Hedelmöityshoitojen vaikutukset IGF2/H19 geeni lokuksen DNA metylaatioon ja vastasyntyneiden fenotyyppiin

Hedelmöityshoidot ovat menetelmiä, jotka mahdollistavat raskauden alkamisen hedelmättömillä pariskunnilla in vitro. Yleisesti käytetyin hedelmöityhoitomenetelmä on koeputkihedelmöitys (in vitro fertilization, IVF), johon voi kuulua myös munasolun mikroinjektio ja/tai alkion pakastus ja pakastetun alkion siirto (frozen embryo trasfer, FET). Hedelmöityshoidot ovat yleisesti hyväksyttyjä menetelmiä länsimaissa ja hedelmöityshoitojen avulla syntyneiden lasten määrä on koko ajan nousussa. Vaikka hoitojen avulla syntyneet lapset ovat yleisesti ottaen terveitä, on ne yhdistetty erityisesti muutoksiin syntymäpainossa ja myös leimautumishäiriöiden kasvaneisiin määriin ja muutoksiin leimattujen geenien epigeneettisissä merkeissä kuten DNA:n metylaatiossa. Epigeneettisesti säädellyillä leimatuilla geeneillä on merkittävä rooli sikiön ja istukan kasvussa sikiönkehityksen aikana ja ympäristötekijöiden tiedetään vaikuttavan niihin. Koska hedelmöityshoidot sijoittuvat alkion epigeneettisen uudelleenohjelmoinnin ajanjaksolle, hoitojen on ajateltu vaikuttavan alkion epigeneettisiin profiileihin vaikuttaen vastasyntyneen fenotyyppiin ja täten mahdollisesti aiheuttaen pitkäaikaisia terveysvaikutuksia. Tämän Pro gradu -tutkielman tarkoituksena oli tutkia vaikuttavatko hedelmöityshoidot istukan DNA:n metylaatioon ja onko hoidoilla myös vaikutusta vastasyntyneen fenotyyppiin. Näiden vaikutusten tutkimiseksi tutkielma keskittyi sikiön kasvuun liittyvään IGF2/H19 geenilokukseen ja sen H19 ICR1:n CTCF6 sekvenssiin. Tarkoituksena oli myös tutkia liittyvätkö muutokset CTCF6 sekvenssissä sijaitsevaan rs10732516 G/A polymorfiaan. Hedelmöityshoitojen avulla ja spontaanisti hedelmöitettyjen vastasyntyneiden DNA:n metylaatiotasot istukassa, ja myös napaveren valkosoluissa, tutkittiin massaspektrometriaan perustuvan Sequenomin MassARRAY® EpiTYPER® -menetelmän ja perinteisen bisulfiittisekvensoinnin avulla. Hedelmöityshoitojen vaikutusta vastasyntyneiden fenotyyppiin tutkittiin tarkastelemalla vastasyntyneiden syntymäpainoa, -pituutta ja päänympärystä kansainvälisten kasvukäyrien avulla. Myös istukoiden painoja vertailtiin. Tämän tutkielman tulokset osoittivat hieman, mutta toistuvasti alentuneita DNA:n metylaatiotasoja rs10732516 patA/matG genotyypin H19 ICR1:ssä istukassa. Muutoksia ei havaittu patG/matA genotyypissä, mikä osoittaa, että muutokset IGF2/H19 lokuksen DNA:n metylaatiossa ovat genotyyppispesifisiä ja yhteydessä rs10732516 polymorfiaan. Samoja muutoksia patA/matG genotyypin paternaalisessa alleelissa ei myöskään havaittu napaveren valkosoluissa, mikä osoittaa, että vaikutukset riippuvat myös solutyypistä. Hedelmöityshoitojen vaikutukset fenotyyppiin havaittiin olevan myös yhteydessä rs10732516 polymorfiaan. Tuoreen alkion siirron avulla syntyneillä A/A genotyypin lapsilla havaittiin olevan pienempi syntymäpaino, kuin G/G genotyypin lapsilla. Lisäksi pakastetun alkion siirron avulla syntyneiden A/A genotyypin lapsien ja niiden istukoiden havaittiin olevan painavampia kuin tuoreen alkion siirron avulla syntyneiden lapsien ja istukoiden. Tässä tutkielmassa hedelmöityshoitojen havaittiin vaikuttavan istukan DNA:n metylaatioon ja vastasyntyneen fenotyyppiin. Muutosten havaittiin olevan yhteydessä rs10732516 polymorfiaan, mikä korostaa kyseisen polymorfian merkittävyyttä tutkittaessa hedelmöityshoitojen vaikutuksia. Kuitenkin lisätutkimuksia tarvitaan tulosten varmistamiseen ja erottamaan johtuvatko muutokset hedelmöityshoitojen menettelytavoista vai taustalla olevasta hedelmättömyydestä.Assisted reproductive technology (ART) refers to treatments used for infertile couples to achieve pregnancy in vitro. The main technology of ART is in vitro fertilization (IVF), which may also include intracytoplasmic sperm injection (ICSI) and/or embryo cryopreservation and frozen embryo transfer (FET). ART treatments are well-accepted in Western countries and there is an increasing number of children being conceived in that way. Even though, majority of ART derived newborns appear healthy, they have been associated with increased risks of adverse perinatal outcomes, especially, alterations in birth size as well as higher frequencies of imprinting disorders and alterations in epigenetic modifications, such as in DNA methylation, of imprinted genes. Epigenetically regulated imprinted genes have crucial roles in fetal and placental growth during development and they are known to be affected by environmental factors. Since ART takes place in the early embryo in vulnerable time-period of epigenetic reprogramming, ART has been suggested to impact on epigenetic profiles of the embryo, consequently, affecting the phenotype of newborns, and therefore potentially causing long-term health effects. This thesis aimed to study whether ART has effects on DNA methylation in the placenta and whether ART has effects on the phenotype of newborns. To study these effects, this thesis focused on the sixth binding sequence of CTCF (CTCF6) of H19 ICR1 of the growth-related imprinted IGF2/H19 gene locus. The aim was also to study whether the possible changes associate with the rs10732516 G/A polymorphism locating at CTCF6 of H19 ICR1. DNA methylation levels of placental tissue as well as white blood cells in umbilical cord blood of ART derived, and spontaneously conceived newborns were explored by mass spectrometry-based Sequenom MassARRAY® EpiTYPER® method and traditional bisulfite sequencing. To study the effects of ART on the phenotype of newborns, the birth weight, length and head circumference of ART and control newborns were explored using international growth standards. Moreover, placental weights were compared. The results of this thesis showed slightly, but consistently decreased DNA methylation levels at H19 ICR1 in the paternal allele of ART derived placentas in rs10732516 patA/matG genotype, but not in patG/matA genotype. Thus, the results suggest that the changes in DNA methylation at IGF2/H19 in the placenta are genotype-specific and associate with the rs10732516 polymorphism. Similar decreased methylation levels in the paternal allele of patA/matG genotype was not detected in white blood cells suggesting that the effects on DNA methylation levels are also cell type-specific. The effects of ART on the phenotype also associated with the rs10732516 polymorphism. Fresh embryo transfer derived newborns with A/A genotype were seen to have smaller birth weight than newborns with G/G genotype. Moreover, in A/A genotype, frozen embryo transfer derived newborns were demonstrated to be heavier and to have heavier placentas than fresh embryo transfer derived newborns. The findings of this thesis suggest that ART has effects on DNA methylation in the placenta and on the phenotype of newborns, and the effects associate with the rs10732516 G/A polymorphism. This underlines the significance of the polymorphism when studying the effects of ART. However, further investigations are needed to confirm these findings and to discern whether the changes are due to the ART procedures or underlying infertility

18q12.3-q21.1 microdeletion detected in the prenatally alcohol-exposed dizygotic twin with discordant fetal alcohol syndrome phenotype

Abstract Background A pair of dizygotic twins discordantly affected by heavy prenatal alcohol exposure (PAE) was reported previously by Riikonen, suggesting the role of genetic risk or protective factors in the etiology of alcohol-induced developmental disorders. Now, we have re-examined these 25-year-old twins and explored genetic origin of the phenotypic discordancy reminiscent with fetal alcohol syndrome (FAS). Furthermore, we explored alterations in DNA methylation profile of imprinting control region at growth-related insulin-like growth factor 2 (IGF2)/H19 locus in twins' white blood cells (WBC), which have been associated earlier with alcohol-induced genotype-specific changes in placental tissue. Methods Microarray-based comparative genomic hybridization (aCGH) was used to detect potential submicroscopic chromosomal abnormalities, and developmental as well as phenotypic information about twins were collected. Traditional bisulfite sequencing was used for DNA methylation analysis. Results Microarray-based comparative genomic hybridization revealed a microdeletion 18q12.3-q21.1. in affected twin, residing in a known 18q deletion syndrome region. This syndrome has been associated with growth restriction, developmental delay or intellectual deficiency, and abnormal facial features in previous studies, and thus likely explains the phenotypic discordancy between the twins. We did not observe association between WBCs? DNA methylation profile and PAE, but interestingly, a trend of decreased DNA methylation at the imprinting control region was seen in the twin with prenatal growth retardation at birth. Conclusions The microdeletion emphasizes the importance of adequate chromosomal testing in examining the etiology of complex alcohol-induced developmental disorders. Furthermore, the genotype-specific decreased DNA methylation at the IGF2/H19 locus cannot be considered as a biological mark for PAE in adult WBCs.Peer reviewe

Development and evaluation of a rapid nucleic acid amplification method to detect influenza A and B viruses in human respiratory specimens

Isothermal nucleic acid amplification methods can potentially shorten the amount of time required to diagnose influenza. We developed and evaluated a novel isothermal nucleic acid amplification method, RT-SIBA to rapidly detect and differentiate between influenza A and B viruses in a single reaction tube. The performance of the RT-SIBA Influenza assay was compared with two established RT-PCR methods. The sensitivities of the RT-SIBA, RealStar RT-PCR, and CDC RT-PCR assays for the detection of influenza A and B viruses in the clinical specimens were 98.8%, 100%, and 89.3%, respectively. All three assays demonstrated a specificity of 100%. The average time to positive result was significantly shorter with the RT-SIBA Influenza assay (90 min). The method can be run using battery-operated, portable devices with a small footprint and therefore has potential applications in both laboratory and near-patient settings. (C) 2018 Elsevier Inc. All rights reserved.Peer reviewe

Prevalence of high-risk human papillomavirus infection and cancer gene mutations in nonmalignant tonsils

Objectives: To analyze the prevalence of high-risk HPV (human papillomavirus) and genetic alterations in nonmalignant tonsils. Methods: We collected benign fresh tonsillar tissue specimens from 477 patients undergoing tonsillectomy because of chronic tonsillitis or tonsillar hypertrophy in 2012 (Group A, n = 237) and in 2015 (Group B, n = 240). Luminex xMAP technique served to detect E6/E7 DNA from 16 different high-risk HPV types. Tonsillar DNA and peripheral blood leukocyte DNA from the infected individuals were analyzed using Nimblegen SeqCap EZ Comprehensive Cancer Design panel. The panel targets 578 different genes that are relevant in carcinogenesis. HPV negative tonsillar specimens from age-and gender matched individuals were used as controls. All specimens harboring high-risk HPV were analyzed using fluorescence in situ hybridization (FISH). Results: Five of 477 (1.0%) patients tested positive for the following HPV types: HPV16 (two cases), HPV52 (one case), HPV66 (one case), HPV52 and HPV68 (coinfection, one case). FISH analyses showed that the appearance of HPV in specimens infected with HPV 16 was episomal. Benign tonsils infected with high-risk HPV harbored mutations in EP300, NF1, PIK3CA, and RB1 which are considered relevant in the development of HPV-associated head and neck squamous cell carcinoma (SCC). Conclusions: The prevalence of high-risk HPV in nonmalignant tonsils is low. High-risk HPV positive tonsils harbored mutations in genes that are commonly altered in HPV-associated head and neck SCC. The role of these mutations in tonsillar carcinogenesis is an interesting target for future research.Peer reviewe

Preperitoneal Fat Grafting Inhibits the Formation of Intra-abdominal Adhesions in Mice

BACKGROUND: Adhesion formation contributes to postoperative complications in abdominal and gynaecological surgery. Thus far, the prevention and treatment strategies have focused on mechanical barriers in solid and liquid form, but these methods are not in routine use. As autologous fat grafting has become popular in treatment of hypertrophic scars because of its immunomodulatory effects, we postulated that fat grafting could also prevent peritoneal adhesion through similar mechanisms.METHODS: This was a control versus intervention study to evaluate the effect of fat grafting in the prevention on peritoneal adhesion formation. An experimental mouse model for moderate and extensive peritoneal adhesions was used (n = 4-6 mice/group). Adhesions were induced mechanically, and a free epididymal fat graft from wild type or CAG-DsRed mice was injected preperitoneally immediately after adhesion induction. PET/CT imaging and scaling of the adhesions were performed, and samples were taken for further analysis at 7 and 30 days postoperation. Macrophage phenotyping was further performed from peritoneal lavage samples, and the expression of inflammatory cytokines and mesothelial layer recovery were analysed from peritoneal tissue samples.RESULTS: Fat grafting significantly inhibited the formation of adhesions. PET/CT results did not show prolonged inflammation in any of the groups. While the expression of anti-inflammatory and anti-fibrotic IL-10 was significantly increased in the peritoneum of the fat graft-treated group at 7 days, tissue-resident and repairing M2 macrophages could no longer be detected in the fat graft at this time point. The percentage of the continuous, healed peritoneum as shown by Keratin 8 staining was greater in the fat graft-treated group after 7 days.CONCLUSIONS: Fat grafting can inhibit the formation of peritoneal adhesions in mice. Our results suggest that fat grafting promotes the peritoneal healing process in a paracrine manner thereby enabling rapid regeneration of the peritoneal mesothelial cell layer.</div

Improved chromosome-level genome assembly of the Glanville fritillary butterfly (Melitaea cinxia) integrating Pacific Biosciences long reads and a high-density linkage map

Background The Glanville fritillary (Melitaea cinxia) butterfly is a model system for metapopulation dynamics research in fragmented landscapes. Here, we provide a chromosome-level assembly of the butterfly's genome produced from Pacific Biosciences sequencing of a pool of males, combined with a linkage map from population crosses. Results The final assembly size of 484 Mb is an increase of 94 Mb on the previously published genome. Estimation of the completeness of the genome with BUSCO indicates that the genome contains 92-94% of the BUSCO genes in complete and single copies. We predicted 14,810 genes using the MAKER pipeline and manually curated 1,232 of these gene models. Conclusions The genome and its annotated gene models are a valuable resource for future comparative genomics, molecular biology, transcriptome, and genetics studies on this species.Peer reviewe

Genome sequencing and population genomic analyses provide insights into the adaptive landscape of silver birch

Silver birch (Betula pendula) is a pioneer boreal tree that can be induced to flower within 1 year. Its rapid life cycle, small (440-Mb) genome, and advanced germplasm resources make birch an attractive model for forest biotechnology. We assembled and chromosomally anchored the nuclear genome of an inbred B. pendula individual. Gene duplicates from the paleohexaploid event were enriched for transcriptional regulation, whereas tandem duplicates were overrepresented by environmental responses. Population resequencing of 80 individuals showed effective population size crashes at major points of climatic upheaval. Selective sweeps were enriched among polyploid duplicates encoding key developmental and physiological triggering functions, suggesting that local adaptation has tuned the timing of and cross-talk between fundamental plant processes. Variation around the tightly-linked light response genes PHYC and FRS10 correlated with latitude and longitude and temperature, and with precipitation for PHYC. Similar associations characterized the growth-promoting cytokinin response regulator ARR1, and the wood development genes KAK and MED5A.Peer reviewe

Author Correction: Genome sequencing and population genomic analyses provide insights into the adaptive landscape of silver birch.

In the version of this article initially published, there was a mistake in the calculation of the nucleotide mutation rate per site per generation: 1 × 10−9 mutations per site per generation was used, whereas 9.5 × 10−9 was correct. This error affects the interpretation of population-size changes over time and their possible correspondence with known geological events, as shown in the original Fig. 4 and supporting discussion in the text, as well as details in the Supplementary Note. Neither the data themselves nor any other results are affected. Figure 4 has been revised accordingly. Images of the original and corrected figure panels are shown in the correction notice

"Ei siinä koskaan oo valmis" : kartoitus kätilöopiskelijan eettisen osaamisen tukemisesta naistentautien vuodeosastolla

Tämän opinnäytetyön tarkoituksena oli selvittää, millaista tukea kätilöopiskelijat kaipaavat eettisessä osaamisessaan, ja miten ohjatun harjoittelun ohjaajat tukevat sitä yliopistollisen sairaalan naistentautien vuodeosastolla. Tavoitteena oli kartoittaa osaston tämän hetkinen tilanne eettisen osaamisen tukemisessa. Opinnäytetyö on osa kansainvälistä Increasing Ethical Competence in Midwifery Education and Practice (INEC) -projektia. Tutkimusaineisto on kerätty puolistrukturoituna yksilöhaastatteluna haastattelemalla naistentautien vuodeosaston sairaanhoitajia sekä osastolla ohjatussa harjoittelussa olleita kätilöopiskelijoita. Aineisto analysoitiin sisällönanalyysi-menetelmää käyttäen. Tutkimustuloksia verrattiin aikaisempiin aiheesta tehtyihin tutkimuksiin. Tutkimustuloksena on, että ohjatun harjoittelun ohjaajat kokevat kätilöopiskelijoiden eettisen osaamisen valmiudet pääsääntöisesti hyviksi. Kätilöopiskelijat kokevat eettiset asiat hankaliksi ja vaikeasti konkretisoitaviksi, ja kaipaavat siksi ohjaajalta erilaisia tukikeinoja. Naistentautien vuodeosastolla tarjotaankin esimerkiksi keskusteluja, materiaalia ja vertaistukea eettisen osaamisen tukikeinoina. Tuloksia voidaan käyttää kehittämään naistentautien vuodeosaston opiskelijaohjausta ja luomaan ohjaajille työkaluja opiskelijoiden eettisen osaamisen tukemiseksi. Esille nousivat ohjaajien vertaistuen, työnohjauksen ja lisäkoulutuksen tarve sekä materiaalin saaminen osastolle esimerkiksi opiskelijakansioon liitettynä. Koska tutkimuksia kätilöopiskelijoiden eettisestä osaamisesta on vähän, aiheesta tarvitaan lisää kansallisia tutkimuksia koskien erityisesti naistentautien erikoisalaa.The purpose of this Bachelor’s thesis was to find out what kind of support midwifery students need to improve their ethical competence taking also in account how preceptors in clinical practice support students. This study took place on a gynecological ward of a university hospital. The aim of this thesis was to survey the current supporting situation of the ethical competence on this ward. The thesis is part of an international Increasing Ethical Competence in Midwifery Education and Practice (INEC) project. The data was collected with a semi-structured individual interview from both the nurses working on the gynecological ward and the midwifery students who completed or were conducting their clinical practices there. The data was analyzed using content analysis method. The findings were compared to previous studies. The result of the study is that in general preceptors find midwifery students’ skills of ethical competence good. Midwifery students find ethical issues tricky and difficult to put into practice. Therefore they require different ways of support from their preceptors. The gynecological ward provides for example discussions, reading material and peer support to improve the ethical competence of students. The results can be used to improve mentoring on the gynecological ward and to create tools to support the ethical competence of students. Attention was paid to the preceptors’ need of peer counseling, clinical supervision, additional training and also the possible material available attached to a student’s folder for example. As the amount of studies and researches about the ethical competence on a field of midwifery is low more studies on national level and particularly focused on gynecology are needed

Uuden nukleiinihappoeristykseen pohjautuvan näytteenkäsittelymenetelmän kehittäminen Bordetella pertussikselle

Bordetella pertussis on pieni gramnegatiivinen sauvabakteeri ja tavallisin hinkuyskän aiheuttaja. Hinkuyskä on merkittävä syy vastasyntyneiden kuolemiin maailmanlaajuisesti. Se on edelleen yleinen kansanterveysongelma jopa maissa, joissa on kattava rokotusohjelma. B. pertussiksen diagnosointiin erityisesti lapsilla ja vastasyntyneillä käytetään kvantitatiivisia reaaliaikaisia PCR-menetelmiä. PCR:ään perustuvan diagnostiikan ongelmana ovat tutkittavista näytteistä peräisin olevat PCR-reaktiota estävät inhibiittorit. Tämä korostaa tehokkaiden näytteenkäsittely- ja nukleiinihappoeristysmenetelmien tarpeellisuutta. Magneettipohjainen nukleiinihappoeristys on yksinkertainen, tehokas ja nopea menetelmä, jota käytetään nykyään yleisesti nukleiinihappojen eristykseen. Orion Diagnosticalla tuotekehityksessä oleva uusi nukleiinihappojen eristykseen pohjautuva näytteenkäsittelymenetelmä perustuu paramagneettisten silikananopartikkeleiden käyttöön. Tämän insinöörityön tarkoituksena oli kehittää kyseistä näytteenkäsittelymenetelmää B. pertussis -bakteerin eristämiseen käyttäen näytemateriaalina nenäsivelynäytettä. Tavoitteena oli kehittää menetelmää mahdollisimman tehokkaaksi eristetyn DNA-määrän suhteen. Kehitettävän menetelmän DNA-saannon mittaamiseksi käytettiin kvantitatiivista reaaliaikaista PCR:ää. Lisäksi yhtenä osa-alueena oli kehitettävän näytteenkäsittelymenetelmän testialustan visuaalisen ilmeen suunnittelu ja käytettävyyden testaus. Menetelmän testialustan visuaalista ilmettä suunniteltiin 3D-mallinnuksella ja -tulostuksella. Uuden näytteenkäsittelymenetelmän kehittämiseksi tehtiin kokeita, joiden perusteella menetelmän todettiin toimivan B. pertussis -bakteerille. Verrattaessa kaupalliseen magneettipartikkelipohjaiseen eristysmenetelmään kehitettävällä menetelmällä päästiin lähes yhtä hyviin DNA-saantoihin. Kokeiden perusteella tunnistettiin myös sellaisia menetelmän osa-alueita, joita DNA-saannon parantamiseksi tulee muun muassa optimoida: näytteenkäsittelymenetelmän pesuliuoksiin jäävä DNA-määrä ja näytemateriaalina olevan nenäliman inhibiittorit vähentävät DNA-saantoa. Tuotekehityksen jatkuessa Orion Diagnosticalla jatkotutkimuksissa tulisi keskittyä menetelmässä käytettävien pesuliuoksien kehittämiseen DNA-saannon parantamiseksi.Bordetella pertussis is a small gram-negative coccobacillus and causative agent of whooping cough. Whooping cough is a significant cause of infant deaths worldwide, and it is a public health concern even in countries with high vaccination coverage. Quantitative real-time PCR methods are used for diagnosis of B. pertussis particularly among children and infants. One challenge with PCR-based diagnostics is impurities of clinical samples that inhibit the PCR reaction. This emphasizes the importance of an effective sample preparation method. Extraction based on magnetic beads is a simple, effective and rapid method which is widely used for extraction of nucleic acids. Orion Diagnostica is developing a new sample preparation method that uses paramagnetic silica-coated nanoparticles for nucleic acid extraction. The purpose of this thesis was to develop the sample preparation method for B. pertussis using nasal mucus as sample material. The aim was to develop the method as effective as possible in terms of DNA yield. The efficiency of the sample preparation method was determined by quantitative real-time PCR. In addition, the aim was to design the visual appearance of the sample preparation product and test its usability in practice as well. The visual appearance of the product was designed by using 3D modeling and printing. On the basis of the results of this thesis project, the new method proved to work for B. pertussis nearly as effectively as a commercial DNA extraction method. According to the results, some critical issues for increasing the DNA yield were identified, for example a large amount of DNA is left in the washing solutions, and the inhibitors of the sample material, nasal mucus, reduces the DNA yield. In future the development work of the sample preparation method should focus on for example developing the washing solutions to further increase the DNA yield of the method