Embryo signals for successful implantation

Human pre-implantation embryos display a high prevalence of aneuploidy and chromosomal mosaicism, unique from any other species. The decreasing incidence of aneuploidy observed between the cleavage and blastocyst stages of preimplantation embryo development infers a degree of 'self-correction' following activation of the embryonic genome. However, contrary to the previous assumption that only euploid embryos should be considered 'normal', new evidence has confirmed that mosaic embryos can result in the birth of healthy babies. Thus, aneuploidy should be viewed as an intrinsic feature of human pre-implantation embryo development, which presents a novel challenge at implantation. The endometrium must implement both positive and negative selection, in order to limit maternal investment to only viable embryos. The ability of the endometrium to act as a 'biosensor' of embryo quality has been well documented yet there is little direct evidence for the key regulators of this process. For the first time, we demonstrate a biological context for embryo biosensoring. Firstly, we discover novel embryo-secreted proteases that are enhanced at the blastocyst stage and relate to implantation outcome upon embryo transfer. Secondly, we identify corresponding protease-sensitive receptors in the endometrium, heightened during the window of implantation. By demonstrating the cleavage and de-activation of endometrial toll-like receptor 4 (TLR4) by embryo conditioned medium, we link successful implantation to a diminished inflammatory response. Furthermore, we demonstrate that TLR4 levels in the endometrium constitute a selectivity checkpoint, which is supressed in women suffering from recurrent miscarriage (RM)

Characterization of pregnancy-associated glycoproteins and progesterone as a predictor of twins and conceptus loss in high-risk pregnancy Holstein cows

Pregnancy loss is a multifactorial condition that compromises reproductive performance in dairy operations. Despite the high oocyte fertilization rate in dairy cows, only 28 % of those maintain a pregnancy to term. Pregnancy loss is estimated to cost U$600.00 per case. Identification of cows losing the pregnancy as early as possible can be helpful in providing timely opportunities for rebreeding, thus potentially minimizing economic losses. Traditionally, early pregnancy diagnosis is performed via ultrasonography, starting at 30 days, which provides information regarding embryo viability, uterine health, and ovarian structures. In addition, this technique allows the diagnosis of twin pregnancy that is three times more likely to be lost than a singleton. Despite its benefits ultrasonography requires well-trained personnel and incurs additional costs involving equipment purchase and maintenance. The use of biomarkers has been studied throughout the years, based on a demand for an easier, less costly, and more accurate test. Pregnancy-associated glycoproteins (PAG) is the most common biomarker marker to assess pregnancy status in cows. Produced and secreted on the maternal circulation by binucleate giant cells. Measurement of PAG in the blood has high sensitivity when performed between 25 32 days of gestation, however, the specificity can be as low as 83%. One of the major components that affect test accuracy is pregnancy loss. It has been reported that cows experiencing early pregnancy loss, present lower plasma concentrations of PAG. Another indirect biomarker to detect pregnancy in cows is progesterone. Cows experiencing pregnancy loss showed lower concentrations of this hormone, in comparison to cows keeping the pregnancy. The development of a threshold for PAG and progesterone that can predict pregnancy loss may aid in management decisions to provide earlier rebreeding opportunities. It was hypothesized that the plasma concentration of PAG and progesterone is reduced and can predict pregnancy loss in cows experiencing a high-risk pregnancy. Additionally, it was hypothesized that the concentration of PAG and progesterone are increased and can predict twins. High-risk pregnancy (HR) were characterized using transrectal ultrasonography 37 days post-AI based on the following criteria: small embryo size (SE, embryo < 15 mm, n=10), slow heartbeat (SH, <60 beats per minute, n = 11), extra amniotic membrane (EM, additional amniotic membrane, n=3). A cohort of twins (TW, n = 41) diagnosed at day 37 post-AI was also enrolled. Twins were also subgroups in unilateral (UT, n=17) and bilateral (BT, n=24). Each HR and TW cow was paired with the same parity cow carrying a normal singleton at d 37 post-AI (CON, n = 65). Blood samples were collected to measure PAG and progesterone at 37, 44, and 51 post-AI. Statistical analysis was performed using ANOVA, logistic regression and receiver operation characteristics (ROC) with JMP. Pregnancy loss at day 51 post-AI was greater (P < 0.01) in HR than CON and TW (CON=1.5%; HR=87.5%; TW=12.2%). Concentration of PAG at day 37 post-AI did not differ (P = 0.75) among groups (CON = 5.3 ± 0.7; HR = 4.8 ± 1.2; TW = 4.0 ± 0.9 ng/ml). The subgroup SE showed a statistical difference regarding the concentration of PAG at day 51 post-AI (P < 0.05), EM showed a tendency (P < 0.10) whereas SH, UT and BT did not when compared to CON. Concentration of progesterone at day 37 post-AI was greater in TW than HR and CON, and lower (P < 0.01) in HR than CON cows (CON = 7.0 ± 0.3; HR = 5.9 ± 0.4; TW = 8.4 ± 0.3 ng/ml). Regression and ROC analysis for PAG at day 37 post-AI did not find a threshold to predict pregnancy loss (P = 0.24) or twins (P = 0.30). Regression and ROC analysis for progesterone at day 37 post-AI found that a threshold of 6.5 ng/ml predicted (P < 0.01) pregnancy loss with an area under the curve (AUC) of 0.65, and threshold of 7.2 ng/ml predicted (P < 0.01) twins with AUC of 0.70. In summary, pregnancy loss and twins were predicted with only moderate accuracy by progesterone concentration at day 37 post-AI and the variability in PAG concentrations at day 37 post-AI was insufficient to generate a threshold to predict pregnancy loss and twins in Holstein lactating cows

Synthesis and characterization of cellulose-derived polymers for the treatment of damaged cartilage

Carboxymethyl cellulose (CMC) is currently the most widely used cellulose derivative and considered as a promising biomaterial due to its biocompatibility, biodegradability and non-toxicity. Polyethylene glycol grafted carboxymethyl cellulose (CMC-g-PEG) has been proved to be a very lubricating polymer. This MSc. thesis work aims to study further the potential application of the lubricating CMC-g-PEG for the treatment of damaged cartilage. CMC-g-PEG with different PEG chain lengths were successfully synthesized and characterized by conductometric titration and Fourier transform infrared spectroscopy. Polyethylene glycol grafted dialdehyde carboxymethyl cellulose (DCMC-g-PEG) was synthesized by oxidation of CMC-g-PEG with sodium periodate. The adsorption of the different polymers on collagen IV films (first approach model for cartilage) was studied by quartz crystal microbalance with dissipation. Compared with dialyzed CMC and CMC-g-PEG, much more DCMC-g-PEG absorbed onto the surface of collagen IV films due to the existence of covalently binding between aldehyde groups of DCMC-g-PEG and amine groups of collagens. The adsorption results of CMC-g-PEG and DCMC-g-PEG provide the evidence that the introduction of aldehyde groups to the backbone of CMC-g-PEG can improve its attaching ability to collagen IV

The Influence of Preovulatory Estradiol on Uterine Transcriptomics and Proteomics Around Maternal Recognition of Pregnancy in Beef Cattle

Preovulatory estradiol has been reported to play a crucial role in pregnancy establishment and maintenance, but the mechanism by which estradiol exerts its effects has not been well characterized. Specifically, the interactions between the maternal uterine environment and the developing conceptus can greatly impact pregnancy success or loss. The objective of this dissertation is to determine the effects of preovulatory estradiol exposure on uterine and trophectoderm transcriptomes, and uterine luminal fluid (ULF) protein composition. Beef cows/heifers were synchronized, artificially inseminated (d 0), and grouped into either high (highE2) or low (lowE2) preovulatory estradiol. Uteri were flushed to collect d16 conceptuses either nonsurgically or following slaughter, and endometrial biopsies (n=29) were collected from the ipsilateral uterine horn. Real-Time PCR (RT-PCR) was performed on trophectoderm (TE; n=21) RNA to measure the relative abundance of IFNT, PTGS2, TM4SF1, C3, FGFR2, and GAPDH. Total cellular RNA was extracted from endometrium for RNA sequencing. ULF pools (n=28) for the following groups: highE2/noconceptus, highE2/conceptus, lowE2/noconceptus, and lowE2/conceptus were analyzed using a 2D LC-MS/MS based 8plex iTRAQ method. RT-PCR data were analyzed using the MIXED procedure in SAS. Transcript abundances in the endometrium were quantified using kallisto, differentially expressed genes (DEGs) were determined using DESeq2 (FDR2), and IPA was used for pathway analysis. Scaffold Q+ was used to quantitate peptide and protein identifications in the ULF. There were no differences in mRNA abundances in TE, but there were 432 DEGs among the highE2/conceptus versus lowE2/conceptus groups, 253 were downregulated (CR2, CDH4, TROAP, COL1A2) and 179 were upregulated (PRSS8, FABP3, IDO1, MUC13, CXCL10) in the highE2/conceptus group. There were 48 differentially expressed proteins (DEPs) among the highE2/conceptus and lowE2/conceptus groups (19 upregulated, 29 downregulated in the highE2/conceptus group), 6 of these were differentially expressed (FD

Characterization of the Immune Response to Lipopolysaccharide in Early Pregnant Ewes as a Model to Study Bacterial Infection Induced Embryonic Loss

An immunological balance has to be established during pregnancy that protects the mother yet tolerates the semi-allogenic fetus. To understand the innate immune response during bacterial infections that may cause early embryonic loss, a lipopolysaccharide (LPS) treated sheep model was used. Two objectives of this study were to examine if omega-3 PUFAs in the form of supplementary whole flaxseed could reduce the inflammatory response to an LPS challenge and to examine if there is a differential immune response to LPS in Suffolk and Dorset ewes. A total of 3 experiments were conducted; two investigating the effect of supplement and one investigating the effect of breed. Estrus was synchronized by CIDR insertion for 5 days, followed by 20 mg of PGF 2alpha at CIDR withdrawal. Early pregnant ewes received via the jugular vein, either phosphate buffered saline (PBS) (3 ml) or LPS (2.5 mug/kg). Blood was collected via jugular venipuncture at hour: 0, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6, 9, 12, and 24. Whole blood samples were used to determine white blood cell counts (WBCs) before centrifugation to collect white blood cells for RNA extraction. Rectal temperature and change in behavior/physical (lethargy, coughing, nasal discharge, absence of eating) appearance were recorded hourly. Real-time PCR was performed for expression of cytokines (CXCL8, IL6, TNFalpha, IFNgamma, IL-10, and TGFbeta), receptors (TLR4, MRC1), enzymes (COX2, SOD2), transcription factors (NF-kappaB, PPARgamma, and Foxp3) and complement component 3. In all experiments, temperature increased in response to LPS, peaking at hour 4 before returning to normal by hours 6-9; WBCs dropped by hour 1 before returning to normal by hours 6-9. In trial 1 of the supplement study (flaxseed versus a control supplement) (Dorset ewes flaxseed + LPS n=3; flaxseed + PBS n=3; control + LPS n=5; control + PBS n=5), LPS increased haptoglobin and cortisol levels and affected gene transcription of CXCL8, IFN?, TLR4, MRC1, SOD2, Foxp3 (by hour), and C3.There was a diet effect with regard to cortisol and gene expression of CXCL8, and TLR4. There was a diet x LPS interaction with regard to temperature, WBCs (by hour), haptoglobin, serum amyloid A and gene expression of CXCL8, IL-6, and TLR4. In trial 2 of the supplement study (Dorset ewes flaxseed + LPS n=11, flaxseed + PBS n=10, control + LPS n=11, control + PBS n=10), LPS increased haptoglobin and cortisol and affected gene expression of CXCL8, TLR4, MRC1, SOD2, PPARgamma, Foxp3, and C3. There was a diet effect on cortisol and gene expression of C3.There was a diet x LPS interaction with regard to temperature and cortisol. In the breed effects study (Dorset + LPS n=11; Dorset + PBS n=10; Suffolk + LPS n=16; Suffolk + PBS n=16), LPS increased cortisol and affected gene transcription of CXCL8, TLR4, MRC1, SOD2, PPARgamma, and C3. There was an effect of breed on temperature; haptoglobin, serum amyloid A (by hour), and cortisol levels; and gene transcription of IL-6, IFNgamma, IL-10, TLR4, COX2, SOD2, PPARgamma, Foxp3, and C3. There was a breed x LPS interaction on change in temperature from hour 0, the frequency of behavior/physical changes; haptoglobin, serum amyloid A (by hour), and cortisol; and gene transcription of IL-6 and C3. Pregnancy status was assessed at 25 dpc with transrectal ultrasound and progesterone was measured in plasma samples. In trial 1 of the supplement study, flaxseed increased progesterone but it did not differ between groups in trial 2. There were no differences in progesterone between the breeds tested. The number of ewes that lambed in each treatment group was not different in any of the experiments. In summary, acute infections may cause embryonic loss by shifting the environment to be pro-inflammatory. There was no clear benefit of supplementary o-3 PUFAs in reducing the inflammatory response. Suffolk ewes had an elevated inflammatory response to LPS compared to Dorset ewes and may be more susceptible to embryonic loss in response to infection

Desenvolvimento de biossensores para diagnóstico precoce de biomarcadores do cancro e aplicabilidade em células fotovoltaícas

Cancer diseases are exponentially growing in Portugal throughout the years, reflecting the same scenario as in Europe. Several efforts have been made to minimize cancer incidence, including adopting a healthier behaviour (as vaccination programs and lifestyle adjustments) and early diagnosis of the disease. Currently, the screening of the disease can be performed by monitoring tumour markers (CEA, CA15-3, CA125, CA19-9, PSA, among others) that circulate in the body fluids, as blood and urine. Thus, the development of new analytical tools for detecting these biomarkers in a clinical context is also of growing interest. This PhD thesis aimed to develop low-cost sensing platforms based on newly biomaterials to screen cancer biomarkers in biological samples towards on-site application. For the purpose, different analyte targets were presented herein, including CRT, which is a potential biomarker in ovarian cancer, and CEA, a tumor marker that despite being an indicator of other types of cancer such as breast, pancreas and lung, offers high specificity in colorectal cancer. Distinct approaches were developed for this purpose, based on the design of plastic antibodies by means of molecular imprinting technology, and on antibody-antigen recognition though the development of immunosensors for the detection of colorectal cancer biomarker. The sensing platforms built on a solid conductive glass support (composed by fluorine-doped tin oxide film (FTO)) allowed understanding the interaction between cancer biomarker and the sensing material through electrical signals. In order to allow the portability of these devices, a new approach was proposed in this project, establishing the integration of a sensing platform in a photovoltaic cell, namely in a DSSC, once these cells are capable of generating electrical energy from solar energy. In addition, efforts were made to improve the overall performance of these photovoltaic cells, modifying the DSSCs configuration by introducing metallic nanoparticles, such as gold nanoparticles, in the composition of photoanodes, or even, by the chemical functionalization of the semiconductor material, followed by the metallic nanoparticles binding. In general, the emerging biosensing materials and platforms out coming from this project may contribute for the development of new non-invasive or minimally invasive and self-sustained devices suitable for the early diagnosis of cancer biomarkers in a clinical application.O cancro é uma doença que tem vindo aumentar de forma exponencial em Portugal ao longo dos anos, à semelhança do que acontece no resto da Europa. Deste modo, diversos esforços têm vindo a ser feitos no sentido de mitigar a sua incidência, entre os quais a adesão a comportamentos saudáveis (programas de vacinação e modificação do estilo de vida) e ao diagnóstico precoce da doença. Atualmente, o rastreio da doença é concretizado através de marcadores tumorais (CEA, CA15-3, CA125, CA19-9, PSA, entres outros), não-invasivos, que circulam no organismo, em fluídos como sangue e urina. Neste sentido, esta tese de doutoramento teve o intuito de desenvolver plataformas sensoras de baixo custo, baseadas em novos biomateriais sensores que facultem o rastreio dos biomarcadores do cancro em amostras biológicas, de forma a serem aplicados num contexto clínico. Para este efeito foram consideradas como moléculas alvo a carnitina (CRT), um potencial biomarcador para o diagnóstico do cancro do ovário, e o antigénio carcinoembrionário (CEA), um marcador tumoral que, apesar de ser indicador de outros tipos de cancro como da mama, do pâncreas e do pulmão, apresenta uma elevada especificidade para o cancro do colorretal. Para a conceção destes biossensores foram concretizadas duas abordagens distintas que se basearam no desenvolvimento de anticorpos plásticos, recorrendo à síntese de polímeros de impressão molecular, e no reconhecimento anticorpo-antigénio, através da construção de imunossensores para a deteção do biomarcador do cancro do colorretal. As plataformas sensoras construídas sobre um suporte sólido de vidro condutor (composto por um filme de óxido de estanho dopado com flúor) permitiram avaliar a interação entre o biomarcador e o material sensor através de sinais de natureza elétrica. De modo a possibilitar a portabilidade destes dipositivos para a deteção de marcadores tumorais, neste projeto foi proposta uma nova abordagem que estabeleceu a integração de uma plataforma sensora numa célula fotovoltaica, nomeadamente, numa célula solar sensibilizada por corante (Dye-Sensitized Solar Cell, DSSC), uma vez que estas são, por si só, células capazes de gerar energia eléctrica por conversão da energia solar. Também foram realizados esforços no sentido de melhorar o desempenho geral destas células fotovoltaicas, alterando a sua configuração, através da implementação de nanopartículas metálicas, como as nanopartículas de ouro, na constituição do fotoânodo, ou até mesmo através da funcionalização química do material semicondutor que compõe estes fotoânodos e, posterior, ligação a estas nanopartículas metálicas. De uma forma geral, os dispositivos sensores desenvolvidos poderão ser uma ferramenta promissora, de baixo custo e portátil, para um diagnóstico precoce, ou para o acompanhamento e a monitorização terapêutica do cancro, de modo não invasivo ou minimamente invasivo, para aplicação num contexto clínico.Programa Doutoral em Nanociências e Nanotecnologi

Uterine regulation of preimplantation embryo development in fertility-classified heifers

Infertility and subfertility represent pervasive problems in domestic animals and humans, and embryonic mortality is a major factor influencing reproductive efficiency. In cattle, the majority of embryonic loss occurs during the first month of gestation that involves the period of blastocyst formation, conceptus elongation, maternal recognition of pregnancy, implantation and beginning of placentation. Pregnancy success and embryonic mortality are affected by paternal, maternal, embryonic, and environmental factors, and the establishment and maintenance of pregnancy are a result of complex conceptus-endometrium interactions that results in adequate conceptuses (embryo/fetus and associated extraembryonic membranes) development, implantation and placentation. Our central hypothesis is that the uterus directly influences embryonic and conceptus development, and we proposed that heifers with consistently high or low fertility have distinct uterine capacity to support pregnancy. To test this hypothesis, serial embryo transfer (3-4 rounds) was used to classify heifers based on pregnancy success on day 28 as high fertile (HF; 100%), subfertile (SF; 25%), or infertile (IF; 0%). Next, a series of experiments were conducted using the fertility-classified heifers to investigate conceptus development and uterine biology in two time points: (1) day 14, to investigate conceptus development prior to the period when pregnancy induce changes are detected in the endometrium transcriptome; (2) at day 17, to evaluate conceptus-endometrial cross talk during the period of maternal recognition of pregnancy. Results from the studies conducted on day 14 supports the idea that: (1) circulating progesterone concentrations are not different among fertility-classified heifers; (2) conceptus development and survival by day 14 is not affected by fertility classification; (3) only minimal differences in endometrium transcriptome are detected among pregnant fertility-classified heifers. Collectively, these results indicated that the biological mechanisms underlying subfertility and infertility manifests between days 14 and 28, when pregnancy recognition signaling and conceptus elongation and implantation must occur for the establishment of pregnancy. Moreover, results from the subsequent experiments conducted at day 17 indicated that: (1) the mechanism of pregnancy loss in fertility-classified heifers start to manifest around the time of maternal recognition of pregnancy; (2) conceptus survival by day 17 is compromised in IF heifers; (3) conceptus development is advanced in HF than SF heifers; (4) conceptus transcriptome is directly influenced by the uterine environment; (5) dysregulated conceptus-endometrial interactions in SF heifers seems to be the major cause of pregnancy loss. Analysis of the uterine luminal fluid (ULF) from fertility classified heifers on day 17 established that: (1) ULF composition is affected by conceptus-endometrium interactions; (2) glucose concentrations in ULF are not different among fertility-classified heifers; (3) pregnancy induced changes in the metabolites found in ULF was diminished in SF heifers, and the majority of the metabolites that increased in the ULF of pregnant HF than SF heifers were associated with energy and amino acid metabolism; (4) increased abundance of proteins involved with energy metabolism, oxidative stress, amino acid metabolism, and cell proliferation and differentiation were detected in ULF of pregnant HF than SF heifers; (5) The lipid content of the ULF is altered by pregnancy and fertility classification; (6) overall concentrations prostaglandins and interferon tau were increased in the uterine lumen of pregnant HF than SF heifers, likely due to differences in conceptus size. Collectively, results from these studies supports the idea that the dysregulated conceptus-endometrium interactions in SF heifers affects the uterine luminal contents and impairs conceptus survival and elongation. Furthermore, knowledge gained from these studies enhances our understanding of the mechanisms regulating pregnancy loss in cattle and provides new information on uterine and conceptus biology during early pregnancy in ruminants.Includes bibliographical reference

Numerical characterisation of label free optical biosensors

There is a significant need for the development and use of numerical methods to simulate advance and complex optical biosensor structures. Finite Element Method (FEM) has been established as one of the most powerful and versatile numerical method and has been implemented in this thesis to characterize, analyse and optimise label-free optical biosensors for the detection of micron size biological objects like bacteria such as E.coli and nanometre size biomolecules such as antibody, nucleic acids and proteins. These sensors are all suitable for deep-probe sensing as large evanescent field can be excited in the sensing medium with substantial penetration depth achieved by techniques like Surface Plasmon Resonance (SPR) and sensor architectures based on nanowires and slot waveguides. This thesis presents three different architectures of label-free optical biosensors. First, a fiber optic surface plasmon resonance (SPR) biosensor for the detection of E.Coli is optically modeled by using the finite-element approach in conjunction with the perturbation technique which is computationally more efficient and can be used for waveguides with low or medium loss values. The same sensing architecture is used when surrounding index is varied from 1.30 -1.44 to cover most of the biological elements that are used in the biosensing applications. Second one is based on evanescent-wave guiding properties of nanowire waveguides a theoretical investigation of silica nanowires employing a wire assembled Mach-Zehnder structure to detect the presence of E.Coli is studied second. Finally, a slot-waveguide based micro-ring resonator is investigated for the detection of DNA Hybridization using H-field FEM based full-vector formulation. It is found that all of the numerical methods provide good agreement with the experimental sensitivities and detection limits

Three-dimensional modelling of the human peri-implantation endometrium

Human embryo implantation is the rate-limiting step for successful pregnancy in assisted reproductive technology, and inappropriate endometrial embryo-quality biosensing is also associated with recurrent pregnancy loss. Elucidating the mechanisms underpinning the peri-implantation endometrium would enable the development of treatments for patients suffering with chronic fertility disorders. However, implantation is difficult to study. Therefore, simple in vitro models of human endometrial cells have been developed. However, the endometrium is a complex tissue made up of stromal cells (EnSC), epithelial glands, vasculature, and immune cells. An in vitro model that encompasses all aspects of the human endometrium would require a complex co-culture system. Therefore, three-dimensional (3D) culturing techniques are the most suitable. This study aimed to establish a protocol for a novel in vitro model of the peri-implantation endometrium by co-culturing primary EnSC and epithelial cells (EEC) using 3D culture techniques. In this study, two approaches were undertaken. The first involved the use of the Endometrial Regenerative Bodies (ERB) model, which was unsuccessful in achieving the study’s aim. However, the second approach, involving the 3D co-culture of EnSC with endometrial gland organoids led to the establishment of a complex organoid culture. The growth conditions of the complex organoids were optimised by modifying the 3D matrix and medium conditions. The standard organoid growth matrix, Matrigel, a basement membrane derived hydrogel, was replaced with a purified Type I collagen hydrogel to provide a more physiological ECM for the EnSC. Direct interaction between the EnSC and EEC in the complex organoids was examined through the establishment of a minimal differentiation medium and single cell RNA-sequencing of a decidual time course of gland organoids and complex organoids. EnSC were able to induce the differentiation of EEC into a secretory phenotype in complex organoid cultures. The established complex organoid protocol offers a starting point for developing a faithful in vitro model of the human peri-implantation endometrium, which will provide a step change in our collective ability to study human embryo implantation. A system that would be patient-specific, and in the long term developed into a high-throughput system for personalised medicine for the treatment of infertility