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.

Des hormones gonadotropiques sont utilisées pour la maîtrise de la reproduction dans le domaine vétérinaire. Ces hormones sont actuellement extraites de tissus ou de fluides animaux. L’entreprise CEVA Santé Animale a récemment fait le choix stratégique de produire ces hormones par voie recombinante. L’objectif de cette étude était d’obtenir une gonadotropine chorionique équine recombinante, reCG, pure et biologiquement active, à partir d’une lignée de cellules mammifères CHO. Les étapes de production, de purification et de caractérisation de l’hormone recombinante ont été développées. Les cellules CHO ont été cultivées en fiole d’Erlenmeyer dans différents milieux de culture. Le suivi de la croissance cellulaire et de la quantité d’hormone produite a permis de sélectionner deux milieux. Le procédé de production, avec ces deux milieux, a été optimisé en bioréacteur en contrôlant les paramètres de culture (température, pH). Les protéines produites dans le surnageant, de ces deux cultures, ont été nommées reCG 1 et reCG 2. Un procédé de purification en 3 étapes a été mis au point pour la reCG 1. Plusieurs résines et conditions chromatographiques ont été criblées en microplaques. Les résines multimodales utilisées ont permis d’éliminer des contaminants majeurs grâce à leur sélectivité. Les agrégats de la reCG ont été éliminés grâce à une résine anionique. Le procédé de purification global a été validé pour la reCG 1 et la reCG 2. Il a permis d’obtenir une pureté de 98 % avec un rendement de 80 %. L’activité biologique de la reCG 1 et la reCG 2, in vitro et in vivo, est comparable à celle de la protéine naturelle. L’activité biologique in vivo des reCG est cohérente avec l’étude réalisée sur les glycosylations des hormones et notamment avec leur degré de sialylation. / The gonadotrophic hormones are used for reproduction control in farming animals. Up to now, these hormones were extracted from animal fluids or tissues. The company CEVA Santé Animal has recently decided to produce recombinant versions of these hormones. The objective of this study was to obtain a pure and biologically active recombinant equine chorionic gonadotropin (reCG) after expression in CHO mammalian cells. The production, purification and characterization steps have been developed. CHO cells were grown in Erlenmeyer flasks with different culture media. Two media were selected based on their cell growth potency and of the amount of reCG produced. By using a bioreactor to control key parameters (temperature, pH), the production process was then optimized. The recombinant proteins that accumulated in the supernatant of the two conditions were called reCG 1 and reCG 2. A 3-steps purification process was then developed using reCG 1. Several resins and chromatographic conditions were screened in microplates. Multimodal resins were used to eliminate the main contaminants thanks to their selectivity. reCG aggregates were efficiently eliminated by a chromatographic step with an anionic resin. The overall purification process was finally validated for reCG 1 and reCG 2. Purity and yield were respectively, 98 % and 80 % for the two reCG. We verified that the in vitro and in vivo activities of reCG 1 and reCG 2 were comparable to those of the CG extracted from natural sources. The in vivo assays also confirmed previous studies showing that the degree of glycosylation of an hormone, and most notably their level of sialytation, is important for their biological activity.

Corpus luteum (CL), a transient endocrine structure formed from the ruptured ovarian follicle after ovulation, secretes progesterone (P4) that is essential for establishment and maintenance of pregnancy in mammals. The biosynthesis and secretion of P4 from CL depends, in general, on trophic hormones of the anterior pituitary gland and on hormones or factors originating from ovary, uterus, embryo and placenta. The structure and function of CL tissue is regulated by intricate interplay between two types of factors, namely, the luteotrophic factors, which stimulate CL growth and function, i.e., P4 secretion, and the luteolytic factors, which inhibit CL function and lead to luteal regression. In monoovulatory species such as higher primates and bovines, a striking diversity in the regulation of CL function exists not only between species, but also within the species during different stages of the luteal phase. In higher primates, unlike other species, one of the important characteristics of CL regulation is that, during non-fertile cycle, circulating LH appears to be the sole trophic factor responsible for maintenance of its function, and during fertile cycle, chorionic gonadotropin (CG), an LH analogue, originating from placenta maintains CL function. In higher primates, the role/involvement of luteolytic factors during luteolysis remains elusive. On the other hand, in the bovine species, the role/involvement of luteolytic factor, prostaglandin (PG) F2α during luteolysis is well established. It should be pointed out that in both the species, the mechanism of luteolysis is still poorly understood and the work presented in this thesis attempts to address these lacunae. Further, in bovines, studies have been carried out to examine potential trophic factor(s) responsible for the maintenance of CL function. Chapter I provides an extensive review of literature on CL structure and function with emphasis on factors that influence its growth, development, function and demise in primates and bovines. In Chapter II, employing bonnet monkey (Macaca radiata) as the representative animal model for higher primates, various studies have been conducted to examine the role of molecular modulators involved in regulation of CL function, particularly during spontaneous luteolysis. Although, it is well established that LH is essential for the maintenance of CL function in higher primates, the mechanism(s) responsible for the decline in serum P4 levels at the end of non-fertile cycles, without a concomitant change in circulating LH milieu, remains to be addressed. Several experiments have been conducted to examine the component(s) of luteotrophic (LH/CG) signaling that is/are modulated during luteolysis in the bonnet monkey CL. To understand the relative lack of responsiveness of CL to the circulating LH during the late luteal phase, LH/CG receptor (R) dynamics (expression of LH/CGR and its various transcript variants) was examined throughout the luteal phase and during different functional states of the monkey CL. The results indicated presence of LH/CGR mRNA, its transcript variants and functional LH/CGR protein in the monkey CL on day 1 of menses. Moreover, the functionality of receptors was tested by confirming the biological response of the CL to bolus administration of exogenous LH preparations, which eventually suggested factor(s) downstream of LH/CGR activation to account for the decline in CL function observed during non-fertile cycle. Studies have been conducted to identify molecular modulators that would selectively exploit intraluteal processes to regulate trophic signaling pathways that are critical to the control of luteal function. Immunoblot and qPCR analyses were carried out to examine presence and activation of Src family of kinases (SFKs) and cAMP-phosphodiesterases (PDEs) during various functional states of CL. The results revealed an increased activation of Src (phosphorylated at Tyr 416) during spontaneous and PGF2α/CET-induced luteolysis that may participate in the regulation of cAMP levels in part by increasing the cAMP-PDE activity observed during spontaneous luteolysis. This observation raised the question on the possible mechanism by which CG, an analog of pituitary LH, rescues CL function during early pregnancy. Thus, subsequent experiments involving LH/hCG administration in CET-treated animals as well as simulated early pregnancy animal model were conducted and the results revealed that, a bolus of LH/hCG decreased Src activation and cAMP-PDE activity accompanying a momentous increase in cAMP levels in both these models that further led to a concomitant increase in P4 secretion. Although the mechanisms of action of LH/CG involve modulation of a number of signaling pathways in the CL, by far, the results from various experiments suggested that it leads to activation of Src kinase and cAMP-PDE, thus causing inhibition of various elements of the primary signaling cascade- AC/cAMP/PKA/CREB during spontaneous luteolysis. One of the consequences of activation of Src kinase and cAMP-PDE was the regulation of expression of genes associated with steroidogenesis and it was observed that expression of SR-B1, a membrane receptor associated with trafficking of HDL-CE into the luteal cells, was lower in the regressed CL. The results taken together suggest that the decrease in responsiveness of CL to LH milieu during non-fertile cycles is not associated with changes in LH/CGR dynamics, but, is instead coupled to the activation of Src kinase and cAMP-PDE, inhibition of molecules downstream of LH signaling, and a decrease in the SR-B1 expression that regulates cholesterol economy of the luteal cell, and in turn, P4 secretion. The control of primate CL function appears to be dominated by the luteotrophic factors (LH/CG) over the luteolytic factors, since the process of luteal regression was overcome by administration of LH/CG. Further, in the primate CL, the molecular modulators of LH/CG signaling (Src kinase and PDE) are maintained in the repressed state by the luteotrophic factor LH/CG for maximum steroidogenic function. In contrast, in non-primate species, without invoking a role for the luteotrophic factor, essentially the synthesis and secretion of luteolytic factor, PGF2α, from the uterus is kept in check during pregnancy by the trophoblast derived IFN- and thus allowing CL to continue to function that is essential for maintenance of pregnancy. In the bovine species, the mechanism of PGF2α-induced luteolysis that involves a change in expression of genes associated with various processes of cellular function is poorly understood. Experiments were conducted utilizing buffalo cows (Bubalus bubalis) as a model system, to determine temporal changes in the global gene expression profile of the CL in response to PGF2α treatment. For this purpose, CL tissues were collected on day 11 of estrous cycle without treatment (designated as 0 h) and at 3, 6 and 18 h post PGF2α treatment for various analyses. Global changes in gene expression pattern in the CL were investigated employing Affymetrix GeneChip bovine genome array and the results are presented in Chapter III. The hybridization intensity values obtained by microarray analysis were subjected to R/Bioconductor tool. Following the application of highly stringent statistical filters to eliminate false positives, a set of differentially expressed genes were identified. The differentially expressed genes were further classified based on a fold change cut-off filter of ≥2, and the analysis revealed 127 genes to be differentially expressed within 3 h of PGF2α administration, of these 64 and 63 genes were up-regulated and down-regulated, respectively. Analysis of microarray data at 6 h post PGF2α administration revealed 774 genes to be differentially expressed, of which 544 genes were up-regulated, while 230 genes were down-regulated. The microarray analysis performed on CL tissues collected at 18 h post PGF2α administration showed that out of the total 939 differentially expressed genes, 571 genes were up-regulated, while 368 genes were down-regulated. Analysis of the ontology report for the biological processes category showed that initially in response to PGF2α administration, genes regulating steroidogenesis, cell survival and transcription were differentially regulated in the CL, but at later time points, differential expression of genes involved in apoptosis, PGF2α metabolism, tissue remodeling and angiogenesis was observed. Further, involvement of molecules downstream of LH/IGF-1 activation was investigated and the results obtained indicated that PGF2α interfered with the LH/IGF-1 signaling since the expression of LH/CGR, GHR and pAkt were down-regulated following PGF2αadministration. Furthermore, the functional luteolysis observed post PGF2αadministration appeared to be due to an interruption in cholesterol trafficking to inner mitochondrial membrane, since StAR expression was inhibited. The results obtained also demonstrated that the expression of AGTR1, VEGFR2 and R3 were down-regulated following PGF 2α administration. Further, the data obtained also suggested modulation of expression of pro- and anti-angiogenic factors upon PGF2α-treatment indicative of an involvement of other autocrine or paracrine factor(s) in the regression of bovine CL. This was an interesting finding as it suggests a novel and potential functional relationship between angiogenesis and the luteolytic response of CL to PGF2α administration. In bovines, despite extensive research being carried out to examine factors involved in the regulation of development and function of the CL, the trophic factor(s) required for maintenance of CL function, especially, P4 biosynthesis and secretion are not well characterized. It was hypothesized that the function of the CL during its finite lifespan must be responsive to LH as well as to various growth factors. Thus, experiments were conducted to examine the effects of increased LH and GH/IGF-I on the maintenance of CL function during mid luteal phase and post PGF2α administration and the results of these studies are presented in Chapter IV. To elucidate the role of LH as a trophic factor in the regulation of CL function, effects of increased endogenous LH through GnRH administration and exogenous hCG injections were examined. The results indicated an absence of noticeable effect of various hCG/GnRH treatments on circulating P4 levels. On the other hand, administration of GH resulted in increased serum IGF-1 and P4 levels. It was further observed that the administration of a combination of hCG and GH increased serum P4 levels better than treatment with GH alone. Further experiments were carried out to examine the complex reciprocal relationship between LH/GH and PGF2α on expression of genes involved in the regulation of luteal structure and function. In buffalo cows, administration of exogenous hCG and/or GH following inhibition of CL function by PGF2α administration did not prevent the PGF2α-induced decline in serum P4 levels, but PGF2-mediated decrease in expression of LH/CGR and GHR genes was prevented upon GH administration. However, the decrease in StAR expression was not restored by hCG and GH treatments, thereby indicating that PGF2 action was not prevented by hCG and/or GH treatments. Taken together, the results of studies carried out in buffalo cows employing various experimental model systems suggest essential role for LH and GH/IGF-1, however, these factors were unable to reverse PGF2α-induced luteolysis. Further, our crucial findings of the effects of increased endogenous LH and IGF-1, in addition to their relationship with luteolytic agents such as PGF2α will open new avenues for studying the mechanisms involved in the regulation of structural and functional properties of the buffalo CL. It is well known that a large number of buffalo cows experience loss of pregnancy and infertility due to inadequate luteal function and/or failure of timely insemination. Results from our studies suggest that the incorporation of PGF2α and hCG or GH/IGF-1 protocols in buffalo cows to be beneficial for improving their breeding efficiency as these protocols are likely to increase luteal function with defined luteolysis. To summarize, the results of studies described in the present thesis provide new insights into the physiological and molecular mechanisms involved in the regulation of CL function during luteolysis in the monoovulatory species. The results suggest that the maintenance of CL function appears to be dependent on both luteotrophic and luteolytic factors, but with a varied degree of dominance between the two species examined. Further, the results indicate that while the luteotrophic factors (LH/CG) dominate the CL regulation in primates, the regulation of CL function in bovines is dominated by the actions of luteolytic factor (PGF2α). In monoovulatory species, the luteotrophic and luteolytic factors following binding to their specific plasma membrane receptors on the luteal cells, would counteract each other and modulate activation of various downstream signaling molecules subsequently leading to regulation of gene expression and P4 secretion (Fig.5.1). LH: luteinizing hormone; CG: chorionic gonadotropin; LH/CGR: LH/CG receptor; Gαs: stimulatory α-subunit of trimeric G-protein; AC: adenylate cyclase; cAMP: cyclic adenosine monophosphate; PKA: protein kinase A; p: phosphorylation: CREB: cAMP response element binding protein; SR-B1: scavenger receptor class B, type I; SF-1: steroidogenic factor 1; LRH-1: liver receptor homologue 1; P4; progesterone; Src; sarcoma; PDE4D: cAMP phosphodiesterase 4D; StAR, steroidogenic acute regulatory protein; PGF2α: prostaglandin F2α; PTGFR: PGF2α receptor; PLC: phospholipase C; CYP19A1: cytochrome P450 aromatase; PTGR1: Prostaglandin reductase 1; AREG: Amphiregulin; RTK: receptor tyrosine kinase; Akt: protein kinase B; FKHR: forkhead transcription factor; DAPL1: death associated protein like 1; ARG2: Arginase, type II Growth factor LH/CGR RR AC Gαs ? Gα TT P? Gα K PKP src cAMP ? P Akt PDE4D P PFKHR FKHR CREB P LRH-1CREB P SF-1 Genes associated with Genes associated with apoptosis ? CYP19A1, apoptosis SR-B1 PTGR1 DAPL1 SF-1, LRH-1 AREG ARG 2 P4 biosynthesis Apoptosis? P4 biosynthesis Apoptosis MONKEY BUFFALO COW Shown here is the diagram depicting intracellular signaling pathways regulated by luteotrophic factor (LH) and luteolytic factor (PGF2α) and their cross talk to counteract changes in the expressions of genes associated with the biosynthesis and secretion of P4 and apoptosis in the CL. In primates, LH/CG activates a multitude of intracellular signaling cascades, primarily Gαs/AC/cAMP/PKA/CREB leading to changes in gene expression. LH during early and mid luteal phase and CG during pregnancy maintain the activation of Src and PDE in an inhibitory state. However, during the late luteal phase of non-fertile cycle, results in present study suggests that activated Src levels and PDE activity increase, with accompanying decrease in cAMP and pCREB levels leading to concomitant decrease in SR-B1 expression, and in turn, P4 secretion. Surprisingly, regulation of apoptotic gene expression and CL regression are still unclear. In bovines, PGF2α of uterine origin mediates changes in luteal gene expression and results in decreased P4 secretion, principally by reduction in StAR level. The present study suggests that during luteolysis PGF2α affects the genes regulated by LH, by interfering with LH (and perhaps IGF-1) signaling leading to alteration in the expression of genes crucial for CL structure and function. (Pl refer the abstract file for figures)

Die Implantation einer Blastozyste im Uterus stellt für den maternalen Organismus eine große Herausforderung dar. Neben morphologischen Veränderungen des Endometriums sind Anpassungen auch im Immun- und Gefäßsystem notwendig. Kenntnisse über die verantwortlichen Faktoren für diese Prozesse sind bei Menschen und anderen Primaten noch immer unvollständig. Dabei sind Implantationsstörungen oftmals Ursache von Fruchtbarkeitsproblemen (SHARKEY und SMITH 2003). Besonders im Bereich der assistierten Reproduktion stellen implantationsbedingte Probleme eine Hauptursache für geringe Erfolgsraten dar (APLIN und KIMBER 2004). Das Ziel der Erforschung von Mechanismen im Zusammenhang mit der Implantation ist es, ein besseres Verständnis für die physiologischen und auch pathologischen Vorgänge zu erlangen. In dessen Folge die Erkenntnisse für die Behandlung von Implantationsstörungen genutzt werden könnten. Anhand des in der reproduktionsbiologischen Forschung etablierten Primatenmodells des Weißbüschelaffen (Callithrix jacchus) wurde in der vorliegenden Arbeit die Expression ausgewählter Faktoren in verschiedenen Zyklusphasen mit dem Schwerpunkt der Implantationsphase untersucht. Dafür wurden zeitlich genau terminierte Uterusproben von konzeptiven und nicht-konzeptiven Tieren entnommen und molekularbiologisch, histologisch und immunhistochemisch analysiert. Erstmals konnte im Uterus von Weißbüschelaffen die zeitliche und räumliche Regulierung von Choriongonadotropin (CG), Östradiolrezeptor-α (ERα), Östradiolrezeptor-ß (ERß), Progesteronrezeptor (PR), Aromatase (ARO), 17β-Hydroxysterois-Dehydrogenase Typ 7 (17β-HSD7) sowie Relaxin (RLN) und Relaxinrezeptor (RXFP1) auf Gen- und Proteinebene in verschiedenen Zyklusphasen in der vorliegenden Vollständigkeit dargestellt werden. Alle in dieser Arbeit untersuchten Faktoren zeigten eine rezeptorspezifische Veränderung der Expressionsmuster, auf räumlicher und zeitlicher Ebene im Verlauf des Implantationsprozesses. Es konnte dargestellt werden, dass die feto-maternale Kommunikation bereits vor dem direkten Kontakt der Blastozyste mit dem Endometrium beginnt. So wurde bei konzeptiven Tieren eine verstärkte Expression von CG, ERα, PR, 17β-HSD7 und RXFP1 in der Implantationsphase nachgewiesen. Diese Faktoren sind geeignete Kandidaten für weiterführende Untersuchungen und Evaluierung von Markern für die Präimplantationsphase und rezeptiven Uteri von Weißbüschelaffen. Die ebenfalls untersuchten Faktoren ERβ, ARO und RLN zeigten Expressionsmuster, welche erst nach der Implantation bei konzeptiven Tieren Unterschiede aufwiesen. Aus diesem Grund sind diese Faktoren nicht als frühe Marker der Implantationsphase geeignet, gleichwohl sie wichtige Funktionen in der Postimplantationsphase übernehmen. Die Ergebnisse dieser Arbeit verdeutlichen, dass nicht alleine ein einzelner Faktor eine herausragende Bedeutung im Implantationsprozess hat, sondern vielmehr ein äußerst komplexes Netzwerk von Signalen nötig ist, um eine erfolgreiche Gravidität zu gewährleisten. Die Aufgabe der Steroidrezeptoren ERα und PR liegt in der grundlegenden Bereitstellung eines rezeptiven Uterus während des Implantationszeitfensters. Gleichzeitig erfolgt eine sehr genau koordinierte Regulation dieser Steroidrezeptoren über die lokal produzierten Faktoren CG, ARO, 17β-HSD7, RLN und der Rezeptoren RXFP1 und ERβ. Neben den morphologischen Veränderungen des Endometriums, sind einzelne Faktoren auch bei der Angiogenese und Entwicklung der maternalen Immuntoleranz beteiligt. Es kann demnach davon ausgegangen werden, dass die präzise Regulation dieser Faktoren eine essentielle Voraussetzung für den Erfolg der Implantation und Gravidität ist.

17 beta-Hydroxy-11 beta(4-dimethylaminophenyl)-17 alpha-(1-propynyl)estra-4, 9-dien-3-one (RU486) inhibited the in vitro development of different stages of mouse preimplantation embryos under study. Two-celled embryos, morulae, and early blastocysts were obtained from B6D2F1 mice. The embryos were grown in Ham F-10 nutrient mixture (with glutamine) supplemented with sodium bicarbonate (2.1 g/L), calcium lactate (282 mg/L), and bovine serum albumin (fraction V, 3 mg/mL) at 37 degrees C in a humidified incubator supplied with 5% CO2 in air. RU486 was added to the culture medium at concentrations of 1, 5, 10, and 20 micrograms/mL. Culture medium with 0.05% ethanol served as the control. In vitro growth of embryos was assessed by the following criteria: (i) two-celled stage embryo development to blastocyst stage after 72 h, (ii) morula stage grown to blastocyst stage after 24 h, and (iii) early blastocyst stage development to hatching blastocyst after 12 h, in culture. RU486 inhibited the in vitro development of two-celled embryos, morulae, and early blastocysts at concentrations of 5, 10, and 20 micrograms/mL culture medium (p less than 0.001). The inhibitory effect of RU486 at these concentrations on the development of all the stages of embryos under study was irreversible. However, RU486 did not affect embryo development at 1 microgram/mL culture medium. The study indicates the direct adverse effect of RU486 at 5 micrograms/mL and higher concentrations in culture medium on the development of mouse preimplantation embryos in vitro, and it encourages its further investigation as a postcoital contraceptive in animal models and humans.

>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.

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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.