Publications by authors named "Outi Hovatta"

188 Publications

Changes in the plasma microvesicle proteome during the ovarian hyperstimulation phase of assisted reproductive technology.

Sci Rep 2020 08 12;10(1):13645. Epub 2020 Aug 12.

Department of Clinical Sciences, Danderyd Hospital, Karolinska Institutet, 18288, Stockholm, Sweden.

The incidence of pulmonary and venous thromboembolism is increased during the first trimester of pregnancies after assisted reproductive technology (ART) compared to spontaneous conception. We previously found that haemostatic plasma variables changed but within normal limits during controlled ovarian hyperstimulation (COH) concomitant with a major increase in plasma microvesicles (MVs) and markers indicating cell activation. We now explored the proteome of these MVs. Thirty-one women undergoing ART were blood sampled at down-regulation (DR) of oestrogen and at high level stimulation (HLS) with its 10-100-fold increased oestrogen level. Samples were analysed by liquid chromatography and tandem mass spectrometry to identify and quantify the proteome. We identified 306 proteins in the MVs and 72 had changed significantly at HLS compared to DR and more than 20% of them were associated with haemostasis. Thus, proteins related to both haemostasis and complement activation altered in plasma MVs in parallel with MV activation during COH. This needs to be further explored in the clinical context.
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http://dx.doi.org/10.1038/s41598-020-70541-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7423945PMC
August 2020

In Vivo Generation of Post-infarct Human Cardiac Muscle by Laminin-Promoted Cardiovascular Progenitors.

Cell Rep 2020 May;31(8):107714

Cardiovascular & Metabolic Disorders Program, Duke-NUS Medical School, National University of Singapore, Singapore 169857, Singapore; Department of Medical Biochemistry and Biophysics, Karolinska Institute, 171 77 Stockholm, Sweden. Electronic address:

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http://dx.doi.org/10.1016/j.celrep.2020.107714DOI Listing
May 2020

Karolinska Institutet Human Embryonic Stem Cell Bank.

Stem Cell Res 2020 05 21;45:101810. Epub 2020 Apr 21.

Department of Clinical Sciences, Intervention and Technology, Karolinska Insitutet, 17177, Stockholm, Sweden; Gynecology and Reproductive Medicine, Karolinska University Hospital, 14186 Stockholm, Sweden; Ming Wai Lau Center for Reparative Medicine, Stockholm node, Karolinska Institutet, 17177, Stockholm, Sweden. Electronic address:

The Karolinska Institutet Human Embryonic Stem Cell Bank (KI Stem Cell Bank) was established at KI, Stockholm, Sweden, when the first human embryonic stem cell (hESC) line was derived by Professor Hovatta and colleagues in 2002. Since then, the bank has grown to include 60 hESC lines. From the very beginning the aim of the bank has been derivation of hESC lines suitable for clinical use. Step by step progress has been made towards this goal, including removal of xeno components, establishment of chemically defined conditions and Good Manufacturing Practice (GMP) compliancy. Today our bank includes such clinical grade hESC line, KARO1, derived and banked according to GMP guidelines. Many of the hESC lines in the bank have been distributed to the scientific community and are deposited in the Stockholm Medical Biobank available for research on collaborative basis.
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http://dx.doi.org/10.1016/j.scr.2020.101810DOI Listing
May 2020

Incidence of pulmonary and venous thromboembolism in pregnancies after in vitro fertilization with fresh respectively frozen-thawed embryo transfer: Nationwide cohort study.

J Thromb Haemost 2020 08 11;18(8):1965-1973. Epub 2020 May 11.

Department of Clinical Sciences, Danderyd Hospital, Karolinska Institutet, Stockholm, Sweden.

Background: The assisted reproductive technique in vitro fertilization (IVF) is associated with an increased risk of venous thromboembolism (VTE) and pulmonary embolism (PE) during the first trimester.

Objectives: To compare the incidence of VTE and PE during the first trimester of IVF pregnancies using fresh or frozen-thawed embryo transfer to that during natural pregnancies.

Patient/methods: Nationwide Swedish registry-based cohort study of women who gave birth (n = 902 891) at the age of 15-50 years to their first child from the 1st of January 1992 until the 31st of December 2012. Exposure groups were IVF with fresh respectively frozen-thawed embryo transfer. Incidences of VTE and PE were calculated, and time-varying hazard ratios estimated for all trimesters after fresh respectively frozen-thawed embryo transfer IVF and compared to natural conception.

Results And Conclusion: Women giving birth after fresh embryo transfer IVF had a more than eightfold increased incidence of venous thromboembolism (hazard ratio [HR] 8.96, 95% CI 6.33 to 12.67) and pulmonary embolism during the first trimester, (HR 8.69, 95% CI 3.83 to 19.71) compared to women giving birth after natural conception. The incidence of VTE in women giving birth after frozen-thawed embryo transfer was not increased during the first trimester. To conclude, fresh embryo transfer IVF was associated with a significantly increased incidence of VTE and PE during the first trimester. These results suggest that frozen-thawed embryo transfer could be a preferred method of IVF with a minimised maternal risk.
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http://dx.doi.org/10.1111/jth.14840DOI Listing
August 2020

Reply: Impact of first-line cancer treatment on follicle quality in cryopreserved ovarian samples.

Hum Reprod 2020 05;35(5):1249-1251

Department of Women's and Children's Health, NORDFERTIL Research Lab Stockholm, Childhood Cancer Research Unit, Department of Women's and Children's Health, Karolinska Institutet and Karolinska University Hospital, 171 64 Solna, Sweden.

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http://dx.doi.org/10.1093/humrep/deaa037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7259367PMC
May 2020

Single-cell analysis of human ovarian cortex identifies distinct cell populations but no oogonial stem cells.

Nat Commun 2020 03 2;11(1):1147. Epub 2020 Mar 2.

Division of Obstetrics and Gynecology, Department of Clinical Science, Intervention and Technology, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden.

The human ovary orchestrates sex hormone production and undergoes monthly structural changes to release mature oocytes. The outer lining of the ovary (cortex) has a key role in defining fertility in women as it harbors the ovarian reserve. It has been postulated that putative oogonial stem cells exist in the ovarian cortex and that these can be captured by DDX4 antibody isolation. Here, we report single-cell transcriptomes and cell surface antigen profiles of over 24,000 cells from high quality ovarian cortex samples from 21 patients. Our data identify transcriptional profiles of six main cell types; oocytes, granulosa cells, immune cells, endothelial cells, perivascular cells, and stromal cells. Cells captured by DDX4 antibody are perivascular cells, not oogonial stem cells. Our data do not support the existence of germline stem cells in adult human ovaries, thereby reinforcing the dogma of a limited ovarian reserve.
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http://dx.doi.org/10.1038/s41467-020-14936-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7052271PMC
March 2020

Human Pluripotent Stem Cells in Reproductive Science-a Comparison of Protocols Used to Generate and Define Male Germ Cells from Pluripotent Stem Cells.

Int J Mol Sci 2020 Feb 4;21(3). Epub 2020 Feb 4.

NORDFERTIL Research Lab Stockholm, Childhood Cancer Research Unit, Department of Women's and Children's Health, Karolinska Institutet, and Karolinska University Hospital, 17164 Solna, Sweden.

Globally, fertility-related issues affect around 15% of couples. In 20%-30% of cases men are solely responsible, and they contribute in around 50% of all cases. Hence, understanding of in vivo germ-cell specification and exploring different angles of fertility preservation and infertility intervention are considered hot topics nowadays, with special focus on the use of human pluripotent stem cells (hPSCs) as a source of in vitro germ-cell generation. However, the generation of male germ cells from hPSCs can currently be considered challenging, making a judgment on the real perspective of these innovative approaches difficult. Ever since the first spontaneous germ-cell differentiation studies, using human embryonic stem cells, various strategies, including specific co-cultures, gene over-expression, and addition of growth factors, have been applied for human germ-cell derivation. In line with the variety of differentiation methods, the outcomes have ranged from early and migratory primordial germ cells up to post-meiotic spermatids. This variety of culture approaches and cell lines makes comparisons between protocols difficult. Considering the diverse strategies and outcomes, we aim in this mini-review to summarize the literature regarding in vitro derivation of human male germ cells from hPSCs, while keeping a particular focus on the culture methods, growth factors, and cell lines used.
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http://dx.doi.org/10.3390/ijms21031028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7038013PMC
February 2020

Human induced pluripotent stem cells from two azoospermic patients with Klinefelter syndrome show similar X chromosome inactivation behavior to female pluripotent stem cells.

Hum Reprod 2019 11;34(11):2297-2310

NORDFERTIL Research Laboratory Stockholm, Childhood Cancer Research Unit, Bioclinicum J9:30, Department of Women's and Children's Health, Karolinska Institutet and Karolinska University Hospital, Solna, Sweden.

Study Question: Does the X chromosome inactivation (XCI) of Klinefelter syndrome (KS)-derived human induced pluripotent stem cells (hiPSCs) correspond to female human pluripotent stem cells (hPSCs) and reflect the KS genotype?

Summary Answer: Our results demonstrate for the first time that KS-derived hiPSCs show similar XCI behavior to female hPSCs in culture and show biological relevance to KS genotype-related clinical features.

What Is Known Already: So far, assessment of XCI of KS-derived hiPSCs was based on H3K27me3 staining and X-inactive specific transcript gene expression disregarding the at least three XCI states (XaXi with XIST coating, XaXi lacking XIST coating, and XaXe (partially eroded XCI)) that female hPSCs display in culture.

Study Design, Size, Duration: The study used hiPSC lines generated from two azoospermic patients with KS and included two healthy male (HM) and one healthy female donor.

Participants/materials, Setting, Methods: In this study, we derived hiPSCs by reprograming fibroblasts with episomal plasmids and applying laminin 521 as culture substrate. hiPSCs were characterized by karyotyping, immunocytochemistry, immunohistochemistry, quantitative PCR, teratoma formation, and embryoid body differentiation. XCI and KS hiPSC relevance were assessed by whole genome transcriptomics analysis and immunocytochemistry plus FISH of KS, HM and female fibroblast, and their hiPSC derivatives.

Main Results And The Role Of Chance: Applying whole genome transcriptomics analysis, we could identify differentially expressed genes (DEGs) between KS and HM donors with enrichment in gene ontology terms associated with fertility, cardiovascular development, ossification, and brain development, all associated with KS genotype-related clinical features. Furthermore, XCI analysis based on transcriptomics data, RNA FISH, and H3K27me3 staining revealed variable XCI states of KS hiPSCs similar to female hiPSCs, showing either normal (XaXi) or eroded (XaXe) XCI. KS hiPSCs with normal XCI showed nevertheless upregulated X-linked genes involved in nervous system development as well as synaptic transmission, supporting the potential use of KS-derived hiPSCs as an in vitro model for KS.

Limitations, Reasons For Caution: Detailed clinical information for patients included in this study was not available. Although a correlation between DEGs and the KS genotype could be observed, the biological relevance of these cells has to be confirmed with further experiments. In addition, karyotype analysis for two hiPSC lines was performed at passage 12 but not repeated at a later passage. Nevertheless, since all XCI experiments for those lines were performed between passage 11 and 15 the authors expect no karyotypic changes for those experiments.

Wider Implications Of The Findings: As KS patients have variable clinical phenotypes that are influenced by the grade of aneuploidy, mosaicism, origin of the X chromosome, and XCI 'escapee' genes, which vary not only among individuals but also among different tissues within the same individual, differentiated KS hiPSCs could be used for a better understanding of KS pathogenesis.

Study Funding/competing Interest(s): This study was supported by grants from the Knut and Alice Wallenberg Foundation (2016.0121 and 2015.0096), Ming Wai Lau Centre for Reparative Medicine (2-343/2016), Ragnar Söderberg Foundation (M67/13), Swedish Research Council (2013-32485-100360-69), the Centre for Innovative Medicine (2-388/2016-40), Kronprinsessan Lovisas Förening För Barnasjukvård/Stiftelsen Axel Tielmans Minnesfond, Samariten Foundation, Jonasson Center at the Royal Institute of Technology, Sweden, and Initial Training Network Marie Curie Program 'Growsperm' (EU-FP7-PEOPLE-2013-ITN 603568). The authors declare no conflicts of interest.
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http://dx.doi.org/10.1093/humrep/dez134DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6894010PMC
November 2019

Impact of first-line cancer treatment on the follicle quality in cryopreserved ovarian samples from girls and young women.

Hum Reprod 2019 09;34(9):1674-1685

Department of Women's and Children's Health, NORDFERTIL Research Lab Stockholm, Childhood Cancer Research Unit, Department of Women's and Children's Health, Karolinska Institutet and Karolinska University Hospital, Solna, Sweden.

Study Question: Does first-line chemotherapy affect the quality of ovarian pre-antral follicles and stromal tissue in a population of young patients?

Summary Answer: Exposure to first-line chemotherapy significantly impacts follicle viability, size of residual intact follicles, steroid secretion in culture and quality of the stromal compartment.

What Is Known Already: First-line chemotherapy is considered to have a low gonadotoxic potential, and as such, does not represent an indication for fertility preservation. Studies investigating the effects of chemotherapy on the quality of ovarian tissue stored for fertility preservation in young patients are limited and the results sometimes contradictory.

Study Design, Size, Duration: We conducted a retrospective cohort study including young patients referred to three centers (Helsinki, Oslo and Tampere) to perform ovarian tissue cryopreservation for fertility preservation between 2003 and 2018.

Participants/materials, Setting, Methods: A total of 43 patients (age 1-24 years) were included in the study. A total of 25 were exposed to first-line chemotherapy before cryopreservation, whereas 18 patients were not. Density and size of follicles divided by developmental stages, prevalence of atretic follicles, health of the stromal compartment and functionality of the tissue in culture were evaluated and related to age and chemotherapy exposure. Activation of dormant follicles and DNA damage were also assessed.

Main Results And The Role Of Chance: Patients exposed to first-line chemotherapy showed a significantly higher density of atretic primordial and intermediary follicles than untreated patients. The intact primordial and intermediary follicles were significantly smaller in size in patients exposed to chemotherapy. Production of steroids in culture was also significantly impaired and a higher content of collagen and DNA damage was observed in the stromal compartment of treated patients. Collectively, these observations may indicate reduced quality and developmental capacity of follicles as a consequence of first-line chemotherapy exposure. Neither increased activation of dormant follicles nor elevated levels of DNA damage in oocyte nuclei were found in patients exposed to chemotherapy.

Limitations, Reasons For Caution: The two groups were not homogeneous in terms of age and the patients were exposed to different treatments, which did not allow us to distinguish the effect of specific agents. The limited material availability did not allow us to perform all the analyses on the entire set of patients.

Wider Implication Of The Findings: This study provides for the first time a comprehensive analysis of the effects of first-line chemotherapy on the health, density and functionality of follicles categorized according to the developmental stage in patients under 24 years of age. When exposed to these treatments, patients were considered at low/medium risk of infertility. Our data suggest a profound impact of these relatively safe therapies on ovarian health and encourages further exploration of this effect in follow-up studies in order to optimize fertility preservation for young cancer patients.

Study Funding/competing Interest(s): This study was funded by the Swedish Childhood Cancer Foundation, the Finnish Cancer Society, the Finnish Pediatric Research Foundation, the Väre Foundation for Pediatric Cancer Research, The Swedish Research Council, the Stockholm County Council (ALF project) and Karolinska Institutet. The authors have no conflict of interest to declare.
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http://dx.doi.org/10.1093/humrep/dez125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6736429PMC
September 2019

Pleomorphic Adenoma Gene 1 Is Needed For Timely Zygotic Genome Activation and Early Embryo Development.

Sci Rep 2019 06 10;9(1):8411. Epub 2019 Jun 10.

Department of Biosciences and Nutrition, Karolinska Institutet, SE-14186, Stockholm, Sweden.

Pleomorphic adenoma gene 1 (PLAG1) is a transcription factor involved in cancer and growth. We discovered a de novo DNA motif containing a PLAG1 binding site in the promoters of genes activated during zygotic genome activation (ZGA) in human embryos. This motif was located within an Alu element in a region that was conserved in the murine B1 element. We show that maternally provided Plag1 is needed for timely mouse preimplantation embryo development. Heterozygous mouse embryos lacking maternal Plag1 showed disrupted regulation of 1,089 genes, spent significantly longer time in the 2-cell stage, and started expressing Plag1 ectopically from the paternal allele. The de novo PLAG1 motif was enriched in the promoters of the genes whose activation was delayed in the absence of Plag1. Further, these mouse genes showed a significant overlap with genes upregulated during human ZGA that also contain the motif. By gene ontology, the mouse and human ZGA genes with de novo PLAG1 motifs were involved in ribosome biogenesis and protein synthesis. Collectively, our data suggest that PLAG1 affects embryo development in mice and humans through a conserved DNA motif within Alu/B1 elements located in the promoters of a subset of ZGA genes.
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http://dx.doi.org/10.1038/s41598-019-44882-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6557853PMC
June 2019

Correction to: Defined serum- and xeno-free cryopreservation of mesenchymal stem cells.

Cell Tissue Bank 2019 06;20(2):329-330

Division of Obstetrics and Gynecology, K57, Department of Clinical Science, Intervention and Technology, Karolinska Institutet, Karolinska University Hospital, Huddinge, 141 86, Stockholm, Sweden.

In the original article, Fig. 1A was by mistakenly duplicated. The corrected image is provided in this correction article.
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http://dx.doi.org/10.1007/s10561-019-09752-zDOI Listing
June 2019

In Vivo Generation of Post-infarct Human Cardiac Muscle by Laminin-Promoted Cardiovascular Progenitors.

Cell Rep 2019 03;26(12):3231-3245.e9

Cardiovascular & Metabolic Disorders Program, Duke-NUS Medical School, National University of Singapore, Singapore 169857, Singapore; Department of Medical Biochemistry and Biophysics, Karolinska Institute, 171 77 Stockholm, Sweden. Electronic address:

Regeneration of injured human heart muscle is limited and an unmet clinical need. There are no methods for the reproducible generation of clinical-quality stem cell-derived cardiovascular progenitors (CVPs). We identified laminin-221 (LN-221) as the most likely expressed cardiac laminin. We produced it as human recombinant protein and showed that LN-221 promotes differentiation of pluripotent human embryonic stem cells (hESCs) toward cardiomyocyte lineage and downregulates pluripotency and teratoma-associated genes. We developed a chemically defined, xeno-free laminin-based differentiation protocol to generate CVPs. We show high reproducibility of the differentiation protocol using time-course bulk RNA sequencing developed from different hESC lines. Single-cell RNA sequencing of CVPs derived from hESC lines supported reproducibility and identified three main progenitor subpopulations. These CVPs were transplanted into myocardial infarction mice, where heart function was measured by echocardiogram and human heart muscle bundle formation was identified histologically. This method may provide clinical-quality cells for use in regenerative cardiology.
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http://dx.doi.org/10.1016/j.celrep.2019.02.083DOI Listing
March 2019

Hormone Production by Human First-Trimester Gonads in a Functional In Vitro System.

Endocrinology 2019 01;160(1):133-142

NORDFERTIL Research Laboratory Stockholm, Solna, Sweden.

In the past, explant tissue-culture methodologies have been used to grow gonads and study their development. Results from in vitro cultures of human gonads showed limited progress toward gonadal cell differentiation and were focused mainly on germ-cell differentiation. Thus, detailed studies focusing on human first-trimester gonadal tissue functionality in vitro are still missing. In this study we investigated the endocrine function of human first-trimester gonads in vitro. We included 27 female and 28 male gonadal samples, derived from a total of 55 cases, at postconceptional ages of 4.5 to 10.5 weeks. Tissues were cultured using an explant tissue-culture system for 14 days. Assays for testosterone (liquid chromatography-tandem mass spectrometry), anti-Müllerian hormone (AMH; ELISA), and inhibin B (ELISA) were performed using media collected after 7 and 14 days of culture. We demonstrated sex- and age-dependent secretion profiles of testosterone, AMH, and inhibin B in the culture media, which resemble the pattern of hormone production in human gonads in vivo, from the few available studies at the same age range. Our study shows that explant tissue-culture conditions are robust for culture of human first-trimester gonadal somatic cells. Thus, it can be used to study human gonadal development and related diseases as well as the effect of potentially hormone-disturbing substances in human gonads during development. However, detailed molecular studies are needed for better understanding of the mechanistic control of the endocrine function of human first-trimester gonads.
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http://dx.doi.org/10.1210/en.2018-00734DOI Listing
January 2019

Advances in the Molecular Pathophysiology, Genetics, and Treatment of Primary Ovarian Insufficiency.

Trends Endocrinol Metab 2018 06 26;29(6):400-419. Epub 2018 Apr 26.

Medical Faculty, Univ. Paris Sud and Paris Saclay, Bicetre Hospital 94275, Le Kremlin Bicêtre, France. Electronic address:

Primary ovarian insufficiency (POI) affects ∼1% of women before 40 years of age. The recent leap in genetic knowledge obtained by next generation sequencing (NGS) together with animal models has further elucidated its molecular pathogenesis, identifying novel genes/pathways. Mutations of >60 genes emphasize high genetic heterogeneity. Genome-wide association studies have revealed a shared genetic background between POI and reproductive aging. NGS will provide a genetic diagnosis leading to genetic/therapeutic counseling: first, defects in meiosis or DNA repair genes may predispose to tumors; and second, specific gene defects may predict the risk of rapid loss of a persistent ovarian reserve, an important determinant in fertility preservation. Indeed, a recent innovative treatment of POI by in vitro activation of dormant follicles proved to be successful.
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http://dx.doi.org/10.1016/j.tem.2018.03.010DOI Listing
June 2018

Laminin 521 Stabilizes the Pluripotency Expression Pattern of Human Embryonic Stem Cells Initially Derived on Feeder Cells.

Stem Cells Int 2018 18;2018:7127042. Epub 2018 Feb 18.

Nordfertil Research Lab Stockholm, Department of Women's and Children's Health, Karolinska Institutet and University Hospital Karolinska Institutet, Stockholm, Sweden.

Human embryonic stem (hES) cells represent an important tool to study early cell development. The previously described use of human recombinant laminin (LN) 521 represented a step forward in generating clinically safe culture conditions. To test the short-term effect of LN521 on cultured hES cells, five male hES cell lines were cultured on human foreskin fibroblasts (hFFs), Matrigel, LN521, and LN121 and characterized by qPCR, immunofluorescence analysis, as well as their potential for three-germ layer differentiation. Variations in gene expression related to pluripotency, stemness, and testicular cells at different passages and culture conditions were evaluated by qPCR. All cell lines expressed pluripotency markers at protein and RNA level and were able to differentiate into cell types of the three germ layers after being cultured on LN521 for nine passages. Reduction in variation of pluripotency marker expression could be observed after culturing the cells on LN521 for nine passages. hES cells cultured on LN521 exhibited less differentiation, faster cell growth, and attachment when compared to hES cells cultured on LN121 or Matrigel. Our results indicate a positive effect of LN521 in stabilizing pluripotency gene expression and might be the first step towards more controllable and robust culture conditions for hES cells.
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http://dx.doi.org/10.1155/2018/7127042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5835285PMC
February 2018

Resveratrol supports and alpha-naphthoflavone disrupts growth of human ovarian follicles in an in vitro tissue culture model.

Toxicol Appl Pharmacol 2018 01 14;338:73-82. Epub 2017 Nov 14.

Swetox, Karolinska Institutet, Unit of Toxicological Sciences, Forskargatan 20, SE-15136 Södertälje, Sweden; Karolinska Institutet, Department of Clinical Sciences, Intervention and Technology, Unit of Obstetrics and Gynecology, K57 Karolinska University Hospital Huddinge, SE-14186 Stockholm, Sweden. Electronic address:

Infertility is a global health problem with an estimated incidence of 15%. Exposure to chemicals is a potential causal factor, and there is a lack of studies examining the effects on female germ cells. Here, we have studied the impact of different aryl hydrocarbon receptor (AHR) modulators on human ovarian follicles using a human ovarian tissue culture model. Expression of AHR was analyzed in tissue samples, and effects of the selected ligands resveratrol (RSVL), 6-formylindolo(3,2-b)carbazole (FICZ), and alpha-naphthoflavone (aNF) on AHR transactivation studied in a granulosa cell tumor line. Cortical human ovarian tissue containing preantral follicles was exposed to the ligands or vehicle (dimethylsulfoxide, DMSO) for seven days in vitro. Follicle growth was assessed by counting and measuring follicles from serial tissue sections, cell death quantified using in situ Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) assay, and steroid hormone production measured using a newly developed ultra-performance liquid chromatography method. AHR was expressed in all donated ovarian tissue samples. FICZ induced AHR transactivation in the granulosa cell line while aNF antagonised it. Compared to DMSO control, FICZ had no effect on follicles in culture, RSVL increased the proportion of growing follicles, and aNF increased cell death, disrupted growth of secondary follicles, increased testosterone, and reduced estradiol levels. We conclude that RSVL supports and aNF disrupts growth of human ovarian follicles in culture. We further conclude that the human ovarian tissue culture model is suitable for studying effects of chemicals on follicular biology.
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http://dx.doi.org/10.1016/j.taap.2017.11.009DOI Listing
January 2018

Phenotypic Screen Identifies a Small Molecule Modulating ERK2 and Promoting Stem Cell Proliferation.

Front Pharmacol 2017 24;8:726. Epub 2017 Oct 24.

Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.

Stem cells display a fundamentally different mechanism of proliferation control when compared to somatic cells. Uncovering these mechanisms would maximize the impact in drug discovery with a higher translational applicability. The unbiased approach used in phenotype-based drug discovery (PDD) programs can offer a unique opportunity to identify such novel biological phenomenon. Here, we describe an integrated phenotypic screening approach, employing a combination of and PDD models to identify a small molecule increasing stem cell proliferation. We demonstrate that a combination of both and screening models improves hit identification and reproducibility of effects across various PDD models. Using cell viability and colony size phenotype measurement we characterize the structure activity relationship of the lead molecule, and identify that the small molecule inhibits phosphorylation of ERK2 and promotes stem cell proliferation. This study demonstrates a PDD approach that employs combinatorial models to identify compounds promoting stem cell proliferation.
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http://dx.doi.org/10.3389/fphar.2017.00726DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5660848PMC
October 2017

A missense mutation in SLC26A3 is associated with human male subfertility and impaired activation of CFTR.

Sci Rep 2017 10 27;7(1):14208. Epub 2017 Oct 27.

Folkhälsan Institute of Genetics, and Molecular Neurology Research Program, University of Helsinki, FI-00014, Helsinki, Finland.

Chloride absorption and bicarbonate excretion through exchange by the solute carrier family 26 member 3 (SLC26A3) and cystic fibrosis transmembrane conductance regulator (CFTR) are crucial for many tissues including sperm and epithelia of the male reproductive tract. Homozygous SLC26A3 mutations cause congenital chloride diarrhea with male subfertility, while homozygous CFTR mutations cause cystic fibrosis with male infertility. Some homozygous or heterozygous CFTR mutations only manifest as male infertility. Accordingly, we studied the influence of SLC26A3 on idiopathic infertility by sequencing exons of SLC26A3 in 283 infertile and 211 control men. A heterozygous mutation c.2062 G > C (p.Asp688His) appeared in nine (3.2%) infertile men, and additionally, in two (0.9%) control men, whose samples revealed a sperm motility defect. The p.Asp688His mutation is localized in the CFTR-interacting STAS domain of SLC26A3 and enriched in Finland, showing a significant association with male infertility in comparison with 6,572 Finnish (P < 0.05) and over 120,000 global alleles (P < 0.0001) (ExAC database). Functional studies showed that while SLC26A3 is a strong activator of CFTR-dependent anion transport, SLC26A3-p.Asp688His mutant retains normal Cl/HCO exchange activity but suppresses CFTR, despite unaffected domain binding and expression. These results suggest a novel mechanism for human male infertility─impaired anion transport by the coupled SLC26A3 and CFTR.
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http://dx.doi.org/10.1038/s41598-017-14606-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5660164PMC
October 2017

RNA Polymerase III Subunit POLR3G Regulates Specific Subsets of PolyA and SmallRNA Transcriptomes and Splicing in Human Pluripotent Stem Cells.

Stem Cell Reports 2017 05;8(5):1442-1454

Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku 20520, Finland.

POLR3G is expressed at high levels in human pluripotent stem cells (hPSCs) and is required for maintenance of stem cell state through mechanisms not known in detail. To explore how POLR3G regulates stem cell state, we carried out deep-sequencing analysis of polyA and smallRNA transcriptomes present in hPSCs and regulated in POLR3G-dependent manner. Our data reveal that POLR3G regulates a specific subset of the hPSC transcriptome, including multiple transcript types, such as protein-coding genes, long intervening non-coding RNAs, microRNAs and small nucleolar RNAs, and affects RNA splicing. The primary function of POLR3G is in the maintenance rather than repression of transcription. The majority of POLR3G polyA transcriptome is regulated during differentiation, and the key pluripotency factors bind to the promoters of at least 30% of the POLR3G-regulated transcripts. Among the direct targets of POLR3G, POLG is potentially important in sustaining stem cell status in a POLR3G-dependent manner.
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http://dx.doi.org/10.1016/j.stemcr.2017.04.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5425787PMC
May 2017

Quality Assurance in Stem Cell Banking: Emphasis on Embryonic and Induced Pluripotent Stem Cell Banking.

Methods Mol Biol 2017 ;1590:11-16

CLINTEC, Karolinska Institute, Flemingsberg, Sweden.

For quality assurance (QA) in stem cell banking, a planned system is needed to ensure that the banked products, stem cells, meet the standards required for research, clinical use, and commercial biotechnological applications. QA is process oriented, avoids, or minimizes unacceptable product defects, and particularly encompasses the management and operational systems of the bank, as well as the ethical and legal frameworks. Quality control (QC ) is product oriented and therefore ensures the stem cells of a bank are what they are expected to be. Testing is for controlling, not assuring, product quality, and is therefore a part of QC , not QA. Like QA, QC is essential for banking cells for quality research and translational application (Schwartz et al., Lancet 379:713-720, 2012). Human embryonic stem cells (hESCs), as cells derived from donated supernumerary embryos from in vitro fertilization (IVF) therapy, are different from other stem cell types in resulting from an embryo that has had two donors . This imposes important ethical and legal constraints on the utility of the cells, which, together with quite specific culture conditions, require special attention in the QA system. Importantly, although the origin and derivation of induced pluripotent stem cells (iPSCs ) differ from that of hESCs, many of the principles of QA for hESC banking are applicable to iPSC banking (Stacey et al., Cell Stem Cell 13:385-388, 2013). Furthermore, despite differences between the legal and regulatory frameworks for hESC and iPSC banking between different countries, the requirements for QA are being harmonized (Stacey et al., Cell Stem Cell 13:385-388, 2013; International Stem Cell Banking Initiative, Stem Cell Rev 5:301-314, 2009).
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http://dx.doi.org/10.1007/978-1-4939-6921-0_2DOI Listing
February 2018

Expression of Pluripotency Markers in Nonpluripotent Human Neural Stem and Progenitor Cells.

Stem Cells Dev 2017 06 27;26(12):876-887. Epub 2017 Mar 27.

1 Division of Neurodegeneration, Center for Alzheimer Research, Department of Neurobiology, Care Sciences and Society, Karolinska Institutet , Stockholm, Sweden .

Nonpluripotent neural progenitor cells (NPCs) derived from the human fetal central nervous system were found to express a number of messenger RNA (mRNA) species associated with pluripotency, such as NANOG, REX1, and OCT4. The expression was restricted to small subpopulations of NPCs. In contrast to pluripotent stem cells, there was no coexpression of the pluripotency-associated genes studied. Although the expression of these genes rapidly declined during the in vitro differentiation of NPCs, we found no evidence that the discrete expression was associated with the markers of multipotent neural stem cells (CD133/CD24), the capacity of sphere formation, or high cell proliferation rates. The rate of cell death among NPCs expressing pluripotency-associated genes was also similar to that of other NPCs. Live cell imaging showed that NANOG- and REX1-expressing NPCs continuously changed morphology, as did the nonexpressing cells. Depletion experiments showed that after the complete removal of the subpopulations of NANOG- and REX1-expressing NPCs, the expression of these genes appeared in other NPCs within a few days. The percentage of NANOG- and REX1-expressing cells returned to that observed before depletion. Our results are best explained by a model in which there is stochastic transient expression of pluripotency-associated genes in proliferating NPCs.
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http://dx.doi.org/10.1089/scd.2016.0346DOI Listing
June 2017

Over Expression of NANOS3 and DAZL in Human Embryonic Stem Cells.

PLoS One 2016 21;11(10):e0165268. Epub 2016 Oct 21.

Division of Obstetrics and Gynecology, Department of Clinical Sciences, Intervention and Technology, Karolinska Institutet and Karolinska University Hospital, Huddinge, SE-141 86, Stockholm, Sweden.

The mechanisms underlying human germ cell development are largely unknown, partly due to the scarcity of primordial germ cells and the inaccessibility of the human germline to genetic analysis. Human embryonic stem cells can differentiate to germ cells in vitro and can be genetically modified to study the genetic requirements for germ cell development. Here, we studied NANOS3 and DAZL, which have critical roles in germ cell development in several species, via their over expression in human embryonic stem cells using global transcriptional analysis, in vitro germ cell differentiation, and in vivo germ cell formation assay by xenotransplantation. We found that NANOS3 over expression prolonged pluripotency and delayed differentiation. In addition, we observed a possible connection of NANOS3 with inhibition of apoptosis. For DAZL, our results suggest a post-transcriptional regulation mechanism in hES cells. In addition, we found that DAZL suppressed the translation of OCT4, and affected the transcription of several genes associated with germ cells, cell cycle arrest, and cell migration. Furthermore, DAZL over expressed cells formed spermatogonia-like colonies in a rare instance upon xenotransplantation. These data can be used to further elucidate the role of NANOS3 and DAZL in germ cell development both in vitro and in vivo.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0165268PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5074499PMC
June 2017

Who benefits from putting family life into ice?

Authors:
Outi Hovatta

Ups J Med Sci 2016 Nov 19;121(4):208-210. Epub 2016 Oct 19.

a Karolinska Institutet, Department of Clinical Science , Intervention and Technology, Karolinska University Hospital Huddinge , Stockholm , Sweden.

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http://dx.doi.org/10.1080/03009734.2016.1219432DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5098482PMC
November 2016

Differentiation of Human Embryonic Stem Cells to Endothelial Progenitor Cells on Laminins in Defined and Xeno-free Systems.

Stem Cell Reports 2016 10 29;7(4):802-816. Epub 2016 Sep 29.

Cardiovascular and Metabolic Disorders Program, Duke-NUS Medical School, Singapore 169857, Singapore; Division of Matrix Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm 17177, Sweden. Electronic address:

A major hurdle for in vitro culturing of primary endothelial cells (ECs) is that they readily dedifferentiate, hampering their use for therapeutic applications. Human embryonic stem cells (hESCs) may provide an unlimited cell source; however, most current protocols deriving endothelial progenitor cells (EPCs) from hESCs use direct differentiation approaches albeit on undefined matrices, yet final yields are insufficient. We developed a method to culture monolayer hESCs on stem cell niche laminin (LN) LN511 or LN521 matrix. Here, we report a chemically defined, xeno-free protocol for differentiation of hESCs to EPCs using LN521 as the main culture substrate. We were able to generate ∼95% functional EPCs defined as VEGFR2CD34CD31VE-Cadherin. RNA-sequencing analyses of hESCs, EPCs, and primary human umbilical vein endothelial cells showed differentiation-related EC expression signatures, regarding basement membrane composition, cell-matrix interactions, and changes in endothelial lineage markers. Our results may facilitate production of stable ECs for the treatment of vascular diseases and in vitro cell modeling.
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http://dx.doi.org/10.1016/j.stemcr.2016.08.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5063508PMC
October 2016

The human PRD-like homeobox gene LEUTX has a central role in embryo genome activation.

Development 2016 10 30;143(19):3459-3469. Epub 2016 Aug 30.

Department of Biosciences and Nutrition, Karolinska Institutet, Novum, Huddinge 141 83, Sweden Science for Life Laboratory, Tomtebodavägen 23 A, Solna 171 21, Sweden Molecular Neurology Research Program, University of Helsinki and Folkhälsan Institute of Genetics, Biomedicum 1, Haartmaninkatu 8, Helsinki 00290, Finland

Leucine twenty homeobox (LEUTX) is a paired (PRD)-like homeobox gene that is expressed almost exclusively in human embryos during preimplantation development. We previously identified a novel transcription start site for the predicted human LEUTX gene based on the transcriptional analysis of human preimplantation embryos. The novel variant encodes a protein with a complete homeodomain. Here, we provide a detailed description of the molecular cloning of the complete homeodomain-containing LEUTX Using a human embryonic stem cell overexpression model we show that the complete homeodomain isoform is functional and sufficient to activate the transcription of a large proportion of the genes that are upregulated in human embryo genome activation (EGA), whereas the previously predicted partial homeodomain isoform is largely inactive. Another PRD-like transcription factor, DPRX, is then upregulated as a powerful repressor of transcription. We propose a two-stage model of human EGA in which LEUTX acts as a transcriptional activator at the 4-cell stage, and DPRX as a balancing repressor at the 8-cell stage. We conclude that LEUTX is a candidate regulator of human EGA.
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http://dx.doi.org/10.1242/dev.134510DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5087614PMC
October 2016

The Hydroxysteroid (17β) Dehydrogenase Family Gene HSD17B12 Is Involved in the Prostaglandin Synthesis Pathway, the Ovarian Function, and Regulation of Fertility.

Endocrinology 2016 Oct 4;157(10):3719-3730. Epub 2016 Aug 4.

Department of Physiology and Turku Center for Disease Modeling (H.K., M.A., J.M.-J., T.D.L., L.S., M.P.), Institute of Biomedicine, University of Turku, FI-20540 Turku, Finland; Department of Clinical Science, Intervention and Technology (P.D., O.H.), Karolinska Institute, 141 52 Huddinge, Sweden; Swedish Toxicology Sciences Research Center (P.D.), Karolinska Institutet, 141 86 Stockholm, Sweden; Department of Biosciences and Nutrition (A.E.D., J.K.), Karolinska Institutet, 171 77 Stockholm, Sweden; Department of Mathematics and Statistics (T.D.L., T.A.), University of Turku, FI-20014 Turku, Finland; Institute for Molecular Medicine Finland (T.A.), University of Helsinki, FI-00014 Helsinki, Finland; Experimental Genetics (J.A.), Center of Life and Food Sciences, Weihenstephan, 85354 Freising, Germany; Institute of experimental Genetics (J.A.), Helmholtz Zentrum, 81377 München, Germany; Genome Analysis Center (J.A.), German Research Center for Environmental Health, 85764 Neuherberg, Germany; Institute of Neuroscience and Physiology (H.R.), Sahlgrenska Academy, University of Gothenburg, SE-405 30 Gothenburg, Sweden; Institute of Medicine (C.O., M.P.), The Sahlgrenska Academy, University of Gothenburg, SE-413 46 Gothenburg, Sweden.

The hydroxysteroid (17beta) dehydrogenase (HSD17B)12 gene belongs to the hydroxysteroid (17β) dehydrogenase superfamily, and it has been implicated in the conversion of estrone to estradiol as well as in the synthesis of arachidonic acid (AA). AA is a precursor of prostaglandins, which are involved in the regulation of female reproduction, prompting us to study the role of HSD17B12 enzyme in the ovarian function. We found a broad expression of HSD17B12 enzyme in both human and mouse ovaries. The enzyme was localized in the theca interna, corpus luteum, granulosa cells, oocytes, and surface epithelium. Interestingly, haploinsufficiency of the HSD17B12 gene in female mice resulted in subfertility, indicating an important role for HSD17B12 enzyme in the ovarian function. In line with significantly increased length of the diestrous phase, the HSD17B females gave birth less frequently than wild-type females, and the litter size of HSD17B12 females was significantly reduced. Interestingly, we observed meiotic spindle formation in immature follicles, suggesting defective meiotic arrest in HSD17B12 ovaries. The finding was further supported by transcriptome analysis showing differential expression of several genes related to the meiosis. In addition, polyovular follicles and oocytes trapped inside the corpus luteum were observed, indicating a failure in the oogenesis and ovulation, respectively. Intraovarian concentrations of steroid hormones were normal in HSD17B12 females, whereas the levels of AA and its metabolites (6-keto prostaglandin F1alpha, prostaglandin D, prostaglandin E, prostaglandin F, and thromboxane B) were decreased. In conclusion, our study demonstrates that HSD17B12 enzyme plays an important role in female fertility through its role in AA metabolism.
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http://dx.doi.org/10.1210/en.2016-1252DOI Listing
October 2016

Characterization and target genes of nine human PRD-like homeobox domain genes expressed exclusively in early embryos.

Sci Rep 2016 07 14;6:28995. Epub 2016 Jul 14.

Biosciences and Nutrition, Karolinska Institutet, Huddinge, Stockholm, Sweden.

PAIRED (PRD)-like homeobox genes belong to a class of predicted transcription factor genes. Several of these PRD-like homeobox genes have been predicted in silico from genomic sequence but until recently had no evidence of transcript expression. We found recently that nine PRD-like homeobox genes, ARGFX, CPHX1, CPHX2, DPRX, DUXA, DUXB, NOBOX, TPRX1 and TPRX2, were expressed in human preimplantation embryos. In the current study we characterized these PRD-like homeobox genes in depth and studied their functions as transcription factors. We cloned multiple transcript variants from human embryos and showed that the expression of these genes is specific to embryos and pluripotent stem cells. Overexpression of the genes in human embryonic stem cells confirmed their roles as transcription factors as either activators (CPHX1, CPHX2, ARGFX) or repressors (DPRX, DUXA, TPRX2) with distinct targets that could be explained by the amino acid sequence in homeodomain. Some PRD-like homeodomain transcription factors had high concordance of target genes and showed enrichment for both developmentally important gene sets and a 36 bp DNA recognition motif implicated in Embryo Genome Activation (EGA). Our data implicate a role for these previously uncharacterized PRD-like homeodomain proteins in the regulation of human embryo genome activation and preimplantation embryo development.
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http://dx.doi.org/10.1038/srep28995DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4944136PMC
July 2016

Microparticles reveal cell activation during IVF - a possible early marker of a prothrombotic state during the first trimester.

Thromb Haemost 2016 08 14;116(3):517-23. Epub 2016 Jul 14.

Nina Olausson, Karolinska Institutet, Department of Clinical Sciences, Danderyd Hospital, SE 18288 Stockholm, Sweden, Tel.: +46 7 052 302 20, Fax: +46 8 123 571 17, E-mail:

Cell-derived microparticles (MPs) are known to be elevated in a number of diseases related to arterial and venous thromboembolism (VTE), such as acute myocardial infarction, VTE (deep-vein thrombosis and pulmonary embolism) and peripheral arterial disease. IVF-associated pregnancies have previously been shown to be associated with an increased incidence of VTE, mechanisms behind being unknown and sparsely studied. Our objective was to assess cell activation during IVF through analysis of MP levels and phenotype following ovarian stimulation. Thirty-one women undergoing IVF were included and blood samples were collected at down regulation of oestrogen and at high level stimulation with 10- to 100-fold increased endogenous oestrogen levels. MPs were analysed by flow cytometry and phenotyped according to size and protein expression. We found that overall phosphatidylserine positive platelet-, endothelial- and monocyte-derived MPs significantly increased following ovarian stimulation with increased levels of platelet activation markers CD40 ligand and P-selectin. Furthermore, there was an increase in endothelial-derived MPs exposing activation marker E-selectin and monocyte-derived MPs, while neutrophil-derived MPs decreased slightly. In conclusion we found a major increase in MPs and markers indicating cell activation in parallel with the profound oestrogen boost during IVF. To assess whether these changes in MPs are associated with thromboembolic events requires extended longitudinal studies.
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http://dx.doi.org/10.1160/TH15-12-0970DOI Listing
August 2016

Derivation of Human Skin Fibroblast Lines for Feeder Cells of Human Embryonic Stem Cells.

Curr Protoc Stem Cell Biol 2016 Feb 3;36:1C.7.1-1C.7.11. Epub 2016 Feb 3.

Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden.

After the first derivations of human embryonic stem cell (hESC) lines on fetal mouse feeder cell layers, the idea of using human cells instead of mouse cells as feeder cells soon arose. Mouse cells bear a risk of microbial contamination, and nonhuman immunogenic proteins are absorbed from the feeders to hESCs. Human skin fibroblasts can be effectively used as feeder cells for hESCs. The same primary cell line, which can be safely used for up to 15 passages after stock preparations, can be expanded and used for large numbers of hESC derivations and cultures. These cells are relatively easy to handle and maintain. No animal facilities or animal work is needed. Here, we describe the derivation, culture, and cryopreservation procedures for research-grade human skin fibroblast lines. We also describe how to make feeder layers for hESCs using these fibroblasts.
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http://dx.doi.org/10.1002/9780470151808.sc01c07s36DOI Listing
February 2016