Abstract Introduction The degradation of plastic waste into smaller micro- and nanoplastic (MNPs) molecules has led to widespread distribution of these particles and accumulation in the environment, making human exposure inevitable. This can result in, or exacerbate, pathological conditions leading to immune dysfunction, neurodegenerative diseases, and infertility. Yet few studies have examined the effects of nanoplastics (NPs) on human health, especially the reproductive system. Reproductive toxicity of plastic particles has been mostly studied in males with most studies investigating microplastics. Therefore, the present study aims to assess the reproductive health consequences of NPs exposure in females by quantifying serum estradiol and progesterone, examining estrous cyclicity, and assessing ovarian reserve (number and quality of follicles) which is a key indicator of female fertility. Materials & Methods The present study was carried out in female mice (C57BL/6) exposed orally to water (control) or one of two solutions containing different concentrations of Polystyrene nanoplastics (PS-NPs; 100 µg/l or 1000 µg/l in water. Exposure occurred daily for 29 days, and vaginal lavage samples were collected for the last 15 days of the exposure phase to check for change in estrous cyclicity. Mice were euthanized at the end of the study and their blood samples and reproductive tissues were collected. Ovaries were fixed in 10% formalin, embedded in paraffin wax, serially sectioned at 5 µm thickness, and stained with hematoxylin and eosin (H&E) for microscopy and follicle analysis. ELISA was also performed to quantify the progesterone and estradiol serum levels. Results There was a significant increase in the estrous cycle length in the high dose (1000 µg/l) PS-NPs exposure group compared to control (5.53±.25 days vs 4.7±0.23 days, P=0.02). Moreover, there was a significant decrease in serum progesterone levels in the high-dose exposure group compared to control (mean difference=1.64 pg/ml, standard error of difference (SED)=0.64, P=0.03). Additionally, it was shown that PS-NPs exposure significantly reduced antral follicles’ diameter in both the low dose (238.61±19.01 µm vs 167.35±19.01 µm, P=0.03) and high dose exposure groups compared to the control group with the higher dose showing a more pronounced reduction in antral follicle' size (238.61±19.01 µm vs 131.95±19.01 µm, P=0.001). Conclusion Oral PS-NPs exposure in female mice appears to induce toxicity by reducing antral follicles size, increasing the estrous cycle length, and decreasing progesterone levels which may result in anovulation and different reproductive issues, such as infertility and polycystic ovary syndrome (PCOS). The effect of PS-NPs on infertility along with NPs’ mechanism of action in female reproductive system should be investigated in future studies. / Thesis / Master of Science (MSc) / Plastics in the environment break down into smaller particles called micro- and nanoplastics. These plastic particles are pollutants present in the aquatic and terrestrial environments entering every level of the food chain and ultimately reaching humans, yet few studies have examined the effects of nanoplastics on human health. A recent World Health Organization (WHO) report on nanoplastics has stressed the urgent need for toxicological studies to assess potential human health effects. Therefore, this study examined the effect of nanoplastics on the female reproductive system. This study was carried out in female mice exposed orally to a solution containing a vehicle control (water) and two different concentrations of nanoplastics (100 and 1,000 µg/l). Exposure occurred daily for a period of 29 days. At the conclusion of the study the mice were humanly euthanized with their blood and reproductive tissues collected for laboratory analysis. Results showed that nanoplastics exposure resulted in irregular reproductive cycle in mice along with a decrease in antral follicle size and progesterone levels which are indicators of anovulation and can lead to disorders, such as infertility and polycystic ovary syndrome (PCOS) which should be further investigated in future studies.

Introduction The degradation of plastic waste into smaller micro- and nanoplastic (MNPs) molecules has led to widespread distribution of these particles and accumulation in the environment, making human exposure inevitable. This can result in, or exacerbate, pathological conditions leading to immune dysfunction, neurodegenerative diseases, and infertility. Yet few studies have examined the effects of nanoplastics (NPs) on human health, especially the reproductive system. Reproductive toxicity of plastic particles has been mostly studied in males with most studies investigating microplastics. Therefore, the present study aims to assess the reproductive health consequences of NPs exposure in females by quantifying serum estradiol and progesterone, examining estrous cyclicity, and assessing ovarian reserve (number and quality of follicles) which is a key indicator of female fertility. Materials & Methods The present study was carried out in female mice (C57BL/6) exposed orally to water (control) or one of two solutions containing different concentrations of Polystyrene nanoplastics (PS-NPs; 100 µg/l or 1000 µg/l in water. Exposure occurred daily for 29 days, and vaginal lavage samples were collected for the last 15 days of the exposure phase to check for change in estrous cyclicity. Mice were euthanized at the end of the study and their blood samples and reproductive tissues were collected. Ovaries were fixed in 10% formalin, embedded in paraffin wax, serially sectioned at 5 µm thickness, and stained with hematoxylin and eosin (H&E) for microscopy and follicle analysis. ELISA was also performed to quantify the progesterone and estradiol serum levels. Results There was a significant increase in the estrous cycle length in the high dose (1000 µg/l) PS-NPs exposure group compared to control (5.53±.25 days vs 4.7±0.23 days, P=0.02). Moreover, there was a significant decrease in serum progesterone levels in the high-dose exposure group compared to control (mean difference=1.64 pg/ml, standard error of difference (SED)=0.64, P=0.03). Additionally, it was shown that PS-NPs exposure significantly reduced antral follicles’ diameter in both the low dose (238.61±19.01 µm vs 167.35±19.01 µm, P=0.03) and high dose exposure groups compared to the control group with the higher dose showing a more pronounced reduction in antral follicle' size (238.61±19.01 µm vs 131.95±19.01 µm, P=0.001). Conclusion Oral PS-NPs exposure in female mice appears to induce toxicity by reducing antral follicles size, increasing the estrous cycle length, and decreasing progesterone levels which may result in anovulation and different reproductive issues, such as infertility and polycystic ovary syndrome (PCOS). The effect of PS-NPs on infertility along with NPs’ mechanism of action in female reproductive system should be investigated in future studies. / Thesis / Master of Science (MSc) / Plastics in the environment break down into smaller particles called micro- and nanoplastics. These plastic particles are pollutants present in the aquatic and terrestrial environments entering every level of the food chain and ultimately reaching humans, yet few studies have examined the effects of nanoplastics on human health. A recent World Health Organization (WHO) report on nanoplastics has stressed the urgent need for toxicological studies to assess potential human health effects. Therefore, this study examined the effect of nanoplastics on the female reproductive system. This study was carried out in female mice exposed orally to a solution containing a vehicle control (water) and two different concentrations of nanoplastics (100 and 1,000 µg/l). Exposure occurred daily for a period of 29 days. At the conclusion of the study the mice were humanly euthanized with their blood and reproductive tissues collected for laboratory analysis. Results showed that nanoplastics exposure resulted in irregular reproductive cycle in mice along with a decrease in antral follicle size and progesterone levels which are indicators of anovulation and can lead to disorders, such as infertility and polycystic ovary syndrome (PCOS) which should be further investigated in future studies.

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-02-17T12:29:16Z No. of bitstreams: 1 Andre Angelo Medeiros Gomes.pdf: 644128 bytes, checksum: cd67aaf23e82d52d2fc876105888dc75 (MD5) / Made available in DSpace on 2017-02-17T12:29:16Z (GMT). No. of bitstreams: 1 Andre Angelo Medeiros Gomes.pdf: 644128 bytes, checksum: cd67aaf23e82d52d2fc876105888dc75 (MD5) Previous issue date: 2013-02-18 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Brazil is one of the biggest producers and consumers of maize (Zea mays L.) in the world and the fusariosis caused by F. verticillioides, on ears and or on stalk, is a recurrent problem when environmental conditions favor epidemics in the field or in storage. The present study aimed to characterize through crosses the F. verticillioides isolates from the three main climatic zones where maize is grown in Brazil. The fertility and effective population size were calculated from data generated by crossing field isolates with female fertile testers of F. verticillioides. For the entire population, 231 out of 300 isolates were cross-fertile with tester isolates. MAT-1 and MAT-2 idiomorphs of the fertile isolates segregated in a 105:126 rate. Female isolates (hermaphrodites) were 96 out of 231 fertile isolates, while 135 were male only isolates. The Ne(mt) was 99% of the count for the Brazilian population when the mating type idiomorphs were used as predictors in the estimative of the population size. But when this calculation was made on the basis of the female fertile isolates the Ne(f) was 83%. When the total population is divided into sub-populations representing climatic zones, the frequencies of female fertile isolates were 33 out of 80 fertile isolates from the Equatorial Tropical Zonal Climate (ETZC); 14 out of 78 fertile isolates from the Temperate Zonal Climate (TZC); and 49 out of 73 fertile isolates from the Tropical Central Brazil Zonal Climate (TCBZ). The effective population size expressed in percentage of the total number of individual in each population for the three populations represented by isolates from the ETZC, TZC and TCBZ were Ne(mt) = 99, 100, 97 and Ne(f) = 83, 52, 96 respectively. The Brazilian population of F. verticillioides shows high fertility and high effective population size, suggesting the possibility of frequent sexual reproduction in the field. Apparently there is a tendency of higher fertility in populations established at low latitudes, represented by the tropical climate. / O Brasil é um dos maiores produtores e consumidores de milho (Zea mays L.) do mundo e a fusariose, causada por Fusarium verticillioides, seja em espigas ou no colmo, é um problema recorrente quando condições ambientes favorecem o surgimento de epidemias no campo ou em armazenamento. O presente trabalho teve como objetivos caracterizar por meio de cruzamentos sexuais, isolados de F. verticillioides dos três principais climas zonais que se cultiva milho no Brasil. Através de cruzamentos de isolados do campo de F. verticillioides, com testadores de G. moniliformis; e determinar a taxa de fertilidade, frequência de hermafroditismo, e tamanho efetivo da população. Dos 300 isolados estudados, 231 tiveram cruzamento fértil. Os tipos de acasalamento MAT-1 e MAT-2 segregaram numa proporção 105:126. Entre os 231 isolados férteis, 96 se comportaram como hermafroditas e 135 como fêmeas estéreis. Na população total, do Brasil, levando em consideração o tipo de acasalamento, o tamanho efetivo da população Ne(mt), foi de 99% da população total, e tomando por base a frequência de hermafrodita , o tamanho efetivo Ne(f) foi de 83% da população total. Quando o número total de isolados é subdividido em três populações distintas, baseado nas condições de clima de local de coleta dos isolados, as frequências de hermafroditas foram; 33 hermafroditas dentre 80 isolados férteis do Clima Zonal Tropical Equatorial (ZTE), com 47 se comportando como fêmea estéril; 14 hermafroditas dentre 78 isolados férteis do Clima Zonal Temperado (ZT), com 64 se comportando como fêmea estéril; e 49 hermafroditas dentre 73 isolados do Clima Zonal Tropical Brasil Central (ZTBC), com apenas 24 se comportando como fêmea estéril. O tamanho efetivo das populações representadas por isolados dos climas ZTE, ZT e ZTBC, foram Ne(mt) = 99, 100, 97 e Ne(f) = 83, 52, 96 respectivamente, expresso em porcentagem do número total de indivíduos de cada população. A população brasileira de F. verticillioides analisada apresenta alto índice de fertilidade e tamanho efetivo, sugerindo a possibilidade de frequente reprodução sexuada no campo. Aparentemente, existe uma tendência de maior fertilidade da população em latitudes menores, representadas pelo clima tropical.

Introdução: Estudos prévios com leveduras evidenciaram um importante papel de substâncias amiloide (SA) na regulação da gametogênese, questionando seu potencial como marcador da qualidade gamética. Contudo, até o presente momento, nenhum estudo avaliou a presença de SA em óocitos e embriões de mamíferos. Objetivos: Descrever a presença, caracterizar o padrão de distribuição e comparar os níveis das SA em óocitos maduros e embriões pré-implantação, utilizando modelo murino. Identificar, caracterizar o padrão de distribuição e quantificar SA em oócitos humanos em diferentes estágios de desenvolvimento (VG- vesícula germinativa, MI- metáfase I e MII- metáfase II) e estabelecer a correlação entre os níveis de SA nos oócitos e as características clínicas de pacientes submetidas a estimulação ovariana controlada (EOC) para fertilização in vitro (FIV). Metodologia: Realizou-se um estudo piloto experimental, prospectivo, utilizando 20 oócitos maduros e 200 embriões pré-implantação de camundongos (1 célula, 2 células, 4 células, 8 células e blastocistos) e 46 oócitos humanos imaturos e in vitro maturados, doados por 11 pacientes submetidas à EOC para FIV. Oócitos em MII e embriões pré-implantação de camundongos congelados, imediatamente após o descongelamento, foram fixados para imunocoloração para visualização de SA por microscopia confocal e por microscopia imunoeletrônica de transmissão. Oócitos humanos imaturos (VG e MI) e maturados in vitro (VG Parado, MI, MI Parado e MII) também foram fixados e imunocorados para avaliação de SA, utilizando o mesmo anticorpo, por microscopia confocal. Para quantificar a imunocoloração de SA, nos oócitos e ao longo do desenvolvimento embrionário inicial, foi utilizado o programa ImageJ. Resultados: Em todas as amostras (camundongos e humanos), a imunocoloração para SA aparece por toda a zona pelúcida, bem como no citoplasma e no núcleo de oócitos, corpúsculos polares e embriões pré- implantação. Em camundongos, o estágio embrionário de 2 células apresentou níveis mais elevados de SA (69000187,4 DP 6733098,1 a.u.), quando comparado aos outros estágios de desenvolvimento (p <0,0001). A microscopia imunoeletrônica confirmou a presença de SA em oócitos e embriões pré- implantação. Em humanos, oócitos no estágio de VG, frescos, apresentaram níveis mais elevados de substância amiloide (4164.7 DP 1573.5 a.u.), quando comparados a oócitos em MI e MII (p = 0,008). Foi encontrada uma associação negativa entre níveis de SA e o índice de massa corporal (IMC) (-0,54; p = 0,0007), o número de dias de estimulação ovariana (-0,44; p = 0,002), a dose de hMG (-0,44; p = 0,002), o tempo entre a captação dos oócitos e a fixação das amostras (-0,33; p = 0,02) e o tempo decorrido após o trigger (-0,33; p = 0,02). Níveis elevados de SA foram encontrados em oócitos de pacientes que apresentaram valores de hormônio anti-mülleriano (AMH) entre 1 e 3 ng/ml, quando comparados a <1 ng/ml (4592,7 DP 2126,3 a.u vs. 737,3 DP 14,7 a.u.; p = 0,0002) e > 3 ng/ml (4592,7 DP 2126,2 a.u. vs. 3197,3 DP 895,0 a.u ; p = 0,006).16 Conclusões: Nós demonstramos pela primeira vez que as SA estão presentes no citoplasma e núcleo de oócitos murinos e humanos e embriões pré- implantação de camundongos. Também mostramos que as SA se distribuem de forma heterogênea ao longo do processo de maturação de oócitos humanos e do desenvolvimento pré-implantacional de embriões de camundongos. Embriões murinos no estágio de 2 células e oócitos humanos no estágio de VG apresentaram os maiores níveis de SA. Evidenciamos também uma correlação entre os níveis de SA nos oócitos humanos e características clínicas de bom prognóstico em pacientes submetidas a FIV, como por exemplo, baixo IMC e valores normais de AMH. Levando em conta que as SA estão envolvidas em mecanismos fundamentais relacionados a alterações celulares patológicas, bem como no processo de divisão celular, estudos futuros devem investigar o seu possível papel como biomarcador do envelhecimento e/ou da qualidade oocitária / Introduction: Previous studies with budding yeasts evidenced an important role of amyloid substances (AS) in the regulation of gametogenesis, questioning its potential as a marker of gametic quality. However, to date, no study has evaluated the presence of AS in mammalian oocytes and embryos. Objectives: To describe the presence, to characterize the distribution pattern and to compare AS levels in mature oocytes and preimplantation embryos using murine model. Identify, characterize the distribution pattern and quantify AS in human oocytes at different stages of development (GV- germinal vesicle, MImetaphase I and MII-metaphase II) and establish the correlation between oocytes AS levels and clinical characteristics of patients undergoing control ovarian stimulation (COE) for in vitro fertilization (IVF). Methods: An experimental prospective pilot study was carried out using 20 mature oocytes and 200 preimplantation mouse embryos (1-cell, 2-cell, 4-cell, 8- cell and blastocysts) and 46 immature and in vitro mature human oocytes donated from 11 consenting patients submitted to COE for IVF. Frozen MII oocytes and mouse preimplantation embryos, immediately after thawing, were fixed for immunostaining for visualization of AS by confocal microscopy and by transmission electron microscopy. Immature (GV and MI) and in vitro mature (GV Arrested, MI, MI Arrested and MII) human oocytes were also fixed and immunostained for AS evaluation, using the same antibody, by confocal microscopy. Fluorescence intensity from immunofluorescent staining and data from confocal microscopy were quantified using ImageJ program. Results: In all samples (mice and humans), immunostaining for AS appears throughout the zona pellucida, as well as in the cytoplasm and nucleus of oocytes, polar bodies, and preimplantation embryos. In mouse, 2-cell embryos exhibited higher levels of AS (69000187,4 SD 6733098,1 a.u.) when compared to the other stages of development (p <0.0001). Electron microscopy confirmed the presence of AS in mouse oocytes and preimplantation embryos. In humans, fresh GV stage oocytes exhibited higher levels of AS (4164.7 SD 1573.5 a.u.) when compared to MI and MII oocytes (p = 0.008). A negative association was found between levels of AS and patient body mass index (BMI) (-0.54; p = 0.0007), number of days of control ovarian stimulation (-0.44; p = 0.002); (-0.34, p = 0.002), dose of gonadotropin used, time between oocyte retrieval and fixation (-0.33; p = 0.02) and time after the hCG trigger (-0.33; p = 0.02). Significantly higher levels of AS were found in patients with AMH between 1 and 3 ng/ml, compared to <1 ng/ml (4592.7 SD 2126.3 a.u. vs. 737, 3 SD 14.7 a.u.; p = 0.0002) and > 3 ng/ml (4592.7 SD 2126.2 a.u. vs. 3197.3 SD 895.0 a.u.; p = 0.006). Conclusions: We demonstrate for the first time the presence, distribution, and change in AS throughout mouse and human oocyte maturation, and mouse embryonic development. Two-cell mouse embryos and GV human oocytes had the highest levels of AS. We also determine associations between AS in human oocytes with good prognosis clinical characteristics in patients submitted to IVF, such as low BMI and normal AMH values. Considering that AS are involved in fundamental mechanisms related to pathological cell changes, as well as in the19 conclusion of cell division process, future studies should investigate their possible role as a biomarker of oocyte aging and/or quality