Naise viljatusega kaasnev immuunsüsteemi aktivatsioon: autoantikehade ja põletikuliste mediaatorite tähendus

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Infertiilsus ehk viljatus on ülemaailmne meditsiiniline ja sotsiaalne probleem, millega puutub kokku kuni 9% reproduktiivses eas paaridest. Kehavälise viljastamise protseduur (IVF) on osutunud kõige efektiivsemaks ravimeetodiks erinevate viljatuse põhjuste korral. IVF ravi tulemuslikkus on otseselt sõltuv follikulogeneesi stimulatsiooni tulemuslikkusest, mille hindamiseks otsitakse jätkuvalt efektiivsemaid ja optimaalsemaid biomarkereid, et nende põhjal paremini ennustada IVF ravi edukust. IVF ravi ebaõnnestumist on seostatud immuunsüsteemi häiretega, sealhulgas suurenenud autoantikehade produktsiooni ja muutunud tsütokiinide profiiliga. Käesolevas doktoriväitekirjas määrati humoraalse immuunsüsteemi aktivatsiooni markereid erineva viljatuse põhjusega naispatsientidel ning hinnati nende tähenduslikkust viljatuse etiopatogeneesis ja viljatusravi tulemuse ennustamisel. Selleks määrati töö esimeses osas patsientide vereseerumis esinevaid nii üldisi organspetsiifikata kui ka spetsiifiliselt reproduktiivorganite vastu suunatud autoantikehi. Tulemused näitasid, et erineva põhjusega viljatutel naistel, kes läbisid IVF protseduuri, esines sagedasti mitmesuguseid organ-spetsiifikata või -spetsiifilisi autoantikehi. Teatud antikehareaktsioonide esinemine oli seotud viljatuse diagnoosi või IVF ravitulemusega. Töö teises osas mõõdeti patsientide follikulaarvedelikust erinevate biomarkerite kontsentratsioone. Follikulaarvedelikust määratud proinflammatoorsete tsütokiinide, kemokiinide ja apoptoosi regulaatorite profiilid erinesid patsientide hulgas haiguspõhiselt. Peale selle leiti biomarkereid, mille esinemine oli seotud viljatusravi erinevate parameetritega. Töö tulemustest võib järeldada, et erineva põhjusega viljatutele IVF naispatsientidele on iseloomulik üldine immuunsüsteemi aktivatsioon. Immuunsüsteemi häired võivad olla seotud neil patsientidel esineva viljatuse patogeneesiga.Infertility has become a global medical and social problem affecting approximately 9% of reproductive-aged couples worldwide. Today, in vitro fertilization (IVF) has become the most successful treatment option for infertile couples with various causes. Despite improvements in fertilization and pregnancy rates, the overall success rate for IVF has remained low. The outcome of IVF procedure is highly dependent on the successfulness of controlled ovarian stimulation, wherefore more effective and optimal biomarkers are sought for better prognosis. Additionally, an underlying mechanism for IVF failure can be immune activation, including an increased production of autoantibodies and alterations in cytokine levels. In this doctoral thesis the presence and significance of humoral immune system activation in serum and follicular fluid of female IVF patients was evaluated in relation to infertility etiology and IVF treatment results. In the first part of the thesis the prevalence of common and infertility related autoantibodies in the blood serum was assessed. Results of the study showed that infertile women with various diagnoses presented with elevated levels of autoantibody reactivity towards several organ-specific and organ-non-specific autoantigens. Furthermore, some of the antibodies associated with cause of infertility or unsuccessful IVF treatment outcome. In the second part of the thesis various follicular fluid biomarkers were measured. We demonstrated that either reduced or elevated levels of certain proinflammatory cytokines, chemokines and apoptosis regulators in the follicular fluid were associated with cause of infertility and correlated with IVF treatment parameters. In conclusion, these results provide further proof that impairment of the immune system is characteristic to infertile women undergoing IVF treatment and that immune system alterations may be involved in the infertility pathogenesis of these patients

Vereseerumi D-vitamiini sisaldus raseduse teisel kolmandikul mõjutab rasedustulemust ja ema tervist

Taust. D-vitamiin mõjutab rohkem kui 200 geeni ekspressiooni, mistõttu on see vitamiin oluline paljudes füsioloogilistes protsessides. Inimene vajab D-vitamiini kogu elu vältel, ent kriitilise tähtsusega on see raseduse ajal. Rasedad naised ja nende vastsündinud moodustavad hüpo-D-vitaminoosi riskirühma ning D-vitamiini puudulikkusest on saanud ülemaailmne probleem.Eesmärk. Analüüsida Eesti naiste rasedusaegset vereseerumi D-vitamiini sisaldust, seda määravaid tegureid ning hinnata D-vitamiini vähese sisalduse mõju ema ning lapse tervisele.Meetodid. Analüüsiti TÜ Kliinikumi naistekliiniku tervete rasedate (n = 118) anamnestilisi ning kliinilisi andmeid raseduse II trimestril ja sünnitusjärgsel perioodil ning andmeid nende vastsündinute loost. Vereseerumi D-vitamiini sisaldus määrati kemoluminestsents- immuunmeetodil. Andmete analüüsiks kasutati lineaarset ja logistilist regressioonanalüüsi ning kohandamist seoseid mõjutavatele teguritele.Tulemused. Eesti rasedate D-vitamiini sisaldus seerumis oli keskmiselt 55,0 ± 22,8 nmol/l, jäädes 79,7%-l (95% uv 71,1–86,3) uuritavatest alla referentsväärtuse (75 nmol/l) ning 17,8%-l (95% uv 11,6–26,1) esines D-vitamiini puudulikkus (≤ 30 nmol/l). D-vitamiini väärtused vereseerumis sõltusid kalendrikuust ja naise raseduseelsest kehamassiindeksist. Rasedusaegne D-vitamiini vähene sisaldus oli seotud suurema rasedusaegse kaaluiibega, vajadusega sekkuda sünnitusse, makrosoomse lapse sünniga, lapse kehvema Apgarihindega, lapse sünnijärgse kohanemisraskusega ning ema rasedusjärgse arteriaalse vererõhu kätevahelise erinevusega üle 10 mm Hg.Järeldused. D-vitamiini väike sisaldus on Eesti rasedate naiste hulgas sage ning selle peamisteks riskiteguriteks on raseduse kandmine vähese päikeseintensiivsusega kalendrikuudel ning raseduseelne rasvumine. D-vitamiini väike sisaldus mõjutab ebasoodsalt raseduse kulgu ning ema ja lapse tervisenäitajaid. Eestis suvekuudel talletatud D-vitamiini varud ei ole talviseks perioodiks piisavad. Lähtudes sellest, soovitame määrata vereseerumi D-vitamiini sisaldust hüpo-D-vitaminoosi riskirühmal ehk rasedatel naistel ning vajaduse korral seda toidulisanditena juurde manustada. Kirjanduse andmetele tuginedes näeme vajadust suurendada D-vitamiini profülaktilist doosi päevas 70–80 IU/kg

Maternal breast milk microbiota and immune markers in relation to subsequent development of celiac disease in offspring

The potential impact of the composition of maternal breast milk is poorly known in children who develop celiac disease (CD). The aim of our study was to compare the microbiota composition and the concentrations of immune markers in breast milk from mothers whose offspring carried the genetic predisposition to CD, and whether they did or did not develop CD during follow-up for the first 3 years of life. Maternal breast milk samples [CD children (n = 6) and healthy children (n = 18)] were collected 3 months after delivery. Enzyme-linked immunosorbent assays were used to measure TGF-beta 1, TGF-beta 2, sIgA, MFG-E8 and sCD14. For microbiota analysis, next generation (Illumina) sequencing, real-time PCR and denaturing gradient gel electrophoresis were used. Phylotype abundance and the Shannon 'H' diversity index were significantly higher in breast milk samples in the CD group. There was higher prevalence of the phyla Bacteroidetes and Fusobacteria, the classes Clostridia and Fusobacteriia, and the genera Leptotrichia, Anaerococcus, Sphingomonas, Actynomyces and Akkermansia in the CD group. The immunological markers were differently associated with some Gram-negative bacterial genera and species (Chryseobacterium, Sphingobium) as well as Gram-positive species (Lactobacillusreuteri, Bifidobacteriumanimalis). In conclusion, the microbiota in breast milk from mothers of genetically predisposed offspring who presented CD showed a higher bacterial phylotype abundance and diversity, as well as a different bacterial composition, as compared with the mothers of unaffected offspring. These immune markers showed some associations with bacterial composition and may influence the risk for development of CD beyond early childhood.Peer reviewe

Multi-ancestry genome-wide association study of gestational diabetes mellitus highlights genetic links with type 2 diabetes

Gestational diabetes mellitus (GDM) is associated with increased risk of pregnancy complications and adverse perinatal outcomes. GDM often reoccurs and is associated with increased risk of subsequent diagnosis of type 2 diabetes (T2D). To improve our understanding of the aetiological factors and molecular processes driving the occurrence of GDM, including the extent to which these overlap with T2D pathophysiology, the GENetics of Diabetes In Pregnancy Consortium assembled genome-wide association studies of diverse ancestry in a total of 5485 women with GDM and 347 856 without GDM. Through multi-ancestry meta-analysis, we identified five loci with genome-wide significant association (P < 5 x 10(-8)) with GDM, mapping to/near MTNR1B (P = 4.3 x 10(-54)), TCF7L2 (P = 4.0 x 10(-16)), CDKAL1 (P = 1.6 x 10(-4)), CDKN2A-CDKN2B (P = 4.1 x 10(-9)) and HKDC1 (P = 2.9 x 10(-8)). Multiple lines of evidence pointed to the shared pathophysiology of GDM and T2D: (i) four of the five GDM loci (not HKDC1) have been previously reported at genome-wide significance for T2D; (ii) significant enrichment for associations with GDM at previously reported T2D loci; (iii) strong genetic correlation between GDM and T2D and (iv) enrichment of GDM associations mapping to genomic annotations in diabetes-relevant tissues and transcription factor binding sites. Mendelian randomization analyses demonstrated significant causal association (5% false discovery rate) of higher body mass index on increased GDM risk. Our results provide support for the hypothesis that GDM and T2D are part of the same underlying pathology but that, as exemplified by the HKDC1 locus, there are genetic determinants of GDM that are specific to glucose regulation in pregnancy.Peer reviewe

Celiac Disease in Children, Particularly with Accompanying Type 1 Diabetes, Is Characterized by Substantial Changes in the Blood Cytokine Balance, Which May Reflect Inflammatory Processes in the Small Intestinal Mucosa

Cytokines play a pivotal role in the maintenance of intestinal homeostasis inducing pro- or anti-inflammatory response and mucosal barrier function in celiac disease (CD) and type 1 diabetes (T1D). We aimed to compare the levels of pro- and anti-inflammatory cytokines in CD patients without and with coexisting T1D, as well as to evaluate its association with the presence of enteroviruses (EV), regulatory T cells (Tregs), and dendritic cells (DCs) in small bowel mucosa. Altogether, 72 patients (median age 10.1 years) who had undergone small bowel biopsy were studied. The study group consisted of 24 patients with CD (median age 6.5 years), 9 patients with CD and concomitant T1D (median age 7.0 years), two patients with T1D (median age 8.5 years), and 37 patients (median age 14.0 years) with functional gastrointestinal disorders (FGD) and a normal small bowel mucosa as controls. The levels of 33 cytokines in serum were measured by multiple analysis using the Milliplex® MAP Magnetic Bead assay. The densities of FOXP3+ Tregs, CD11c+ DC, indoleamine 2,3-dioxygenase+ (IDO+) DC, langerin+ (CD207+) DCs, and EV were evaluated by immunohistochemistry as described in our previous studies. Circulating anti-EV IgA and IgG were evaluated using ELISA. The most important finding of the study is the significant increase of the serum levels of IL-5, IL-8, IL-13, IL-15, IL-17F, IL-22, IL-27, IP-10, MIP-1β, sIL-2Rα, sTNFRII, and TNFα in CD patients compared to controls and its correlation with the degree of small bowel mucosa damage graded according to the Marsh classification. The leptin level was higher in females in all study groups. The levels of IL-2, IL-6, IL-12 (P70), IL-15, IP-10, and IFNγ correlated significantly with the density of FOXP3+ Tregs in lamina propria of the small bowel mucosa, which supports the evidence about the signaling role of these cytokines in the peripheral maintenance of FOXP3+ Tregs. At the same time, a significant negative correlation occurred between the level of IL-4 and density of FOXP3+ Tregs in controls. Another important finding of our study was the correlation of IL-17F, IP-10, sTNFRII, MCP-1, and GM-CSF with the density of EV-positive cells in the lamina propria of the small bowel mucosa. Correlation of MIP-1 (CCL-4) with CD103+ DC and langerin+ DC densities may point to their significance in the recruitment of immune cells into the lamina propria and in driving the inflammatory response in CD patients. Our results suggest the predominance of Th1 and Th17 immune responses over EV VP1 protein in CD and T1D patients. The significant elevation of Th2 cytokines, like IL-5 and IL-13, but not IL-4, in CD patients and its correlation with the degree of small bowel mucosa damage could reflect the role of these cytokines in gut defense and inflammation

Animal reproduction, technology and welfare, December 3-4, 2018, Estonian University of Life Sciences, Tartu, Estonia : [presentations]

PresentationsPresentations of the final conference of the SEARMET "Animal reproduction, technology and welfare"

Multi-ancestry genome-wide association study of gestational diabetes mellitus highlights genetic links with type 2 diabetes

Abstract Gestational diabetes mellitus (GDM) is associated with increased risk of pregnancy complications and adverse perinatal outcomes. GDM often reoccurs and is associated with increased risk of subsequent diagnosis of type 2 diabetes (T2D). To improve our understanding of the aetiological factors and molecular processes driving the occurrence of GDM, including the extent to which these overlap with T2D pathophysiology, the GENetics of Diabetes In Pregnancy Consortium assembled genome-wide association studies of diverse ancestry in a total of 5485 women with GDM and 347 856 without GDM. Through multi-ancestry meta-analysis, we identified five loci with genome-wide significant association (P &lt; 5 x 10-8) with GDM, mapping to/near MTNR1B (P = 4.3 x 10-54), TCF7L2 (P = 4.0 x 10-16), CDKAL1 (P = 1.6 x 10-14), CDKN2A-CDKN2B (P = 4.1 x 10-9) and HKDC1 (P = 2.9 x 10-8). Multiple lines of evidence pointed to the shared pathophysiology of GDM and T2D: (i) four of the five GDM loci (not HKDC1) have been previously reported at genome-wide significance for T2D; (ii) significant enrichment for associations with GDM at previously reported T2D loci; (iii) strong genetic correlation between GDM and T2D and (iv) enrichment of GDM associations mapping to genomic annotations in diabetes-relevant tissues and transcription factor binding sites. Mendelian randomization analyses demonstrated significant causal association (5% false discovery rate) of higher body mass index on increased GDM risk. Our results provide support for the hypothesis that GDM and T2D are part of the same underlying pathology but that, as exemplified by the HKDC1 locus, there are genetic determinants of GDM that are specific to glucose regulation in pregnancy