Um Projeto Temático de Pesquisa foi desenvolvido no Laboratório de Poluição Ambiental do Departamento de Patologia da Faculdade de Medicina da Universidade de São Paulo com o objetivo de avaliar os efeitos da exposição aguda/crônica ao ar ambiente de um grande centro urbano sobre a saúde. Dentro deste projeto, uma linha de pesquisa foi dedicada ao estudo dos efeitos dessa exposição sobre a saúde reprodutiva feminina. Evidências de estudos epidemiológicos e experimentais implicam os fatores ambientais na infertilidade humana e resultado obstétrico adverso. Contudo, poucos estudos foram conduzidos até o presente para avaliar um possível efeito da exposição à poluição ambiental particulada sobre a saúde reprodutiva feminina. Portanto, o objetivo dos projetos da minha linha de pesquisa é fornecer dados que possam demonstrar os possíveis efeitos da exposição crônica à poluição ambiental particulada sobre a função ovariana e o desenvolvimento embrionário inicial. O objetivo do primeiro projeto desta tese foi avaliar diferentes metodologias utilizadas para a coloração diferencial das linhagens celulares do blastocisto, um método mais adequado para a avaliação de sua qualidade e normalidade. As células de blastocistos intactos de camundongo obtidos através de fertilização in vitro (FIV) foram permeabilizadas e coradas utilizando-se diferentes concentrações de um detergente (TX-100; 0,5% ou 1%) e de iodeto de propídeo (IP; 50 g/mL ou 100 g/mL) e depois disso, incubadas durante a noite em uma solução contendo diferentes fixadores (etanol, metanol, paraformaldeído PFA1% ou 4%) e bisbenzimida. Para a avaliação da qualidade de coloração e contagem diferencial dos núcleos, os blastocistos foram montados em lâminas de vidro e analisados em um microscópio de epifluorescência. O escore de qualidade de coloração foi significativamente diferente (p<0,05) entre todas as soluções fixadoras, sendo maior para o etanol, seguido pelo metanol, PFA1% e PFA4%. Mudanças da concentração do IP e o uso de diferentes soluções de fixação revelaram efeitos significativos na contagem de células da massa celular interna (MCI) e na razão MCI/trofectoderma (TE). Concentrações diferentes do detergente utilizado para a permeabilização celular apresentaram efeitos significativos sobre a contagem de células TE e razão MCI/TE. Concluímos que o protocolo que utiliza TX-100 1% para a permeabilização celular, 50 g/mL de IP para coloração das células TE e etanol como solução de fixação representa o método mais eficiente para a coloração diferencial e contagem das células das linhagens celulares do embrião no estágio de blastocisto. No segundo projeto que compõe esta, o objetivo foi avaliar os efeitos da exposição pré e/ou pós-natal ao ar ambiente sobre a fertilização, desenvolvimento embrionário e segregação das linhagens celulares em blastocistos pré-implantacionais, utilizando o modelo de FIV de camundongo. Fêmeas de camundongo com idade de seis semanas tiveram a ovulação estimulada e foram expostas no período pré- e/ou pós-natal ao ar filtrado (AF-AF), ar filtrado ar ambiente (AF-AA) ou ar ambiente ar ambiente (AA-AA) em câmaras de exposição 24 horas por dia, sete dias na semana, durante nove semanas. Os pontos de avaliação reprodutivos analisados incluíram a duração da gestação, tamanho e peso da prole, índice de nascidos vivos, razão sexual, resposta ovariana à estimulação, taxa de fertilização, desenvolvimento embrionário, taxas de formação e de eclosão dos blastocistos, contagem celular total e proporção da alocação celular à MCI e TE. A duração da gestação, tamanho e peso da prole, índice de nascidos vivos e razão sexual foram similares nos diferentes grupos de exposição. A resposta ovariana não foi afetada pelo protocolo de exposição. Um efeito multivariável para a exposição pré e/ou pós-natal ao material particulado fino ambiente sobre a FIV, o desenvolvimento embrionário e a coloração diferencial dos blastocistos foi observado. A contagem celular na MCI e a razão MCI/TE dos blastocistos produzidos no protocolo AF-AF foram significativamente maiores do que aquelas em blastocistos produzidos nos protocolos FA-AA e AA-AA. Nenhuma diferença na contagem celular total foi observada. Baseando-se nessas observações, nosso estudo sugere que a exposição ao material particulado fino presente no ar ambiente de um grande centro urbano pode afetar negativamente a saúde reprodutiva feminina através da alteração da especificação das linhagens celulares do embrião no estágio de blastocisto. Finalmente, o propósito do terceiro projeto que compõe esta tese foi de avaliar os efeitos da exposição pré e/ou pósnatal ao ar ambiente no final da vida reprodutiva sobre a fertilização, desenvolvimento embrionário e segregação das linhagens celulares em blastocistos pré-implantacionais, utilizando o modelo de FIV de camundongo. Fêmeas de camundongo com idade de cinco meses tiveram a ovulação estimulada e foram expostas no período pré e/ou pós-natal ao ar filtrado (AF-AF), ar filtrado ar ambiente (AF-AA) ou ar ambiente ar ambiente (AA-AA) em câmaras de exposição 24 horas por dia, sete dias na semana, durante seis meses. Os pontos de avaliação reprodutivos foram os mesmos que aqueles utilizados no segundo projeto. A duração da gestação, tamanho e peso da prole, índice de nascidos vivos e razão sexual foram similares nos diferentes grupos de exposição. A resposta ovariana não foi afetada pelo protocolo de exposição. Um efeito multivariável para a exposição pré e/ou pós-natal ao material particulado fino ambiente sobre coloração diferencial dos blastocistos, mas não sobre a FIV e o desenvolvimento embrionário, foi observado. A contagem celular na MCI e a razão MCI/TE dos blastocistos produzidos no protocolo AF-AF foram significativamente maiores do que aquelas em blastocistos produzidos nos protocolos FA-AA e AA-AA. A contagem celular do TE dos blastocistos produzidos no protocolo FA-FA foi significativamente menor do que aquela em blastocistos produzidos nos protocolos FA-AA e AA-AA. A contagem celular total foi similar entre os grupos. Nosso estudo sugere que a exposição à poluição ambiental particulada de um grande centro urbano não altera a função ovariana, mas pode afetar negativamente a saúde reprodutiva feminina no período final do menacme, através da alteração da especificação das linhagens celulares do embrião no estágio de blastocisto / A thematic research project to evaluate the health effects of acute/chronic exposure to ambient air in a large urban center was developed at the Air Pollution Laboratory in the Department of Pathology at the University of São Paulo School of Medicine. Within this project a specific research line was committed to the study of the effects of this exposure on female reproductive health. Evidence from epidemiological and experimental studies implied environmental factors as possible contributors to human infertility and poor obstetric outcome. However, very few studies evaluating a possible effect of exposure to particulate air pollution on female reproductive health have been conducted so far. Thus, the aim of the projects in my research line was to provide data that could show the possible effects of chronic exposure to particulate air pollution on ovarian function and early embryo development. The objective of the first project was to assess different methodologies used in cell lineage differential staining of the blastocyst, a method for more accurate evaluation of its quality and normality. Cells of zona-intact mouse blastocysts obtained from in vitro fertilization (IVF) were permeabilized and stained using different concentrations of a detergent (TX-100; 0.5% or 1%) and propidium iodide (PI; 50 g/mL or 100 g/mL) followed by overnight incubation in a solution containing different fixatives (ethanol, methanol, paraformaldehyde - PFA 1% or 4%) and bisbenzimide. To evaluate the staining quality and count the nuclei differentially, blastocysts were mounted and viewed using epifluorescence microscopy. Staining quality scores were significantly different (P < 0.05) among all fixative solutions with the highest for ethanol followed by methanol, PFA1%, and PFA4%. Changes in PI concentration and use of different fixative solutions revealed significant effects on inner cell mass (ICM) cell count and ICM/trophectoderm (TE) ratio. Different concentrations of the detergent used for cell permeabilization showed significant effects on TE cell counts and ICM/TE ratio. I concluded that the protocol using 1% TX-100 for cell permeabilization, 50 g/mL of PI for TE cell staining, and ethanol as a fixative solution is the most efficient method for cell lineage differential staining and counting at the blastocyst stage. In the second project the objective was to evaluate the effects of preand/ or postnatal exposure to ambient air on fertilization, embryo development, and cell lineage segregation in preimplantation blastocysts using the IVF mouse model. Six-week old superovulated mice were preand/ or postnatally exposed to filtered air (FA-FA), filtered-ambient air (FAAA), or ambient air (AA-AA) in exposure chambers 24/7 for nine weeks. Reproductive endpoints evaluated included gestation length, litter size, litter birth weight, live birth index, sex ratio, ovarian response to superovulation, fertilization rate, embryo development, blastocyst and hatching rates, total cell count, and proportion of cell allocation to ICM and TE. Gestation length, litter size, litter birth weight, live birth index, and sex ratio were similar among exposure groups. Ovarian response was not affected by the exposure protocol. A multivariate effect for pre- and/or postnatal exposure to ambient fine particulate matter on IVF, embryo development, and blastocyst differential staining was found. Cell counts in ICM and ICM/TE ratios in blastocysts produced in the FA-FA protocol were significantly higher than in blastocysts produced in FA-AA and AA-AA protocols. No difference in the total cell count was observed. Based on these observations the study suggests that exposure to ambient fine particulate matter in a large urban center may negatively affect female reproductive health by disrupting the lineage specification at the blastocyst stage. Finally, the purpose of the third project was to evaluate the effects of pre- and/or postnatal exposure to particulate air pollution on fertilization, embryo development, and cell lineage segregation in preimplantation blastocysts during the late-life reproductive period using the IVF mouse model. Five-month-old superovulated mice were pre- and/or postnatally exposed to filtered air (FA-FA), filtered-ambient air (FA-AA), or ambient air (AA-AA) in exposure chambers 24/7 during six months. Reproductive endpoints were the same as the ones selected for the second project. Gestation length, litter size, litter birth weight, live birth index, and sex ratio were similar among exposure groups. Ovarian response was not affected by the exposure protocol. A multivariate effect for pre- and/or postnatal exposure to ambient air on blastocyst differential staining but not on IVF and embryo development was found. Cell counts in ICM and ICM/TE ratios in blastocysts produced in the FA-FA protocol were significantly higher than in blastocysts produced in FA-AA and AA-AA protocols. Cell counts in TE cells in blastocysts produced in the FA-FA protocol were significantly lower than in blastocysts produced in FA-AA and AA-AA protocols. The total cell count was similar among groups. This study suggests that exposure to particulate air pollution in a large urban center has no effect on ovarian function but may negatively affect female reproductive health in the late-life period by disrupting the lineage specification at the blastocyst stage.

Um Projeto Temático de Pesquisa foi desenvolvido no Laboratório de Poluição Ambiental do Departamento de Patologia da Faculdade de Medicina da Universidade de São Paulo com o objetivo de avaliar os efeitos da exposição aguda/crônica ao ar ambiente de um grande centro urbano sobre a saúde. Dentro deste projeto, uma linha de pesquisa foi dedicada ao estudo dos efeitos dessa exposição sobre a saúde reprodutiva feminina. Evidências de estudos epidemiológicos e experimentais implicam os fatores ambientais na infertilidade humana e resultado obstétrico adverso. Contudo, poucos estudos foram conduzidos até o presente para avaliar um possível efeito da exposição à poluição ambiental particulada sobre a saúde reprodutiva feminina. Portanto, o objetivo dos projetos da minha linha de pesquisa é fornecer dados que possam demonstrar os possíveis efeitos da exposição pré-concepcional de curta duração às partículas de exaustão do diesel (PED) e à poluição ambiental particulada sobre a função ovariana, o desenvolvimento embrionário inicial e resultado gestacional utilizando um modelo experimental e um epidemiológico. O objetivo do primeiro projeto desta tese foi avaliar os efeitos de dois meios de cultura comerciais no desenvolvimento de oócitos de camundongo fertilizados in vitro até o estágio de blastocisto. Zigotos obtidos de fêmeas de camundongo de 8 semanas de idade submetidas à indução da ovulação foram cultivados in vitro até o estágio de blastocisto em meio simples otimizado enriquecido com potássio (KSOM) ou meio G1/G2. A porcentagem de zigotos que se desenvolveu até o estágio de blastocisto 96 e 120 horas após a inseminação e que sofreu eclosão parcial ou completa no quinto dia de cultivo foi significativamente maior no grupo KSOM. O número médio de células da massa celular interna (MCI) foi 11,7 ± 4,0 e 9,2 ± 5,2 para os zigotos cultivados nos grupos KSOM e G1/G2, respectivamente, mostrando um número significativamente maior de células MCI em blastocistos derivados da cultura no meio KSOM. Concluímos que o meio KSOM comercialmente disponível é superior ao meio seqüencial G1/G2 para o cultivo de zigotos até o estágio de blastocisto no modelo de fertilização in vitro (FIV) em camundongos. No segundo projeto que compõe esta tese, o objetivo foi avaliar os efeitos da exposição de curta duração às PED sobre a fertilização, desenvolvimento embrionário e segregação das linhagens celulares em blastocistos pré-implantacionais utilizando o modelo de FIV em camundongo. A instilação intranasal de água destilada (grupo controle), de PED nativas (grupo PED-N) ou de PED ácidoextraídas (grupo PED-AE), realizada uma vez ao dia por três dias, iniciada no primeiro dia de administração de gonadotrofinas, foi realizada em fêmeas de camundongo com oito semanas de idade. Os pontos de avaliação reprodutivos analisados incluíram a resposta ovariana a estimulação, taxa de fertilização, desenvolvimento embrionário, taxas de formação e de eclosão dos blastocistos, contagem celular total e proporção da alocação celular à MCI e trofoectoderma (TE), e a morfologia da MCI. A resposta ovariana não foi afetada pelo protocolo de exposição. Um efeito multivariado para a exposição às PED-N e PED-AE na coloração diferencial de blastocistos e na morfologia da MCI, mas não para a FIV ou desenvolvimento embrionário, foi observado. A contagem celular da MCI e a razão MCI/TE em blastocistos produzidos no grupo controle foram significativamente maiores do que em blastocistos produzidos nos grupos PED-N e PED-AE. O número total de células dos blastocistos foi similar entre os grupos. O escore que representa a morfologia da massa celular interna foi significativamente maior no grupo controle quando comparado àquele encontrado nos grupos PED-N e PEDAE. Baseando-se nesses resultados, nosso estudo sugere que a exposição de curta duração às PED pode afetar negativamente o processo reprodutivo através do distúrbio da especificação das linhagens celulares do embrião em estágio de blastocisto. Finalmente, a exposição a toxinas ambientais pode ser inevitável durante o período pré-concepcional em grandes centros urbanos e seus efeitos são desconhecidos. Portanto, o propósito do terceiro projeto que compõe esta tese foi avaliar os potenciais efeitos da exposição de curta duração à poluição ambiental particulada durante a fase folicular sobre os resultados clínicos, laboratoriais e gestacionais de casais submetidos à fertilização in vitro e transferência de embriões (FIVETE). Trezentos e quarenta e oito mulheres submetidas ao seu primeiro ciclo de FIVETE foram avaliadas retrospectivamente neste estudo coorte, casocontrole casado. A exposição ao material particulado ambiental (MP) durante a fase folicular de cada paciente foi estimada baseando-se em dados da poluição ambiental (1997-2006) categorizados em período Q1-3 ( 56,72 g/m3) e Q4 (>56,72 g/m3). Desse grupo, 177 pacientes que engravidaram (casos) foram comparadas com 354 mulheres que conceberam espontaneamente (controles). Os principais pontos de avaliação incluíram a resposta ovariana às gonadotrofinas, o número de oócitos recuperados e as taxas de fertilização, de clivagem, de qualidade embrionária, de implantação, de gestação, de abortamento e de nascidos vivos. Nenhum efeito da exposição a níveis elevados de MP durante a fase folicular foi observado nos resultados clínico e laboratorial, na transferência embrionária ou no sucesso dos ciclos de tratamento das pacientes submetidas à FIVETE. Mulheres expostas ao período Q4 durante a fase folicular do ciclo de concepção apresentaram um risco significativamente maior de abortamento, independentemente do método de concepção (razão de chance: 2,58; intervalo de confiança de 95%: 1,63 4,07), quando comparado àquele de mulheres expostas ao período Q1-3. O risco de perda da gestação aumentou 3% por unidade de aumento do valor médio do MP na fase folicular (p= 0,000). Os resultados apresentados aqui fornecem evidências para uma relação causal entre a breve exposição a níveis elevados de MP ambiente durante o período pré-concepcional e a perda gestacional inicial, independentemente do método de concepção e está associada a um aumento de 2,6 vezes no risco de abortamento. Apesar da ausência de efeitos dessa exposição sobre os resultados clínicos e laboratoriais e sobre o sucesso do tratamento, a FIVETE foi incapaz de reduzir esse risco / A thematic research project to evaluate the health effects of acute/chronic exposure to ambient air in a large urban center was developed at the Air Pollution Laboratory in the Department of Pathology at the University of São Paulo School of Medicine. Within this project a specific research line was committed to the study of the effects of this exposure on female reproductive health. Evidence from epidemiological and experimental studies implied environmental factors as possible contributors to human infertility and poor obstetric outcome. However, very few studies evaluating a possible effect of exposure to particulate air pollution on female reproductive health have been conducted so far. Thus, the aim of the projects in my research line was to provide data that could show the possible effects of short-term preconceptional exposure to diesel exhaust particles and particulate air pollution on ovarian function, early embryo development and pregnancy outcome using experimental and epidemiological models. The objective of the first project was to examine the effects of two commercial media on the development of mouse ova fertilized in vitro to the blastocyst stage. One-cell embryos obtained from eight-week old superovulated mice were cultured in vitro up to the blastocyst stage in potassium-enriched simplex optimized medium (KSOM) or G1/G2 media. The percentage of zygotes that developed to the blastocyst stage 96 and 120 hours after insemination and that partially or completely hatched by day five of culture was significantly higher in the KSOM group. The mean number of inner cell mass (ICM) cells was 11.7 ± 4.0 and 9.2 ± 5.2 for zygotes cultured in KSOM and G1/G2 groups respectively, revealing a significantly higher cell number in the ICM of blastocysts derived from culture in KSOM medium. I concluded that commercially available KSOM medium is superior to sequential G1/G2 media for culturing one-cell embryos up to the blastocyst stage in the mouse IVF model. In the second project the objective was to evaluate the effects of short-term exposure to diesel exhaust particles on fertilization, embryo development, and cell lineage segregation in preimplantation blastocysts using the mouse IVF model. Intranasal instillation of distilled water (control group), native diesel exhaust particles (N-DEP group) or acid-extracted diesel exhaust particles (AE-DEP) once a day, for three days starting on the first day of gonadotrophin administration was performed on eight-week old female mice. Reproductive endpoints evaluated included ovarian response to superovulation, fertilization rate, embryo development, blastocyst and hatching rates, total cell count, and proportion of cell allocation to ICM and trophectoderm (TE), and ICM morphology. Ovarian response was not affected by the exposure protocol. A multivariate effect for exposure to NDEP and AE-DEP on blastocyst differential staining and ICM morphology but not on IVF or embryo development was found. Cell counts in ICM and ICM/TE ratios in blastocysts produced in the control group were significantly higher than in blastocysts produced in N-DEP and AE-DEP groups. The total cell count was similar among groups. The score that represents ICM morphology was significantly higher in the control group when compared to that found in N-DEP and AE-DEP groups. Based on these results this study suggests that short-term exposure to DEP may negatively affect the reproductive process by disrupting the lineage specification at the blastocyst stage. Finally, exposure to environmental toxins may be unavoidable during the preconceptional period in large urban centers and its effects are unknown. Thus, the purpose of the third project was to assess the potential effects of short-term exposure to particulate air pollution during the follicular phase on clinical, laboratory, and pregnancy outcomes for couples undergoing IVF/ET. Three hundred forty-eight patients undergoing their first IVF/ET cycle were evaluated in this retrospective cohort-matched casecontrolled single-center study. Exposure to ambient particulate matter (PM) during the follicular phase for each patient was estimated based on air pollution data (1997-2006) categorized in Q1-3 ( 56.72 g/m3) and Q4 (>56.72 g/m3) periods. From this group 177 women who became pregnant (cases) were compared with 354 who had conceived spontaneously (controls). Main outcome measures included response to gonadotrophins, number of oocytes retrieved, fertilization, cleavage, embryo quality, implantation, pregnancy, miscarriage, and live birth rates. No effects of follicular phase exposure to high levels of PM on clinical, laboratory, embryo transfer or treatment outcome were found in women undergoing IVF/ET. Women exposed to Q4 level PM during the follicular phase of the conception cycle had significantly higher risk of miscarriage, regardless of the method of conception (odds ratio, 2.58; 95% confidence interval: 1.63-4.07) when compared to women exposed to Q1-3 level PM. The risk of miscarriage increased 3% per unit increase in follicular phase PM average level (p=0.000). The results presented here provide evidence of a causal role for brief exposure to high levels of ambient PM during the preconceptional period in early pregnancy loss, regardless of the method of conception, with a 2.6-fold increase in risk of miscarriage. Despite the absence of effects of this exposure on clinical, laboratory, and treatment outcome, IVF/ET was unable to reduce this risk

<p>Uvod: Infertilitet se defini&scaron;e kao bezuspe&scaron;na koncepcija nakon jedne godine seksualnih odnosa bez upotrebe kontracepcije u fertilnoj fazi menstrualnog ciklusa. Metode asistirane reprodukcije predstavljaju efektivan način lečenja infertiliteta. Ispitivanje i identifikacija kratkoročnih i dugoročnih efekata arteficijalnih reproduktivnih tehnologija je veoma izazovan zadatak. Prvenstveni razlog tome je velika heterogenost u načinu sakupljanja, obrade, klasifikacije i tumačenja, sada već, obilja informacija koje su prikupljene u različitim istraživanjima. Individualni pristup lečenju neplodnosti, brz napredak i stalne promene u metodologiji arteficijalnih reproduktivnih tehnologija, uz ranije navedene pote&scaron;koće u vezi sa prikupljanjem i analizom podataka, značajno otežavaju precizno sagledavanje svih mogućih rizika i posledica arteficijanog začeća. Uprkos brojnim istraživanjima, naučnim publikacijama i akumuliranim dokazima, ostale su mnoge dileme u vezi odgovora na pitanja - da li su arteficijalno začete trudnoće u većoj meri praćene rizicima za neadekvatan razvoj ploda, lo&scaron;ijim perinatalnim ishodom i kakve su dugoročne posledice po decu, kao i da li su ovi rizici podjednako zastupljeni u jednoplodnim i vi&scaron;eplodnim trudnoćama.<br />Cilj rada: Ciljevi rada su bili da se utvrdi struktura morbiditeta kod prevremeno rođene dece začete vantelesnom oplodnjom (iz jednoplodnih i vi&scaron;eplodnih trudnoća) u prve dve godine života, te da se identifikuju perinatalni faktori koji su povezani sa pojavom akutnih i hroničnih komplikacija i oboljenja kod prevremeno rođene dece začete vantelesnom oplodnjom. Takođe, cilj rada je bio da se utvrde karakteristike psihomotornog razvoja kod prevremeno rođene dece začete vantelesnom oplodnjom na kraju dvanestog, osamnaestog i dvadesetčetvrtog meseca života, kao i da se identifikuju specifični faktori rizika za nepovoljan telesni, neurolo&scaron;ki i psiholo&scaron;ki ishod lečenja kod prevremeno rođene dece začete vantelesnom oplodnjom.<br />Materijal i metode: U studiju su uključena prevremeno rođena deca koja su bila hospitalizovana u Službi za neonatologiju i intenzivnu i poluintenzivnu negu i terapiju, i koja su nakon toga, tokom prve dve godine života redovno praćena u neonatolo&scaron;koj ambulanti Instituta za zdravstvenu za&scaron;titu dece i omladine Vojvodine u Novom Sadu. Retrospektivnim delom studije su obuhvaćena deca koja su lečena u Službi i praćena u neonatolo&scaron;koj ambulanti, a koja su rođena počev od 01. 01. 2011. do 31.12.2012. godine i praćena do navr&scaron;ena puna 24 meseca života. Podaci o pacijentima koji su uključeni u retrospektivni deo istraživanja prikupljani su pregledom medicinske dokumentacije. U prospektivni deo studije su uključena deca koja su lečena u Službi i koja su praćena u neonatolo&scaron;koj ambulanti, a koja su rođena između 01.01. 2013.godine i 31.12.2014. godine i potom praćena do navr&scaron;enih 24 meseca života. Iz navedene kohorte, formirane se dve grupe: Ispitivana grupa (Grupa 1) je obuhavatila svu prevremeno rođenu decu začetu vantelesnom oplodnjom koja su bila hospitalizovana i praćena na Institutu u navedenom periodu. Kontrolna grupa (Grupa 2) obuhvatila je prevremeno rođenu decu začetu prirodnim putem. Deca iz kontrolne grupe izabrana su iz kohorte tako da njihov broj bude jednak broju dece iz ispitivane grupe. Ispitanici iz ove grupe su ujednačeni (&#39;&#39;mečovani&#39;&#39;) sa decom iz ispitivane grupe prema gestacijskoj starosti i datumu rođenja. Gestacijska starost ispitanika iz kontrolne grupe se ne razlikuje za vi&scaron;e od &plusmn; 4 dana u odnosu na decu iz ispitivane grupe. Datum rođenja ispitanika koji su uključeni u kontrolnu grupu se ne razlikuje za vi&scaron;e od &plusmn; 3 meseca u odnosu na decu iz ispitivane grupe.<br />U momentu uključivanja u studiju uzimani su sledeći anamezni podaci:<br />Podaci u vezi sa majkom, trudnoći i porođaju: starost majke u momentu koncepcije, broj prethodnih poku&scaron;aja asistirane koncepcije, stručna sprema, mesto stanovanja, hronične bolesti dijagnostikovane pre trudnoće, akutne i hronične bolesti dijagnostikovane tokom trudoće (hipertenzija, pre-eklampsija, eklampsija, o&scaron;tećenje jetre), prevremena ruptura plodovih ovojaka, primena lekova tokom trudnoće, jednoplodna ili vi&scaron;eplodna trudnoća. Podaci o poremećajima posteljice i ovojaka: ablacija, placenta previja, horioamnionitis. Podaci u vezi sa detetom: intrauterina infekcija, intrauterina restrikcija rasta, način porođaja, Apgar skor. Antropometrijski parametri (telesna masa, telesna dužina, obim glave) na rođenju i tokom perioda ambulantnog praćenja deteta. Dužina inicijalne hospitalizacije deteta. Dužina invazivne i/ili neinvazivne respiratorne potpore i oksigenoterapije. Dijagnoze na otpustu iz bolnice: prisustvo te&scaron;kih posledica prematuriteta, &scaron;to podrazumeva: intrakranijalnu hemoragiju 3. i 4. stepena (definisanu u međunarodnoj klasifikaciji bolesti &ndash; deseta revizija (MKB10) pod &scaron;ifrom P52.2), cističnu periventrikularnu leukomalaciju, retinopatiju prematuriteta, bronhopulmonalnu displaziju, nekrotizirajući enterokolitis, sepsu i/ili meningitis (mikrobiolo&scaron;ki ili klinički dijagnostikovanu). Prisustvo urođenih anomalija ili genetskih sindroma i bolesti (definisanih u MKB10 pod &scaron;iframa Q00 do Q99), kao i prisustvo urođenih bolesti metabolizma (definisanih u MKB10 pod &scaron;iframa E00 do E90).<br />U retrospektivnom delu studije, pregledani su specijalistički izve&scaron;taji iz neonatolo&scaron;ke ambulante pri posetama deteta u uzrastu deteta od 12, 18 i 24 meseca, i beleženi su sledeći podaci: sve prethodno postavljene dijagnoze koje su navedene na specijalističkim izve&scaron;tajima iz neonatolo&scaron;ke ambulante, antropometrijski prametri u momentu pregleda (telesna dužina, telesna masa i obim glave), neurolo&scaron;ki nalaz (tonus, trofika, kožni i tetivni refleksi, prisustvo lateralizacije u neurolo&scaron;kom nalazu), nalaz oftalmologa (uredan nalaz/patalo&scaron;ki nalaz), procena fine i grube motorike, govora, kognitivne funkcije i socijalnog kontakta i zbirna procena psihomotornog razvoja. U prospektivnom delu studije, pri kontrolnim pregledima u neonatolo&scaron;koj ambulanti, u uzrastu deteta od 12, 18 i 24 meseca, određivano je i beleženo sledeće: ranije postavljene dijagnoze koje su navedene u medicinskoj dokumentaciji, antropometrijski prametri u momentu pregleda (telesna dužina, telesna masa i obim glave), neurolo&scaron;ki nalaz (tonus, trofika, kožni i tetivni refleksi, prisustvo lateralizacije u neurolo&scaron;kom nalazu), nalaz oftalmologa (uredan nalaz/patalo&scaron;ki nalaz), procena fine i grube motorike, govora, kognitivne funkcije i socijalnog kontakta i zbirna procena psihomotornog razvoja.<br />Rezultati: Prosečna vednost TM ispitanika iz Grupe 1, u uzrastu od 12 meseci, bila je statistički značajno manja u odnosu na ispitanike iz Grupe 2 (Studentov t test). Prosečne vednosti TD ispitanika iz obe grupe, u uzrastu od 12 meseci, nisu se statistički značajno razlikovale (Studentov t test). Prosečne vednosti OGL ispitanika iz obe grupe, u uzrastu od 12 meseci, nisu se statistički značajno razlikovale (Studentov t test). Udeo ispitanika sa patolo&scaron;kim oftalmolo&scaron;kim nalazom nije se statistički značajno razlikovao između Grupe 1 i Grupe 2 (Fi&scaron;erov test tačne verovatnoće). Udeo ispitanika sa patolo&scaron;kim neurolo&scaron;kim nalazom nije se statistički značajno razlikovao između Grupe 1 i Grupe 2 (Hi kvadrat test). Prosečne vrednosti globalnog koeficijenta razvoja (RQ), kao i prosečne vrednosti skora za pojedine elemente za procenu razvoja (motorika, koordinacija, govor i dru&scaron;tvenost) po Brunet -L&eacute;zine skali, nisu se statistički značajno razlikovale između grupa (Studentov t test). U Grupi 1 bilo je 92 (59,740%) deteta čiji je nekorigovani RQ bio ispod 90, dok je u Grupi 2 bilo 61 (39,610%) dete čiji je nekorigovani RQ bio ispod 90. Ova razlika u broju dece sa RQ koji je ispod proseka za kalendarski uzrast je statistički značajna (Hi kvadrat test, p=0,0004). Relativni rizik za ispodprosečno postignuće na testu za procenu psihomotornog razvoja (RQ&lt;90), za decu iz Grupe 1 bio je vi&scaron;i, u odnosu na decu iz Grupe 2 (RR = 1,495; 95% CI 1,181 &ndash; 1,892). U Grupi 1, bilo je 87 (56,494%) dece koja su postigla ispodprosečne korigovane vrednosti skora na testu za procenu psihomotornog razvoja (korigovani RQ&lt;90). U Grupi 2 bilo je 69 (44,805%) dece koja su postigla ispodprosečne korigovane vrednosti skora na testu za procenu psihomotornog razvoja (korigovani RQ&lt;90). Ova razlika je statistički značajna (Hi kvadrat test, p =0,040). Relativni rizik za ispodprosečno postignuće na testu za procenu psihomotornog razvoja (korigovani RQ&lt;90), za decu iz Grupe 1 bio je vi&scaron;i, u odnosu na decu iz Grupe 2 (RR = 1,261; 95%CI 1,008 &ndash; 1,577). U kategoriji dece, koja su i pored korekcije u odnosu na GS imala ispodprosečno postignuće na testu za procenu psihomotornog razvoja, u Grupi 1 čak 81/87 (93,310%) dece je imalo vrednost korigovanog RQ &ge; 85, a u Grupi 2 ovu vrednost korigovanog RQ imalo je 60/69 (86,956%) dece.<br />Prosečne vednosti TM ispitanika iz obe grupe, u uzrastu od 18 meseci, nisu se statistički značajno razlikovale (Studentov t test). Prosečne vednosti TD ispitanika iz obe grupe, u uzrastu od 18 meseci, nisu se statistički načajno razlikovale (Studentov t test). Prosečne vednosti OGL ispitanika iz obe grupe, u uzrastu od 18 meseci, nisu se statistički značajno razlikovale (Studentov t test). Udeo ispitanika sa patolo&scaron;kim oftalmolo&scaron;kim nalazom nije se statistički značajno razlikovao između Grupe 1 i Grupe 2 (Hi kvadrat test). Udeo ispitanika sa patolo&scaron;kim neurolo&scaron;kim nalazom nije se statistički značajno razlikovao između Grupe 1 i Grupe 2 (Hi kvadrat test). Prosečne vrednosti RQ, kao i prosečne vrednosti skora za pojedine elemente za procenu razvoja (motorika, koordinacija, govor i dru&scaron;tvenost) po Brunet -L&eacute;zine skali su se statistički značajno razlikovale između grupa u uzrastu od 18 meseci (Studentov t test). U Grupi 1 bilo je 57 (37,013%) dece čiji je nekorigovani RQ bio ispod 90, dok je u Grupi 2 bilo 31 (20,130%) dete čiji je nekorigovani RQ bio ispod 90. Udeo dece sa RQ koji je ispod proseka za kalendarski uzrast je statistički značajno različit između grupa (Hi kvadrat test, p = 0,010). Relativni rizik za ispodprosečno postignuće na testu za procenu psihomotornog razvoja (nekorigovani RQ&lt;90), za decu iz Grupe 1 bio je vi&scaron;i, u odnosu na decu iz Grupe 2 (RR = 1,288; 95%CI 1,181 &ndash; 2,730). Statistički značajna razlika postojala je i kada je upoređen broj dece sa vrednostima korigovanog RQ ispod 90 u Grupi 1 i Grupi 2 (36 naspram 19 po redosledu navođenja; Hi kvardat test, p = 0,011). Relativni rizik za ispodprosečno postignuće na testu za procenu psihomotornog razvoja (korigovani RQ&lt;90), za decu iz Grupe 1 bio je vi&scaron;i, u odnosu na decu iz Grupe 2 (RR = 1,895; 95%CI 1,139 &ndash; 3,152).<br />Prosečne vednosti TM ispitanika iz obe grupe, u uzrastu od 24 meseca, nisu se statistički značajno razlikovale (Studentov t test). Prosečne vednosti TD ispitanika iz obe grupe, u uzrastu od 24 meseca, nisu se statistički značajno razlikovale (Studentov t test). Prosečne vednosti OGL ispitanika iz obe grupe, u uzrastu od 24 meseca, nisu se statistički značajno razlikovale (Studentov t test). Udeo ispitanika sa patolo&scaron;kim oftalmolo&scaron;kim nalazom nije se statistički značajno razlikovao između Grupe 1 i Grupe 2 (Hi kvadrat test). Udeo ispitanika sa patolo&scaron;kim neurolo&scaron;kim nalazom nije se statistički značajno razlikovao između Grupe 1 i Grupe 2 (Hi kvadrat test). Prosečne vrednosti RQ, kao i prosečne vrednosti skora za pojedine elemente za procenu razvoja (motorika, koordinacija, govor i dru&scaron;tvenost) po Brunet -L&eacute;zine skali, nisu se statistički značajno razlikovale između grupa, u uzrastu od 24 meseca (Studentov t test). U Grupi 1 bilo je 21 dete (13,636%) čiji je nekorigovani RQ bio ispod 90, dok je u Grupi 2 bilo 17 (11,049%) dece čiji je nekorigovani RQ bio ispod 90. Razlika u broju dece sa RQ koji je ispod proseka za kalendarski uzrast nije statistički značajna (Hi kvadrat test, p= 0,488). Statistički značajna razlika nije postojala ni kada je upoređen broj dece sa vrednostima korigovanog RQ ispod 90 u Grupi 1 i Grupi 2 (12 naspram 9 po redosledu navođenja; Hi kvardat test, p = 0,497).<br />Logističkom regresionom analizom pokazano je da su ve&scaron;tačko začeće, vi&scaron;eplodnost trudnoće i IUGR nezavisni faktori rizika za manju TM u kalendarskom uzrastu od 12 meseci. Logističkom regresionom analizom dobijena je statistički značajna korelacija između vrednosti RQ u uzrastu od 18 meseci i sledećih nezavisnih varijabli: arteficijalno začeta trudnoća i vi&scaron;eplodna trudnoća. Isptanici iz Grupe 1 i Grupe 2 nisu se statistički značajno razlikovali ni po jednom od posmatranih pokazatelja telesnog i psihomotornog razvoja u uzrastu od 24 meseca.<br />Struktura morbiditeta kod dece, tokom dvogodi&scaron;njeg perioda praćenja, nije se značajno razlikovala između grupa. Jedina razlika između grupa, konstatovana je u uzrastu od 12 i 18 meseci, bila je u učestalosti akutnih respiratornih infekcija, čija je pojava, pak, bila direktno povezana sa vi&scaron;eplodnim trudnoćama, odnosno brojem siblinga u domaćinstvu.<br />Zaključak: Prosečna starost majki dece koja su začeta IVF-om je veća od prosečne starosti majki dece koja su spontano začeta. Struktura morbiditeta majki dece koja su začeta IVF-om i majki dece koja su začeta spontanom koncepcijom je ista, ali je stopa morbiditeta veća kod majki dece koja su začeta IVF-om. Vi&scaron;eplodne trudnoće su veoma zastupljene kod začeća IVF-om. Trudnoće začete IVF-om se dominantno i skoro ekskluzivno okončavaju carskim rezom. Prevremena ruptura ovojaka ploda je česta komplikacija trudnoća koje su začete IVF-om. Stopa morbiditeta prevremeno rođene dece začete vantelesnom oplodnjom nije veća u odnosu na prevremeno rođenu decu začetu prirodnim putem. U strukturi morbiditeta kod dece koja su začeta vantelesnom opodnjom, zastupljena su ista oboljenja i komplikacije kao kod prevremeno rođene dece začete prirodnim putem. Incidencija pojedinih oboljenja je ista, sa izuzetkom bronhopulmonalne displazije koja se javlja če&scaron;če kod dece začete vantelesnom oplodnjom i retinopatije prematuriteta koja se javlja če&scaron;če kod dece začete prirodnim putem. Porođajna telesna masa, intrauterina restrikcija rasta, starost majke, stručna sprema majke, prethodna hronična oboljenja majke, bolesti majke dijagnostikovane tokom trudnoće, jednoplodna i vi&scaron;eplodna trudnoća, način porođaja i PROM su potencijalni faktori rizika za lo&scaron;iji postnatalni ishod kod dece iz arteficijalno začetih trudnoća. U uzrastu od 12 meseci, prevremeno rođena deca začeta tehnikama in vitro fetrilizacije, sem po dostignutoj telesnoj masi, ne razlikuju se značajno po drugim telesnim karakteristikama, od prevremeno rođene dece koja su začeta prirodnim putem. Faktori rizika za manju telesnu masu kod prevremeno rođene dece, u uzrastu od 12 meseci su: arteficijalno začeće, vi&scaron;eplodne trudnoće i intrauterina restrikcija rasta. U uzrastu od 12 meseci, prevremeno rođena deca začeta in vitro fertilizacijom, imaju blago lo&scaron;ije (ali ne i značajno niže) postignuće na testovima za procenu psihomotornog razvoja, odnosno imaju vi&scaron;i rizik da postignu ispodprosečne vrednosti skora na testu za procenu psihomotornog razvoja. U uzrastu od 18 meseci, nema razlike u pokazateljima telesnog razvoja između prevremeno rođene dece koja su arteficijalno začeta i dece koja su rođena iz spontano začetih trudnoća. U uzrastu od 18 meseci, prevremeno rođena deca iz arteficijalno začetih trudnoća imaju niže postignuće na testovima za procenu psihomotornog razvoja u odnosu na prevremeno rođenu decu iz spontano začetih trudnoća. Faktori rizika koji su povezani sa lo&scaron;ijim postignućem na testu za procenu psihomotornog razvoja kod prevremeno rođene dece su arteficijalno začeće trudnoće i vi&scaron;eplodnost trudnoće. U uzrastu od 24 meseca nema razlike u telesnim parametrima između prevremeno rođene dece koja su arteficijalno začeta i prevremeno rođene dece koja su začeta prirodnim putem. U uzrastu od 24 meseca nema razlike u postignuću na testu za procenu psihomotornog razvoja kod prevremeno rođene dece su arteficijalno začeće trudnoće i vi&scaron;eplodnost trudnoće. U uzrastu od 24 meseca, prevremeno rođena deca, i iz arteficijalno, i iz spontano začetih trudnoća, na testu za procenu psihomotornog razvoja postižu rezultate koji su u skladu sa njihovim kalendarskim uzrastom.</p> / <p>Introduction: Infertility is defined as an unsuccessful conception after one year of sexual intercourse without the use of contraception in the fertilizing phase of the menstrual cycle. Assisted reproduction methods represent an effective way of treating infertility. Examination and identification of short-term and long-term effects of artificial reproductive technologies is a very challenging task. The primary reason for this is the great heterogeneity in the way of collecting, processing, classifying and interpreting, now, the abundance of information that has been gathered in various studies. Individual approach to the treatment of infertility, rapid progress and constant changes in the methodology of the artificial reproductive technologies, in addition to the aforementioned difficulties associated with the collection and analysis of data, significantly hamper accurate assessment of all possible risks and consequences artificial conception. Despite numerous studies, scientific publications and the accumulated evidence, many doubts about the question whether artificially conceived pregnancies are accompanied by the higher risks or inadequate fetal development, poor perinatal and long-term outcomes still remained.<br />The Aim: The objectives of this work were to determine the structure of morbidity in prematurely born children conceived by artificial reproductive technologies (from single and multiple pregnancies) in the first two years of life, and to identify perinatal factors that are associated with the occurrence of acute and chronic complications and diseases in prematurely born children from this pregnancies. In addition, the aim of the study was to determine the characteristics of psychomotor development in prematurely born children conceived by artificial reproductive technologies at the end of the twelfth, eighteenth and twenty-fourth month of life, as well as to identify specific risk factors for the unfavorable physical, neurological and psychological outcome of those children.<br />Materials and Methods: The study included premature born newborns who were hospitalized in the Department for neonatology and intensive and semi-intensive care unit, and are thereafter, during the first two years of life. The retrospective part of the study included children who were hospitalized at the Institute, and who were born from January 1st 2011. to December 31st 2012. and were followed up to 2 years of life. Data on patients included in the retrospective part of the survey were collected through a review of medical records. The prospective part of the study included children who were treated and followed up at the Institute, and who were born between January 1st 2013 and December 31st 2014. and then followed up to 2 years of life. From this cohort two groups were formed: The tested group (Group 1) included all preterm infants who were conceived by ART. The control group (Group 2) included naturally conceived prematurely born children. The children in the control group were selected from the cohort so that their number was equal to the number of children in the study group. The gestational age of the examinees from the control group does not differ for more than &plusmn; 4 days from the children from the study group. The date of birth of subjects included in the control group does not differ for more than &plusmn; 3 months from the children in the study group.<br />At the moment of inclusion in the study, the following individual data were taken:<br />Maternal data, pregnancy and childbirth: the age of the mother at the moment of conception, the number of previous attempts at assisted conception, professional care, place of residence, chronic diseases diagnosed before pregnancy, acute and chronic diseases diagnosed during pregnancy (hypertension, pre-eclampsia, eclampsia, liver damage), premature rupture of the fetuses, the use of medication during pregnancy, single or multiple pregnancy. Data on placental disorders and abnormalities: ablation, placenta overdose, horioamnionitis. Child-related data: intrauterine infection, intrauterine growth restriction, delivery method, Apgar score. Anthropometric parameters (body weight, body length, head circumference) at birth and during the period of outpatient monitoring of the child. Length of initial hospitalization of the child. Length of invasive and / or non-invasive respiratory support and oxygen therapy. Diagnosis on discharge from the hospital: the presence of severe consequences of prematurity, which implies intracranial hemorrhage of 3rd and 4th degree (defined in International Classification of Disease - Tenth Revision (MKB10) under code P52.2), cystic periventricular leukomalacia, retinopathy of prematurity, bronchopulmonary dysplasia , necrotizing enterocolitis, sepsis and / or meningitis (microbiologically or clinically diagnosed). Presence of congenital anomalies or genetic syndromes and diseases (defined in MKB10 under codes Q00 to Q99), as well as the presence of congenital metabolic diseases (defined in MKB10 under codes E00 to E90).<br />In the retrospective part of the study, specialist reports from a neonatological clinic were examined for child visits at the age of 12, 18 and 24 months, and the following data were ecorded: all pre-diagnosis reported on specialist reports from a neonatological clinic, anthropometric arms at the moment examination (body length, body weight and head circumference), neurological findings (tone, trophic, skin and tendon reflexes, presence of lateralization in neurological findings), ophthalmologist findings (neat / patial findings), assessment of fine and coarse motoring, speech, cognitive functions and social contact and a collective assessment of psychomotor development. In the prospective part of the study, during control examinations in a neonatological clinic, at the age of 12, 18 and 24 months, the following were determined and recorded: previously set out in the current medical documentation, anthropometric parameters at the moment of examination (body length, body weight and the volume of the head), neurological findings (tone, trophic, skin and tendon reflexes, presence of lateralization in neurological findings), ophthalmologist findings, assessment of fine and grose motor functions, speech, cognitive functions, social contact and psychomotor development.<br />Results: The average BW of subjects in Group 1 at the age of 12 months, was statistically significantly lower in relation to respondents from Group 2 (Student&#39;s T test). The average length of subjects from both groups at the age of 12 months did not statistically differ (Student&#39;s T test). The average head circumference between children from both groups, at the age of 12 months, did not statistically differ (Student&#39;s T test). The proportion of subjects with pathological ophthalmological findings did not statistically significantly differ between Group 1 and Group 2 (Fischer&#39;s exact probability test). The proportion of subjects with pathological neurological findings did not statistically significantly differ between Group 1 and Group 2 (Hi square test). The average values of the global development coefficient (RQ), as well as the average score values for individual elements of development evaluation test - Brunet-L&eacute;zine scale (motor function, coordination, speech and sociability) did not differ significantly between groups (Student t test). In Group 1 there were 92 (59.740%) of children whose uncorrected RQ was under 90, while in Group 2 there were 61 (39.610%) children whose uncorrected RQ was below 90. This difference in the number of children with RQ below the average for calendar age is statistically significant (Hi square test, p = 0.0004). The relative risk of under-achievement in the psychomotor evaluation test (RQ &lt;90) for children from Group 1 was higher than in children from Group 2 (RR = 1.495; 95% CI 1.181 - 1.922). In Group 1, there were 87 (56.494%) children who achieved sub-optimal corrected score values for the assessment of psychomotor development (corrected RQ &lt;90). In Group 2, there were 69 (44.805%) children who achieved sub-optimal corrected score values for the assessment of psychomotor development (corrected RQ &lt;90). This difference is statistically significant (Hi square test, p = 0.040). The relative risk for the suboptimal achievement in the psychomotor evaluation test (corrected RQ &lt;90) for children from Group 1 was higher than in Group 2 (RR = 1.261; 95% CI 1.008 - 1.577). In Group 1, as many as 81/87 (93.310%) of children had a corrected RQ value of &ge; 85, while in Group 2 this value of the corrected RQ there were 60/69 (86.956%) children.<br />At the age of 18 months, the average BW of subjects from both groups did not differ significantly (Student&#39;s T test). The average length of subjects from both groups, at the age of 18 months, did not statistically differ (Student&#39;s T test). The average head circumference of children from both groups, at the age of 18 months, did not statistically differ (Student&#39;s T test). The proportion of subjects with pathological ophthalmological findings did not statistically significantly differ between Group 1 and Group 2 (Hi square test). The proportion of subjects with pathological neurological findings did not statistically differ between Group 1 and Group 2 (Hi square test). The average RQ values, as well as the average scores for individual elements of psychomotor development (motor function, coordination, speech and sociability) according to the Brunet-L&eacute;zine scale, have been statistically significantly different between groups, at the age of 18 months (Student&#39;s T test). In Group 1 there were 57 (37.013%) children whose uncorrected RQ was below 90, while in Group 2 there were 31 (20,130%) children whose uncorrected RQ was below 90. The share of children with RQ below the average value for the calendar age is statistically significantly different between groups (Hi square test, p = 0.010). The relative risk for the suboptimal achievement in the Psychomotor Development Assessment (uncorrected RQ &lt;90) for Group 1 children was higher than in Group 2 (RR = 1.288; 95% CI 1.181 - 2.730). A statistically significant difference between Group 1 and Group 2 existed when the number of children with corrected RQ below 90 was compared (36 naspram 19 respectively, Hi quadrate test, p = 0.011). The relative risk for the suboptimal achievement on the Psychomotor Evaluation Test (corrected RQ &lt;90) for the children from Group 1 was higher when compared to children in Group 2 (RR = 1.895; 95% CI 1.139 &ndash; 3.152).<br />At the age of 24 months the average BW, body length and head circumference of subjects in both groups were not significantly different (Student&#39;s T test). The proportion of subjects with pathological ophthalmological findings did not statistically significantly differ between Group 1 and Group 2 (Hi square test). The proportion of subjects with pathological neurological findings did not statistically significantly differ between Group 1 and Group 2 (Hi square test). The average RQ values, as well as the average score values for individual elements for development evaluation (motor function, coordination, speech and sociability) according Brunet-L&eacute;zine scale, did not significantly differ between groups at the age of 24 months (Student&#39;s T test). In Group 1, there were 21 children (13.636%) whose uncorrected RQ was under 90, while in Group 2 there were 17 (11.049%) of children whose uncorrected RQ was below 90. The difference in the number of children with RQ below the average for the calendar age was not statistically significant (Hi square test, p = 0.488). A statistically significant difference did not exist even when the number of children with values of the corrected RQ below 90 in Group 1 and Group 2 (12 naspram 9 respectively, Hi quadrate test, p = 0.497) was compared.<br />Logistic regression analysis has shown that artificial conception, multiple pregnancy and IUGR are independent risk factors for lesser BW in a calendar age of 12 months. By logistic regression analysis, a statistically significant correlation between RQ values at 18 months of age and the following independent variables was obtained: artificially started pregnancy and multiple pregnancy. Group 1 and Group 2 patients did not significantly differ by any of the indicators of physical and psychomotor development at the age of 24 months.<br />The structure of morbidity in children, during the two-year follow-up period, did not differ significantly between groups. The only difference between the groups was found in the rates of acute respiratory infections at the age of 12 and 18 months (rate of infections was higher in Group 1), whose occurrence, however, was directly related to multiple pregnancies, or the number of sibling in the household.<br />Conclusion: The average age of mothers of children conceived by the IVF is higher than the average age of mothers of children who were conceived spontaneously. The structure of the morbidity of mothers of children who were artificially conceived and mothers of children born after spontaneous conception is the same, but the morbidity rate is higher in the mothers of children who were conceived by IVF. Pregnancies concieved by IVF almost exclusively ended by cesarean section. Premature rupture of the membranes is a common complication of IVF pregnancies. The rate of morbidity of prematurely born children conceived by ART is not higher than that of prematurely born children conceived naturally. The structure of morbidity in children from ART pregnancies was the same as in naturally conceived prematurely born children. The incidence of specific illnesses is the same, with the exception of bronchopulmonary dysplasia that occurs more frequently in children born from ART pregnancies, and retinopathy of prematurity that occurs more frequently in spontaneously conceived children. Maternal birth weight, intrauterine growth restriction, mother&#39;s age, maternal care, previous mother&#39;s chronic illness, mother&#39;s disease diagnosed during pregnancy, single and multiple pregnancies and PROM are potential risk factors for worse postnatal outcome in children from artificially initiated pregnancies. Risk factors for lower body weight in premature babies, at the age of 12 months, are: artificial conception, multiple pregnancy and intrauterine growth restriction. At the age of 12 months, prematurely born children from IVF pregnancies, have slightly worse (but not significantly lower) psychomotor achievements. At the age of 18 months, there is no difference in the indicators of physical development between prematurely born children who are artificially conceived and children born from spontaneous pregnancies. At the age of 18 months, prematurely born children from ART pregnancies have lower achievement on tests for assessing psychomotor development compared to prematurely born children from spontaneously initiated pregnancies. Risk factors associated with a poor performance on the psychomotor development assessment tests, in preterm infants, are an artificial conception of pregnancy and a multi fertile pregnancy. At the age of 24 months, there is no difference in the physical parameters between prematurely born children from ART and naturally conceived pregnancies. At the age of 24 months, there is no difference in the achievement on the test for the assessment of psychomotor development between children from ART and spontaneous pregnancies. At the age of 24 months, on the psychomotor development assessment, prematurely born children achieve the results consistent with their calendar age.</p>

A síndrome de hiperestímulo ovariano (SHO) pode ocorrer de forma espontânea na gestação ou durante indução da ovulação para fertilização in vitro. Com o objetivo de identificar fatores clínicos e hormonais de risco para a SHO iatrogênica e analisar o papel das variantes alélicas do FSHR na sua etiologia, estudamos 29 pacientes com SHO iatrogênica moderada e grave. Idade < 33,5 anos, FSH basal < 5,2 UI/L e resposta de estradiol > 1757 pg/mL durante a estimulação ovariana atuaram como preditores independentes do risco de desenvolvimento da SHO iatrogênica, sendo a associação dos três fatores capaz de predizer a síndrome com acurácia de 78%. Não foram encontradas mutações nas 29 pacientes com SHO iatrogênica. Entretanto, o alelo Asn680 do polimorfismo Ser680Asn se associou às formas mais graves da SHO iatrogênica / Ovarian hyperstimulation syndrome (OHSS) can occur spontaneously during early pregnancy or as an iatrogenic complication in assisted reproductive medicine. The aim of this study was to identify clinical and hormonal risk factors for iatrogenic OHHS and to evaluate the role of allelic variants of the FSHR in the etiology of this syndrome. We studied 29 patients with moderate and severe iatrogenic OHHS. Age < 33.5 y, basal FSH < 5.2 U/L and estradiol response during ovarian stimulation > 1757 pg/mL were independent risk factors for iatrogenic OHSS and these three factors in association were able to predict the syndrome with accuracy of 78%. We did not find mutations in any patient studied. However, there was a predominance of the Asn680 allele of Ser680Asn polymorphism in patients with the most severe forms of the syndrome

<p>Uvod: Infertilitet pogađa 10-15% parova reproduktivnog doba. Vanetesna oplodnja (VTO) je najefikasniji vid tret-mana infertiliteta, ali uprkos značajnom napretku stopa uspeha VTO u proseku iznosi oko 30% po ciklusu. Glavnim razlogom neuspeha smatra se neadekvatan kvalitet embriona, dok se pretpostavlja da u 10-20% slučajeva razlog neuspeha leži u neadekvatnoj receptivnosti uterusa. Na osnovu inicijalnih istraživanja histeroskopija, koja predstvalja zlatni standard u dijagnostici i tretmanu patologije kavuma uterusa, se često izvodi u svakodnevnoj kliničkoj praksi kako bi se povećala uspe&scaron;nost VTO. Uprkos &scaron;irokoj primeni i dalje ne postoji dovoljno kvalitetnih dokaza o realnoj ulozi histeroskopije na ishod VTO kako kod patolo&scaron;kih stanja kavuma tako i rutinski, pre prvog ili rekurentnog poku&scaron;aja VTO. Cilj disertacije bio je da se utvrdi uticaj sprovođenja histeroskopije na ishod VTO, ustanovi učestalost prethodno neprepoznate patologije kavuma uterusa, kao i da se ispitaju stavovi pacijenata o primeni rutinske histeroskopije pred VTO. Materijal i metode: Istraživanje je sprovedeno u Kliničkom centru Vojvodine, u formi prospektivne studije u dve sukcesivne etape od 01.01.2015. do 01.04.2017. U prvoj etapi poređen je ishod VTO kod pacijentkinja kojima pred postupak VTO nije sprovedena histeroskopija (Grupa A), pacijentkinja kod kojih je dobijen uredan nalaz histeroskopije pred postupak VTO (Grupa B) i pacijentkinja gde je pred postupak VTO dobijen patolo&scaron;ki nalaz kavuma na histeroskopiji koji je u istom aktu tertian (Grupa C). Druga etapa istraživanja predstavljala je randomiziranu kontrolisanu studiju (RCT &ndash; randomised controlled trial). Nakon verifikacije urednog ultrazvučnog nalaza pred prvi postupak VTO, pacijentkinje su randomizirane u Grupu A2 kojima pred postupak VTO nije sprovedena histeroskopija i Grupu B2 kojima je pred postupak VTO sprovedena rutinska histeroskopija. Statistička analiza sprovedena je upotrebom odgovarajućeg softvera (JMP Ver. 9). Poređeni su podaci o osnovnim karakteristikama pacijenata, toka i ishoda ciklusa VTO. Primarni parametar ishoda bila je stopa kliničke trudnoće po embriotransferu. Pored analize ishoda primarno konstruisanih grupa, urađena je analiza i naknadno konstruisanih subgrupa, kao i predikcioni model uspeha VTO baziran na logističkoj regresiji. Rezultati: Studija je uključila 253 pacijentkinje (52 pacijentkinja iz Grupe A, 50 iz Grupe B, 50 iz Grupe C, 51 iz Grupe A2 i 50 iz Grupe B2). Nije postojala statistički značajna razlika u karakteristikama pacijentkinja, parametrima ovarijalne rezerve, broju dobijenih jajnih ćelija ni drugim parametrima toka postupka VTO među posmatranim grupama. U prvoj etapi istraživanja dobijena je statistički značajno (p=0,013) veća stopa kliničkih trudnoća kod pacijentkinja kojima je pred postupak VTO sprovedena histeroskopija - 50 % za Grupu B i 42% za grupu C u odnosu na 30,77% kod pacijentkinja bez histeroskopije (Grupa A), bez statistički značajne razlike među histeroskopskim grupama. U drugoj etapi istraživanja stopa kliničkih trudnoća prilikom upotrebe rutinske histeroskopije pred prvu VTO (Grupa B2) iznosila je 46% naspram 31,37% kod pacijentkinja bez histeroskopije pred prvu VTO (Grupa A2), iako uočena razlika nije dostigla statističku značajnost (p =0,089), uz relativan rizik (RR) za ostvarivanje kliničke trudnoće nakon primene histeoskopije uiznosio od 1,47 (95% CI 0,88-2,43) (p=0,13). Analizon subgrupa kod 100 pacijentkinja sa rutinski sprovedenom histeroskopijom pred VTO i 103 pacijentkinje bez histeroskopije pred VTO, dobijena je statistički značajnao veća stopa kliničkih trudnoća (48% naspram 31,07%, istim redom), uz RR od 1,54 (95% CI 1,08-2,20) (p=0,013), kao i stopa tekućih trudnoća od RR 1,49 (CI 1,01-2,19) (p= 0,039). Analiza ukupnog uticaja izvođenja histeroskopije pred VTO dobila je statistički značanjno veću stopu kliničkih trudnoća po ET za grupu histeroskopije uz RR 1,48 (CI 1,06-2,07) (p=0,017). Histeroskopijom je nakon urednog ultrazvučnog nalaza ustanovljeno postojanje patolo&scaron;kog nalaza kod 34,65% pacijenata i to 22,7% major patologije i 11,88% minor patologije kavuma. Nije postojala statistički značajna razlika u uspehu VTO u odnosu na sam nalaz histeroskopije. 98,67% pacijenata podržalo je rutinsku upotrebu histeroskopije pred prvi postupak VTO, dok je 83% pacijenata podržavlo rutinsku upotrebu histeroskopije pred svaki postupak VTO. U finalnom predikcionom modelu se uz AUC od 0,748 jedino postojanje visokokvalitetnog embriona uz odnos &scaron;ansi (OR) 7,91 (95% CI 1,80-56,06; p=0,0047), transfer blastociste uz OR 3,80 (95% CI 1,90-7,98; p=0,0001) i izvođenje histeroskopije pred VTO uz OR 2,13 (95% CI 1,14-4,08, p=0,0169) pokazalo statistički značajnim prediktorima trudnoće. Diskusija: Studija je dobila pozitivan uticaj histeroskopije na ishod postupka VTO, iskazan pre svega povećanjem stope kliničkih trudnoća nakon sprovođenja histeroskopije (bilo da je na histeroskopiji nađen uredan ili patolo&scaron;ki nalaz). Dodatna prednost histeroskopije predstavljala je i i detekcija prethodno nepropoznate patologije kavuma. Umeren efekat na ukupno pobolj&scaron;anje stope kliničkih trudnoća prilikom rutinskog sprovođenja histeroskopije pred prvu VTO, koji je statističku značajnost dostigao tek analizom subgrupa u skladu je sa nalazima novijih dobro dizajniranih studija koji donekle limitiraju nekritičku upotrebu histeroskopije. Biolo&scaron;ko obja&scaron;njenje potencijalnog pozitivnog uticaja histeroskopije najverovatnije leži u detekciji i tretmanu prethodno nepropoznate patologije kavuma, olak&scaron;avanju procedure embriotransfera, kao i humoralnim i molekularnim promenama koje nastaju u endometrijumu kao posledica odgovarajuće histeroskopske traume a koji su u dosa&scaron;anjim istraživanjima apostrofirani kao faktori koji mogu povećati receptivnost uterusa. Zaključak: Histeroskopija je efikasna, bezbedna i visoko prihvatljiva procedura koja dovodi do povećanja uspeha VTO u standardnim kliničkim indikacijama (prethodnog neuspelog postupka VTO i sumnje na patolo&scaron;ki nalaz kavuma uterusa) bilo da se na samoj histeroskopiji nađe uredan ili patolo&scaron;ki nalaz. Rutinska primena histeroskopije pred prvi postupak VTO se na osnovu rezultata studije ne može smatrati apsolutno opravdanom usled statistički nedovoljno značajnog povećanja stope kliničke trudnoće. Uzev&scaron;i u obzir visoku prihvatljivost od strane pacijenata i najverovatniji pozitivan efekat na stopu trudnoće primena rutinske histeroskopije pred prvu VTO bila bi opravdana ukoliko se implementira koncept ambulantne histeroskopije.</p> / <p>Introduction: Infertility affects 10-15% of all couples. In vitro fertilisation (IVF) is the most effective method of infertility treatment, but despite a significant improvement, success rate of IVF is still around 30% per cycle. The main reason for the IVF failure is inadequate embryo quality, but in 10-20% of cases the cause of IVF failure lies in impaired uterine receptivity. Based on earlier studies hysteroscopy, gold standard in the diagnosis and treatment of uterine cavity pathology, is often performed to increase IVF success. Despite its wide use, there is lack of high quality evidence regarding real contribution of hysteroscopy on IVF outcome in situations of uterine cavity pathology or routinely prior to first IVF or after recurrent implantation failure. The aim of this dissertation was to determine the influence of performing hysteroscopy on IVF outcome, as well as the incidence of previously unrecognized uterine pathology, and to examine patient&#39;s attitudes about performing routine hysteroscopy prior to IVF. Material and methods: The research was conducted in a prospective manner in two successive stages at Clinical Center of Vojvodina from 01.01.2015. until 01.04.2017. During first stage of the study IVF outcome was compared between patients who did not have a hysteroscopy prior to IVF (group A), patients with normal hysteroscopic finding prior to the IVF (Group B) and patients with abnormal hysteroscopic findings prior to IVF which was treated at the same time (Group C). The second stage of the study was a randomized controlled trial (RCT). After verification of normal ultrasound findings prior to the first IVF, patients were randomized to group A2 in who me hysteroscopy was not performed and group B2 who had routine hysteroscopy prior to first IVF. Statistical analysis was carried out using the appropriate statistical software (JMP Ver. 9). Patient characteristics, course and outcome of IVF cycle were compared between groups. The primary outcome was clinical pregnancy rate (CPR) per embryotransfer. In addition to analyzing the IVF outcomes in primarily defined groups, subgroup analysis was also performed, as well as IVF success pre-diction model based on logistic regression. Results: The study included 253 patients (52 patients in Group A, 50 in Group B, 50 in Group C, 51 in Group A2 and 50 in Group B2). There was no statistically significant difference in patient characteristics, ovarian reserve parameters, number of retrieved oocytes, or other relevant parameters of IVF course between the observed groups. In the first stage of the study there was statistically significant (p = 0.013) higher clinical pregnancy rate in patients who had a hysteroscopy before IVF - 50% for Group B and 42% for group C versus 30,77 % in patients without hysteroscopy before IVF (Group A), without statistically significant difference between hysteroscopic groups. In the second stage of the study, routine hysteroscopy prior to first IVF (Group B2) led to clinical pregnancy rate 46% versus 31.37% in patients without hysteroscopy prior to first IVF (Group A2), although without statistical significance (p = 0.089. Relative risk (RR) for achieving clinical pregnancy after performing hysteroscopy was 1.47 (95% CI 0.88-2.43) (p = 0.13). Subgroup analysis of 100 patients with routinely performed hysteroscopy before IVF and 103 patients without hysteroscopy prior to the IVF showed statistically significant higher rates of clinical pregnancies (48% versus 31.07%, in the same order), with RR of 1.54 (95% CI 1.08-2.20), (p = 0.013), and for ongoing pregnancies RR was 1.49 (95% CI 1.01-2.19) (p = 0.039). Overall effect of performing hysteroscopy prior to IVF resulted in a statistically significant increase in the clinical pregnancy with RR 1.48 (95% CI 1.06-2.07) (p = 0.017). After normal ultrasound finding hysteroscopy revealed 34.65% of pathological finding, 22.7% of major and 11.88% of minor pathology of the cavity). There was no statistically significant difference in IVF outcome based on hysteroscopy findings. 98.67% of patients supported the routine use of hysteroscopy before the first IVF procedure, while 83% of patients supported the routine use of the hysteroscopy before every IVF procedure. In the final prediction model, with the AUC of 0.748, only the presence of high quality embryos with odds ratio (OR) 7,91 (95% CI 1,80-56,06; p=0,0047), blastocyst transfer with OR 3,80 (95% CI 1,90-7,98; p=0,0001) and performing hysteroscopy prior to IVF with OR 2,13 (95% CI 1,14-4,08, p=0,0169) proved to be statistically significant predictors of pregnancy. Discussion: The study shoved a positive influence of hysteroscopy on the IVF outcome by increasing clinical pregnancy rate after performing hysteroscopy (whether hysteroscopy revealed normal or pathological finding). Additional benefit of hysteroscopy was detection of previously unrecognized uterine pathology. A moderate effect on the overall improvement in clinical pregnancy rate with use of routine hysteroscopy, which reached statistical significance only by subgroup analysis, is in line with findings of recent well designed studies that somewhat limit the noncritical use of hysteroscopy. A biological explanation of the potential positive effect of hysteroscopy is most likely due to detection and treatment of the previously unrecognized uterine pathology, facilitating embryotransfer procedure, as well as the humoral and molecular changes that occur in the endometrium as a consequence of the hysteroscopic trauma. Those changes were hypothesized as factors that can increase uterine receptivity by numerous research. Conclusion: Hysteroscopy is an effective, safe and highly acceptable procedure that increases IVF success when performed for accepted clinical indications (previous IVF failures, pathological findings of uterine cavity), whether hysteroscopy reveals normal or pathological finding. The routine use of hysteroscopy prior to first IVF based on this study can not be considered justified since increase in clinical pregnancy rate did not reach statistical significance. Given the high acceptance of this concept by the patients and moderate but probable positive effect on IVF outcome, implementation of routine hysteroscopy prior to first VTO would be justified only in office hysteroscopy setting.</p>

MSCAGR (Animal Science) / Department of Animal Science / The objective of the study was to determine the effects of fertilisation with bio-digester slurry and the inclusion of carbohydrate additives at ensiling on the fermentation characteristics, chemical composition, ruminal degradability, and in vitro digestibility of Napier grass silage. Napier grass planted at the School of Agriculture Experimental Farm, University of Venda in 5 m x 4 m plots replicated three times in a completely randomised design and was irrigated with either biodigester slurry or no bio-digester slurry (tap water) for a period of 12 weeks. After 12 weeks, the Napier was freshly cut and ensiled for 90 days in 1 litre glass jars in a 2 (Control - tap water and slurry irrigation) x 4 (No additive, molasses, maize meal and brown sugar) factorial arrangement. Fermentation quality and nutritive composition were determined using standard protocols. The dry matter (DM) and crude protein (CP) ruminal degradability was determined in sacco by incubating feed samples in nylon bags (external dimension: 6 × 12 cm, pore size of 46 μm) in the rumen in three Bonsmara steers fitted with rumen cannulae for 0, 6, 12, 24, 48, 72, 96 and 120 hours (h). Parameters to describe the dynamics of ruminal degradability of DM and CP were obtained by fitting the data on the exponential equation P = a + b (1 - e-ct) using the NEWAY computer program, where “a” is the rapidly degradable fraction, “b” is the slowly degradable fraction and “c” is the outflow rate. The in vitro DM and CP degradability of rumen undegradable residue collected after 12, 24 and 48 h incubation was determined by sequential digestion in pepsin (abomasal) and pancreatin (small intestine) solutions. Fertilisation with bio-digester slurry increased (P <0.05) CP content of fresh cut Napier grass pre-ensilage. Bio-digester slurry fertilisation with molasses inclusion improved (P <0.05) the silage DM content which improved (P >0.05) fermentation characteristics with pH of 4.2 and lowest NH3-N of 13.3 g/kg. Other chemical compositions and fermentation characteristics were not affected (P >0.05) due to fertilisation x additives treatment combinations. No bio-digester slurry fertilisation with maize meal inclusion increased (P <0.01) DM degradability at 0 h incubation. As time progressed to 24 h, no biodigester slurry fertilisation with no additive included reduced (P <0.01) DM degradability with no difference (P >0.05) on other treatments. Potential DM degradability (a + b) of no bio-digester slurry fertilisation with no additive inclusion silage was reduced (P <0.01). The reduction was associated with low levels (P <0.01) of slowly degradable fraction “b”. In vitro DM and CP digestibility were not affected (P >0.05) due to fertilisation x additives treatment combinations. In conclusion, bio-digester slurry application improved the quality of fresh cut Napier grass, with the combination of bio-digester slurry fertilisation and molasses addition yielding the best silage quality.