Targeted single molecule sequencing methodology for ovarian hyperstimulation syndrome

Background

One of the most significant issues surrounding next generation sequencing is the cost and the difficulty assembling short read lengths. Targeted capture enrichment of longer fragments using single molecule sequencing (SMS) is expected to improve both sequence assembly and base-call accuracy but, at present, there are very few examples of successful application of these technologic advances in translational research and clinical testing. We developed a targeted single molecule sequencing (T-SMS) panel for genes implicated in ovarian response to controlled ovarian hyperstimulation (COH) for infertility.

Results

Target enrichment was carried out using droplet-base multiplex polymerase chain reaction (PCR) technology (RainDance®) designed to yield amplicons averaging 1 kb fragment size from candidate 44 loci (99.8% unique base-pair coverage). The total targeted sequence was 3.18 Mb per sample. SMS was carried out using single molecule, real-time DNA sequencing (SMRT® Pacific Biosciences®), average raw read length = 1178 nucleotides, 5% of the amplicons >6000 nucleotides). After filtering with circular consensus (CCS) reads, the mean read length was 3200 nucleotides (97% CCS accuracy). Primary data analyses, alignment and filtering utilized the Pacific Biosciences® SMRT portal. Secondary analysis was conducted using the Genome Analysis Toolkit for SNP discovery l and wANNOVAR for functional analysis of variants. Filtered functional variants 18 of 19 (94.7%) were further confirmed using conventional Sanger sequencing. CCS reads were able to accurately detect zygosity. Coverage within GC rich regions (i.e.VEGFR; 72% GC rich) was achieved by capturing long genomic DNA (gDNA) fragments and reading into regions that flank the capture regions. As proof of concept, a non-synonymous LHCGR variant captured in two severe OHSS cases, and verified by conventional sequencing.

Conclusions

Combining emulsion PCR-generated 1 kb amplicons and SMRT DNA sequencing permitted greater depth of coverage for T-SMS and facilitated easier sequence assembly. To the best of our knowledge, this is the first report combining emulsion PCR and T-SMS for long reads using human DNA samples, and NGS panel designed for biomarker discovery in OHSS.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1451-2) contains supplementary material, which is available to authorized users.     

Kallistatin prevents ovarian hyperstimulation syndrome by regulating vascular leakage

Angiogenesis and increased permeability are essential pathological basis for the development of ovarian hyperstimulation syndrome (OHSS). Kallistatin (KS) is an endogenous anti‐inflammatory and anti‐angiogenic factor that participates in a variety of diseases, but its role in OHSS remains unknown. In this study, treating a human ovarian granulosa‐like tumour cell line KGN and human primary granulosa cells (PGCs) with human chorionic gonadotropin (hCG) reduced the expression of KS, but increased the expression of VEGF. Furthermore, we found that KS could attenuate the protein level of VEGF in both KGN cells and human PGCs. More interestingly, we observed that exogenous supplementation of KS significantly inhibited a series of signs of OHSS in mice, including weight gain, ovarian enlargement, increased vascular permeability and up‐regulation of VEGF expression. In addition, KS was proved to be safe on mice ovulation, progression of normal pregnancy and fetus development. Collectively, these findings demonstrated that KS treatment prevented OHSS, at least partially, through down‐regulating VEGF expression. For the first time, these results highlight the potential preventive value of KS in OHSS.     

The implication of single‐nucleotide polymorphisms in ovarian hyperstimulation syndrome

Ovarian hyperstimulation syndrome (OHSS) is an undesirable complication in the course of ovarian stimulation. This kind of stimulation is aimed at acquiring a sufficient number of high‐quality oocytes in in vitro fertilization (IVF). Whereas the predisposition to OHSS could be impacted by genetic polymorphisms in susceptible genes, the present study has been jointly conducted with an Iranian cohort to scrutinize its relevant implication. Genomic DNA was extracted from blood samples of patients with a normal ovarian response (NOR) or with OHSS. Samples were analyzed to detect polymorphisms MTHFR rs1801131, MTHFR rs1801133, AMHR2 rs2002555, LHCGR rs2293275, PGR rs10895068, and SERPINE1 rs1799889. Variations of MTHFR, AMHR2, LHCGR, and PGR genes were significantly associated with the developing OHSS. After correction for multiple analysis, this difference was not evident for PGR genotypes. The polymorphic alleles of MTHFR (rs1801131 C‐allele and rs1801133 T‐allele), AMHR2 (rs2002555 G‐allele), and LHCGR (rs2293275 G‐allele) were significantly more prevalent among patients with OHSS compared to those in the NOR group. In contrast, the minor allele of PGR single‐nucleotide polymorphism (SNP) (rs10895068, A‐allele) was more prominent among patients with a NOR than those with OHSS. No significant difference was observed in genotypes or alleles of SERPINE1 rs1799889. The observations indicated that the minor alleles of MTHFR, AMHR2, and LHCGR genes could be considered an independent risk factor in susceptibility to OHSS. Nevertheless, polymorphic allele in the PGR rs10895068 SNP contributes to preventing OHSS occurrence. Therefore, it can be argued that these genes have a significant impact on OHSS.     

Elective frozen-thawed embryo transfer (FET) in women at risk for ovarian hyperstimulation syndrome

Elective cryopreservation of cultured embryos has become a treatment option for women at risk for ovarian hyperstimulation syndrome (OHSS). The aim of our study was to investigate the outcome of elective cryopreservation and consecutive frozen-thawed embryo transfer (FET) in a large IVF clinic in Austria. A total of 6104 controlled ovarian hyperstimulation cycles (COH) were performed on 2998 patients including 200 patients (6.7%) who were undergoing elective cryopreservation and FET due to high risk of OHSS. We estimated the cumulative live birth rate using the Kaplan-Meier method and evaluated independent predictors for successful live births with a Cox model. A total of 270 frozen-thawed embryo transfers were performed on 200 patients with up to 4 transfers per patient. The first embryo transfer showed a live birth rate of 42.0%, the second transfer showed a cumulative rate of 58.5%. After a total of 4 FETs from the same COH cycle, a cumulative live birth rate of 61.0% per COH cycle could be achieved. Four cases of OHSS occurred amongst these patients (2.0%), all of them of moderate severity. Multivariate analysis identified maternal age, the use of assisted hatching and the number of embryos transferred at the blastocyst stage as independent predictors for cumulative live birth. Our study clearly suggests that elective FET is safe and shows excellent cumulative live birth rates. This concept can, therefore, be used to avoid the severe adverse events caused by COH and the inefficient use of cultured embryos.     

New insights into plant development in New England

This year, the biannually organized FASEB meeting 'Mechanisms in Plant Development' took place in August in Vermont, USA, organized by Martin Hulskamp (University of Koln, Koln, Germany) and John Schiefelbein (University of Michigan, Ann Arbor, MI, USA). The meeting covered numerous topics, ranging from patterning and differentiation to the evolution of developmental mechanisms. Despite apparent distinctions between the sessions, many of the talks were broad ranging and most highlighted unifying developmental concepts.     

Exosomes: New insights into cancer mechanisms

Exosomes are mobile extracellular vesicles with a diameter 40 to 150 nm. They play a critical role in several processes such as the development of cancers, intercellular signaling, drug resistance mechanisms, and cell-to-cell communication by fusion onto the cell membrane of recipient cells. These vesicles contain endogenous proteins and both noncoding and coding RNAs (microRNA and messenger RNAs) that can be delivered to various types of cells. Furthermore, exosomes exist in body fluids such as plasma, cerebrospinal fluid, and urine. Therefore, they could be used as a novel carrier to deliver therapeutic nucleic-acid drugs for cancer therapy. It was recently documented that, hypoxia promotes exosomes secretion in different tumor types leading to the activation of vascular cells and angiogenesis. Cancer cell-derived exosomes (CCEs) have been used as prognostic and diagnostic markers in many types of cancers because exosomes are stable at 4°C and −70°C. CCEs have many functional roles in tumorigenesis, metastasis, and invasion. Consequently, this review presents the data about the therapeutic application of exosomes and the role of CCEs in cancer invasion, drug resistance, and metastasis.     

New insights into Chlamydia intracellular survival mechanisms

Chlamydia sp. are responsible for a wide range of diseases of significant clinical and public health importance. In this review, we highlight how recent cellular and functional genomic approaches have significantly increased our knowledge of the pathogenic mechanisms used by these genetically intractable bacteria. As the extensive repertoire of chlamydial proteins that are translocated into the mammalian host is identified and characterized, a molecular understanding of how Chlamydiae co-opt host cellular functions and block innate immune pathways is beginning to emerge.     

High ovarian GDF-8 levels contribute to elevated estradiol production in ovarian hyperstimulation syndrome by stimulating aromatase expression

Rationale: Growth differentiation factor-8 (GDF-8), also known as myostatin, belongs to the transforming growth factor-beta (TGF-β) superfamily. GDF-8 is expressed in the ovary and regulates various ovarian functions. Ovarian hyperstimulation syndrome (OHSS) is one of the most serious disorders during in vitro fertilization treatment. Aromatase, encoded by the CYP19A1 gene, is the enzyme that catalyzes the final step in estradiol (E2) biosynthesis. It has been demonstrated that high serum E2 levels are associated with the development of OHSS. However, the effects of GDF-8 on aromatase expression and its roles in the pathogenesis of OHSS remain unclear.Methods: The effect of GDF-8 on aromatase expression and the underlying mechanisms were explored by a series of in vitro experiments in primary human granulosa-lutein (hGL) and KGN cells. Rat OHSS model and human follicular fluid samples were used to examine the roles of the GDF-8 system in the pathogenesis of OHSS.Results: We demonstrate that GDF-8 stimulates aromatase expression and E2 production in hGL and KGN cells. In addition, TGF-β type I receptor ALK5-mediated SMAD2/3 signaling is required for GDF-8-induced aromatase expression and E2 production. Using a rat OHSS model, we show that the aromatase and GDF-8 levels are upregulated in the ovaries of OHSS rats. Blocking the function of ALK5 by the administration of its inhibitor, SB431542, alleviates OHSS symptoms and the upregulation of aromatase. Clinical results reveal that the protein levels of GDF-8 are upregulated in the follicular fluid of OHSS patients. Moreover, the expression of GDF-8 is increased in hGL cells of OHSS patients.Conclusions: This study helps to elucidate the mechanisms mediating the expression of aromatase in human granulosa cells, which may lead to the development of alternative therapeutic approaches for OHSS.     

New insights into the mechanisms of protein palmitoylation

Since its discovery more than 30 years ago, protein palmitoylation has been shown to have a role in protein-membrane interactions, protein trafficking, and enzyme activity. Until recently, however, the molecular machinery that carries out reversible palmitoylation of proteins has been elusive. In fact, both enzymatic and nonenzymatic S-acylation reaction mechanisms have been proposed. Recent reports of protein palmitoyltransferases in Saccharomyces cerevisiae and Drosophila provide the first glimpse of enzymes that carry out protein palmitoylation. Equally important is the mechanism of depalmitoylation. Two major classes of protein palmitoylthioesterases have been described. One family is lysosomal and is involved in protein degradation. The second is cytosolic and removes palmitoyl moieties preferentially from proteins associated with membranes. This review discusses recent advances in the understanding of mechanisms of addition of palmitate to proteins and removal of palmitate from proteins.     

Estrogen-induced abnormally high cystic fibrosis transmembrane conductance regulator expression results in ovarian hyperstimulation syndrome

Ovarian hyperstimulation syndrome (OHSS) remains one of the most life-threatening and potentially fatal complications of assisted reproduction treatments, arising from excessive stimulation of the ovaries by exogenous gonadotropins administrated during in vitro fertilization procedures, which is characterized by massive fluid shift and accumulation in the peritoneal cavity and other organs, including the lungs and the reproductive tract. The pathogenesis of OHSS remains obscure, and no definitive treatments are currently available. Using RT-PCR, Western blot, and electrophysiological techniques we show that cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel expressed in many epithelia, is involved in the pathogenesis of OHSS. Upon ovarian hyperstimulation, rats develop OHSS symptoms, with up-regulated CFTR expression and enhanced CFTR channel activity, which can also be mimicked by administration of estrogen, but not progesterone, alone in ovariectomized rats. Administration of progesterone that suppresses CFTR expression or antiserum against CFTR to OHSS animals results in alleviation of the symptoms. Furthermore, ovarian hyperstimulation does not induce detectable OHSS symptoms in CFTR mutant mice. These findings confirm a critical role of CFTR in the pathogenesis of OHSS and may provide grounds for better assisted reproduction treatment strategy to reduce the risk of OHSS and improve in vitro fertilization outcome.     

Met and the microenvironment: New insights for ovarian cancer metastasis

Ovarian cancer often has few symptoms, which makes it difficult to detect at an early stage. Therefore, most of the women will already have metastasis at the time of diagnosis. In their search of undercovering the mechanisms underlying ovarian cancer invasion, Mitra and collaborators demonstrate that the fibronectin receptor (α₅β₁-integrin) can directly activate the receptor tyrosine kinase Met, independently of its ligand. By linking the extracellular matrix with Met activation, and the invasion of ovarian cancer cells, Mitra et al. confirm the crucial role played by Met in ovarian cancer and open new perspectives in the development of ovarian cancer targeted therapies.Key words: Met, independent-ligand activation, ovarian cancer metastasis, integrin, fibronectin, FAKWith 21,880 new cases estimated in 2010 and 13,850 deaths, ovarian cancer is one of the most lethal cancers for women in the United States (National Cancer Institute, www.cancer.gov/cancertopics/types/ovarian). Because most of the deaths occurred at advance stages of the disease, it is crucial to develop a better understanding of the mechanisms leading to the spreading of ovarian tumor cells. This has been the focus of the Ernst Lengyel laboratory for several years now.In their recent work published in Oncogene, Mitra et al. demonstrate a novel pathway linking the fibronectin receptor (α₅β₁-integrin) to Met.¹ Both α₅-integrin and Met have previously been reported to induce invasion in models of ovarian cancer invasion, and to be potential therapeutic targets.^2,3 In the path of several reports showing that the receptor tyrosine kinase Met can be activated by other receptors tyrosine kinases (RTK)⁴, this report demonstrates that α₅-integrin can directly activate Met, independently of its ligand, the hepatocyte growth factor/scatter factor (HGF/SF).     

New insights into mechanisms of immune glomerular injury.

Although glomerular disease remains the most common cause of end-stage renal disease worldwide, major advances have been made recently in understanding the cellular and molecular mechanisms that mediate these disorders. The nephrotic syndrome in noninflammatory lesions such as minimal change or focal sclerosis and membranous nephropathy results from disorders of the glomerular epithelial cell that can be simulated in animal models by antibodies to various epithelial cell membrane epitopes. Clarification of how these antibodies affect epithelial cells to induce a loss of glomerular barrier function should substantially improve understanding of the pathogenesis of minimal change or focal sclerosis. In membranous nephropathy, proteinuria is mediated primarily by the C5b-9 complex through similar mechanisms that also involve glomerular epithelial cells as targets. Inflammatory glomerular lesions are induced by circulating inflammatory cells or proliferating resident glomerular cells. Understanding of how these cells induce tissue injury has also evolved considerably over the past decade. Neutrophil-induced disease involves leukocyte adhesion molecules in regulating neutrophil localization; proteases, oxidants, and myeloperoxidase in mediating injury; and platelets in augmenting these processes. The activated mesangial cell exhibits altered phenotype and proliferation with the release of oxidants and proteases. Mesangial cell proliferation may be initiated by basic fibroblast growth factor and is maintained by an autocrine mechanism involving platelet-derived growth factor. Transforming growth factor beta is important in the subsequent development of sclerosis.     

New insights into mechanisms of resistance to microtubule inhibitors

Mechanisms to explain tumor cell resistance to drugs that target the microtubule cytoskeleton have relied on the assumption that the drugs act either to suppress microtubule dynamics or to perturb the balance between assembled and nonassembled tubulin. Recently, however, it was found that these drugs also alter the stability of microtubule attachment to centrosomes, and do so at the same concentrations that are needed to inhibit cell division. Based on this new information, a new model is presented that explains resistance resulting from a variety of molecular changes that have been reported in the literature. The improved understanding of drug action and resistance has important implications for chemotherapy with these agents.