Abstract The aim of the present study was to evaluate the efficacy of the first trimester screening for Down syndrome (DS) in an unselected low-risk Finnish population. The study involved 4,617 women who attended screening between the 8th and 14th weeks of pregnancy in 1998-2000. They gave a blood sample for the measurement of pregnancy associated plasma protein A (PAPP-A) and free beta human chorionic gonadotrophin (β-hCG). Of these women, 3,178 also had an ultrasound examination for the measurement of fetal nuchal translucency (NT). The risk figure for every screened woman was calculated using a computerized risk figure program. The risk 1 in 250 was used as a cut-off. The subgroup of screen positives comprised 5.8% of the study group. There were 16 DS cases. The combined method (maternal age, NT and the biochemical markers) detected 77% of the affected pregnancies. NT combined with maternal age gave a detection rate of 69%. Serum markers without NT combined with maternal age found 75% of the Down's. In 49 consecutive singleton in-vitro-fertilization pregnancies, the β-hCG value was more often elevated compared to spontaneous pregnancies, increasing the false positive rate. In 67 twin pregnancies, the serum marker levels were approximately double those in singletons. Smoking reduced PAPP-A by 20% making the smokers more likely to get a positive screening result. To determine the impact of the screening on the live born incidence of DS, two historical populations were compared. The first group was screened by second trimester serum samples (β-hCG and AFP) and the second group by first trimester ultrasound examination. When detection rates were at the same level, the second trimester screening reduced the number of live born Down's children more effectively. In conclusion, the first trimester combined method (maternal age, NT, β-hCG and PAPP-A) for Down syndrome screening is efficient in an unselected low risk population. The biochemical screening is not recommended in IVF-pregnancies.

>Magister Scientiae - MSc / Glucose oxidase (GOx) and horseradish peroxidase (HRP) are important enzymes for the development of amperometric enzyme linked immunosensors. The selectivity of each enzyme towards its analyte deepens its importance in determining the sensitivity of the resultant immunosensor. In designing immunosensors that have customized transducer surfaces, the incorporation with FAD and iron based enzymes ensures that electron kinetics remains optimal for electrochemical measurement. Various different immobilization strategies are used to produce response signals directly proportional to the concentration of analyte with minimal interferences. The combination of self-assembled monolayers and supramolecular chemistry affords stability and simplicity in immunosensor design. In this work, two electrochemical strategies for the detection of human chorionic gonadotropin(hCG) is presented. This involves the modification of a gold surface with a thiolated β-cyclodextrin epichlorohydrin polymer (βCDPSH) to form a supramolecular inclusion complex with ferrocene (Fc)-functionalised carboxymethyl cellulose polymer (CMC). Cyclic voltammetry indicated that ferrocene is in close proximity to the electrode surface due to the supramolecular complex formed with βCDPSH. Furthermore, strategy (a) for the detection of hCG used α-antihCG labelled (HRP) as reporter conjugate. Strategy (b) maintained the CMC bifunctionalised with Fc and recognition antibody for hCG hormone. However, the system was functionalised with a HRP enzyme and detection is done by using GOx reporter conjugates for in situ production of hydrogen peroxide. The reduction of H2O2 was used for the amperometric detection of hCG by applying a potential of 200 mV. The sensitivity and limit of detection of both strategies were calculated from calibration plots. For strategy (a) the LOD was found to be 3.7283 ng/mL corresponding to 33.56 mIU/mL and a sensitivity of 0.0914 nA ng-1 mL-1. The corresponding values for strategy (b) are 700 pg/mL (6.3 mIU/mL) and 0.94 nA ng-1 mL-1.

La glycosylation est la forme la plus courante de modification post-traductionnelle (PTM) des protéines humaines, puisque plus de 70% d’entre elles sont glycosylées. Celle-ci régule de nombreuses propriétés biologiques comme leur stabilité, leur demi-vie et leur activité. Néanmoins, les protéines peuvent également présenter d'autres types de PTM, ce qui peut conduire pour une protéine donnée à un très grand nombre d'isoformes variant par leur masse, leurs propriétés biologiques et physico-chimiques et leur concentration dans les échantillons biologiques. Ainsi, caractériser une glycoprotéine comporte de nombreux défis et nécessite la mise en œuvre de méthodes séparatives très performantes et de détection très sensibles et informatives.La gonadotrophine chorionique humaine (hCG) est l’hormone spécifique de la grossesse humaine. Elle est essentielle au développement du placenta et du fœtus. Elle est composée de deux sous-unités hCGα et hCGβ qui sont fortement glycosylées (4 sites de N-glycosylation et 4 sites d’O-glycosylation). Récemment, des travaux ont montré une corrélation entre sa glycosylation et une bonne implantation du fœtus. Une caractérisation des ces glycoformes s’avère donc nécessaire.Par conséquent, de nouvelles méthodes en LC/CE-MS ont été développées pour la caractérisation de la hCG à l’échelle intacte en utilisant deux médicaments à base de hCG ayant des glycosylations différentes. Alors que la méthode en CZE-MS (TQ) a permis de différencier les profils des glycoformes de la sous-unité hCGα des deux médicaments, la complémentarité des méthodes RP- et HILIC-MS (qTOF) a conduit à leur identification.Pour limiter les erreurs potentielles d’identification dues au chevauchement des profils isotopiques, le profil de chaque isoforme a été résolu par FT-ICR MS. Dans ce but, une séparation au format nanoLC en mode RP a été développée, améliorant ainsi la sensibilité de la méthode d’un facteur 500 par rapport au format conventionnel. Cette méthode a permis de confirmer l’identification des glycoformes de la sous-unité hCGα. D’autre part, il a été possible d’obtenir des profils différents de glycosylation de la sous-unité hCGβ en favorisant leur ionisation par réduction de la hCG. Enfin, un traitement à la PNGase a conduit à l’élimination des N-glycanes pour l’obtention des profils d’O-glycosylation de la sous-unité hCGβ. / Glycosylation is the most common form of post-translational modifications (PTMs) of human proteins, since more than 70% are glycosylated. It regulates numerous biological properties including their stability, half-life, and activity. Nevertheless, proteins can also exhibit other types of PTMs that lead to a very large number of isoforms, varying in mass, properties and concentration in the biological samples. Therefore, the characterization of a glycoprotein is highly challenging and requires the use of powerful separation techniques and sensitive and informative detection modes.The human chorionic gonadotropin (hCG) is the hormone specific to human pregnancy. It is essential for the development of placenta and fetus. It is based on two heavily glycosylated subunits, hCGα and hCGβ, having 8 glycosylation sites (4 N- and 4 O-glycosylation sites). Some recent studies demonstrated that here is a correlation between the hCG glycosylation state and the fetus implantation. This is why the characterization of the hCG glycoformes is needed.Therefore, new LC/CE-MS methods were developed for the characterisation of hCG at the intact level using two hCG-based drugs having different glycosylation profiles. While the CZE-MS (TQ) method showed its potential for glycosylation fingerprinting, the complementarity of LC-(qTOF) MS methods in RP and HILIC modes allowed the identification of the glycoforms of the hCGα subunit.To limit the identification errors due to the overlapping of isotopic distribution patterns, the profile of each isoform was resolved by FT-ICR MS. For this purpose, a nanoLC separation in RP mode was developed, thus improving the sensitivity of the method by a factor 500 compared to the conventional format. This method allowed the confirmation of the identification of hCGα glycoforms. Then, it was possible to obtain different glycosylation patterns of the hCGβ by promoting its ionization after hCG reduction. Then, a PNGase treatment was carried out to remove the N-glycans in order to obtain the O-glycoprofiles of hCGβ isoforms.

>Magister Scientiae - MSc / Glucose oxidase (GOx) and horseradish peroxidase (HRP) are important enzymes for the development of amperometric enzyme linked immunosensors. The selectivity of each enzyme towards its analyte deepens its importance in determining the sensitivity of the resultant immunosensor. In designing immunosensors that have customized transducer surfaces, the incorporation with FAD and iron based enzymes ensures that electron kinetics remains optimal for electrochemical measurement. Various different immobilization strategies are used to produce response signals directly proportional to the concentration of analyte with minimal interferences. The combination of self-assembled monolayers and supramolecular chemistry affords stability and simplicity in immunosensor design. In this work, two electrochemical strategies for the detection of human chorionic gonadotropin(hCG) is presented. This involves the modification of a gold surface with a thiolated β-cyclodextrin epichlorohydrin polymer (βCDPSH) to form a supramolecular inclusion complex with ferrocene (Fc)-functionalised carboxymethyl cellulose polymer (CMC). Cyclic voltammetry indicated that ferrocene is in close proximity to the electrode surface due to the supramolecular complex formed with βCDPSH. Furthermore, strategy (a) for the detection of hCG used α-antihCG labelled (HRP) as reporter conjugate. Strategy (b) maintained the CMC bifunctionalised with Fc and recognition antibody for hCG hormone. However, the system was functionalised with a HRP enzyme and detection is done by using GOx reporter conjugates for in situ production of hydrogen peroxide. The reduction of H2O2 was used for the amperometric detection of hCG by applying a potential of 200 mV. The sensitivity and limit of detection of both strategies were calculated from calibration plots. For strategy (a) the LOD was found to be 3.7283 ng/mL corresponding to 33.56 mIU/mL and a sensitivity of 0.0914 nA ng-1 mL-1. The corresponding values for strategy (b) are 700 pg/mL (6.3 mIU/mL) and 0.94 nA ng-1 mL-1.

No description available.

An equilibrium between proinflammatory and anti-inflammatory immune responses is essential for maternal tolerance of the fetus throughout gestation. To study the participation of fetal tissue-derived factors in this delicate immune balance, we analyzed the effects of human chorionic gonadotropin (hCG) on murine Treg cells and Th17 cells in vitro, and on pregnancy outcomes, fetal and placental growth, blood flow velocities and remodeling of the uterine vascular bed in vivo. Compared with untreated CD4+CD25+ T cells, hCG increased the frequency of Treg cells upon activation of the LH/CG receptor. hCG, with the involvement of IL-2, also interfered with induced differentiation of CD4+ T cells into proinflammatory Th17 cells. In already differentiated Th17 cells, hCG induced an anti-inflammatory profile. Transfer of proinflammatory Th17 cells into healthy pregnant mice promoted fetal rejection, impaired fetal growth and resulted in insufficient remodeling of uterine spiral arteries, and abnormal flow velocities. Our works show that proinflammatory Th17 cells have a negative influence on pregnancy that can be partly avoided by in vitro re-programming of proinflammatory Th17 cells with hCG.

Proteins, participating in a myriad of biological function, are at the core of all cellular activities occurring within living organisms. Therapeutic proteins, hence constitute a major part of the pharmaceutical industry. The glycoprotein hormones follicle stimulating hormone (FSH), luteinizing hormone (LH), thyroid stimulating hormone (TSH) and human chorionic gonadotropin (CG) regulate various reproductive and metabolic functions in humans and hence have high therapeutic potentials. The increasing demand of recombinant proteins for therapeutic uses drives the development of better expression systems. The methylotrophic yeast Pichia pastoris, has been termed as an industrial workhorse for heterologous protein expression. However, the N-glycosylation in yeast is of the high mannose type, resulting in a reduced serum half-life of the recombinant proteins. In the current work, we have re-engineered the Pichia N-glycosylation pathway to mimic the human type of N-glycosylation. Towards this end, we abolished the yeast native N-glycosylation and introduced enzymes from various eukaryotic sources into the system. These modifications resulted in the conversion of the yeast Man9-20GlcNAc2 glycan structure to a more human like GlcNAc2Man3GlcNAc2 form on over 70 % of the heterologous expressed proteins. In order to demonstrate the application of these strains as efficient protein expression hosts, the glycoengineerd Pichia was used for large scale expression of the glycoprotein hormones, hCG and FSH. The purified recombinant hormones were found to have binding affinities and structure similar to that of the natural hormones. These recombinant hormones were also able to elicit over two fold responses in animal models compared to buffer controls and the activity was comparable to the natural counterparts. Thus, we report the generation of a glycoengineered Pichia pastoris, which can be considered as a serious contender for the expression of glycosylated proteins of therapeutic importance.

L’exposition prénatale à différents agents toxiques est généralement étudiée en considérant le placenta comme une barrière entre la mère et le fœtus ; nous le considérons en tant qu’organe cible des agents toxiques. Pour ce faire, nous avons sélectionné un modèle cellulaire de trophoblastes adapté aux études toxicologiques. En clinique, des pathologies de la grossesse sont associées à des modifications de la sécrétion de l’hormone placentaire lactogène hPL et de l’hormone gonadotrope chorionique hCG. Nos travaux in vitro ont permis de faire le lien entre une exposition à des micropolluants (le mono(2-ethylhexyl) phtalate, un perturbateur endocrinien, et le benzo(a)pyrene, un carcinogène) et ces observations cliniques. Les biomarqueurs de sécrétion hormonale (hPL et hCG hyperglycosylée) et de dégénérescence (activation du purinorécepteur P2X7) que nous avons identifiés permettent de détecter l’exposition et le risque suite à une exposition à des polluants. / Prenatal exposure to pollutants is commonly evaluated using placenta as a barrier between mother and fetus. Here, we consider placenta as a target organ for toxic agents. To achieve this, we selected a trophoblastic cell model, which is adapted to toxicological studies. In clinical studies, pregnancy pathologies are associated to changes in human placental lactogen (hPL) and human chorionic gonadotropin (hCG) secretions. Our in vitro work links exposure to micropollutants (mono(2-ethylhexyl)phthalate, an endocrine disruptor, and benzo(a)pyrene, a carcinogen) and clinical observations. We identified biomarkers of hormonal secretion (hPL and hyperglycosylated hCG) and degeneration (P2X7 receptor activation), which enable the evaluation of exposure and risk attached to exposure to pollutants.

Abstract The purpose of this study was to evaluate extended first trimester screening for severe chromosomal disorders and adverse pregnancy outcomes in singleton pregnancies among the general population in Finland. Maternal serum biochemical markers, pregnancy associated plasma protein-A (PAPP-A) and free beta human chorionic gonadotropin (fβ-hCG), and fetal nuchal translucency (NT) thickness were measured during the gestational weeks 8+0–13+6. A computerized risk figure program was used to calculate an individual risk figure for chromosomal disorders. It was investigated whether the screening parameter, PAPP-A, is associated with adverse pregnancy outcomes. The prevalence of Down syndrome (DS) cases in Finland during the years 2002–2006 was 1:364 (N=795). The proportion of women aged 35 years or older increased from 5–10% in the years 1980–1990 to 19.1% during the study period. Most DS cases (61.1%) presented in that age group. The first trimester combined screening for Down syndrome yielded a detection rate (DR) of 81.9% for a 4.3% false positive rate (FPR). The performance was evaluated among 76949 voluntary women during the study period of 01.05.2002–31.12.2008. There were 188 cases of DS. The screening worked better among the older women. The number of invasive procedures needed to detect one case of DS was higher among the younger women. Adding specific algorithms for screening of other chromosomal abnormalities yielded DR of 74.0% for trisomy 18 (T18) and 54.5% for trisomy 13 (T13) with an additional increase of 0.3% FPR. For chromosomal abnormalities other than T18 and T13, the specific algorithms did not improve the screening performance. Low first trimester maternal serum levels of PAPP-A (≤0.30 MoM) were significantly associated with small for gestational age (SGA) newborns and stillbirths (SBs). The combined screening method for DS works well in practice and has been standardized in Finland. In screening for trisomies 18 and 13 a specific algorithm is reasonable. Low first trimester levels of PAPP-A could be used as an independent marker for pregnancies at high risk for SGA babies and SBs. / Tiivistelmä Tutkimuksen tarkoituksena oli arvioida laajennetun ensimmäisen raskauskolmanneksen kromosomipoikkeavuuksien seulonnan toimivuutta yksisikiöisissä raskauksissa suomalaisessa normaaliväestössä. Äidin seerumin biokemialliset merkkiaineet, raskauteen liittyvä valkuaisaine A (PAPP-A) ja raskaushormoni (fβ-hCG) sekä sikiön niskaturvotus mitattiin raskausviikoilla 8+0–13+6. Yksilöllinen riskiluku kromosomipoikkeavuuksille laskettiin käyttäen tietokoneen riskinlaskentaohjelmaa. Seulonnan merkkiaineen, PAPP-A:n, matalien pitoisuuksien yhteyttä epäsuotuisiin raskauden lopputuloksiin tutkittiin. Downin oireyhtymän esiintyvyys Suomessa oli 1:364 (N=795) vuosina 2002–2006. 35-vuotiaiden tai sitä vanhempien naisten osuus oli tutkimusaikana 19.1 %, mikä on huomattavasti suurempi kuin vuosien 1980–1990: 5–10 %. Näiden naisten sikiöiden joukosta löytyi suurin osa Down oireyhtymistä (61.1 %). Ensimmäisen raskauskolmanneksen yhdistelmäseulonnan toimivuutta tutkittiin aikana 01.05.2002–31.12.2008. Tutkimukseen osallistui 76 949 vapaaehtoista naista. Joukossa oli 188 Downin oireyhtymätapausta. Seulonnan herkkyys Downin oireyhtymälle oli 81.9 % ja tarkkuus 4.3 %. Seulonta toimi parhaiten vanhempien naisten joukossa. Niiden kajoavien toimenpiteiden määrä, jotka tarvittiin yhden Down-sikiön löytämiseksi, oli suurempi nuorten naisten joukossa. Tutkimuksessa Downin oireyhtymän algoritmiin lisättiin spesifiset algoritmit trisomioille 18 ja 13, jolloin saavutettiin 74.0 %:n herkkyys trisomialle 18 ja 54.5 %:n herkkyys trisomialle 13. Väärien positiivisten seulontatulosten määrä kasvoi 0.3  %:n verran. Seulonnan toimivuus muiden kromosomipoikkeavuuksien joukossa ei parantunut spesifisten algoritmien avulla. Lisäksi matalan PAPP-A-pitoisuuden yhteys pienipainoisuuten ja kuolleena syntyneisyyteen oli tilastollisesti merkittävä. Tutkimus osoitti, että esimmäisen raskauskolmanneksen yhdistelmäseulonta toimii hyvin käytännössä. Trisomioiden 18 ja 13 seulonnassa spesifisten algoritmien käyttö on järkevää. Matalaa ensimmäisen raskauskolmanneksen PAPP-A-arvoa voitaisiin käyttää itsenäisenä riskimerkkiaineena raskauksille, joissa pienipainoisuuden ja kuolleena syntymisen riski on kohonnut.