Experimental and Computational Approaches to Optimizing Bovine Gamete Cryopreservation

Cryopreservation uses freezing to suspend the metabolic activity of biological specimens for increased longevity of biotic materials like gametes. Cryopreservation has been known to affect both the functionality (performance of activities) and the viability (survival) of biological specimens during the freezing and thawing process due to four types of damage: (1) thermal, (2) ice, (3) osmotic stress, and (4) cytotoxic. Cryoprotective agents (CPAs) are known to reduce thermal and ice damage but cause osmotic stress and cytotoxic damage. Osmotic stress occurs when the addition of CPAs causes a rapid expulsion of water from the cell as the extracellular environment has become hypertonic. Cytotoxic damage occurs when a cell is exposed for too long to CPAs that may be damaging to the cell at high temperatures, but aid in preservation at low temperatures. The purpose of my project is to minimize osmotic stress in bovine embryos and cytotoxic damage in bovine sperm caused by CPAs using novel algorithmically guided techniques. To minimize osmotic stress in bovine embryos, I aim to facilitate the equilibration of embryos with cryoprotective agents isochorically (constant volume). Isochoric cryoprotectant equilibration, requires a feedback control system that in our case will use real-time image analysis developed in this thesis to estimate current embryo volume and then adjusts the concentration of CPAs being administered to the system. I implemented a colour-based image analysis software that was able to process images of bovine embryos as they were exposed to CPAs at a sub-second rate. The sub-second processing rates include cell volume estimates that are comparable to manual cell volume estimates. To minimize cytotoxic damage in bovine sperm, I optimized cryopreservation media (CPM) composition to maximize post-thaw motility. The composition of CPM can contain many ingredients that have the potential to interact and are infeasible to test only empirically. Here, I combined empirical experiments, data-driven optimization algorithms, and machine learning to optimize the composition of CPM. I used differential evolution and Gaussian process regression to optimize CPM composition that are on par with commercial media after 9 iterations. During the optimization process I determined that Gaussian process regression model was superior to artificial neural networks when predicting post-thaw motility for a given CPM composition. By optimizing these cryopreservation processes, cellular damage can be reduced, improving functionality and viability of gametes used in assisted reproductive technology that can be applied across animal husbandry and biomedical fields

Mathematical modelling and image processing for some challenges in the In Vitro Fertilisation clinic

In Vitro Fertilisation (IVF) is a treatment involving the fertilisation of human egg cells in the laboratory to create embryos, which are transferred to the uterus of the patient in the hope that they become pregnant. Only 19% of treatments worldwide result in a live birth. This thesis involves interdisciplinary collaboration with experimentalists and the London Women’s Clinic, to identify and tackle a series of mathematical challenges and barriers to improving success rates in IVF clinics. We first explore the use of Differential Dynamic Microscopy (DDM) as a non-invasive oocyte health assessment tool. Identifying challenges that limit the clinical usefulness of DDM, we develop a methodology, using synthetic data, to enhance and validate parameter fitting in DDM. We optimise the selection of synthetic data parameters, and present a new pipeline for generating parameter fitting. After showing existing non-linear curve fitting algorithms are inaccurate in DDM applications, we establish a new machine learning parameter fitting pipeline, trained exclusively on synthetic data and applied in real datasets. We, subsequently, explore the application of DDM to phase-contrast microscopy. Phase-contrast images exhibit shadowing, leading to anisotropy in the DDM matrix and invalidating a key assumption of DDM. We derive an analytic expression describing this anisotropy, and conclude for isotropic motions that shadowing does not affect parameter fitting. For anisotropic motion, we also outline conditions on the microscope setup and imaged behaviour that affect fitting error. The second part of this thesis considers challenges related to cryopreservation of oocytes and embryos through a rapid-cooling technique, vitrification. We numerically simulate the process of vitrification (rapid freezing) and show that cooling rates are unaffected by the number or arrangement of embryos or oocytes on the device, which validates current protocol. Additionally, the challenge of predicting embryo viability from time-lapse images of post-thaw embryo re-expansion is tackled. We implement machine-learning image segmentation to measure the cross-sectional area of the embryo, and identify non-linear re-expansion as a new 5 metric indicating lower implantation rates by as much as 3% in a sample of clinical data.

The enigmatic morula: mechanisms of development, cell fate determination, self-correction and implications for ART

Background: Assisted reproduction technology offers the opportunity to observe the very early stages of human development. However, due to practical constraints, for decades morphological examination of embryo development has been undertaken at a few isolated time points at the stages of fertilisation (day 1), cleavage (day 2-3) and blastocyst (day 5-6). Rather surprisingly, the morula stage (day 3-4) has been so far neglected, despite its involvement in crucial cellular processes and developmental decisions. Objective and rationale: The objective of this review is to collate novel and unsuspected insights into developmental processes occurring during formation of the morula, highlighting the key importance of this stage for a better understanding of preimplantation development and an improvement of ART. Search method: PubMed was used to search the MEDLINE database for peer-reviewed English-language original articles and reviews concerning the morula stage in mammals. Searches were performed by adopting ‘embryo’, ‘morula’, ‘compaction’, ‘cell fate’ and ‘IVF/assisted reproduction’ as main terms, in association with other keywords expressing concepts relevant to the subject (e.g. cell polarity). The most relevant publications, i.e. those concerning major phenomena occurring during formation of the morula in established experimental models and the human species, were assessed and discussed critically.Outcomes: Novel live cell imaging technologies and cell biology studies have extended our understanding of morula formation as a key stage for the development of the blastocyst and determination of the inner cell mass (ICM) and the trophectoderm (TE). Cellular processes, such as dynamic formation of filopodia and cytoskeleton-mediated zippering cell-to-cell interactions, intervene to allow cell compaction (a geometrical requisite essential for development) and formation of the blastocoel, respectively. At the same time, differential orientation of cleavage planes, cell polarity and cortical tensile forces interact and cooperate to position blastomeres either internally or externally, thereby influencing their cellular fate. Recent time lapse microscopy (TLM) observations also suggest that in the human the process of compaction may represent an important checkpoint for embryo viability, through which chromosomally abnormal blastomeres are sensed and eliminated by the embryo.Wider implications: In clinical embryology, the morula stage has been always perceived as a ‘black box’ in the continuum of preimplantation development. This has dictated its virtual exclusion from mainstream ART procedures. Recent findings described in this review indicate that the morula, and the associated process of compaction, as a crucial stage not only for the formation of the blastocyst, but also for the health of the conceptus. This understanding may open new avenues for innovative approaches to embryo manipulation, assessment and treatment

A review of different deep learning techniques for sperm fertility prediction

Sperm morphology analysis (SMA) is a significant factor in diagnosing male infertility. Therefore, healthy sperm detection is of great significance in this process. However, the traditional manual microscopic sperm detection methods have the disadvantages of a long detection cycle, low detection accuracy in large orders, and very complex fertility prediction. Therefore, it is meaningful to apply computer image analysis technology to the field of fertility prediction. Computer image analysis can give high precision and high efficiency in detecting sperm cells. In this article, first, we analyze the existing sperm detection techniques in chronological order, from traditional image processing and machine learning to deep learning methods in segmentation and classification. Then, we analyze and summarize these existing methods and introduce some potential methods, including visual transformers. Finally, the future development direction and challenges of sperm cell detection are discussed. We have summarized 44 related technical papers from 2012 to the present. This review will help researchers have a more comprehensive understanding of the development process, research status, and future trends in the field of fertility prediction and provide a reference for researchers in other fields

Molecular, morphological, and kinetic diagnosis of human preimplantation embryo viability

There have been phenomenal advances in the field of reproductive medicine and success rates following in vitro fertilisation have improved dramatically in recent years. The aim of this project was to improve our understanding of human preimplantation embryo development by identifying potential markers of viability that may aid us in selecting the best embryo for uterine transfer in the clinical embryology laboratory. Investigations into the distribution of cytoskeletal F-actin in human embryos demonstrated that a highly organised actin cortex is important for embryo cleavage and continued development to the blastocyst stage. Whilst they are polarised in the mouse from the oocyte to the blastocyst, the regulatory proteins leptin and STAT3 are co-localised only at the oocyte stage in humans, and are distributed within different cytoplasmic domains in human cleavage stage embryos and blastocysts. Whether polarity in humans is predetermined in the oocyte remains elusive, but none of the evidence generated in this thesis supports this idea. Leptin transiently activates STAT3 via the long form of the leptin receptor, and most significantly in the ICM of human day 6 blastocysts. Morphological features of blastocysts that can be visualised microscopically, such as a double ICM and cytoplasmic projections connecting the ICM to the TE, provide clues to their viability and may help us to choose the most suitable embryo from a cohort when deciding which to transfer. Nuclear volumes may in future contribute to this selection. Using time lapse technology to study cleavage patterns is now a routine occurrence in the clinical embryology laboratory. The results in this thesis show that distinctive patterns of divisions and the site at which blastocysts hatch can provide us with more information than a snap-shot morphological evaluation. Finally, contributing to the development of modelling software and predictive algorithms for the study of human embryos, particularly in time lapse imaging, means that our understanding of this fascinating area of medicine will continue to progress

BONE REGENERATION OF THE CRANIOFACIAL SKELETON WITH BIOENGINEERED SCAFFOLDS

La ricostruzione dei difetti ossei del massiccio facciale rappresenta una sfida chirurgica e attualmente la miglior soluzione, in uso nella pratica clinica, consiste nell’impiego di lembi liberi microchirurgici. Il campo della bioingegneria sta crescendo notevolmente e fornisce alcune alternative alla ricostruzione ossea. Durante il dottorato sono stati condotti una serie di studi preclinici al fine di valutare la performance degli scaffold bioingegnerizzati nella rigenerazione ossea del distretto craniofacciale. Nello studio pilota due tipi di scaffold (un idrogelo costituito da polietilen glicole-chitosano [HyCh] e uno scaffold nato dalla combinazione di acido polilattico, policaprolattone e HyCh [PLA-PCL-HyCh]), seminati con cellule staminali mesenchimali umane (hMSCs) in diverse concentrazioni (i.e. 1000, 2000, and 3000 cells/mm 3 ), sono stati testati per la ricostruzione di difetti mandibolari non critici nel modello animale di coniglio. Le proprietà rigenerative degli scaffold bioingegnerizzati sono state analizzate con studi radiologici in vivo ed analisi radiologiche ed istologiche ex vivo. E’ stato dimostrato che la rigenerazione ossea può essere incrementata significativamente attraverso l’utilizzo di una ricostruzione con scaffold o con scaffold seminato, ottenendo rispettivamente una rigenerazione ossea del 50% e del 70% del sito chirurgico in 120 giorni, confrontata con il 40% che si ottiene con la rigenerazione spontanea. Questi risultati hanno permesso di stabilire un riferimento di base per ulteriori esperimenti volti all’ottimizzazione della performance rigenerativa. E’ stato avviato un primo tentativo di ottimizzare il modello di base con un trapianto allograft di cellule staminali (i.e. rabbit mesenchymal stromal cells [rMSCs] in sostituzione delle hMSCs) e con la somministrazione topica di BMP-2 (i.e. scaffolds seminati con hMSCs a cui si aggiunge BMP-2). Purtroppo i risultati sperimentali così ottenuti non hanno superato il riferimento di base. Studi successivi sono stati dedicati alla progettazione e sviluppo di un nuovo tipo di scaffold composito con architettura core-shell. Tale scaffold grazie alla struttura reticolare del core prodotto mediante stampante 3D presenta notevole versatilità ed eccellenti proprietà meccaniche. Il nuovo scaffold è stato introdotto per testare la possibilità di rigenerare difetti con dimensioni critiche. I risultati ottenuti non sono stati completamente soddisfacenti a causa di problematiche legate al fissaggio subottimale dello scaffold a livello del difetto chirurgico. Infine sono stati eseguiti diversi studi ancillari per fare chiarezza in alcune problematiche della rigenerazione ossea: 1) è stata dimostrata la fattibilità della ricostruzione con scaffold bioriassorbibile di un difetto segmentale di mandibola nel coniglio con un lungo follow up (i.e. 3 months) nessuna complicanza maggiore; 2) è stato sviluppato un valido modello di osteoradionecrosi mandibolare con ottima correlazione dose-effetto biologico anche se, dal punto di vista rigenerativo, lo scaffold seminato con hMSCs non è riuscito a promuovere il processo neo-osteogenetico; 3) è stato condotto uno studio preliminare per investigare l’interazione tra le hMSCs e le cellule tumorali e al momento non sono emerse evidenze che supportino la teoria che le hMSCs possano favorire la crescita tumorale.Reconstruction of maxillofacial skeleton defects is a surgical challenge, and microvascular reconstruction is the current gold standard. The field of tissue bioengineering has been providing an increasing number of alternative strategies for bone reconstruction. We performed a series of preclinical studies to assess the performance of bioengineered scaffolds in craniofacial bone regeneration. In our pilot study an hydrogel made of polyethylene glycol-chitosan (HyCh) and a core-shell combination of poly(L-lactic acid)/poly(ε-caprolactone) and HyCh (PLA-PCL-HyCh), seeded with different concentrations of human mesenchymal stromal cells (hMSCs) (i.e. 1000, 2000, and 3000 cells/mm3), has been explored in non-critical size mandibular defects in a rabbit model. The bone regenerative properties of the bioengineered scaffolds were analyzed by in vivo radiological examinations and ex vivo radiological, histomorphological, and immunohistochemical analyses. We demonstrate that bone regeneration can be boosted by scaffold- and seeded scaffold-reconstruction, achieving, respectively, 50% and 70% restoration of presurgical bone density in 120 days, compared to 40% restoration seen in spontaneous regeneration. These results helped to establish a baseline reference for further experiments and we started to search for an optimization of the regenerative performance. We tried to optimize the baseline model though the application of an allograft model (i.e. seeding rabbit mesenchymal stromal cells [rMSCs] instead of hMSCs) and though the local administration of BMP-2 (i.e. bioengineered scaffolds seeded with hMSCs, implemented with BMP-2). Unfortunately the experimental results could not overcome the outcome of 70% restoration of presurgical bone density in 120 days. Afterwards our team developed and produced a new hybrid core-shell composite scaffolds in 3D-printed PLA-HyCH with excellent mechanical properties. We introduced a the new composite scaffold in our experiments, testing the possibility of regenerating of critical size defects, but once again we could not obtain a better performance than our reference value. This finding probably was consequence of suboptimal fixation of the scaffolds at level of the surgical defects. Moreover, some ancillary studies were performed in order to clarify some issues of the bone regeneration: 1) the feasibility of the reconstruction of a segmental mandibular defect though a bioresorbable hybrid core-shell composite scaffold was proved with a long follow up (i.e. 3 months) of the animal without any major complication; 2) a valid animal model of mandibular osteoradionecrosis with an excellent correlation between dose and biological damage was developed, but, from the regenerative standpoint, the scaffold-hMSC model could not highly catalyze the bone repair of bone defects in this particular setting; 3) a preliminary investigation focused on the interaction between hMSCs and tumor cells was accomplished, and, from the few data obtained at the moment, no evidence supported the hypothesis that hMSCs could promote tumor growth

Analyses génomiques et phénotypiques contrastant les embryons bovins des races Holstein et Jersey

Au cours des dernières années, la production laitière a été en croissance constante en raison de plusieurs facteurs tels que l’utilisation d’individus plus performants. L’augmentation du nombre d’enregistrements de vaches de la race Jersey confirme l’intérêt économique des producteurs pour cette race en raison de la production élevée de protéines et de gras du lait par rapport aux autres races. Cependant, les embryons de la race Jersey ont montré des difficultés dans le processus de cryopréservation lequel peut aider à une commercialisation massive de matériel génétique de cette race. Il a été observé que la race Jersey présente un faible taux de gestation lors du transfert des embryons après la cryopréservation en comparaison aux embryons de la race Holstein. Nous proposons que cette différence entre les deux races bovines soit due à des caractéristiques spécifiques de la race au niveau du phénotype et du génome. Dans un premier temps, les résultats de cette étude ont mis en évidence des différences au niveau du phénotype et du profil lipidique et génomique de l’embryon Jersey. Celui-ci est caractérisé par un contenu de lipides associé à une faible fonction mitochondriale laquelle déterminera le faible succès à la cryopréservation. Par la suite, nous avons évalué au niveau du phénotype et de la génomique, l’utilisation de la L-carnitine dans le milieu de culture in vitro afin de compenser cette caractéristique des embryons de la race Jersey. Cette étude sur la supplémentation de L-carnitine a permis d'identifier une faible réponse chez les embryons Jersey qui est expliqué par le faible effet de la L-carnitine sur l’activité mitochondriale. Afin de définir l’impact de la fonction mitochondriale sur la viabilité de l’embryon lors de nos études, nous avons mis au point une méthode pour compenser la dysfonction mitochondriale sur le développement embryonnaire chez le bovin. Enfin, l’application de la vitamine K2 dans le milieu de culture in vitro montre un impact positif sur la fonction mitochondriale qui a mené à des changements phénotypiques et génomiques chez les embryons bovins. En conclusion, ce projet a permis de caractériser et d’identifier la race comme un facteur qui limite la cryopréservation des embryons et peut influencer sur le métabolisme embryonnaire au niveau des mitochondries. La fonction mitochondriale est une caractéristique importante sur le développement embryonnaire bovin qui peut ouvrir sur des perspectives d’amélioration de la viabilité embryonnaire.For the past decades, milk production has been increasing due to several factors such as the use of high genetic merit individuals. In this regard, Jersey cows have been of interest for the producers because of high protein and fat indexes in their milk compared to others breeds. However, there are some challenges associated with Jersey particularly poor results using embryo cryopreservation which could help to massively commercialize the genetic material of this breed. It was observed that the Jersey breed have low pregnancy rates following embryo transfer of cryopreserved Jersey embryos compared to the Holstein breed. Here, we hypothesised that those differences between these two breeds in embryo cryopreservation are due to specific phenotypic and genotypic characteristics at the embryo level. Initially, the results of this study showed differences on the phenotype, lipid profile and genomic differences of Jersey embryo characterized by the higher lipid droplets content associated with low mitochondrial function which will determine the low success with cryopreservation. Subsequently, we assessed the phenotype and genotype of embryos using L-carnitine supplementation in the in vitro embryo culture medium in order to compensate those characteristics in Jersey embryos. The results of this study revealed moderate beneficial effects of L-carnitine supplementation in Jersey embryos through low effect of L-carnitine on mitochondrial activity. To define the impact of mitochondrial function on the embryo viability during our study, we developed a method to compensate the mitochondrial dysfunction during early embryo development in bovine model. To do that, Vitamin K2 supplementation in the in vitro embryo culture medium was applied which showed a positive effect on the mitochondrial function leading to satisfactory phenotypic and genotypic changes in the embryos. In conclusion, this study resulted in identification and characterization of the cattle breeds effects as a critical factor on cryopreservation performance and embryonic metabolism of the mitochondria. Our results emphasized that mitochondrial function is an important feature of embryonic development in cattle, which can provide opportunities to improve embryonic viability

Modern Approaches To Quality Control

Rapid advance have been made in the last decade in the quality control procedures and techniques, most of the existing books try to cover specific techniques with all of their details. The aim of this book is to demonstrate quality control processes in a variety of areas, ranging from pharmaceutical and medical fields to construction engineering and data quality. A wide range of techniques and procedures have been covered

Effective treatment of solid tumors via Cryosurgery

Ph.DDOCTOR OF PHILOSOPH