E18.5. Fetal thymuses were pooled from each litter. Placentae were examined histologically, stained by immunohistochemistry (IHC) for CD34 (hematopoietic progenitor cell antigen) and vascular channels quantified. RNA from E7.5 and E12.5 placentae and E7.5 fetal thymuses was subjected to RNA sequencing and pathway analysis. Placental weights were decreased in litters inoculated with influenza at E3.5 and E7.5. Placentae from E7.5 and E12.5 inoculated litters exhibited decreased labyrinth development and the transmembrane protein 150A gene was upregulated in E7.5 placentae. Fetal weights were decreased in litters inoculated at E7.5 and E12.5 compared to controls. RNA sequencing of E7.5 thymuses indicated that 957 genes were downregulated ≥2-fold including Mal, which is associated with Toll-like receptor signaling and T cell differentiation. There were 28 upregulated genes. It is concluded that maternal influenza A virus infection impairs fetal thymic gene expression as well as restricting placental and fetal growth.

A multiplex PCR genotyping assay to distinguish XX and XY tissues in sheep.

Sex is an important biological variable as many physiological as well as disease processes differ between females and males. The fundamental biological distinction between females and males starts with chromosomal sex, and the establishment of XX and XY cells and tissues. Polymerase Chain Reaction (PCR) is a simple and effective method to easily determine chromosomal or genetic sex of cells and tissues. The goal of this study was to develop a simple multiplex PCR genotyping assay to distinguish XX and XY tissues in sheep. Primers were designed to amplify a fragment of the autosomal gene myogenin (MYOG) and sex determining region on the Y chromosome (SRY). PCR analysis was performed on a variety of genomic DNA samples isolated from fetal sheep skeletal muscle, brain, liver, and placenta, and revealed a single 259 bp band for MYOG in XX females, and a 259 bp band for MYOG and a 167 bp band for SRY in XY males. Amplicons were clearly distinguishable by gel electrophoresis, and their sequences revealed 100% identity to the known ovine MYOG and SRY sequence. The reported multiplex PCR genotyping assay provides a rapid means to distinguish XX and XY sheep tissues using low volume samples.

The Role of LIN28-let-7-ARID3B Pathway in Placental Development.

Placental disorders are a major cause of pregnancy loss in humans, and 40-60% of embryos are lost between fertilization and birth. Successful embryo implantation and placental development requires rapid proliferation, invasion, and migration of trophoblast cells. In recent years, microRNAs (miRNAs) have emerged as key regulators of molecular pathways involved in trophoblast function. A miRNA binds its target mRNA in the 3'-untranslated region (3'-UTR), causing its degradation or translational repression. Lethal-7 (let-7) miRNAs induce cell differentiation and reduce cell proliferation by targeting proliferation-associated genes. The oncoprotein LIN28 represses the biogenesis of mature let-7 miRNAs. Proliferating cells have high LIN28 and low let-7 miRNAs, whereas differentiating cells have low LIN28 and high let-7 miRNAs. In placenta, low LIN28 and high let-7 miRNAs can lead to reduced proliferation of trophoblast cells, resulting in abnormal placental development. In trophoblast cells, let-7 miRNAs reduce the expression of proliferation factors either directly by binding their mRNA in 3'-UTR or indirectly by targeting the AT-rich interaction domain (ARID)3B complex, a transcription-activating complex comprised of ARID3A, ARID3B, and histone demethylase 4C (KDM4C). In this review, we discuss regulation of trophoblast function by miRNAs, focusing on the role of LIN28-let-7-ARID3B pathway in placental development.

Trophectoderm-Specific Knockdown of LIN28 Decreases Expression of Genes Necessary for Cell Proliferation and Reduces Elongation of Sheep Conceptus.

LIN28 inhibits let-7 miRNA maturation which prevents cell differentiation and promotes proliferation. We hypothesized that the LIN28-let-7 axis regulates proliferation-associated genes in sheep trophectoderm in vivo. Day 9-hatched sheep blastocysts were incubated with lentiviral particles to deliver shRNA targeting LIN28 specifically to trophectoderm cells. At day 16, conceptus elongation was significantly reduced in LIN28A and LIN28B knockdowns. Let-7 miRNAs were significantly increased and IGF2BP1-3, HMGA1, ARID3B, and c-MYC were decreased in trophectoderm from knockdown conceptuses. Ovine trophoblast (OTR) cells derived from day 16 trophectoderm are a useful tool for in vitro experiments. Surprisingly, LIN28 was significantly reduced and let-7 miRNAs increased after only a few passages of OTR cells, suggesting these passaged cells represent a more differentiated phenotype. To create an OTR cell line more similar to day 16 trophectoderm we overexpressed LIN28A and LIN28B, which significantly decreased let-7 miRNAs and increased IGF2BP1-3, HMGA1, ARID3B, and c-MYC compared to control. This is the first study showing the role of the LIN28-let-7 axis in trophoblast proliferation and conceptus elongation in vivo. These results suggest that reduced LIN28 during early placental development can lead to reduced trophoblast proliferation and sheep conceptus elongation at a critical period for successful establishment of pregnancy.

Transfer of MicroRNAs From Epididymal Epithelium to Equine Spermatozoa.

All epididymal regions are lined with multiple epithelial cell types, each with different functions to provide the luminal environment for spermatozoal maturation. Epithelial cells also create apical blebs, which are released from the apical surface via apocrine secretion and disintegrate in the lumen, thereby releasing epididymosomes. Epididymosomes transport proteins to spermatozoa and contain microRNAs. We hypothesized that epididymosomes also transfer miRNA from epididymal epithelium to spermatozoa. Quantitative real-time polymerase chain reaction was used to determine miRNA profiles of epididymal tissue from caput and cauda, epididymal spermatozoa from caput and cauda, and epididymosomes and from caput, proximal corpus, distal corpus, and cauda. Pathway analysis was performed using DIANA tools on the miRNA unique to caudal spermatozoa. We found 66 newly acquired miRNAs in spermatozoa located in the caudal epididymis. Predicted pathways targeted by these miRNAs suggest a role in cell motility and viability and factors in oocyte and embryo maturation and development. These findings suggest that miRNAs are transported to spermatozoa from epididymal epithelium via epididymosomes.