Single-cell RNA sequencing identifies molecular targets associated with poor in vitro maturation performance of oocytes collected from ovarian stimulation.

STUDY QUESTION: What is the transcriptome signature associated with poor performance of rescue IVM (rIVM) oocytes and how can we rejuvenate them? SUMMARY ANSWER: The GATA-1/CREB1/WNT signalling axis was repressed in rIVM oocytes, particularly those of poor quality; restoration of this axis may produce more usable rIVM oocytes. WHAT IS KNOWN ALREADY: rIVM aims to produce mature oocytes (MII) for IVF through IVM of immature oocytes collected from stimulated ovaries. It is not popular due to limited success rate in infertility treatment. Genetic aberrations, cellular stress and the absence of cumulus cell support in oocytes could account for the failure of rIVM. STUDY DESIGN, SIZE, DURATION: We applied single-cell RNA sequencing (scRNA-seq) to capture the transcriptomes of human in vivo oocytes (IVO) (n = 10) from 7 donors and rIVM oocytes (n = 10) from 10 donors. The effects of maternal age and ovarian responses on rIVM oocyte transcriptomes were also studied. In parallel, we studied the effect of gallic acid on the maturation rate of mouse oocytes cultured in IVM medium with (n = 84) and without (n = 85) gallic acid. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human oocytes were collected from donors aged 28-41 years with a body mass index of <30. RNA extraction, cDNA generation, library construction and sequencing were performed in one preparation. scRNA-seq data were then processed and analysed. Selected genes in the rIVM versus IVO comparison were validated by quantitative real-time PCR. For the gallic acid study, we collected immature oocytes from 5-month-old mice and studied the effect of 10-µM gallic acid on their maturation rate. MAIN RESULTS AND THE ROLE OF CHANCE: The transcriptome profiles of rIVM/IVO oocytes showed distinctive differences. A total of 1559 differentially expressed genes (DEGs, genes with at least 2-fold change and adjusted P < 0.05) were found to be enriched in metabolic processes, biosynthesis and oxidative phosphorylation. Among these DEGs, we identified a repression of WNT/ß-catenin signalling in rIVM when compared with IVO oocytes. We found that oestradiol levels exhibited a significant age-independent correlation with the IVO mature oocyte ratio (MII ratio) for each donor. rIVM oocytes from women with a high MII ratio were found to have over-represented cellular processes such as anti-apoptosis. To further identify targets that contribute to the poor clinical outcomes of rIVM, we compared oocytes collected from young donors with a high MII ratio with oocytes from donors of advanced maternal age and lower MII ratio, and revealed that CREB1 is an important regulator. Thus, our study identified that GATA-1/CREB1/WNT signalling was repressed in both rIVM oocytes versus IVO oocytes and in rIVM oocytes of lower versus higher quality. Consequently we investigated gallic acid, as a potential antioxidant substrate in human rIVM medium, and found that it increased the mouse oocyte maturation rate by 31.1%. LARGE SCALE DATA: Raw data from this study can be accessed through GSE158539. LIMITATIONS, REASONS FOR CAUTION: In the rIVM oocytes of the high- and low-quality comparison, the number of samples was limited after data filtering with stringent selection criteria. For the oocyte stage identification, we were unable to predict the presence of oocyte spindle, so polar body extrusion was the only indicator. WIDER IMPLICATIONS OF THE FINDINGS: This study showed that GATA-1/CREB1/WNT signalling was repressed in rIVM oocytes compared with IVO oocytes and was further downregulated in low-quality rIVM oocytes, providing us the foundation of subsequent follow-up research on human oocytes and raising safety concerns about the clinical use of rescued oocytes. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Collaborative Research Fund, Research Grants Council, C4054-16G, and Research Committee Funding (Research Sustainability of Major RGC Funding Schemes), The Chinese University of Hong Kong. The authors have no conflicts of interest to declare.

Ruptured Superficial Femoral Artery Anastomotic Pseudoaneurysm after 30 Years.

INTRODUCTION: Anastomotic pseudoaneurysms are a complication of vascular reconstructive surgery with the majority in the femoral region. Although rare, ruptured femoral anastomotic pseudoaneurysms have high mortality and require emergency surgery. CASE PRESENTATION: A 60-year-old male with a history of a left leg crush injury was treated with a superficial femoral artery interposition vein graft 30 years ago. He presented nowadays with a three-day history of severe pain in his left thigh. CT angiography demonstrated a ruptured anastomotic pseudoaneurysm with contrast extravasation into an intramuscular hematoma. He had significant scarring from his previous surgeries which made the leg hostile for an open repair. Therefore, percutaneous access selectively cannulated the left iliofemoral vasculature. An angiogram showed a distal superficial femoral artery pseudoaneurysm. Subsequently, two 10mmx15cm Viabahn covered stents (Gore & Associates, Flagstaff, AZ) were placed bridging healthy superficial femoral artery. A completion angiogram demonstrated no extravasation into the pseudoaneurysm. The patient recovered and was discharged home two days postoperatively. CONCLUSION: Ruptured femoral anastomotic pseudoaneurysms are traditionally repaired with open pseudoaneurysm excision and arterial reconstruction, although endovascular repair has been reported. Furthermore, most femoral anastomotic pseudoaneurysms form less than 10 years after initial operation. We present a unique case of ruptured superficial femoral artery pseudoaneurysm, 30 years after the initial operation. Endovascular stents offer effective treatment for ruptured anastomotic pseudoaneurysms.

Case reports of atrial and pericardial rupture from blunt cardiac trauma.

BACKGROUND: Blunt cardiac trauma is diagnosed in less than 10% of trauma patients and covers the range of severity from clinically insignificant myocardial contusions to lethal multi-chamber cardiac rupture. The most common mechanisms of injury include: motor vehicle collisions (MVC), pedestrians struck by motor vehicles and falls from significant heights. A severe complication from blunt cardiac trauma is cardiac chamber rupture with pericardial tear. It is an exceedingly rare diagnosis. A retrospective review identified only 0.002% of all trauma patients presented with this condition. Most patients with atrial rupture do not survive transport to the hospital and upon arrival diagnosis remains difficult. CASE PRESENTATION: We present two cases of atrial and pericardial rupture. The first case is a 33-year-old female involved in a MVC, who presented unresponsive, hypotensive and tachycardic. A left sided hemothorax was diagnosed and a chest tube placed with 1200 mL of bloody output. The patient was taken to the OR emergently. Intraoperatively, a laceration in the right pericardium and a 3 cm defect in the anterior, right atrium were identified. Despite measures to control hemorrhage and resuscitate the patient, the patient did not survive. The second case is a 58-year-old male involved in a high-speed MVC. Similar to the first case, the patient presented unresponsive, hypotensive and tachycardic with a left sided hemothorax. A chest tube was placed with 900 mL of bloody output. Based on the output and ongoing resuscitation requirements, the patient was taken to the OR. Intraoperatively, a 15 cm anterior pericardial laceration was identified. Through the defect, there was brisk bleeding from a 1 cm laceration on the left atrial appendage. The injury was debrided and repaired using a running 3-0 polypropylene suture over a Satinsky clamp. The patient eventually recovered and was discharged home. CONCLUSIONS: We present two cases of uncontained atrial and pericardial rupture from blunt cardiac trauma. Contained ruptures with an intact pericardium present as a cardiac tamponade while uncontained ruptures present with hemomediastinum or hemothorax. A high degree of suspicion is required to rapidly diagnose and perform the cardiorrhaphy to offer the best chance at survival.

Morphometric sex determination of Milky and Painted Storks in captivity.

Logistic regression was applied to develop a morphometric sexing method of two closely related stork species that were previously sexed through amplification of the CHD gene. Tarsus length (TL) and bill length (BL) measurements were recorded from captive populations of adult Milky Stork (Mycteria cinerea) (n = 60) and Painted Stork (Mycteria leucocephala) (n = 58) at Zoo Negara Malaysia. Despite having monomorphic plumages, both stork species exhibited normal sexual size dimorphism in which males were significantly larger than females in the tested variables. Based on logistic regression analysis, BL correctly classified the sex of sampled individuals from Painted and Milky stork with an overall predicted accuracy of 94.8 and 90.0%, respectively. However, TL measurements generated a lower predicted accuracy level of 86.2% and a same accuracy level of 90% on the sex classification of individuals from Painted and Milky stork, respectively. By comparing the measurements of both species, only the average BL measurements of the Milky storks were significantly lower than that of Painted storks (t-test, P80.001). The logistic regression equation in this study may serve as a simple and more practical option for sexing Milky and Painted storks for their breeding and conservation programmes.

An evolutionarily conserved nested gene pair - Mab21 and Lrba/Nbea in metazoan.

The embedding of one gene in another as a nested gene pair is a unique phenomenon of gene clustering in the metazoan genome. A gene-centric paralogous genomic sequence comparison strategy was used in this study to align these paralogous nested pairs, Mab21l2-Lrba and Mab21l1-Nbea, to identify the associated paralogous non-coding elements (pNEs) they shared. A majority of these pNEs in the Mab21l2-Lrba locus display tissue-specific enhancer activities recapitulating the expression profiles of Mab21l2 and Mab21l1. Since these enhancers are spread into the introns of Lrba, dissociation of the two genes will likely disrupt the function of at least one of them. Phylogenetic analysis of this complex locus in different species suggests that Mab21 was probably locked in the Lrba/Nbea intron in the ancestral metazoan species, in which the cis-elements uncovered in this study may act as a selective force to prevent the dissociation of this gene pair in vertebrates.

Calamine lotion: mimicking intramammary calcifications.

Detection of microcalcifications is important in detecting early breast cancer. We present a case of screening mammogram in which calamine lotion mimics intramammary calcifications.

mab-7 encodes a novel transmembrane protein that orchestrates sensory ray morphogenesis in C. elegans.

The tapered sensory rays of the male Caenorhabditis elegans are important for successful male/hermaphrodite copulation. A group of ram (ray morphology abnormal) genes encoding modifying enzymes and transmembrane protein have been reported as key regulators controlling ray morphogenesis. Here we report the characterization of another component essential for this morphogenetic process encoded by mab-7. This gene is active in the hypodermis, structural cells, the body seam and several head neurons. It encodes a novel protein with a hydrophobic region at the N-terminus, an EGF-like motif, an ShKT motif and a long C-terminal tail. All these domains are shown to be critical to MAB-7 activity except the EGF-like domain, which appears to be regulatory and dispensable. MAB-7 is shown to be a type II membrane protein, tethered on the cell surface by the N-terminal transmembrane domain with the remainder of the protein exposed to the extracellular matrix. Since ectopic mab-7 expression in any ray cell or even in touch neurons of non-ray lineage can rescue the mutant phenotype, mab-7 is probably acting non-autonomously. It may facilitate intercellular communication among ray cells to augment normal ray morphogenesis.

C. elegans SIN-3 and its associated HDAC corepressor complex act as mediators of male sensory ray development.

Mab21 gene family members are required for embryonic development and sensory organ formation in both invertebrates and vertebrates. However, their mechanistic role on differentiation is largely unexplored. We report here the isolation of SIN-3 as a MAB-21 interacting molecule. sin-3 is co-expressed with mab-21 in the ray structural cells and genetically interacts with mab-21 to control sensory organ development. Using pharmacological and RNAi approaches, we demonstrated that histone deacetylase and conserved SIN-3-associated components are required for ray patterning. Conserved physical interactions between these components were also observed, implicating the recruitment of HDAC complex by MAB-21/SIN-3 may occur to determine ray identity in males.

The species, sex, and stage specificity of a Caenorhabditis sex pheromone.

Four species in the ELEGANS group of subgenus the Caenorhabditis are distinguished by two very different mating systems: androdioecy in C. elegans and Caenorhabditis briggsae with males and self-fertilizing hermaphrodites and dioecy in Caenorhabditis remanei and Caenorhabditis sp. strain CB5161 with males and females. Using chemotaxis assays, we demonstrate that females secrete a potent sex pheromone that attracts males from a distance, whereas hermaphrodites do not. The female sex pheromone is not species-specific, with males of all four species attracted to both the C. remanei and Caenorhabditis sp. female sex pheromones. The pheromone is, however, sex-specific, with only females secreting the pheromone and attracting only males. Furthermore, the sex pheromone is stage-specific, with female secretion and male detection of the pheromone beginning near adulthood. Females lose their attractiveness immediately after mating but regain it several hours after mating ceases. Finally, the female somatic gonad is required for sex-pheromone production, and the male-specific cephalic neurons (CEM) are required for male response.

Postembryonic expression of Caenorhabditis elegans mab-21 and its requirement in sensory ray differentiation.

The male tail sensory rays in Caenorhabditis elegans are complex copulatory structures, the normal patterning of which requires a number of regulatory genes. Among them, mab-21 specifies the identity of sensory ray 6. By using green fluorescent protein reporters, we identify multiple cis-acting elements that control the developmental expression of mab-21. Traced with a functional mab-21:gfp gene driven by authentic regulatory sequences, mab-21 expression could be detected in hypodermal, neuronal, muscle, and ray cells. We showed here that the expression of mab-21 in the hypodermis and neuronal cells was dispensable for its function in ray 6. In contrast, its expression in the ray 6 structural cell and neurons as conferred by its 3' enhancer was crucial for determining the correct ray 6 identity.

Intraarterial chemotherapy for brain tumors by using a spatial dose fractionation algorithm and pulsatile delivery.

PURPOSE: To evaluate the cause of complications in intraarterial chemotherapy for brain tumors and validate a dosage algorithm based on arterial territory. MATERIALS AND METHODS: Four hundred sixty-two procedures were performed in 113 patients. Technique included pulsatile infusion of a chemotherapeutic agent. Dosage was calculated per hemisphere and divided per arterial territory according to a spatial dose fractionation algorithm based on the vascular territories of major cerebral arteries: middle cerebral artery, 60%; anterior cerebral artery, 20%; posterior cerebral artery, 15%; and perforator arteries, 5%. Hospital charts of all patients were retrospectively reviewed for complications, with specific attention given to the angiograms to determine a cause. Then, subgroup analysis of the chemotherapy protocol with the largest patient population was performed to evaluate predictors of complications. RESULTS: Six (1.3%) complications were asymptomatic; 12 (2.6%), transient neurologic; three (0.6%), permanent minor neurologic; three (0.6%), permanent major neurologic; and 32 (7.0%), seizures. In the subgroup analysis, the hemispheric dose administered according to the algorithm was strongly predictive of seizure and neurologic deficit. CONCLUSION: Neurotoxicity of intraarterial cerebral chemotherapy can be minimized by using pulsatile injection and the described spatial dose fractionation algorithm.

Embryonic XMab21l2 expression is required for gastrulation and subsequent neural development.

Cell fate determining gene mab-21 regulates the proper establishment of neural cell fate and sensory organ identity in nematode. Mammalian homologs of mab-21 have also been implicated to play critical roles in mid-, hindbrain and craniofacial differentiation. We report here the isolation of a mab-21 homolog, XMab21l2, from Xenopus. We showed that its expression in Xenopus was initiated at gastrulation and prominent signal was detected in neurulating embryos at the neural tube, the optic tissue, the developing midbrain, and the pharyngeal pouches. We demonstrated by RNA interference (RNAi), together with other antisense approaches, that XMab21l2 expression is required for the completion of gastrulation and subsequent neural development.

Expression of ram-5 in the structural cell is required for sensory ray morphogenesis in Caenorhabditis elegans male tail.

Tissue morphogenesis requires complex cellular interaction and communication. The sensory ray in the Caenorhabditis elegans male tail has a simple cellular make-up and a non-essential function, thus providing an ideal model for studying the mechanisms guiding morphogenesis. We present here the analysis of a novel gene, ram-5, mutations of which are characterized by abnormal lumpy rays in the male tail. Microscopic analysis and behavioral studies revealed that lumpy rays contain operational sensory neurons. However, abnormalities were observed in the hypodermis and structural cells as well as in appositions between these two cell types. Molecular cloning and expression studies revealed that the ram-5 gene encodes a transmembrane protein localized in sensory ray support cells, the structural cells. Expression of ram-5 in these cells is required for normal ray morphogenesis. ram-5-dependent cell-cell communication is implicated in organizing the structural cell and the hypodermis, potentially through adhesion at the structural cell-hypodermal cell border.

Mutations with sensory ray defect unmask cuticular glycoprotein antigens in Caenorhabditis elegans male tail.

Caenorhabditis elegans male tail has nine pairs of bilaterally symmetric ray processes extended into a cuticular fan. The formation of these structures involves both cell lineage differentiation and cellular morphogenesis. Nine mutations were examined, all of which presented an amorphous ray phenotype. Glycoconjugates carrying an N-acetylglucosamine (GlcNAc) epitope were detected at a high level in their male bursa. It was shown that these antigens are not responsible for the morphological defects. It was further demonstrated that these ram and mab gene products represent critical components for male tail cuticle organization. Mutations of them abolish the integrity of the male bursal cuticle and unmask the underlying GlcNAc epitope.

Prognostic factors in recurrent glioblastoma multiforme and anaplastic astrocytoma treated with selective intra-arterial chemotherapy.

BACKGROUND AND PURPOSE: Factors predictive of primary brain tumor outcome have been studied extensively, although the prognostic value of radiologic data, such as MR imaging and angiographic characteristics, has not been studied in depth. The purpose of this study was to determine whether radiologic data were prognostic factors among patients with recurrent glioblastoma multiforme and anaplastic astrocytoma treated with selective intra-arterial chemotherapy. METHODS: Forty-six patients were enrolled in a Phase II study of intra-arterial chemotherapy with carboplatin and Cereport (Alkermes Inc.; Cambridge, MA), a bradykinin analog that selectively increases permeability of the blood-tumor barrier. MR imaging volumes of enhancing tumor, resection cavity, and T2 signal abnormality were measured with T1-weighted and T2-weighted sequences. Volumes were analyzed individually and in various combinations. Tumor vascularity was graded on angiograms. Outcome was measured by time to tumor progression and survival. RESULTS: Of 46 patients included in this study, 41 underwent evaluation. Thirty were male and 11 were female; mean age was 48.5 years. Karnofsky scores ranged from 70 to 100. Thirty-two patients had glioblastoma multiforme, whereas nine had anaplastic astrocytoma. Twenty-eight patients had tumor progression and 13 had stable disease. Twenty-three patients died after an average of 205 days; 18 were surviving at an average of 324 days from the start of intra-arterial chemotherapy. In multivariate analysis, time from diagnosis to intra-arterial chemotherapy was predictive both of time to tumor progression and survival. Net tumor volume and vascularity also were significant for survival. Age, Karnofsky performance status, histologic findings, gender, MR imaging area, resection cavity volume, T2 signal abnormality volume, and various combined volumes were not significant. CONCLUSION: If confirmed by further studies, radiologic factors such as tumor volume and angiographic vascularity should be considered in design and stratification of future chemotherapy trials.

Stress-induced phenocopy of C. elegans defines functional steps of sensory organ differentiation.

The differentiation of male specific sensory rays in the nematode Caenorhabditis elegans is a complex process regulated by multiple genetic components. A novel approach with heat shock treatment was employed to show that multistep regulation is involved in this process. Intervention in this stepwise regulation resulted in phenocopy of specific gene mutations. The results suggest that differential gene function acting at a precise time frame is necessary to guide the normal differentiation of sensory rays.

Genomic cloning and chromosomal localization of the mouse Mab21l2 locus.

A second mouse gene related to the nematode mab-21 gene has been isolated. This gene, Mab21l2, encodes a transcript with an open reading frame discretely organized in a single exon. It shares 93.3% and 55.5% amino acid identity with the human and worm mab-21 respectively. FISH analysis determined that this gene is on chromosome 3 at a position between bands 3E3 and 3F1. This newly identified mouse gene will be useful in future examination of mab-21 gene function in vertebrate models.

Developmental expression of Mab21l2 during mouse embryogenesis.

mab-21 has been identified as a critical component required for sensory organ identity establishment in Caenorhabditis elegans. [Chow, K.L., Emmons, S.W., 1994. Development 120, 2579-2592; Chow, E. L., Hall, D.H., Emmons, S.W., 1995. Development 121, 3615-3625]. Human and mouse homologs of this gene have been isolated and their transcripts are predominantly detected in the eye and cerebellum [Margolis, R.L., Stine, O.C., McInnis, M.G., et al., 1996. Hum. Mol. Genet 5, 607-616; Mariani, M., Corradi, A., Baldessari, D., et al., 1998. Mech. Dev. 79, 131-135. We report here the expression profile of a second murine mab-21 homolog, Mab21l2 [Wong, R.L.Y., Wong, H.T., Chow, K.L., 1999. Cyto. Cell Genet., [in press]. Whole mount in situ hybridization data from embryonic day 8.5 to day 15 revealed that Mab21l2 expression patterns partially overlapped with that of Mab21l1. In addition, its strong expression in the mid- and hindbrain, otic vesicle, optic vesicle, maxillary and mandibular process, paraxial mesoderm, dorsal midline, limb bud and developing digits suggest that Mab21l2 has more diverse functions in vertebrate development.

Regulation of body length and male tail ray pattern formation of Caenorhabditis elegans by a member of TGF-beta family.

We have identified a new member of the TGF-beta superfamily, CET-1, from Caenorhabditis elegans, which is expressed in the ventral nerve cord and other neurons. cet-1 null mutants have shortened bodies and male tail abnormal phenotype resembling sma mutants, suggesting cet-1, sma-2, sma-3 and sma-4 share a common pathway. Overexpression experiments demonstrated that cet-1 function requires wild-type sma genes. Interestingly, CET-1 appears to affect body length in a dose-dependent manner. Heterozygotes for cet-1 displayed body lengths ranging between null mutant and wild type, and overexpression of CET-1 in wild-type worms elongated body length close to lon mutants. In male sensory ray patterning, lack of cet-1 function results in ray fusions. Epistasis analysis revealed that mab-21 lies downstream and is negatively regulated by the cet-1/sma pathway in the male tail. Our results show that cet-1 controls diverse biological processes during C. elegans development probably through different target genes.

The mab-21 gene of Caenorhabditis elegans encodes a novel protein required for choice of alternate cell fates.

The gene mab-21, which encodes a novel protein of 386 amino acids, is required for the choice of alternate cell fates by several cells in the C. elegans male tail. Three cells descended from the ray 6 precursor cell adopt fates of anterior homologs, and a fourth, lineally unrelated hypodermal cell is transformed into a neuroblast. The affected cells lie together in the lateral tail epidermis, suggesting that mab-21 acts as part of a short-range pattern-formation mechanism. Each of the changes in cell fate brought about by mab-21 mutants can be interpreted as a posterior-to-anterior homeotic transformation. mab-21 mutant males and hermaphrodites have additional pleiotropic phenotypes affecting movement, body shape and fecundity, indicating that mab-21 has functions outside the tail region of males. We show that the three known alleles of mab-21 are hypomorphs of a new gene. Mosaic analysis revealed that mab-21 acts cell autonomously to specify the properties of the sensory ray, but non-autonomously in the hypodermal versus neuroblast cell fate choice. Presence of cell signalling in the choice of the neuroblast fate was confirmed by cell ablation experiments. Mutations in mab-21 were shown previously to be genetic modifiers of the effects of HOM-C/Hox gene mutations on ray identity specification. The results presented here support the conclusion that mab-21 acts as part of a mechanism required for correct cell fate choice, possibly involving the function of HOM-C/Hox genes in several body regions.