Academic literature on the topic 'Amniotic fluid'

Our aim was to determine fetal and maternal endocrine and fluid-balance responses to prolonged loss of amniotic and allantoic fluids in sheep. In seven sheep, amniotic and allantoic fluids were drained [379.1 +/- 20.1 (SE) ml/day] from 107 to 135.3 +/- 0.6 days of gestation (term: 145 days). The results from these sheep were compared with those from seven control sheep. Maternal water intake, urine production, and urine osmolality were not altered by fluid drainage, nor were fetal and maternal arterial blood gases, pH, or plasma osmolalities. Fluid drainage increased amniotic, but not allantoic, fluid osmolality. Maternal plasma cortisol concentration increased with fluid drainage, but maternal plasma concentrations of prolactin and arginine vasopressin were unchanged. Fluid drainage increased prolactin concentrations in fetal plasma and amniotic fluid, but fetal plasma concentrations of cortisol (hydrocortisone), arginine vasopressin, norepinephrine, and epinephrine were unchanged. Our results show that the fetus is capable of maintaining its plasma osmolality despite prolonged loss of fluid from its amniotic and allantoic sacs and that this is associated with alterations in the production rate and the composition of amniotic fluid.

The effects of porcine fetal fluids (amniotic and allantoic) on microsomal prostaglandin G/H synthase (PGHS) activity were assessed. Both amniotic and allantoic fluids obtained from late-gestation sows stimulated PGHS activity (as indicated by increased formation of radiolabelled prostaglandin) in a dose-dependent manner. At the maximum dose tested, amniotic and allantoic fluids stimulated prostaglandin (PG) formation by 55.5 +/- 1.5 and 58.5 +/- 4.7%, respectively (n = 3, P less than 0.01). Based upon ED50 values, amniotic fluid was approximately threefold more effective than allantoic fluid in stimulating PG formation. The stimulatory effect of amniotic but not allantoic fluid increased significantly (P less than 0.01) during gestation (Days 47-112). The observed changes in the stimulatory effect of amniotic fluid on microsomal PG formation parallels the in vivo changes that occur in intra-uterine PG synthesis. Amniotic fluid stimulatory activity may contribute to this gestational increase in PG synthesis.

Amniotic fluid assessment is vital to fetal well-being. Accurately diagnosing an amniotic fluid abnormality can aid in the proper management of a pregnancy at risk for an adverse outcome. Sonography is the most common way to assess amniotic fluid volume throughout a pregnancy; however, the most accurate technique, amniotic fluid index or maximum vertical pocket, is yet to be determined. Dye-dilution techniques are the most accurate way to measure amniotic fluid volume, but they are invasive and can be performed only at the time of a cesarean delivery. Multiple studies have been performed to determine the accuracy of the amniotic fluid index and maximum vertical pocket methods when diagnosing amniotic fluid volume abnormalities. Based on the studies reviewed in this article, neither method stands out as superior to the other when it comes to diagnosing amniotic fluid abnormalities during pregnancy. However, the maximum vertical pocket should also always be considered when an amniotic fluid abnormality is suspected because the amniotic fluid index overdiagnoses amniotic fluid abnormalities, leading to increased rates of pregnancy intervention and the potential for adverse pregnancy outcomes.

Abstract We examined whether the 33-kDa serine protease prostate-specific antigen (PSA) is present in amniotic fluid and, if so, whether its concentration changes with gestational age. Analyzing 115 amniotic fluids with a highly sensitive immunofluorometric procedure, we found PSA in all the amniotic fluids examined and established that its concentration increases with increasing gestational age from 11 to 21 weeks, decreasing at delivery. PSA in amniotic fluid is present predominantly in the free (33 kDa) form; a minor fraction (< 20%) is present bound to alpha 1-antichymotrypsin. No significant correlation was seen between PSA and alpha-fetoprotein (AFP) in amniotic fluid or maternal serum in samples with high AFP. Amniotic fluid PSA was also measurable by two different established methods for PSA. Pregnant women had higher concentrations of serum PSA than nonpregnant women. The highest PSA concentration in amniotic fluid was associated with a pregnancy that was complicated by the Rhesus incompatibility syndrome but the source of the PSA was not established. From recent literature reports, and the association of PSA with prostate and breast tumors, we think PSA may serve as a growth factor regulator in cancer and in normal fetal development during pregnancy.

Abstract Disorders in amniotic fluid production occur frequently in pregnancy, and are associated with increased perinatal mortality and morbidity. This article will discuss amniotic fluid physiology, and will summarize noninvasive methods of measuring amniotic fluid. The definitions, common causes and possible therapeutic interventions available to treat oligohydramnios and polyhydramnios will be discussed. Learning objectives To have an understanding of the mechanisms underlying the regulation of amniotic fluid volumes To understand the limitations of current clinical measurements of amniotic fluid volumes To know the definitions, causes and potential treatments of disorders in amniotic fluid.

Acute Kidney Disease (CKD) is a major public health problem that affects some 3-7% of patients admitted to the hospital and approximately 25-30% of patients in the intensive care unit. None of the existing therapies are exempt from side effects and kidney physiological functionality is never restored. Transplantation has been reported as the preferred cure for CKD management but organ shortage and risks due to the immunosuppressive therapy makes it far from being the perfect treatment for ESRD. In this study we have focused our attention on finding novel strategies, in vitro and in vivo, to obtain kidney regeneration in case of acute and chronic kidney damage. First we have demonstrated the ability of hAFSCs to survive, proliferate and integrate into the embryonic kidney, while it undergoes organ development, in an in vitro culture system. We observed the presence of hAFSCs within kidney primordial, including tubules and developing nephrons. Thus, hAFSCs seem to have the capacity to undergo the expected mesenchymal to epithelial transition that occurs in normal renal development and are induced to express important early kidney markers such as GDNF, ZO-1 and Claudin. Moreover, hAFSCs do not appear to require prior genetic modification or exogenous production of kidney proteins for their differentiation to occur. This is a very important advantage that hAFSCs have for potential future regenerative or bioengineering application. With the in vivo experiments, we have demonstrated that early direct injection of hAFSCs into the kidney strongly ameliorates ATN injury as reflected by more rapid resolution of tubular structural damage and by normalization of creatinine and BUN levels. In addition, our data show evidence of immunomodulatory and antinflammatory effect of hAFSCs, at an early time point, comparable in magnitude to endogenous cytokine production. Understanding how donor and host cells combine to attenuate tubular damage may lead eventually to the application of hAFSCs for therapeutic purposes in acute kidney diseases. Nonetheless, beside the presence of a small number (1%) of cells with pluripotent characteristics, the composition of the other 99% of Amniotic Fluid cells is diverse, with a great amount of cells exhibiting commitment to a defined germ line or cellular endpoint. There seems to be clear evidence for the existence of progenitor cells in Amniotic Fluid, which can give rise to different cell types of mature organs. By 17 weeks of gestation is notable an increase tissue specific cellular presence and this data may indicate that the choice of the time point for cell selection is fundamental. In addition, we demonstrated in the amniotic fluid, the presence of a renal population with specific traits of commitment. In particular, the presence of podocytes at both undifferentiated and almost mature stages could favour their use for kidney regeneration in vitro and in vivo animal models. The presence and identification of specific renal progenitor cells in the Amniotic Fluid, committed to different compartments of the kidney environment, could represent a valuable new tool for regenerative purposes with regards to the treatment of a broad range of renal diseases. The discovery of renal specific progenitor cells within Amniotic Fluid could bring a breakthrough in the study for novel and more selective approaches in the renal therapy. However, the real pluripotential capability of these progenitors cells, in particular the kidney progenitors presenting more differentiation characteristics, has to be established. Moreover, their potential for survival, proliferation, integration, and differentiation needs to be assessed in in vivo models involving different types of renal damage.
L’insufficienza renale terminale ha raggiunto ormai proporzioni epidemiche in tutto il mondo e, tutt’oggi, non sono ancora state trovate terapie sostitutive o rigenerative efficaci a lungo termine. Attualmente la terapia dialitica e il trapianto allogenico rimangono le uniche alternative valide da utilizzare in questi pazienti nonostante se ne conoscano i numerosi limiti e complicanze. Recenti dati epidemiologici, in America e in Europa, mostrano che l’insufficienza renale colpisce circa l’8% della popolazione. [1] L’aumentata domanda di organi, in aggiunta all’insufficiente disponibilita’ di donatori, sta spingendo sempre piu’ i ricercatori di tutto il mondo a sviluppare nuove alternative terapeutiche per la sostituzione dei reni non funzionanti. [2] La creazione di organi bio-artificiali, attraverso l’utilizzo delle tecniche di ingegneria tissutale, ha finora dimostrato grandi difficolta’ specialmente nel riprodurre quegli organi e tessuti la cui struttura e funzione risultino particolarmente complesse, come nel caso dei reni. Storicamente gli ingegneri tissutali che si sono cimentati in questo campo hanno potuto utilizzare esclusivamente linee cellulari adulte dando origine a costrutti bidimensionali caratterizzati da limitata funzione e difficile applicabilita’ in vivo. [3] Nell’ultima dacade le cellule staminali stanno ricevendo sempre maggiore attenzione scientifica grazie al loro crescente impiego nella medicina rigenerativa per la ricostruzione e rigenerazione di tessuti bio-artificiali ed organi. Le cellule Staminali Embrionali (SE), derivate dalla blastocisti, hanno come caratteristiche peculiari il fatto che si replichino ampliamente e che siano capaci di formare aggregati (corpi embrioidi) che possono dar luogo ad una varietà di cellule specializzate come, ad esempio, cellule neurali, cardiache e pancreatiche. [3, 4] Il reclutamento di questo tipo di cellule staminali, tuttavia, comporta la distruzione di embrioni umani creando spinosi problemi etici e morali che portano, in molti Paesi, a vietarne l’utilizzo e il progresso scientifico. Per evitare questo tipo di controversie ricercatori di varie discipline hanno identificato potenziali fonti di cellule staminali alternative. [4, 5] E’ ormai ben noto che in molti tessuti adulti esistono cellule progenitrici con il compito di rigenerare o riparare l'organo a seguito dei fisiologici processi di senescenza o in caso di danno. [6, 7] Ci sono sempre piu’ evidenze che questi progenitori d’organo abbiano caratteristiche di plasticità piu’ elevate di quanto si pensasse originariamente. Parallelamente molti ricercatori credono che la rigenerazione di organi adulti derivi principalmente dalla mobilizzazione di cellule staminali provenienti dal midollo osseo. E’ stato dimostrato che cellule staminali del midollo osseo possono attraversare la barriera endolteliale e dar luogo a differenti linee cellulari differenziate, trasformando cellule circolanti in fegato, cervello, pancreas, pelle, intestino e anche rene. [27, 29] Il liquido amniotico e’stato usato per anni come uno strumento sicuro e valido per la ricerca di malattie genetiche e congenite del feto. Tuttavia, il liquido amniotico contiene un grande numero di cellule progenitrici che posono avere un importante ruolo nelle applicazioni della bioingegneria tissutale. Streubel et al. [8] hanno riportato l’utilizzo di cellule non emopoietiche per la conversione di amniociti in miociti. Recentemente una popolazione di cellule staminali c-Kit+, isolate nel liquido amniotico umano e murino, e’ stata caratterizzata e differenziata in tessuti originati dai tre foglietti embrionali: muscolare, neuronale, adipocitario, epatico, osseo ed endoteliale [9] Nel laboratorio diretto dal dr. R.E. De Filippo, Assistant Professor presso il Childrens Hospital di Los Angeles, abbiamo ampiamente studiato e utilizzato questa nuova popolazione di cellule staminali derivate dal liquido amniotico focalizzando le nostre ricerche sul loro utilizzo nella rigenerazione renale. Abbiamo dimostrato che questa popolazione totipotente di cellule mesenchimali e’ capace di riprodurre alcune tappe essenziali della nefrogenesi dopo essere state iniettate in reni embrionici. Tuttavia, le cellule staminali da liquido amniotico rapresentano meno dell’1% dell’intera popolazione cellulare e forse esistono altri progenitori cellulari, nel liquido stesso, gia’ orientati e piu’ proni alla differenziazione di particolari linee cellulari renali che possano essere utilizzate per gli stessi scopi rigenerativi ma con risultati migliori. Il volume e la composizione del liquido amniotico cambia durante la gravidanza e dall’ottava settimana di gestazione i reni fetali iniziano a produrre liquido che rapidamente aumenta di volume durante il secondo trimestre. [10] Il contatto tra il liquido amniotico e i diversi tessuti fetali sembra giustificare la presenza dei differenti tipi cellulari disciolti nel liquido stesso. La presenza di cellule mature derivanti dai tre foglietti germinali e’ stata gia’ dimostrata in passato come pure la presenza di progenitori cellulari di origine mesenchimale ed emopoietica prima della 12ma settimana gestazionale nell’uomo. [11,12,13] Cellule esprimenti proteine e markers genetici tipici di tessuti diversi come cervello, cuore, e pancreas sono state ritrovate nel liquido amniotico ma ulteriori indagini sono necessarie per completare la caratterizzazione dei diversi tipi cellulari presenti alle diverse eta’ gestazionali. [14, 15, 16] Lo sviluppo renale e’ un complesso processo di attivazione genica, interazioni cellulari ed extracellulari che devono aver luogo secondo un ordine spazio-temporale preciso e nella quantita’ adeguata. Durante l’embriogenesi, il rene metanefrico origina dall’invasione da parte della gemma ureterale, derivata dal dotto epiteliale di Wolffian, nel mesenchima metanefrico. [17] La gemma ureterale inizia la sua arborizzazione all’interno del mesenchima e portera’ alla formazione dell’intero sistema escretore, dall’uretere ai dotti collettori, mentre il mesenchima metanefrico dara’ luogo alle strutture epiteliali costituenti i nefroni (dal tubulo distale alla capsula di Bowman). CD-24 e Caderina 11 sono due markers di superficie che vengono usati per identificare cellule staminali ancora indifferenziate ma presenti nel mesenchima metanefrico prima di ricevere l’induzione da parte della gemma ureterale. [18] In aggiunta, altri markers di superficie che identificano una sottopopolazione di cellule appartenenti al mesenchima metanefrico nei vari stadi dell’induzione verso la nefrogenesi sono stati recentemente descritti in letteratura: Caderine E, PDGFRα, PDGFRβ, e NGFR ad alta affinita’. [19] Cellule Staminali derivate da liquido amniotico e differenziazione renale in vitro e in vivo Negli ultimi sette anni il gruppo di ricerca di cui ho fatto parte per due anni negli Stati Uniti (University of Southern California - Childrens Hospital Los Angeles) sta studiando una popolazione di cellule staminali ricavate da liquido amniotico (Amniotic Fluid Stem Cells, AFSC), umano e murino. Caratterictiche peculiari di questa popolazione cellulare sono: l’espressione di geni e marcatori di superficie comuni a cellule staminali di origine embrionale e mesenchimale; propagazione in vitro senza necessita’ di feeder-layer; mantenimento della lunghezza dei telomeri; capacità di differenziarsi in vitro e in vivo in molti tipi differenti di cellule e tessuti provenienti da tutti e tre i foglietti embrionali. [7] In particolare, negli ultimi 4 anni, il nostro gruppo si e’ concentrato sull’utilizzo di questa particolare popolazione di cellule staminali derivate da liquido amniotico nella rigenerazione renale in vitro e in vivo. [20, 21] Brevemente, siamo stati in grado di dimostrare, basandoci su un sistema in vitro, come le hAFSC abbiano la capacità di differenziarsi in parenchima renale dopo iniezione diretta in reni embrionici (12.5-16 giorni di gestazione) coltivati in vitro fino a 10 giorni. Le cellule staminali da liquido amniotico erano state precedentemente transfettate con il gene codificante una proteina fluorescente verde (GFP) e un secondo gene codificante per il Lac-Z. Mediante queste transfezioni siamo stati in grado di distinguere le cellule iniettate e dopo aver coltivato gli organi, anche a lungo termine (10 giorni), e’ stato possibile dimostrare la loro integrazione ed assimilazione nelle differenti tappe dello sviluppo renale. Analisi istologica dei reni iniettati con le staminali ha rivelato quanto questa popolazione di cellule sia capace di contribuire alle strutture primordiali del rene a partire dalla vescicola renale fino alle ultime fasi della nefrogenesi (tubuli e glomeruli). Mediante RT-PCR abbiamo quindi dimostrato la neoespressione, da parte delle AFSC iniettate, di geni attivi nelle diverse fasi dello sviluppo embrionale del nefrone. [20] Dopo aver dimostrato questa abilità di integrazione nel tessuto renale in via di sviluppo e la compartecipazione a tutte le tappe utili alla formazione del nefrone maturo in vitro, la nostra idea e’ stata quella di procedere all’applicazione in vivo delle cellule staminali da liquido amniotico. L’obiettivo e’ stato quello di dimostrare la loro reale capacità di sopravvivere, replicarsi ed integrarsi attivamente nei reni danneggiati di un modello di topo immunodepresso. Cellule staminali da liquido amniotico di topo (mouse Amniotic Fluid Stem Cells, mAFSC), esprimenti Lac-z e Luciferasi come marcatori, sono quindi state iniettate per via endovenosa (vena della coda) in un modello di topi immunodepressi con tubulonecrosi acuta. Il nostro ultimo obiettivo e’ stato quello di dimostrare se le cellule staminali venissero utilizzate dai reni danneggiati per riparare il danno e quindi fossero in grado di velocizzare la ripresa funzionale dell’organo. I risultati di tali esperimenti hanno dimostrato che le AFSC hanno una buona capacita’, anche in vivo, di integrarsi e partecipare attivamente alla riparazione del danno. Esse hanno iniziato ad esprimere GDNF, un fattore di trascrizione precoce presente nello sviluppo renale e in particolare nella formazione tubulare e glomerulare, e diversi altri markers tubulari quali Acquaporina-2, Agglutinina P, Agglutinina DB. Dagli esperimenti in vivo e’ quindi emerso che la popolazione di cellule staminali totipotenti, derivata da liquido amniotico (hAFSC), e’ capace di differenziarsi in diversi tipi cellulari appartenenti sia a strutture glomerulari (capsula di Bowman) che tubulari (tubulo distale e prossimale) senza dimostrare una chiara specificita’ per una delle due strutture. [9] In accordo con recenti pubblicazioni, abbiamo dimostrato un effetto immuno-modulartorio delle cellule staminali. Lo studio approfondito delle citochine, endogene ed esogene (prodotte dalle hAFSC iniettate), e il loro effetto nel migliorare la porzione infiammatoria del danno renale sono il passo successivo delle nostre ricerche. Un limite potenziale all’utilizzo terapeutico di questa popolazione cellulare totipotente risiede nel fatto che la maggior parte delle malattie renali che portano ad insufficienza renale terminale, colpiscono primariamente le strutture tubulari o quelle glomerulari, ma difficilmente entrambe contemporaneamente. Utilizzando dunque cellule staminali troppo indifferenziate, e quindi totipotenti, si rischierebbe di perdere efficacia terapeutica a causa del fatto che esse riceverebbero troppi segnali contemporaneamente in senso differenziativo e sarebbero indotte a seguire petterns riparativi non mirati e meno efficaci nella riparazione del danno principale. Se infatti avessimo bisogno di trattare selettivamente un danno tubulare piuttosto che uno glomerulare, l’utilizzo di cellule staminali totipotenti non sarebbe cosi’ ottimale come invece l’utilizzo di progenitori tubulo specifici opportunamente espansi ed eventualmente modificati. Questo concetto insieme al fatto che il liquido amniotico e’ composto da differenti popolazioni cellulari ha spinto a considerare la possibilita’ che ci possano essere linee cellulari maggiormente orientate in senso renale (progenitori organo specifici) che possano essere utilizzate in modo piu’ vantaggioso per la rigenerazione di strutture renali specifiche (id. cellule tubulari prossimali o distali, podociti, cellule mesangiali, cellule endoteliali e altro) Caratterizzazione cellulare del liquido amniotico e ricerca di progenitori renali specifici o gia’ parzialmente differenziati L’ultima parte della tesi si e’ concentrata nello studiare ed identificare le varie popolazioni cellulari presenti nel liquido amniotico a diverse settimane di gestazione. I campioni, di eta’ compresa tra le 15 e le 20 settimane di gestazione, sono stati ottenuti tramite amniocentesi, tecnica usata per studiare il cariotipo del feto durante lo sviluppo. Sono stati valutati differenti terreni di coltura, indagando proliferazione e conservazione della morfologia nei campioni ottenuti. L’analisi e la caratterizzazione della popolazione totale presente nel liquido amniotico e’ stata effettuata utilizzando RT-PCR, Real Time PCR e Western Blotting, analizzando l’espressione specifica di geni che sono coinvolti nel mantenimento della pluripotenzialita’, geni che identificano specificatamente i tre foglietti embrionali ed infine geni che identificano progenitori organo-specifici. Sono state inoltre identificate popolazioni specifiche renali, tramite immunoseparazione con biglie magnetiche (MASC). L’espressione di marcatori per i foglietti embrionali endoderma e mesoderma e’ piu’ alta in campioni piu’ giovani rispetto a campioni con tempo di gestazione maggiore mentre, per l’ectoderma, rimane pressoche’ invariata nel tempo. La presenza di cellule pluripotenti e’ costante cosi’ come le cellule staminali mesenchimali mentre le cellule progenitrici ematopoietiche, investigate tramite CD34, fanno la loro comparsa successivamente alle 17 settimane di gestazione. La presenza di progenitori tessuto specifici già “committed” e’ evidente nei campioni di gestazione più avanzata sia per quantitita’ che per specificità dell’organo preso in esame. E’ stata approfondita l’analisi di cellule progenitrici renali, utilizzando un ampio pannello di marcatori che identificano sia la componente tubulare che quella glomerulare del nefrone, struttura fondamentale per la filtrazione renale. I risultati ottenuti confermano la presenza di cellule progenitrici renali dopo le 18 settimane di gestazione. E’ stata identifica e studiata una popolazione esprimente CD24 e Caderin 11 isolata da campioni di liquido amniotico di 18 o piu’ settimane. CD24 e OB-cadehrin sono stati identificati nel topo come co-espressi in vivo nel mesenchima metanefrico. Dal mesenchima metanefrico ha origine il nefrone ed e’ una delle due strutture embrionali fondamentali per lo sviluppo del rene. Da questa popolazione principale sono state ottenute 4 nuove sottopopolazioni che identificano sottocompartimenti del glomerulo, come per esempio le cellule corticali stromogeniche (tramite selezione per la Tyrosin Kinase, TrKA), i podociti (selezionati per la Nefrina), le cellule del mesangio (con selezione positiva per PDGFR Alpha) e le cellule in transizione mesenchima-epitelio (con selezione per la Cadherina-E). Tramite PCR e Real Time PCR e’ stata dimostrata la forte specificita’ di ogni singola linea cellulare. E’ necessario uno studio approfondito che preveda per le AKPC differenziazioni in vitro ed in vivo, utilizzando fattori di crescita nefro-specifici e diversi modelli di danno renale acuto e cronico, in modo tale da confermare la loro possibile completa differenziazione in cellule renali mature. Un approfondimento sul meccanismo d’azione e sulle migliori tempisitiche di somministrazione, infine, sono i punti fondamentali da chiarire per comprendere il meccanismo d’azione delle hAFSC in vivo. Queste ricerche sono una base fondamentale per future applicazioni cliniche in pazienti che soffrono di nefropatie acute e croniche

Doutoramento em Química
O trabalho apresentado nesta tese teve como principais objectivos contribuir para o conhecimento da composição do líquido amniótico humano (LA), colhido no 2º trimestre de gravidez, assim como investigar possíveis alterações na sua composição devido à ocorrência de patologias pré-natais, recorrendo à metabonómica e procurando, assim, definir novos biomarcadores de doenças da grávida e do feto. Após uma introdução descrevendo o estado da arte relacionado com este trabalho (Capítulo 1) e os princípios das metodologias analíticas usadas (Capítulo 2), seguida de uma descrição dos aspectos experimentais associados a esta tese (Capítulo 3), apresentam-se os resultados da caracterização da composição química do LA (gravidez saudável) por espectroscopia de ressonância magnética nuclear (RMN), assim como da monitorização da sua estabilidade durante o armazenamento e após ciclos de congelamento-descongelamento (Capítulo 4). Amostras de LA armazenadas a -20°C registaram alterações significativas, tornando-se estas menos pronunciadas (mas ainda mensuráveis) a -70°C, temperatura recomendada para o armazenamento de LA. Foram também observadas alterações de composição após 1-2 ciclos de congelamento-descongelamento (a ter em conta aquando da reutilização de amostras), assim como à temperatura ambiente (indicando um período máximo de 4h para a manipulação e análise de LA). A aquisição de espectros de RMN de 1H de alta resolução e RMN acoplado (LC-NMR/MS) permitiu a detecção de 75 compostos no LA do 2º trimestre, 6 dos quais detectados pela primeira vez no LA. Experiências de difusão (DOSY) permitiram ainda a caracterização das velocidades de difusão e massas moleculares médias das proteínas mais abundantes. O Capítulo 5 descreve o estudo dos efeitos de malformações fetais (FM) e de cromossomopatias (CD) na composição do LA do 2º trimestre de gravidez. A extensão deste trabalho ao estudo dos efeitos de patologias no LA que ocorrem no 3º trimestre de gravidez é descrita no Capítulo 6, nomeadamente no que se refere ao parto pré-termo (PTD), pré-eclampsia (PE), restrição do crescimento intra-uterino (IUGR), ruptura prematura de membranas (PROM) e diabetes mellitus gestacional (GDM). Como complemento a estes estudos, realizou-se uma análise preliminar da urina materna do 2º trimestre para o estudo de FM e GDM, descrita no Capítulo 7. Para interpretação dos dados analíticos, obtidos por espectroscopia RMN de 1H, cromatografia líquida de ultra eficiência acoplada a espectrometria de massa (UPLC-MS) e espectroscopia do infravermelho médio (MIR), recorreu-se à análise discriminante pelos métodos dos mínimos quadrados parciais e o método dos mínimos quadrados parciais ortogonal (PLS-DA e OPLS-DA) e à correlação espectral. Após análise por validação cruzada de Monte-Carlo (MCCV), os modelos PLS-DA de LA permitiram distinguir as FM dos controlos (sensibilidades 69-85%, especificidades 80-95%, taxas de classificação 80-90%), revelando variações metabólicas ao nível do metabolismo energético, dos metabolismos dos aminoácidos e glícidos assim como possíveis alterações ao nível do funcionamento renal. Observou-se também um grande impacto das FM no perfil metabólico da urina materna (medido por UPLC-MS), tendo no entanto sido registados modelos PLS-DA com menor sensibilidade (40-60%), provavelmente devido ao baixo número de amostras e maior variabilidade da composição da urina (relativamente ao LA). Foram sugeridos possíveis marcadores relacionados com a ocorrência de FM, incluindo lactato, glucose, leucina, valina, glutamina, glutamato, glicoproteínas e conjugados de ácido glucurónico e/ou sulfato e compostos endógenos e/ou exógenos (<1 M) (os últimos visíveis apenas na urina). No LA foram também observadas variações metabólicas devido à ocorrência de vários tipos de cromossomopatias (CD), mas de menor magnitude. Os perfis metabólicos de LA associado a pré- PTD produziram modelos que, apesar do baixo poder de previsão, sugeriram alterações precoces no funcionamento da unidade fetoplacentária, hiperglicémia e stress oxidativo. Os modelos obtidos para os grupos pré- IUGR pré- PE, pré- PROM e pré-diagnóstico GDM (LA e urina materna) registaram baixo poder de previsão, indicando o pouco impacto destas condições na composição do LA e/ou urina do 2º trimestre. Os resultados obtidos demonstram as potencialidades da análise dos perfis metabólicos do LA (e, embora com base em menos estudos, da urina materna) do 2º trimestre para o desenvolvimento de novos e complementares métodos de diagnóstico, nomeadamente para FM e PTD.
The work presented in this thesis aimed to contribute to knowledge of 2nd trimester human amniotic fluid (AF) composition and to investigate the possible metabolic effects of prenatal disorders on AF composition through metabonomics, in order to define new potential disorder biomarkers. After an introduction describing the state-of-the-art (Chapter 1), the analytical methodologies used (Chapter 2) and the description of the experimental details of the work performed (Chapter 3), the results from the chemical characterization of AF (healthy pregnancy) by nuclear magnetic resonance (NMR) spectroscopy are presented, as well as the results of AF stability assessment during storage and after freeze-thaw cycles (Chapter 4). AF samples stored at -20°C registered significant compositional changes, less marked (but still measurable) at -70°C, the latter temperature being then recommended for AF storage. In addition, significant compositional changes were also observed after 1-2 freeze-thaw cycles (to be considered when sample re-usage is necessary), and at room temperature (indicating a maximum period of 4h for handling and analysis of AF). High resolution 1H NMR and hyphenated NMR (LC-NMR/MS) analysis enabled the detection of 75 different compounds in 2nd trimester AF, 6 of which were detected for the first time in AF. Moreover, diffusion-edited spectroscopy (DOSY) experiments allowed the characterization of the main AF proteins in terms of diffusivity and, hence, average molecular weight. Chapter 5 describes the study of the effects of fetal malformations (FM) and chromosomal disorders (CD) on the composition of 2nd trimester AF. In Chapter 6, this approach is extended, to the effects of 3rd trimester disorders, namely preterm delivery (PTD), preeclampsia (PE), intrauterine growth restriction (IUGR), premature rupture of the membranes (PROM) and gestational diabetes mellitus (GDM). These studies were complemented by a preliminary analysis of 2nd trimester maternal urine to study FM and GDM (Chapter 7). Interpretation of the analytical data obtained by 1H NMR spectroscopy, ultra performance liquid chromatography-mass spectrometry (UPLC-MS) and mid-infrared spectroscopy (MIR) was performed through partial least squares and orthogonal partial least squares - discriminant analysis (PLS-DA and OPLS-DA) and statistical correlation spectroscopy. Monte-Carlo cross-validated (MCCV) PLS-DA models of AF revealed separation of FM cases from controls (sensitivities 69-85%, 80-95% specificities, classification rates: 80-90%), revealing disturbances in energy metabolism, amino acids and sugar metabolisms and possibly abnormal kidney function. A high impact of FM on maternal urine was also observed (by UPLC-MS), however, the models obtained were of lower sensitivity (40-60%), probably due to the low sample numbers and higher variability of urine composition (in relation to AF). Possible markers of FM were suggested including lactate, glucose, leucine, valine, glutamine, glutamate, glycoproteins and conjugation products of glucuronic acid and/or sulfate with endogenous and/or exogenous metabolites (<1 M). In addition, metabolite variations were found in AF related to the occurrence of several types of chromosomal disorders (CD), although of smaller magnitude. Second trimester AF profiling associated with pre-PTD produced models, which, despite their low predictive power, enabled the detection of metabolite variations suggestive of early fetal-placental dysfunction, hyperglycaemia and oxidative stress. The models obtained for pre-IUGR, pre-PE, pre-PROM and pre-diagnostic GDM (both AF and urine) showed low predictive power, reflecting the small impact of these disorders in 2nd trimester AF and/or urine composition. The results presented demonstrate the potential of metabolic profiling of 2nd trimester AF (and, although based on less studies, maternal urine) for the development of new complementary prenatal diagnosis methods, namely for FM and PTD.

The study objective was to determine whether early 2 nd trimester amniotic fluid (AF) erythropoietin (EPO) was associated with and predictive of (a) development of maternal gestational diabetes (GDM) and (b) the infant outcome parameters of (i) gestational age at birth (GAAB) assessed exclusively among spontaneous vaginal deliveries or (ii) birth weight (measured in grams and percentiles). Enzyme-linked-immunosorbent assay was used to determine the EPO concentration of 170 biobanked AF samples. Student's t-test revealed no difference between GDM and non-GDM subjects. AF EPO was not predictive of GAAB despite being significantly greater among preterm infants compared to post-term infants. In contrast, AF EPO was significantly higher among the smallest infants using both birth weight classification schemes. However, following inclusion of known covariates AF EPO was predictive of gram birth weight only. Early 2nd trimester AF EPO may emerge as a useful biomarker of fetal nutritional status and/or growth.

The ability of the early intrauterine environment to program fetal neurodevelopment has been documented in several species, and there is evidence to suggest that a similar process may occur in humans. Glucocorticoids are the primary candidates for fetal hypothalamic-pituitary-adrenal (HPA) programming, but testosterone and DHEAS may also be involved. This thesis examines the relationship between these amniotic fluid honnones, particularly in the context of prenatal stress. Human studies lack the precision of animal experimental paradigms, and most stress protocols in humans have involved laboratory stressors poorly standardised for duration and nature of exposure. I aimed to use a standardised clinical stressor to examine the endocrine response in maternal plasma and amniotic fluid. I hypothesised that amniocentesis in a clinical setting elicits a maternal stress response associated with alterations in amniotic fluid steroidal hormones. I found amniocentesis to be associated with raised Spielberger state anxiety scores, validating the use of this procedure as a clinical model of psychological stress. Despite high state scores, only a modest rise of maternal cortisol (variance of 4%) was noted suggesting the response of an attenuated maternal HPA axis. I observed a positive correlation between maternal plasma cortisol and amniotic fluid cortisol that increased with gestational age. The magnitude of the correlation between maternal and amniotic fluid cortisol increased markedly with maternal state anxiety. A significant, positive correlation was observed between amniotic fluid cortisol and amniotic fluid testosterone. Amniotic fluid DHEAS correlated negatively with state anxiety scores but positively with maternal cortisol. This thesis characterises the relationships between the steroidal hormones cortisol, testosterone and DHEAS in amniotic fluid and in maternal plasma, especially in the context of prenatal stress generated by imminence of amniocentesis. The observations of a positive correlation between maternal and amniotic fluid cortisols, the magnitude increasing with gestation and maternal state anxiety scores, together with positive correlations between amniotic fluid cortisol and testosterone suggest more then one mechanism by which prenatal stress may influence fetal development.

There is a constant demand for haematopoietic stem cells (HSC) for clinical applications. Amniotic fluid stem (AFS) cells serve as a potential autologous cell source for therapy. Previously murine and sheep AFS have shown to have significant haematopoietic activity after transplantation in immune deficient mice. The haematopoietic potential of Human AFS have never been established in vivo, and its use has been limited by the presence of debris and low cell number at sample collection. My thesis explored the (1) isolation of human amniotic fluid (AF), (2) haematopoietic potential of human AF (CD117/c-Kit+; AFSC) by reconstituting the haematopoietic system of NOD-SCID/IL2rγnull (NSG) mice in vivo and (3) expansion of haematopoietic human AFSC in vitro. Human AF samples (2nd and 3rd trimester, n=110) were collected for the study under an ethically approved project from women undergoing amniocentesis for prenatal diagnosis of congenital disease, or amniodrainage procedures for fetal abnormality. I have employed several strategies to eliminate the large amount of cellular debris from the collected human AF and provide a more homogeneous cell population. Percoll density centrifugation demonstrated a reduction in cell debris and enrichment of the CD117+ population. The haematopoietic potential of human AFSC was explored in vivo. Human AF (2nd and 3rd trimester) and cord blood (CB; control) were selected for CD117 and CD34 respectively. Sorted cells (104 in 200μl PBS) were injected intravenously into sub-lethally irradiated NSG mice (~n=6/group). Human AFSC engrafted the haematopoietic system of NSG mice at levels similar to those achieved with CB-HSC post-primary and secondary transplantation. Importantly, multi-lineage haematopoietic reconstitution was observed at 16 weeks post-primary and secondary transplantation. Moreover, the possibility of expanding haematopoietic progenitors from human AF in vitro was demonstrated with the use of a cytokine-based media and the generation of haematopoietic progenitors by AF derived-induced pluripotent stem cell (AF-iPS) lines. In conclusion, I showed that human AF could be isolated, have long-term multi-lineage haematopoietic potential that is similar to the current “gold-standard” stem cell source for haematopoietic transplantation as well as demonstrates haematopoietic expansion. These findings make human AFSC to be an alternative novel fetal cell source for pre- and post-natal cell or cell-based gene therapy for the treatment of haematological disorders in the future.

The amniotic fluid is a rich source of fetal mesenchymal stem cells with broad differentiation capacity. Human amniotic fluid stem cells (AFSCs) have a high expansion potential, fast growth kinetics and harbour therapeutic potential to treat a variety of conditions. Moreover, they are easily isolated at mid-trimester or at delivery and can be used without ethical restrictions. Osteogenesis imperfecta (OI) is a genetic disease characterised by bone fragility, due to production of abnormal collagen type I. In this study, human AFSCs were transplanted into immunocompetent oim mice (OI mouse model, n=28) at birth. Bones were harvested after 8 weeks and analysed for mechanical properties, micro-structure, engraftment of donor cells and gene expression. Non-transplanted oim and wild-type mice were used as controls. Human AFSC injection decreased bone fracture rate and increased bone strength. Donor cells migrated to the bones, engrafted into sites of active bone formation and appeared to differentiate into osteoblasts, producing normal collagen. Moreover, transplantation improved the microarchitecture of the bones, although bone volume remained unaffected. Transplantation also promoted endogenous osteogenesis, with mouse genes involved in osteoblast differentiation and skeletal development significantly up-regulated, compared to non-transplanted mice. As a side project, a protocol for the isolation and differentiation of human fetal osteoblasts from the calvaria was optimised and a biobank of 24 samples, from gestational ages ranging from 9 to 21 weeks post-conception, was established. The data presented in this thesis indicate that human AFSCs are a promising source for cell therapy in OI. Donor cells may exert their therapeutic effects both by normalising the ECM and by influencing the maturation of resident osteoblasts. Ongoing work is focused on the further understanding of the mechanism of action of donor cells, using co-culture experiments with human AFSCs and human fetal osteoblasts.

ABSTRACT Background: Meconium aspiration syndrome refers to the aspiration of meconium and amniotic fluid by the fetus. It can occur when the fetus is still in the uterus, passing through the birth canal or when it takes its first breath after birth. Meconium aspiration is a serious condition with high morbidity and mortality. This study aimed to examine the effect of meconium stained amniotic fluid on the risk of infants asphyxia. Subjects and Method: Meta analysis and systematic review was conducted by collecting published articles from PubMed, Google Scholar, Clinical Key, Science Direct, and Springer Link databases. Keywords used risk factor, asphyxia, birth asphyxia, meconium stained amniotic, meconium stained liquor, and cross sectional. The inclusion criteria were full text, using English language, using cross-sectional study design, and reporting adjusted odds ratio. The study population was infants. Intervention was meconium stained amniotic liquid with comparison clean amniotic liquid. The study outcome was asphyxia. The collected articles were selected by PRISMA flow chart. The quantitative data were analyzed by fixed effect model using Revman 5.3. Results: 7 studies from Ethiopia reported that meconium stained amniotic fluid increased the risk of asphyxia in infants 5.83 (aOR= 5.83; CI 95%= 4.15 to 8.20; p <0.001). Conclusion: Meconium stained amniotic fluid increases the risk of asphyxia in infants. Keywords: meconium, amniotic fluid, asphyxia, infants Correspondence: Alfiati Nanda Widiyaningrum. Masters Program in Public Health, Universitas Sebelas Maret. Jl. Ir. Sutami 36A, Surakarta 57126, Central Java. Email: widiyaningruma@gmail.com. Mobile:081327524537. DOI: https://doi.org/10.26911/the7thicph.03.130

Many forms of pediatric and adult heart disease are accompanied by high morbidity and mortality, as the heart muscle has limited regenerative potential. Cell therapy has been proposed as a means to promote the regeneration of injured heart muscle. We have established lines of broad spectrum multipotent stem cells derived from primitive fetal cells present in human amniotic fluid (hAFS) cells (1). AFS cells offer several advantages: They are easy to isolate and grow (no feeder layers needed), are highly expansive including clonal growth and they can differentiate into all germ layers. In the current study, we demonstrate that AFS cells can differentiate into cardiac muscle cells and be used for cardiac tissue regeneration.

Review question / Objective: To identify the types and examine the range of available evidence of vertical transmission of SARS-CoV-2 from mother to newborn. To clarify the key concepts and criteria for diagnosis of SARS-CoV-2 vertical infection in neonates. To summarize the existing evidence and advance the awareness on SARS-CoV-2 vertical infection in pregnancy. Background: Severe Acute Respiratory Syndrome Virus 2 (SARS-CoV-2), the virus that causes 2019 coronavirus disease (COVID-19), has been isolated from various tissues and body fluids, including the placenta, amniotic fluid, and umbilical cord of newborns. In the last few years, much scientific effort has been directed towards studying SARS-CoV-2, focusing on the different features of the virus, such as its structure and mechanisms of action. Moreover, much focus has been on developing accurate diagnostic tools and various drugs or vaccines to treat COVID-19.

The specific objectives of this research were the study of the physical and nutritional properties of the In Ovo Feeding (IOF) solution (i.e. theosmostic properties and the carbohydrate: protein ratio composition). Then, using the optimal solution for determining its effect on hatchability, early nutritional status and intestinal development of broilers and turkey during the last quarter of incubation through to 7 days post-hatch (i.e. pre-post hatch period) by using molecular, biochemical and histological tools. The objective for the last research phase was the determination of the effect of in ovo feeding on growth performance and economically valuable production traits of broiler and turkey flocks reared under practical commercial conditions. The few days before- and- after hatch is a critical period for the development and survival of commercial broilers and turkeys. During this period chicks make the metabolic and physiological transition from egg nutriture (i.e. yolk) to exogenous feed. Late-term embryos and hatchlings may suffer a low glycogen status, especially when oxygen availability to the embryo is limited by low egg conductance or poor incubator ventilation. Much of the glycogen reserve in the late-term chicken embryo is utilized for hatching. Subsequently, the chick must rebuild that glycogen reserve by gluconeogenesis from body protein (mostly from the breast muscle) to support post-hatch thermoregulation and survival until the chicks are able to consume and utilize dietary nutrients. Immediately post-hatch, the chick draws from its limited body reserves and undergoes rapid physical and functional development of the gastrointestinal tract (GIT) in order to digest feed and assimilate nutrients. Because the intestine is the nutrient primary supply organ, the sooner it achieves this functional capacity, the sooner the young bird can utilize dietary nutrients and efficiently grow at its genetic potential and resist infectious and metabolic disease. Feeding the embryo when they consume the amniotic fluid (IOF idea and method) showed accelerated enteric development and elevated capacity to digest nutrients. By injecting a feeding solution into the embryonic amnion, the embryo naturally consume supplemental nutrients orally before hatching. This stimulates intestinal development to start earlier as was exhibited by elevated gene expression of several functional genes (brush border enzymes an transporters , elvated surface area, elevated mucin production . Moreover, supplying supplemental nutrients at a critical developmental stage by this in ovo feeding technology improves the hatchling’s nutritional status. In comparison to controls, administration of 1 ml of in ovo feeding solution, containing dextrin, maltose, sucrose and amino acids, into the amnion of the broiler embryo increased dramatically total liver glycogen in broilers and in turkeys in the pre-hatch period. In addition, an elevated relative breast muscle size (% of broiler BW) was observed in IOF chicks to be 6.5% greater at hatch and 7 days post-hatch in comparison to controls. Experiment have shown that IOF broilers and turkeys increased hatchling weights by 3% to 7% (P<0.05) over non injected controls. These responses depend upon the strain, the breeder hen age and in ovo feed composition. The weight advantage observed during the first week after hatch was found to be sustained at least through 35 days of age. Currently, research is done in order to adopt the knowledge for commercial practice.