Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Martina Michalská Supervisor: doc. PharmDr. Martina Čečková, Ph.D. Title of diploma thesis: Expression of selected membrane transporters in placentas of pregnant women diagnosed for preterm rupture of membranes Placenta is a key organ for pregnancy maintenance. One of its main functions is transport of compounds between mother and her fetus. The transplacental penetration is ensured due to membrane transporters that are present in the apical or basal side of trophoblast. Their expression level is affected by many physiological and pathological factors, among others it can be influenced by infection and inflamatory reaction. Inflammation is also one of the risk factors of preterm deliveries and it can be therefore assumed that these pathological states are accompanied by changes in expression of placental transporters. This study was performed using 51 placentas obtained from Faculty hospital in Hradec Králové from women who underwent preterm delivery and on 15 placentas delivered in term. The study employed quantitative RT-PCR approach. The mRNA expression of membrane transporters ABCB1, ABCG2, OATP1A2, OATP1B3, OATP2A1, OATP2B1, OATP3A1, OATP4A1 was assessed and the results were compared to...

The aim of the research was to find new potential biomarkers of preterm premature rupture of membranes. The amniotic fluid and fetal membranes peptidic composition was analyzed using a fully automated 2D liquid chromatographic system coupled to mass spectrometry. A comparison of peptidomes of amniotic fluid and amniochorionic membranes with preterm premature rupture and term intact membranes was performed. Ten proteins from amniotic fluid were identified as potential biomarkers for PPROM. The created map of amniotic fluid peptides and proteins depending on the gestational age is important for proteomics-based identification of biomarkers for fetal abnormalities and other pregnancy complications. / Mokslinio darbo metu siekta nustatyti potencialius priešlaikinio neišnešioto vaisiaus dangalų plyšimo biožymenis. Panaudojant dvidimensinę skysčių chromatografiją bei masių spektrometriją išanalizuota vaisiaus vandenų ir dangalų peptidinė sudėtis. Ištirti ir palyginti amniochorioninės membranos ir vaisiaus vandenų peptidai bei su jais siejami baltymai, kai prieš laiką plyšta neišnešioto vaisiaus dangalai arba vaisius išnešiojamas iki numatyto gimdymo termino ir dangalai išlieka sveiki. Išanalizavus skirtumus, nustatyti nauji galimi priešlaikinio neišnešioto vaisiaus dangalų plyšimo biožymenys.

Structured summary Aim of the study: To compare the diagnostic reliability, accuracy, and safety of amniocentesis and amniotic fluid Interleukin-6 testing in the diagnosis of intrauterine inflammation of patients with preterm premature rupture of membranes. Type of study: Prospective cohort study Name and location of study site: Department of Gynaecology and Obstetrics, Faculty of Medicine, Charles University in Pilsen Set and methodology: We prospectively examined patients with pPROM between the 23rd and 34th week of gestation in 2014 - 2017. All of them underwent amniocentesis and determination of IL-6 levels in amniotic fluid, leukocytes and bacteria in amniotic fluid as well as maternal blood examination for inflammation parameters. The results were compared to histological examination of the placenta after delivery for the presence of chorioamnionitis. Based on the values mentioned above the sensitivity, specificity, negative and positive predictive value, false positive and negative predictive value and accuracy of the test were determined together with an assessment of statistical significance. Furthermore, the feasibility and incidence of perioperative complications as well as the risk of secondary infection when pregnancy continued were evaluated by serial aniocenteses at weekly intervals. The...

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Martina Michalská Supervisor: doc. PharmDr. Martina Čečková, Ph.D. Title of diploma thesis: Expression of selected membrane transporters in placentas of pregnant women diagnosed for preterm rupture of membranes Placenta is a key organ for pregnancy maintenance. One of its main functions is transport of compounds between mother and her fetus. The transplacental penetration is ensured due to membrane transporters that are present in the apical or basal side of trophoblast. Their expression level is affected by many physiological and pathological factors, among others it can be influenced by infection and inflamatory reaction. Inflammation is also one of the risk factors of preterm deliveries and it can be therefore assumed that these pathological states are accompanied by changes in expression of placental transporters. This study was performed using 51 placentas obtained from Faculty hospital in Hradec Králové from women who underwent preterm delivery and on 15 placentas delivered in term. The study employed quantitative RT-PCR approach. The mRNA expression of membrane transporters ABCB1, ABCG2, OATP1A2, OATP1B3, OATP2A1, OATP2B1, OATP3A1, OATP4A1 was assessed and the results were compared to...

Background: In preterm infants with premature rupture of membranes, antibiotic treatment is frequently started but rates of early onset sepsis are lower. In line with national guidelines, a stratified approach in the decision to start antibiotic treatment using maternal history, clinical impression and biomarkers has been implemented in our level III neonatal center and its results are evaluated. - Methods: Retrospective cohort study of all preterm newborns with rupture of membranes at least 1 h prior to delivery admitted to our tertiary neonatal intensive care unit. Data on antibiotic exposure, mortality and major neonatal complications were extracted from the electronic patient charts to evaluate the effects and safety of our stratified approach. - Results: Four hundred fifty-six infants met the inclusion criteria. 120 (26%) received primary antibiotics whereas 336 (74%) did not. Of those receiving primary antibiotics, 13 (11%) had a blood culture positive sepsis, 46 (38%) met the criteria of clinical sepsis and in 61 (51%) sepsis was ruled out and antibiotics were stopped after 48-96 h. All infants with blood culture positive sepsis were identified and treated within the first 24 h of life using this approach. None of the 336 infants who were not started on antibiotics primarily needed antibiotic therapy within the first 5 days of life. There were no deaths or major neonatal complications in the group that did not receive empiric antibiotics. - Conclusions: Our stratified approach for preterm infants with premature rupture of membranes allows a safe reduction of antibiotic exposure even in this high risk population. As a result, only 25% of high risk preterm newborns are treated with antibiotics of which more than half receive less than 5 days of treatment. To treat one infant with blood culture positive sepsis, only 9 infants receive empiric antibiotics.

The vaginal microbial community is critical to a woman’s health and the health of her family. Bacterial vaginosis (BV) is a polymicrobial syndrome characterized by a shift of the vaginal microbiota from a Lactobacillus dominated community to a dense biofilm containing a complex mixture of organisms. Although BV is an important risk factor for poor reproductive health outcomes, the etiology of BV is poorly understood. Gardnerella vaginalis is a hallmark species of BV. Phylogenetic analysis of cpn60 universal target sequences from metagenomic studies of the vaginal microbiome and from G. vaginalis isolates resolved four subgroups within the species. This subdivision, supported by whole genome similarity comparisons, demonstrated that these subgroups might represent different species. Among a group of African women, only G. vaginalis subgroup B was significantly more abundant in women with BV relative to women with Nugent scores not consistent with BV. To characterize the subgroups further, several phenotypic and molecular factors of G. vaginalis subgroups were assessed. Proteomic profiles of isolates within each subgroup formed unambiguous clusters. Sialidase gene sequences were detected in all subgroups, however enzymatic activity was detected only in subgroup B. Two isolates of subgroup B isolates (N153 and N101) were incapable of growth in 7% CO2. Given the well-known relationship between an anaerobic microbiota and BV, anaerobic isolates of G. vaginalis are potentially important players in the vaginal microbial community. To determine genome content differences that could account for the phenotypic difference, whole genome sequences of four G. vaginalis subgroup B isolates representing facultative and anaerobic phenotypes were determined. Comparison of genomes led to the identification of genes predicted to encode proteins involved in cell wall biogenesis and protection from oxidative damage that might account for the observed phenotypes. The cpn60 universal target based methodology that improved resolution of the vaginal microbiota including G. vaginalis was applied in a prospective study of the vaginal microbiome of women with preterm premature rupture of membranes (PPROM). The objectives were to characterize the vaginal microbiota of women following PPROM, and to determine if microbiome composition at the time of rupture predicts latency duration and perinatal outcomes. Only 13/70 samples collected from 36 women were dominated by Lactobacillus spp., the expected profile for healthy women, while Megasphaera type 1 and Prevotella spp. were detected in all samples. Microbiome profiles at the time of membrane rupture did not cluster by gestational age at PPROM, or latency duration. Microbial profiles were unstable over the latency period, with dramatic shifts in composition between weekly samples, and an overall decrease in Lactobacillus abundance. Mollicutes were detected by PCR in 81% (29/36) of women, and these women had significantly lower gestational age at delivery and correspondingly lower birth weight infants than Mollicutes negative women. Taken together, the results presented in this thesis demonstrate the value of high resolution profiling of the vaginal microbiome using cpn60 UT sequences. The resolution of subgroups within G. vaginalis has potentially significant implications for women's health diagnostics, requiring a shift away from considering G. vaginalis as a single entity. The PPROM study provides foundational information that may lead to the identification of informative sequence patterns, providing clinicians with better tools for expectant management following PPROM.

Abstract The aim of the present study was to evaluate whether bacterial vaginosis (BV) diagnosed in early pregnancy and treated with vaginal clindamycin affects pregnancy outcome, and to investigate the predictive value of interleukins-6 (IL-6) and -8 (IL-8), and insulin-like growth factor-binding protein-1 (IGFBP-1) in cervical secretions, separately and combined by cervical measurement with transvaginal ultrasonography, on preterm delivery. A further aim was to analyze retrospectively the significance of absent or reversed end-diastolic velocity (AREDV) in the umbilical artery on perinatal outcome, and to investigate the short- and long-term outcome of infants born prematurely as a result of various causes (indicated preterm birth, preterm premature rupture of the membranes=PPROM). Bacterial vaginosis (BV) was screened in 1956 women in a low-risk population at the first antenatal visit, using Gram stain. One hundred and one of 143 BV-positive women were randomized to receive vaginal clindamycin or placebo. Seventy-seven women at 22-32 gestational weeks with premature uterine contractions, and 78 controls were recruited for assay of cervical IL-6, IL-8-, and IGFBP-1, and ultrasonographic measurements, which were repeated twice at two-week intervals. Eighty-three women with AREDV in the umbilical artery in high-risk pregnancies at less than 34 gestational weeks (e.g. pre-eclampsia, small-for-gestational age [SGA]) between the years 1988-95 were analyzed retrospectively as regards perinatal outcome. Further, for 103 women between the 24th and the 33rd week of pregnancy, delivered by cesarean section because of maternal or fetal indications, and for 103 matched women, between the years 1990-97, their infants were analyzed as regards neonatal mortality and morbidity, and the outcome at one year of corrected age. Similarly, 78 women with PPROM at gestational weeks 17-30, and 78 controls were also analyzed. The prevalence of BV was 7.3% (143/1956) and the preterm birth rate in women with BV was 9.9%. Preterm birth occurred in 21% vs. 0% according to whether or not BV persisted. The preterm birth rate was 14% in the clindamycin group vs. 6% in the placebo group. Cervical IL-6 at a concentration of 128 ng/L had a 73% sensitivity and 77% specificity in predicting preterm birth (35% vs. 6%). The combination of IL-6 and a cervical index of > 0.2 increased the specificity to 97%, the sensitivity falling to 45%. Concentrations of IGFBP-1 were most elevated (> 21 μg/mL) in cases with neonatal infections (36% vs. 2%). In cases of absent end-diastolic velocity (AEDV) the perinatal mortality (PNM) rate was 9%, compared with 36% in the reversed end-diastolic velocity (REDV) group. Respiratory distress (RDS) and hypoglycemia, and chronic lung disease (CLD; 15% vs. 3%) occurred significantly more often in the indicated than in the spontaneously preterm infants. The PPROM infants had more limb contractures (8% vs. 0%) and pulmonary hypoplasia (12% vs. 5%) and more chronic lung problems up to one year of age than the spontaneously preterm born infants without PPROM. The persistence of pregnancy BV is a risk factor for preterm birth, but vaginal clindamycin used in a low-risk population in early pregnancy is of no use in reducing the preterm birth rate in cases of BV. The level of IL-6 has a relatively low sensitivity and a limited role as a single method in clinical decision making but in combination with cervical examination by ultrasonography it seems to have a predictive role in cases of threatened preterm birth. A finding of AREDV in the umbilical artery is a warning signal of threatened fetal asphyxia. Infants born after indicated preterm delivery (for fetal or maternal reasons) or PPROM are at risk of later chronic lung disease.

Abstract Prematurity is the main cause of morbidity and mortality in infants. In 25–40% of the cases preterm birth is associated with intrauterine inflammation. Surfactant proteins (SPs) A, C, and D have roles in innate immunity. In the female reproductive tract and in amniotic fluid (AF), these proteins may modulate the inflammatory responses leading to preterm birth. The aim of the present study was to establish a mouse model of lipopolysaccharide (LPS)-induced preterm birth of live-born pups and to study the activation of the innate immune system. By using mice overexpressing either rat SP-A (rSP-A) or rSP-D the roles of SP-A and SP-D in inflammatory responses were investigated. In addition, the expression of SP-C in gestational tissues was analyzed. The association of SP-C single nucleotide polymorphism (Thr138Asn) with spontaneous preterm birth and preterm premature rupture of membranes (PPROM) was investigated in a homogenous northern Finnish population of mothers and infants. Wild-type (WT) mice were injected with a single dose of intraperitoneal LPS at 16 or 17 days of gestation (term 19–20 days) leading to preterm delivery of live-born fetuses. After LPS, cytokine levels increased rapidly in maternal serum and in the uterus. This maternal inflammatory response was followed by the modest inflammatory activation in fetal and feto-maternal compartments. In fetal lung the expression of SP-A and SP-D was downregulated. The overexpression of SP-A or SP-D was evident in gestational tissues of rSP-A or rSP-D mice, respectively. In addition, excess of these proteins was detected in AF. Overexpression of either rSP-A or rSP-D modulated the LPS-induced inflammatory response related to preterm birth. Most notably, the expression of IL-4 and IL-10 in uteri and IL-10 in fetal membranes was lower in overexpressing animals. SP-C was detected in mouse and human placentas, fetal membranes, and in uteri of pregnant mice. The fetal SP-C polymorphism strongly associated with the duration of PPROM. The present study provides new information about the molecular events in inflammation induced preterm birth, particularly about the roles of cytokines and SPs in this process. Understanding of the mechanisms involved in preterm parturition may provide means for prevention and management of preterm births in the future. / Tiivistelmä Ennenaikaisuus on suurin vastasyntyneiden terveyttä ja henkeä uhkaava vaara. Kohdunsisäiset tulehdusreaktiot ovat ennenaikaisten synnytysten yleisimpiä aiheuttajia. Surfaktanttiproteiinit (SP:t) A, C ja D osallistuvat synnynnäisen immuniteetin säätelyyn. Voidaan olettaa, että synnytyskanavassa ja lapsivedessä surfaktanttiproteiinit säätelevät ennenaikaiseen synnytykseen johtavia tulehdusreaktioita. Tutkimuksen tavoitteena oli luoda hiirimalli, jossa ennenaikainen synnytys saadaan aikaan lipopolysakkaridin (LPS:n) injektiolla vatsaonteloon. Hiirimallin avulla tutkittiin puolustusjärjestelmän aktivaatiota sekä äidin että sikiön kudoksissa. Rotan SP-A:ta (rSP-A:ta) tai rSP-D:tä yli-ilmentävien hiirten avulla selvitettiin, muuttavatko nämä proteiinit ennenaikaiseen syntymään johtavaa vastetta. Lisäksi määritettiin SP-C:n ilmentyminen hiiren ja ihmisen kohdussa, sikiökalvoilla ja istukassa. SP-C-geenin yhden emäksen polymorfian (Thr138Asn) liittymistä ennenaikaiseen synnytykseen tai sikiökalvojen puhkeamiseen tutkittiin homogeenisessä pohjoissuomalaisessa tutkimuspopulaatiossa. Villin tyypin hiirille raskauden 16. tai 17. päivänä annettu LPS-annos sai aikaan elävien poikasten ennenaikaisen syntymisen. Emon seerumissa havaittiin sytokiinipitoisuuksien nopea nousu LPS:n vaikutuksesta. Emon tulehdusvaste johti synnynnäisen immuniteetin aktivaatioon istukassa, sikiökalvoilla ja kohdussa, kun taas muutokset sikiön kudoksissa olivat pieniä. Sikiön keuhkoissa SP-A:n ja SP-D:n ilmentyminen väheni. SP-A:ta tai SP-D:tä yli-ilmentävillä hiirillä havaittiin lisääntynyt SP-A:n tai SP-D:n määrä kohdussa, sikiökalvoilla, istukassa ja lapsivedessä. SP-A:n tai SP-D:n yli-ilmentyminen muutti LPS:n aiheuttamaa tulehdusvastetta. Erityisesti IL-4:n ja IL-10:n ilmentyminen kohdussa ja IL-10:n ilmentyminen sikiökalvoilla vähenivät. SP-C:n ilmentyminen havaittiin hiiren ja ihmisen istukassa ja sikiökalvoilla sekä hiiren kohdussa raskauden aikana. Sikiön SP-C-geenin polymorfia liittyi sikiökalvojen ennenaikaisen puhkeamisen kestoon. Tutkimus antaa lisätietoa tulehduksen aiheuttaman ennenaikaisen synnytyksen mekanismista sekä sytokiinien ja SP-A:n SP-D:n osuudesta synnytystapahtumassa. Mekanismin ymmärtäminen on erittäin tärkeää, jotta ennenaikaiset synnytykset voitaisiin tulevaisuudessa ehkäistä tehokkaammin.

Changes of immune parameters induced by intraamnial inflammation Abstract Intraamniotic infection plays an important role in the etiology of preterm birth and can lead to a serious threat to fetal health. The diagnostic approach is based on direct microbiological detection of an infectious agent or indirect detection by determining various biomarkers, which concentration increases during intraamniotic infection. Due to the nature of the infection, these parameters are determined from amniotic fluid, which makes this diagnosis difficult for both the doctor and the mother and routinely unavailable. The dissertation comments the published results of a scientific team whose aim was to identify suitable markers of infection, determine their concentration in amniotic fluid and test their diagnostic potential in cervical fluid, ie biological material that can be collected non- invasively. Amniotic and cervical fluid samples were taken from women with singleton pregnancies with preterm labor and that were complicated by intraamniotic inflammation and infection in part of the cohort. It was found that among the tested molecules there is a statistically higher concentration of calreticulin, cathepsin G, CD11b, FcgammaBP and MIP1α in amniotic fluid during intraamniotic infection. Significantly higher levels of...

MicroRNAs (miRNAs) are small non-coding RNAs with a length of 18 to 25 nucleotides playing a pivotal role in post-transcriptional regulation of gene expression. There are miRNAs whose expression is limited to a certain tissue type and diseases which are characterized by a unique miRNA expression profile. I assumed spontaneous preterm birth (PTB) and preterm prelabor rupture of membranes (PPROM) would be characterized by a unique miRNA expression profile. I observed the gene expression of 15 placental specific miRNAs (miR-512-5p, miR-515-5p, miR-516b-5p, miR-517-5p, miR-518b, miR-518f-5p, miR-519a-5p, miR-519d-3p, miR-519e- 5p, miR-520a-5p, miR-520h, miR-524-5p, miR-525-5p, miR-526a and miR-526b-5p) in placental tissue of pacients with PTB, PPROM and women with term in labor pregnancies (FG). PTB group consisted of 24 pacients, PPROM group of 75 pacients and FG group of 20 pacients. Quantitative real-time PCR was used to quantify gene expression. In the group of PTB pregnancies I identified 3 significantly upregulated miRNAs (miR-516b-5p, miR-519d-3p and miR-524-5p) and 4 miRNAs (miR-518b, miR-519a-5p, miR-520h and miR-526a) with a trend to upregulation compared to controls (FG). In the group of PPROM pregnancies I identified 3 miRNAs (miR-519d-3p, miR-520h and miR-256b-5p) with a trend to...