Chromosome segregation and recombination in human meiosis: Clinical applications and insight into disjunction errors

Chromosome copy number errors (or aneuploidy) of gametes and embryos occurs in humans more frequently than in any other studied species, with a spectrum of manifestations from implantation failure to affected live births. It is predominantly problem arising in maternal meiosis with at least 20% of oocytes being aneuploid, a proportion that increases dramatically with advancing maternal age. Currently the only intervention to reduce the chances of transmitting aneuploidy is by invasive embryo biopsy procedures in high-risk groups (mainly patients with advanced maternal age) undergoing in-vitro fertilisation. Despite the severity of this problem, aneuploidy of the human preimplantation embryo is relatively poorly understood. With this in mind the purpose of this thesis is to explore the premise underpinning the use of preimplantation genetic screening (PGS) in human embryos and investigate its clinical applications and current methodologies. A series of published works demonstrate what I believe to be a significant contribution to the development of applications for studying human preimplantation aneuploidy, also providing insight into its origins and mechanisms at the earliest stages of human development. Specifically, I present a novel standard set of protocols as a general reference work from practitioners in the fields of embryo biopsy and array comparative genomic hybridisation (CGH - the current ‘gold standard’ for preimplantation aneuploidy screening). I present a summary of work encapsulated in three published clinical papers using a linkage based analysis of Single Nucleotide Polymorphism (SNP) karyotypes (Karyomapping). Karyomapping was designed as a near-universal approach for the simultaneous detection of chromosomal and monogenic disorders in a PGS setting and these results demonstrate the utility of the technique in three separate scenarios. In order to study the underlying mechanisms of female meiosis I present my findings on the use of a calcium ionophore to activate human oocytes artificially. An algorithm based on Karyomapping (termed MeioMapping) is demonstrated for the first time specifically to investigate human female meiosis. By recovering all three products of human female meiosis (oocyte, and both polar biopsies – herein termed “Trios”) using calcium ionophore, I present a novel protocol (commissioned by Nature Protocols) to allow exploration of the full extent of meiotic chromosome recombination and segregation that occurs in the female germline. Finally I present a published set of experiments using this protocol to provide new insight into meiotic segregation patterns and recombination in human oocytes. This work uncovers a previously undescribed pattern of meiotic segregation (termed Reverse Segregation), providing an association between recombination rates and chromosome mis-segregation (aneuploidy). This work demonstrates that there is selection for higher recombination rates in the female germline and that there is a role for meiotic drive for recombinant chromatids at meiosis II in human female meiosis. The work presented in this thesis provides deeper understanding of meiotically derived maternal aneuploidy and recombination. More importantly it provides a vehicle within an ethical framework to continue to expand our knowledge and uncover new insights into the basis of meiotic errors that may aid future reproductive therapies

Unravelling chromosomal instability in mammalian preimplantation embryos using single-cell genomics

https://www.ester.ee/record=b5163270*es

Kromosomaalne ebastabiilsus imetajate varajastes embrüotes

Väitekirja elektrooniline versioon ei sisalda publikatsiooneViljatus on kogu maailmas kiiresti kasvavaks probleemiks ning iga kuues paar seisab silmitsi olukorraga, et soovitud rasedust ei teki. Esmasünnitajate keskmine vanus kasvab samuti, kuid naise vananedes tema viljakus langeb ning seetõttu pöördub tänapäeval aina rohkem paare viljatusravi poole, et leida oma probleemile lahendus. Kehaväline viljastamine (in vitro fertilization, IVF), mille korral toimub munarakkude viljastamine ning embrüote areng katseklaasis, on maailmas kõige sagedamini kasutatav lastetusravi meetod. Paraku, IVF embrüote sagedased kromosomaalsed aberratsioonid on kõige suurem väljakutse IVF ravil, sest see on üks peamisi raseduse varajase katkemise (kuni 75%) ja IVF ebaõnnestumise põhjuseid. Samas, embrüotega seotud teadustöö on piiratud eetiliste põhjuste tõttu, seega sõltuvad inimese embrüote uuringud suurel määral sobivatest loommudelitest. Kasutades uudset üksikraku kogu-genoomi analüüsi metoodikat, näidati antud doktoritöös, et ka veise IVF embrüotes esinevad väga sagedased kromosomaalsed aberratsioonid, mille tulemusena on suurem osa varajasi embrüoid mosaiiksed. Lisaks avastati, et embrüo on võimeline segregeerima ema- ja isapoolse genoomi eraldi rakuliinidesse, mis võib-olla aluseks eriploidsuse (mixoploidy) ja kimääride tekkele, kuid selle nähtuse esinemissagedus on hetkel teadmata. Kromosoomi aberratsioonid eksisteerivad samuti loomulikul teel saadud embrüotes, kuid munarakkude ja embrüote in vitro manipuleerimine suurendab genoomi ebastabiilsuse sagedust veise varajastes embrüotes, vähendades nende elujõulisust. Kui sama trend kehtib ka inimese embrüote puhul, peaks see julgustama teadus- ja meditsiinikogukonda mitte ainult parandama in vitro kultuuri tingimusi, vaid ka tõstma inimeste teadlikkust viljakuse ja IVF ravi osas. Selleks, et tuvastada embrüo aneuploidiat kasutatakse tänapäeval embrüote sõeluuringut, analüüsides kas kolmanda päeva või viienda päeva biopsiat. Viimasel ajal on aga hakatud pöörama suuremat tähelepanu ka blastotsööli vedeliku (blastotsüsti sees olev vedelik) analüüsile, kuid antud töö käigus selgus, et blastotsööli vedeliku analüüsi tulemused on vastuolulised ning seda meetodit ei saa hetkel kasutada embrüote genoomi analüüsis.Fertility issues have become a plague of modern society, as one in six couples will encounter problems to achieve clinical pregnancy. Nowadays, natural conception is often taken for granted, leading to an increased number of couples that turn to assisted reproduction to help conceive and ultimately give birth to a healthy baby. In vitro fertilization (IVF) and embryo transfer is the most commonly performed procedure in ART. However, the uprising era of single-cell research has highlighted the alarming fact that human IVF embryos have high prevalence of chromosomal instability (CIN), which represents one of the most serious challenges in IVF. At the same time, the use of in vitro fertilization (IVF) treatment provides a unique opportunity to study the fundamentals of preimplantation human development, from fertilization to blastocyst stage of development. At the same time, embryo-related research is ethically one of the most complex areas of reproductive science that greatly relies on the use of appropriate animal models. By using novel single-cell genomics approaches, the current thesis has demonstrated that the nature and frequency of genomic abnormalities in in vitro cleavage-stage embryos is highly conserved between cattle and human. Additionally, heterogoneic cell division was discovered, by which the zygote segregates entire parental genomes into separate blastomere lineages, giving rise to mixoploidy and chimaerism, although the frequency of this phenomenon and the fate of mixoploid embryos remain to be investigated. By using the established bovine model it was demonstrated that chromosomal abnormalities are also present in naturally conceived embryos, but in vitro procedures exacerbate CIN in early embryos, compromising their survival rate. If this is the same for human this should encourage scientific and medical communities not only to refine and improve in vitro culture conditions, but also raise awareness among men and women of reproductive age. To tackle the issues of embryonic aneuploidy, embryo chromosome screening is performed, by analysing either day 3 or day 5 biopsy. Recently, the use of blastocoel fluid (BF) biopsy was proposed, but the data presented here shows that BF-DNA is not suitable for diagnostic purposes, and day 5 biopsy remains the safest option with the most reliable results

Investigating the consequences of chromosome abnormalities arising during pre-implantation development of the mouse

The majority of human pre-implantation embryos created through in vitro fertilization (IVF) are mosaic as they are constituted of a mixture of diploid and aneuploid cells. Chromosome abnormalities are widely believed to contribute towards the relatively low success rates of IVF treatment. Consequently major efforts have been undertaken to develop effective tools to aid the selection of embryos with minimal abnormalities with the aim of improving clinical outcomes. However, the ultimate fate of mosaic embryos is not known. Human embryo research is limited by practical and ethical constraints, and directly relevant animal studies are sparse. To circumvent many of these limitations, a mouse model for pre-implantation chromosome mosaicism was developed. Acute chromosome segregation errors were induced in cleavage stage mouse blastomeres by bypassing the spindle assembly checkpoint (SAC). This model was used to investigate the fate of abnormal cells within the developing pre-implantation embryo, and the ultimate developmental outcome of mosaic embryos. Time-lapse imaging of pre-implantation development revealed that cells with chromosome abnormalities were progressively depleted during blastocyst maturation; inner cell mass (ICM) cells exhibited higher rates of apoptosis, while in the trophectoderm (TE) lineage effects on the cell-cycle predominated. Depletion continued throughout post-implantation development. Significantly, the presence of a critical number of control blastomeres within the embryo could rescue the early post-implantation lethality that occurred in embryos containing high rates of abnormalities. Thus it was demonstrated that mosaic embryos can achieve full developmental potential and that abnormal cells are progressively depleted as development proceeds. Finally, the mechanisms responsible for eliminating the abnormal cells from the embryo were investigated, revealing that embryos containing chromosome abnormalities may have increased metabolic requirements which could contribute to their clonal depletion; a feature previously characterised in aneuploid cells in the context of cancer research.This work was sponsored by a Wellcome Trust Clinical PhD Fellowshi

Mechanisms behind the fate of early chromosomal and transcriptional heterogeneities in the mouse embryo

A series of events during the first four days of mouse embryo development leads to the formation of a blastocyst. The blastocyst consists of neatly segregated three lineages: the epiblast (EPI), which will form the fetus, the extra-embryonic primitive endoderm and the outer layer of the extra-embryonic trophectoderm (TE). This organization prepares the embryo for implantation and subsequent development. This study aims to explore two broad questions: 1) what mechanisms dictate the fate of the progenies of the chromosomally abnormal cells generated during the 4-8 cell stage division; 2) how the transcriptional heterogeneities between the blastomeres of the 4-cell stage embryo affect subsequent lineage segregation. A high incidence of aneuploidy in the early cleavage divisions is considered the principal cause for low human fecundity and developmental defects. However, there is a dramatic decline in the prevalence of aneuploidy as gestation progresses. To understand the fate of aneuploid cells, a mouse model of chromosome mosaicism was used. In vitro culture system and live imaging demonstrated that aneuploid cells were eliminated from the EPI by apoptosis both during pre- and peri-implantation development. Also, aneuploid cells displayed chronic proteotoxic stress. Subsequently, p53-mediated autophagy eliminated aneuploid cells from the EPI. Unlike aneuploid embryos, 1:1 diploid-aneuploid mosaic embryos show developmental potential equivalent to diploids. Their peri-implantation development was followed, and it was found that while aneuploid cells in the EPI underwent apoptosis, the diploid cells over-proliferated to regulate the overall EPI size. These results elucidate the cellular and molecular mechanisms used by mouse embryo to refine the EPI cell population and ensure only the chromosomally fit cells proceed through the development of the fetus. The second part of the study investigates into the early molecular players that bias cell fate decisions. Sox21 was earlier identified as the most heterogeneous gene at the 4-cell stage that can influence cell fate decision. The deep sequencing of Sox21 knockout and wild-type embryos was carried out at the 4-cell stage and compared. Klf2 and Tdgf1 were found to be important downstream targets of Sox21 that influence lineage segregation. Depletion of both these genes predisposed cells to the TE lineage. Co-overexpression of both these genes rescued the effect of Sox21 knockdown on cell fate. These results demonstrate the mechanism by which Sox21 heterogeneity, from as early as the 4-cell stage, biases cell fate. Together, these findings indicate the fundamental mechanisms used by mouse embryo to ensure developmental plasticity

Relationship of morphokinetics and gene expression in the generation of aneuploidies in the human embryo

INTRODUCCIÓN Durante los últimos veinte años, el número de procedimientos de reproducción asistida ha aumentado drásticamente e, igualmente, se espera que continúe aumentando debido al retraso existente en la maternidad. En Europa se realizan más de medio millón de ciclos de fecundación in vitro (FIV) cada año, dando lugar a aproximadamente 100.000 recién nacidos, lo cual equivale al 1.5% de niños nacidos en Europa. Junto con la expansión en el campo de la reproducción asistida, el número de avances, tanto técnicos como científicos, también ha ido en aumento. Sin embargo, a pesar de los esfuerzos, las tasas de embarazo por ciclo han permanecido constantes durante los últimos diez años. Las características de la población que hace uso de estos tratamientos, cuya edad sobrepasa los 35 años en más del 70% de los casos, puede estar correlacionada con la existencia de este techo de cristal, ya que se ha descrito que la tasa de anomalías cromosómicas aumenta significativamente con la edad materna. Como consecuencia, los embriones fecundados in vitro a partir de ovocitos de mujeres de edad materna avanzada tienen una mayor probabilidad de ser cromosómicamente anormales, lo cual tiene como consecuencia un aumento en las tasas de aborto y una disminución en las de recién nacido vivo. Puesto que no existe la posibilidad de modificar las características de la población que requiere tratamientos de FIV, al menos desde un punto de vista clínico, ni tampoco la de modificar la calidad embrionaria a nivel genético, el objetivo actual debería estar dirigido a mejorar las técnicas empleadas durante los tratamientos de FIV para minimizar el posible efecto sobre la calidad embrionaria. Para el desarrollo de nuevas herramientas de cultivo y selección embrionaria es necesario un mejor conocimiento de la biología de los embriones humanos. Sin embargo, debido al uso limitado de estos para fines científicos, el número de estudios en embriones completos y de buena calidad es muy limitado. Y esto, junto a la naturaleza multifactorial de las anomalías cromosómicas, ha hecho aún más complejo encontrar los mecanismos relacionados con las causas y consecuencias de la existencia de aneuploidías durante el desarrollo embrionario. Los avances tecnológicos más recientes en análisis de imagen, análisis molecular y genético se presentan como una prometedora estrategia para desvelar la maquinaria implicada en la generación de aneuploidías. Así, nuestra hipótesis es que tanto la morfología, la cinética, como la transcriptómica del embrión están relacionados con la existencia de aneuploidías, ya sea como causa o como efecto, y un estudio simultáneo de todos estos parámetros en un mismo embrión podría permitir entender mejor la biología del desarrollo OBJETIVOS El objetivo general de este estudio fue investigar en detalle el origen y las consecuencias de la aparición de aneuploidías durante el desarrollo preimplantacional del embrión humano. Para ello, se llevaron a cabo los siguientes objetivos específicos: O1. Realización de un análisis descriptivo del embrión humano a cuatros niveles diferentes: morfología, cinética, transcriptómica y tasa de aneuploidías. O2. Estudio de las posibles relaciones entre morfología, patrones cinéticos, expresión génica y existencia de aneuploidías. O3. Desarrollo de un modelo de predicción de aneuploidías basado en las diferencias más significativas encontradas entre embriones aneuploides y euploides. O4. Integración de los datos sobre morfocinética, transcriptómica y aneuploidías para crear un atlas único sobre el desarrollo preimplantacional humano. MÉTODOS Se descongelaron ciento diecisiete (117) cigotos humanos donados a investigación provenientes de 19 parejas con una edad media materna de 33.7 ± 4.3 años. De ellos, sobrevivieron Ochenta y cinco cigotos, los cuales fueron cultivados usando la tecnología de time-lapse, que permite crear una película del desarrollo embrionario a partir de fotografías tomadas cada 5 minutos. Los embriones fueron cultivados durante diferentes tiempos incluyendo el estadio pronuclear y las siete primeras divisiones mitóticas. Tras el cultivo, cada embrión se desagregó en células individuales, incluyendo los corpúsculos polares en aquellos en estadio de cigoto. La mitad de las células de cada embrión se amplificaron usando la técnica de WGA (Whole Genome Amplification), que permite la amplificación de ADN a nivel de célula única, para ser analizadas posteriormente mediante arrays de CGH (Comparative genomic hybridization) y determinar así su dotación cromosómica. Por otro lado, la otra mitad de las células de cada embrión se analizaron mediante PCR cuantitativa mediante un sistema de microfluidos que permitía el análisis a nivel de célula única. Así, se estudió la expresión de 86 genes relacionados con la existencia de aneuploidías en la bibliografía previa para determinar el perfil transcriptómico de los embriones. Finalmente, se correlacionaron los parámetros morfocinéticos, obtenidos en las películas de time-lapse, el estadio cromosómico y los niveles de expresión génicos para cada embrión. CONCLUSIONES C1. Los parámetros morfológicos pueden tener un comportamiento dinámico durante el desarrollo embrionario. En concreto, los fragmentos celulares, los cuales aparecen mayoritariamente durante la primera división mitótica del embrión pueden dividirse, fusionarse a otros fragmentos o ser reabsorbidos por blastómeras. C2. Los parámetros cinéticos se ven alterados por la existencia de divisiones irregulares durante el desarrollo embrionario, por lo cual estos deben calcularse de manera independiente según el tipo de división para evitar conclusiones confusas. C3. Más de la mitad de los embriones humanos en este estudio, provenientes de pacientes de técnicas de reproducción asistida fueron aneuploides. Además, según los resultados, la incidencia de aneuploidías está correlacionada con la existencia de multinucleación, aunque no con la existencia de fragmentación o vacuolas en el embrión. C4. Durante el desarrollo embrionario, encontramos grupos de genes con diferentes patrones de expresión según su origen de transcripción: materno, embrionario o ambos. Además, se observó que la activación embrionaria ocurre desde el estadio más temprano del embrión humano, el cigoto. C5. La cinética de los embriones aneuploides está alterada en comparación con los euploides. En concreto, el tiempo entre la desaparición pronuclear y el comienzo de la primera citocinesis fue más largo en los embriones aneuploides. Además, el tiempo entre los estadios de tres y cuatro células fue también más largo en los embriones aneuploides, aunque esta diferencia se debió a la mayor proporción de embriones aneuploides con divisiones irregulares, las cuales alteran el patrón cinético embrionario. C6. El perfil transcriptómico de los embriones aneuploides mostró diferencias estadísticamente significativas durante las primeras 30 horas de desarrollo frente al de los embriones euploides. Gracias a estas diferencias, se comprobó que la existencia de aneuploidías en los embriones en estadio de células se puede predecir haciendo uso de una firma transcriptómica basada en 12 genes. C7. El atlas del embrión humano generado con los datos de este estudio muestra una alta variabilidad durante el desarrollo preimplantacional embrionario. Mientras que los embriones euploides parecen seguir una única ruta para desarrollarse satisfactoriamente sin mucha variabilidad en su morfocinética, los embriones aneuploides siguen múltiples rutas, solapándose y camuflándose en ocasiones con el comportamiento de los embriones euploides, dificultando así su diferenciación mediante técnicas no-invasivas.Over the last 20 years, the number of assisted reproduction procedures has drastically increased, and this trend is expected to continue as parenthood is postponed. In Europe, more than half a million in vitro fertilization (IVF) cycles are performed annually, resulting in 100,000 newborns. With the increased use of assisted reproduction, a concomitant increase in success rates would also be expected. However, the pregnancy rate per cycle has remained constant over the same 10-year range, despite important progress in the field. The majority of women using assisted reproduction are over 35 years old, and this needs to be taken into consideration since the rate of chromosomal abnormalities in the oocytes increases with age. As a consequence, advanced maternal age may result in an increase in the number of aneuploid embryos, which would be translated into high miscarriage rates and low live-birth rates. Since we are not able to modify the IVF population or to change embryo quality, our goal should be to focus on improving IVF techniques. Such improvement necessitates a better understanding of the etiology of embryo aneuploidies. Recent advances in imaging and molecular and genetic analyses are postulated as promising strategies to unveil the mechanisms involved in aneuploidy generation. Thus, our goal was to analyse, simultaneously in the same human embryo, morphology, kinetics, transcriptomics and genetics to find a correlation between these parameters in the origin of aneuploidies. Here we combine time-lapse, complete chromosomal assessment and single-cell real-time quantitative PCR to simultaneously obtain information from all cells that compose a human embryo until the approximately eight-cell stage. Our data indicate that the chromosomal status of aneuploid embryos correlates with significant differences in the kinetic pattern when compared with euploid embryos, especially in the duration of the first mitotic phase. We also demonstrate that embryo kinetics is affected by the existence of irregular divisions during development, and this should be taken into consideration for future studies. Gene expression analyses reveal that embryonic genome activation starts as early as the zygote stage. Moreover, gene expression profiling suggests that a subset of genes is differentially expressed in aneuploid embryos during the first 30 hours of development. Thus, we propose that the chromosomal fate of an embryo is likely determined as early as the pronuclear stage and may be predicted by a 12-gene transcriptomic signature. Finally, the atlas generated from the data obtained in this study shows the high variability underlying human embryo development. While euploid embryos seem to follow a uniform development without high variability for morphokinetics, aneuploid embryos may follow different pathways, overlapping with and mimicking euploid embryos in some cases, making them hard to differentiate

EmbryoGenetics

The science of human genetics has advanced at an exponential pace since the double-helix structure of DNA was identified in 1953. Within only 25 years of that discovery, the first gene was sequenced. Subsequent efforts in the span of a few decades have brought advanced next-generation sequencing and new tools for genome editing, allowing scientists to write and rewrite the code of life. We are now realizing that genetics represents yet another system of information technology that follows Moore’s law, stating that computer processing power roughly doubles every two years. Importantly, with such rapid and sophisticated advancements, any tools or studies applicable to adult genetics can now also be applied to embryos.Genetic disorders affect 1% of live births and are responsible for 20% of pediatric hospitalizations and 20% of infant mortality. Many disorders are caused by recessive or X-linked genetic mutations carried by 85% of humans. Because assisted reproduction has armed us with technologies like in vitro fertilization that provide access to human embryos, we began to screen some genetic diseases simply by selecting sex. The first live births following preimplantation genetic testing (PGT) to identify sex in X-linked disease were reported by Alan Handyside in 1990. This groundbreaking work used the identification of male embryos and selective transfer of unaffected normal or carrier females as proof-of-concept to avoid genetic diseases, paving the way to extend the concept to PGT for monogenic diseases (PGT-M), including Mendelian single-gene defects (autosomal dominant/recessive, X-linked dominant/recessive), severe childhood lethality or early-onset disease, cancer predisposition, and HLA typing for histocompatible cord-blood stem cells’ transplantation. Later, we moved onto the identification and selection of euploid embryos by analysing all 23 pairs of chromosomes in 4–8 cells from the trophectoderm, called PGT for aneuploidy (PGT-A). PGT-A currently leverages next-generation sequencing technologies to uncover meiotic- and mitotic-origin aneuploidies affecting whole chromosomes, as well as duplications/deletions of small chromosome regions. A step forward was the use of structural chromosome rearrangements (PGT-SR) to identify Robertsonian and reciprocal translocations, inversions, and balanced vs. unbalanced rearrangements. Another advancement came with PGT for polygenic risk scoring (PGT-P). This technique takes us from learning how to read simple words to starting to understand poetry (i.e., evolving from PGT-M/A/SR to PGT-P for multifactorial, polygenic risk prediction). Moreover, we are moving from embryo selection to intervention because the genetic code is not only readable, but also re-writeable. Indeed, gene editing is now possible using tools like CRISPR/Cas9, which are applicable to all species, including human embryos

Telomere Length and Distribution in Three Developmental Stages

Telomeres are specialised nucleoprotein structures present at the ends of each chromatid that function to maintain genome stability. It is well established that a gradual decline in telomere length is associated with the process of cellular ageing, and thereby to the pathobiology of age-related diseases. In addition, the localisation of the telomere at the nuclear periphery plays an important role in the spatio-temporal organisation of the genome and in ensuring faithful segregation of chromosomes during meiosis. The aims of this thesis were to investigate telomere localisation in the nucleus, and telomere length in three hitherto early stages of development, gametogenesis, preimplantation embryogenesis and the neonatal period. Specifically: 1. To test the hypothesis that telomeres localised at the nuclear periphery in sperm cells and that this organisation was altered in sub-fertile men 2. To optimise a means of assessing average telomere length using DNA from small sample sizes and using whole genome amplified DNA from single cells 3. To investigate the role of telomere length in reproductive ageing and aneuploidy generation in women by testing the hypothesis that telomere length is significantly shorter in the first polar bodies and cleavage stage embryos of older women 4. To test the hypothesis that “preterm at term” babies (i.e. premature babies assessed at the time of their due date) displayed genetic signs of premature ageing (as manifested by significantly shorter telomeres than their term born counterparts) alongside the already established clinical signs (characterised by hypertension, diabetes and altered body fat distribution) Results confirmed the peripheral distribution of telomeres in the sperm heads of normally fertile males (using both 2D and 3D imaging) plus the novel finding that telomere distribution patterns are altered in the sperm heads of infertile males. Secondly, a reliable means of measuring telomere length was optimised in order to assess average telomere length using DNA from small sample volumes (down to single cells). Using this technology, average telomere length analysis in polar bodies and embryos found no evidence to support the hypothesis that telomere length is associated with either advanced maternal age or aneuploidy generation. Similarly, results suggest that telomere length is not significantly shorter in “preterm at term” infants compared to term born controls, thus providing no evidence that telomere attrition is involved in the pathobiology of the ‘aged phenotype’ observed in preterm infants. Taken together, results from this thesis provide some novel insights into the function of these highly important features of the genome, but also highlight that a great deal remains to be uncovered in the complex molecular mechanisms that contribute to the regulation of telomere length and nuclear distribution