La présente thèse par articles aborde différentes facettes du dépistage prénatal de certaines aneuploïdies au premier trimestre de la grossesse. L’introduction retrace l’historique du dépistage prénatal et énonce les différents marqueurs biochimiques et échographiques associés aux aneuploïdies. La première publication démontre que le tabagisme maternel abaisse significativement les niveaux sanguins maternels de PAPP-A et de la fraction libre de la β-hCG et augmente significativement la clarté nucale, confirmant la nécessité de contrôler cette co-variable dans le calcul de risque final, du moins pour la trisomie 18. Le deuxième article identifie des seuils de clarté nucale au-delà desquels la biochimie génétique n’apporte aucune valeur additionnelle au dépistage prénatal de la trisomie 21 et de la trisomie 18. Pour les fœtus avec clarté nucale supérieure aux seuils établis, un diagnostic prénatal intrusif devrait être offert sans délai. Le troisième et dernier article porte sur la première détermination des niveaux plasmatiques maternels de la protéine FLRG (follistatin-related gene) au premier trimestre de grossesse et sur son rôle potentiel à titre de marqueur biochimique dans le dépistage prénatal de la trisomie 21. Bien que détectables, les niveaux plasmatiques maternels de FLRG ne sont pas significativement altérés en présence de fœtus avec syndrome de Down. Dans la discussion générale, les trois articles sont abordés sous un angle plus spécifique au Québec. Des données complémentaires et originales y sont présentées. Une discussion sur l’évolution future du dépistage prénatal est entamée et des axes de recherche sont proposés. / In this thesis by articles, we explore different facets of first trimester prenatal screening of aneuploidy. Introduction retraces the origin of prenatal screening and enunciates current biochemical and ultrasound markers associated with aneuploidy. In the first article, impact of maternal smoking on first-trimester prenatal screening results is assessed for Down syndrome and trisomy 18. Both maternal blood levels of PAPP-A and free β-hCG are significantly decreased by maternal smoking while fetal nuchal translucency (NT) thickness is significantly increased. Without adjustment, this results in an increase of false positives, at least for trisomy 18. Based on these results, adjustment for smoking should be mandatory in first-trimester prenatal screening. In the second article, we identify NT threshold values above which biochemical screening provides no additional benefit. In pregnancies in which NT is above the proposed upper cut-offs, invasive prenatal screening should be offered without undue delay. In the third and last article, maternal plasma levels of follistatin- related gene protein (FLRG) are determined for the first time in first trimester of pregnancy. Its potential role as a new marker for Down syndrome is assessed. Although FLRG can be successfully detected in maternal plasma, its levels are not significantly altered by the presence of Down syndrome fetuses. In the general discussion, articles are mainly addressed under a Quebec standpoint. Additional and complementary original data are presented and different clinical research avenues are proposed.

<p>Premenstrual dysphoria, so severe that it affects the lives of the women afflicted, is the condition studied in this thesis. Physiological and pharmacological mechanisms of pathogenetic relevance were investigated. </p><p>Women with premenstrual dysphoria showed a stronger and less dampened response of LH to an estradiol challenge than asymptomatic women, indicating an altered neuroendocrine regulation. In women with premenstrual dysphoria, the LH response was correlated to the severity of irritability and bloating, and the early FSH response was correlated to the severity of depressed mood. </p><p>The positron-emission study showed strong, consistent correlations between worsening of mood symptoms and a decrease in brain trapping of the immediate serotonin precursor, from the mid-follicular to the late luteal phase in women with premenstrual dysphoria. The strongest correlations were seen for the cardinal mood symptoms of premenstrual dysphoria, and for their opposites. Physical symptoms showed weaker or no correlations with the exception of nociceptive symptoms from erogenous body regions which showed positive correlations to serotonin precursor trapping in the right caudate nucleus. The findings are consistent with the serotonin hypothesis of premenstrual dysphoria, and might possibly explain the observed effects of serotonin-augmenting drugs in this condition.</p><p>The partial 5-HT_1A receptor agonist buspirone was superior to placebo in the treatment of premenstrual dysphoria. The weak SRI and 5-HT₂ receptor antagonist nefazodone was not superior to placebo. For women with premenstrual dysphoria in need of medication and who do not tolerate SRIs because of the sexual sideeffects, buspirone may be an alternative drug, since it had no adverse effects on sexual function. </p><p>The prevalence of polycystic ovaries and serum levels of androgens were not higher in women with premenstrual dysphoria than in their asymptomatic counterparts. The findings are not consistent with the hypothesis that irritability in women with premenstrual dysphoria is induced by elevated testosterone levels. </p><p>Thesis results, which are in line with the serotonin hypothesis of premenstrual dysphoria, may imply that increased brain sensitivity is one of the factors underlying severe premenstrual mood symptoms, thereby further supporting a common serotonergic dysregulation in this condition.</p>

Premenstrual dysphoria, so severe that it affects the lives of the women afflicted, is the condition studied in this thesis. Physiological and pharmacological mechanisms of pathogenetic relevance were investigated. Women with premenstrual dysphoria showed a stronger and less dampened response of LH to an estradiol challenge than asymptomatic women, indicating an altered neuroendocrine regulation. In women with premenstrual dysphoria, the LH response was correlated to the severity of irritability and bloating, and the early FSH response was correlated to the severity of depressed mood. The positron-emission study showed strong, consistent correlations between worsening of mood symptoms and a decrease in brain trapping of the immediate serotonin precursor, from the mid-follicular to the late luteal phase in women with premenstrual dysphoria. The strongest correlations were seen for the cardinal mood symptoms of premenstrual dysphoria, and for their opposites. Physical symptoms showed weaker or no correlations with the exception of nociceptive symptoms from erogenous body regions which showed positive correlations to serotonin precursor trapping in the right caudate nucleus. The findings are consistent with the serotonin hypothesis of premenstrual dysphoria, and might possibly explain the observed effects of serotonin-augmenting drugs in this condition. The partial 5-HT1A receptor agonist buspirone was superior to placebo in the treatment of premenstrual dysphoria. The weak SRI and 5-HT2 receptor antagonist nefazodone was not superior to placebo. For women with premenstrual dysphoria in need of medication and who do not tolerate SRIs because of the sexual sideeffects, buspirone may be an alternative drug, since it had no adverse effects on sexual function. The prevalence of polycystic ovaries and serum levels of androgens were not higher in women with premenstrual dysphoria than in their asymptomatic counterparts. The findings are not consistent with the hypothesis that irritability in women with premenstrual dysphoria is induced by elevated testosterone levels. Thesis results, which are in line with the serotonin hypothesis of premenstrual dysphoria, may imply that increased brain sensitivity is one of the factors underlying severe premenstrual mood symptoms, thereby further supporting a common serotonergic dysregulation in this condition.

La présente thèse par articles aborde différentes facettes du dépistage prénatal de certaines aneuploïdies au premier trimestre de la grossesse. L’introduction retrace l’historique du dépistage prénatal et énonce les différents marqueurs biochimiques et échographiques associés aux aneuploïdies. La première publication démontre que le tabagisme maternel abaisse significativement les niveaux sanguins maternels de PAPP-A et de la fraction libre de la β-hCG et augmente significativement la clarté nucale, confirmant la nécessité de contrôler cette co-variable dans le calcul de risque final, du moins pour la trisomie 18. Le deuxième article identifie des seuils de clarté nucale au-delà desquels la biochimie génétique n’apporte aucune valeur additionnelle au dépistage prénatal de la trisomie 21 et de la trisomie 18. Pour les fœtus avec clarté nucale supérieure aux seuils établis, un diagnostic prénatal intrusif devrait être offert sans délai. Le troisième et dernier article porte sur la première détermination des niveaux plasmatiques maternels de la protéine FLRG (follistatin-related gene) au premier trimestre de grossesse et sur son rôle potentiel à titre de marqueur biochimique dans le dépistage prénatal de la trisomie 21. Bien que détectables, les niveaux plasmatiques maternels de FLRG ne sont pas significativement altérés en présence de fœtus avec syndrome de Down. Dans la discussion générale, les trois articles sont abordés sous un angle plus spécifique au Québec. Des données complémentaires et originales y sont présentées. Une discussion sur l’évolution future du dépistage prénatal est entamée et des axes de recherche sont proposés. / In this thesis by articles, we explore different facets of first trimester prenatal screening of aneuploidy. Introduction retraces the origin of prenatal screening and enunciates current biochemical and ultrasound markers associated with aneuploidy. In the first article, impact of maternal smoking on first-trimester prenatal screening results is assessed for Down syndrome and trisomy 18. Both maternal blood levels of PAPP-A and free β-hCG are significantly decreased by maternal smoking while fetal nuchal translucency (NT) thickness is significantly increased. Without adjustment, this results in an increase of false positives, at least for trisomy 18. Based on these results, adjustment for smoking should be mandatory in first-trimester prenatal screening. In the second article, we identify NT threshold values above which biochemical screening provides no additional benefit. In pregnancies in which NT is above the proposed upper cut-offs, invasive prenatal screening should be offered without undue delay. In the third and last article, maternal plasma levels of follistatin- related gene protein (FLRG) are determined for the first time in first trimester of pregnancy. Its potential role as a new marker for Down syndrome is assessed. Although FLRG can be successfully detected in maternal plasma, its levels are not significantly altered by the presence of Down syndrome fetuses. In the general discussion, articles are mainly addressed under a Quebec standpoint. Additional and complementary original data are presented and different clinical research avenues are proposed.

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&#160;%:n herkkyys trisomialle 18 ja 54.5 %:n herkkyys trisomialle 13. Väärien positiivisten seulontatulosten määrä kasvoi 0.3&#160; %: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.