Medicine, Faculty of
Obstetrics and Gynaecology, Department of
DSpace
UBCV
Watt, Katherine
Master of Science - MSc
University of British Columbia
Assisted Reproductive Technology (ART) has been used for nearly four decades to help infertile couples achieve pregnancy. Such pregnancies are at a greater risk of restricted fetal growth and preterm birth, though whether these risks arise from environmental factors imposed on embryos cultured in vitro, or from genetic or epigenetic factors related to underlying infertility of the parents, remains unclear. Embryos generated using ART are frequently mosaic – having at least two genetically distinct cell populations within the same embryo. Mosaicism that persists in the cells that form the placenta can be observed as confined placental mosaicism (CPM), wherein genetic imbalances that may be fatal for a fetus are tolerated in the placenta, albeit with consequences such as restricted fetal growth, premature birth, and fetal demise. To investigate whether genetic imbalances and mosaicism occur more frequently for ART conceptions, term placentas from in-vitro fertilization (IVF) conceptions were analyzed at three distinct sites for large variations in chromosomal copy number. The incidence of CPM (4/82, 4.9%) was not significantly greater than what has previously been observed in term placentas from spontaneous conceptions (8/528, 1.5%, P=0.064, Fisher’s Test). The presence of constitutional genetic abnormalities and mosaicism were also investigated by karyotyping live-born infants from ART and natural conception pregnancies, and abnormalities were detected at similar rates between IVF (3/220, 1.36%), intracytoplasmic sperm injection (ICSI) (4/255, 1.57%), and naturally conceived cases (5/336, 1.49%). My results support observations that ART pregnancies are not at significantly greater risk of chromosomal abnormality or mosaicism than spontaneous pregnancies, and suggest that the poorer perinatal outcomes observed for ART conceptions may be attributable to other factors, such as small copy number variation or epigenetics.
INVESTIGATION OF ANEUPLOIDY AND MOSAICISM THROUGHOUT PREGNANCIES CONCEIVED THROUGH ASSISTED REPRODUCTIVE TECHNOLOGY (ART) by Katherine Watt BSc,. The University of British Columbia, 2009 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in The Faculty of Graduate and Postdoctoral Studies (Reproductive and Developmental Sciences) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) June 2019 © Katherine Watt, 2019 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, a thesis/dissertation entitled: “Investigation of Aneuploidy and Mosaicism Throughout Pregnancies Achieved Through Assisted Reproductive Technology (ART)” submitted by Katherine Leigh Watt in partial fulfillment of the requirements for the degree of Master of Science in Reproductive and Developmental Sciences Examining Committee: Alexander Beristain Supervisor Wendy Robinson Chair Carolyn Brown Supervisory Committee Member Evica Separovic Additional Examiner Additional Supervisory Committee Members: Patrice Eydoux Supervisory Committee Member Timothy Rowe Supervisory Committee Member iii Abstract Assisted Reproductive Technology (ART) has been used for nearly four decades to help infertile couples achieve pregnancy. Such pregnancies are at a greater risk of restricted fetal growth and preterm birth, though whether these risks arise from environmental factors imposed on embryos cultured in vitro, or from genetic or epigenetic factors related to underlying infertility of the parents, remains unclear. Embryos generated using ART are frequently mosaic – having at least two genetically distinct cell populations within the same embryo. Mosaicism that persists in the cells that form the placenta can be observed as confined placental mosaicism (CPM), wherein genetic imbalances that may be fatal for a fetus are tolerated in the placenta, albeit with consequences such as restricted fetal growth, premature birth, and fetal demise. To investigate whether genetic imbalances and mosaicism occur more frequently for ART conceptions, term placentas from in-vitro fertilization (IVF) conceptions were analyzed at three distinct sites for large variations in chromosomal copy number. The incidence of CPM (4/82, 4.9%) was not significantly greater than what has previously been observed in term placentas from spontaneous conceptions (8/528, 1.5%, P=0.064, Fisher’s Test). The presence of constitutional genetic abnormalities and mosaicism were also investigated by karyotyping live-born infants from ART and natural conception pregnancies, and abnormalities were detected at similar rates between IVF (3/220, 1.36%), intracytoplasmic sperm injection (ICSI) (4/255, 1.57%), and naturally conceived cases (5/336, 1.49%). My results support observations that ART pregnancies are not at significantly greater risk of chromosomal abnormality or mosaicism than spontaneous pregnancies, and suggest that the poorer perinatal outcomes observed for ART conceptions may be attributable to other factors, such as small copy number variation or epigenetics. iv Lay Summary Assisted Reproductive Technologies (ARTs) are medical techniques that help infertile couples achieve pregnancy. Despite their continued use over four decades, the long-term safety with regards to the genetic health of offspring produced using these methods is poorly understood. In the current study, the genetic constitution of pregnancies – including both the infant and the placenta – from ART conceptions was compared with conceptions achieved from natural intercourse. This study found that genetic abnormalities were no more likely to be detected in ART pregnancies than natural conceptions, in either the placenta or the resulting infant. v Preface In Chapter 3, I contributed to the results of 75 of the 97 cases for which placental biopsies were analyzed by CGH or MLPA, and flow cytometry. The remaining 22 cases were prepared and analyzed by Edgar Chan Wong and comprise a chapter of his thesis, and a previous publication (Chan Wong et al., 2012). The MLPA-generated fragments described in Chapter 3 were run at the BC Children’s Hospital Research Institute’s (BCCHR) Bioanalyzer Core Facility on the Applied Biosystems 3130 Genetic Analyzer under the supervision of Elizabeth Hui. In Chapter 4, I contributed to the results of 526 of the 853 cases used for analysis. Of these, 408 include cases where I performed methods necessary to prepare cells from cord or peripheral blood to be analyzed by karyotyping for chromosomal abnormalities. The remaining 118 were cases for which I performed methods necessary to prepare DNA for analysis by CGH to detect aneuploidy. Karyotyping and CGH for the remainder of cases was performed by previous members of Dr. Sai Ma’s lab including Ina Lazarov, Chelsea McCall, Lucy Fei, Edgar Chan Wong, Andrew Wilson, Lina Wang, Agata Minor and Sai Ma. Cytogenetic results from selected cases among the complete collection included in my dissertation have been previously published by Dr. Ma’s research group (Lam et al., 2001; Ma et al., 2001; Minor et al., 2007). The data collected by SNP array for Chapter 4 were generated at The Centre for Applied Genomics (TCAG) at The Hospital for Sick Children (Toronto, ON) under the supervision of Chao Lu. The work I performed to collect data for my thesis occurred in the lab of and under the supervision of Dr. Sai Ma. After March 2018, Drs. Alexander Beristain and Wendy Robinson advised me throughout the analysis of my collected data as well as the composition of my thesis. vi The entirety of the data collected for my thesis, including collection, processing and analyzing of samples, was performed under the approval of the Research Ethics Board of the University of British Columbia (H13-02161). vii Table of Contents Abstract .......................................................................................................................................... iii Lay Summary ................................................................................................................................. iv Preface ..............................................................................................................................................v Table of Contents .......................................................................................................................... vii List of Tables ...................................................................................................................................x List of Figures ................................................................................................................................ xi List of Abbreviations .................................................................................................................... xii Acknowledgements ........................................................................................................................xv Chapter 1: Introduction ............................................................................................................... 1 1.1 Thesis Context and Overview ..................................................................................... 1 1.2 Assisted reproductive technology (ART) ................................................................... 3 1.2.1 Infertility ............................................................................................................. 3 1.2.1.1 Female factor infertility .................................................................................. 4 1.2.1.2 Male factor infertility ...................................................................................... 6 1.2.1.3 Idiopathic infertility ........................................................................................ 8 1.2.2 ART conception methods ................................................................................... 9 1.2.2.1 Intrauterine Insemination (IUI) ....................................................................... 9 1.2.2.2 In vitro fertilization (IVF) ............................................................................. 10 1.2.2.3 Intracytoplasmic sperm injection (ICSI) ....................................................... 13 1.2.3 ART-associated risks ........................................................................................ 15 1.3 Aneuploidy and Chromosomal Rearrangements ...................................................... 24 1.3.1 Origins of aneuploidy and rearrangements ....................................................... 25 viii 1.3.2 Preimplantation testing ..................................................................................... 29 1.3.3 Prenatal testing .................................................................................................. 32 1.3.4 Cytogenetic and molecular testing .................................................................... 35 1.4 Confined Placental Mosaicism (CPM) ..................................................................... 37 1.4.1 Origins of CPM ................................................................................................. 38 1.4.2 Pathology of CPM ............................................................................................ 42 1.4.3 Diagnosis of CPM ............................................................................................. 44 1.5 Hypothesis and Objectives ........................................................................................ 47 Chapter 2: Materials and Methods ............................................................................................ 49 2.1 Patient Recruitment ................................................................................................... 49 2.2 Placenta Biopsy ......................................................................................................... 50 2.3 Blood Culture and Harvest ........................................................................................ 51 2.4 Karyotype, Counting and Sexing .............................................................................. 51 2.5 DNA Extraction ........................................................................................................ 52 2.6 Chromosomal CGH .................................................................................................. 53 2.7 MLPA ....................................................................................................................... 55 2.8 Flow Cytometry ........................................................................................................ 57 2.9 FISH .......................................................................................................................... 58 2.10 SNP array .................................................................................................................. 60 2.11 Validation of MLPA for aneuploidy detection ......................................................... 62 Chapter 3: Confined Placental Mosaicism in IVF and IUI term pregnancies .......................... 68 3.1 Introduction ............................................................................................................... 68 3.2 Case Ascertainment .................................................................................................. 68 ix 3.3 Case Sampling .......................................................................................................... 70 3.4 Statistical Analysis .................................................................................................... 70 3.5 Results ....................................................................................................................... 71 3.6 Discussion ................................................................................................................. 79 Chapter 4: Aneuploidy and Mosaicism in ART pregnancies ................................................... 85 4.1 Introduction ............................................................................................................... 85 4.2 Case description ........................................................................................................ 85 4.3 Results ....................................................................................................................... 87 4.4 Discussion ................................................................................................................. 93 Chapter 5: Conclusion ............................................................................................................. 101 References ....................................................................................................................................108 x List of Tables Table 1.1: Examples of chromosomal regions associated with imprinting disorders ........... 22 Table 2.1: Expected copy number for Dosage Quotient ranges generated by MLPA ......... 57 Table 2.2: Samples submitted for SNP array ........................................................................... 61 Table 2.3: Results from CGH and MLPA for methodology validation. ................................ 64 Table 3.1: Studies on the incidence of CPM in normal and IUGR natural conception term pregnancies .......................................................................................................................... 69 Table 3.2: Maternal Clinical Information for CPM study cohorts ........................................ 71 Table 3.3: Abnormalities detected in cases examined for CPM ............................................. 72 Table 3.4: FISH confirmation of monosomy X in cytotrophoblast cells of SM10-523 biopsies............................................................................................................................................... 74 Table 3.5: FISH confirmation of tetraploidy detected by flow cytometry in mesenchymal cells of two IVF pregnancies ....................................................................................................... 75 Table 3.6: Results for CPM detection in IVF and IUI cohorts ............................................... 78 Table 4.1: Maternal Clinical Information – Range and (Mean) ............................................ 86 Table 4.2: Breakdown of Maternal Ethnicity per Conception Cohort .................................. 87 Table 4.3: Summary of cases analyzed – by cohort and analysis method ............................. 88 Table 4.4: Summary of results from karyotyped cases ........................................................... 88 Table 4.5: Cytogenetic Abnormalities Detected in Natural Conception Pregnancies .......... 88 Table 4.6: Cytogenetic Abnormalities Detected in IVF Pregnancies ..................................... 91 Table 4.7: Cytogenetic Abnormalities Detected in ICSI Pregnancies .................................... 92 xi List of Figures Figure 1.1: Balanced translocations and meiotic segregation patterns. ................................ 28 Figure 1.3: Chorionic villi - the functional unit of the placenta. ............................................ 40 Figure 2.1: Illustration depicting typical site selection for placenta biopsy. ......................... 50 Figure 2.2: Non-congruent results for SM10-620 S3. .............................................................. 65 Figure 2.3 Analysis of mosaic aneuploidy for SM02-8 site 3. .................................................. 66 Figure 3.1: Confined Placental Mosaicism for male IVF baby SM09-478. ........................... 73 Figure 3.2: Monosomy X mosaicism for villi of SM10-523. .................................................... 73 Figure 3.3: FISH confirmation of monosomy X in cytotrophoblast cells of SM10-523 biopsies................................................................................................................................................ 74 Figure 3.4: CGH results for SM09-447 suggest partial trisomy 13. ....................................... 75 Figure 3.5: Flow cytometry results for two IVF cases suggesting tetraploidy. ..................... 75 Figure 3.6: FISH confirmation of tetraploidy detected by flow cytometry in mesenchymal cells of two IVF pregnancies. ............................................................................................. 76 Figure 3.7: Results suggesting partial mosaic loss for SM12-841 site 4. ................................ 76 Figure 3.8: Results suggesting partial mosaic gain for SM12-842 site 9. ............................... 77 Figure 4.1: Karyotypes showing mosaicism for marker chromosome. .................................. 89 Figure 4.2: Molecular testing to identify marker chromosome for SM13-1144 cord blood. 90 Figure 4.3: SNP array using peripheral blood DNA for SM14-1246 to identify marker chromosome. ........................................................................................................................ 91 Figure 4.4: Karyotype for male IVF case (SM04-74) with Y deletion. .................................. 93 xii List of Abbreviations aCGH array comparative genome hybridization ART assisted reproductive technology BWS Beckwith-Wiedemann syndrome cffDNA cell-free fetal DNA CBAVD congenital bilateral absence of vas deferens CEP centromeric enumeration probe CFTR cystic fibrosis transmembrane receptor (gene) CGH comparative genomic hybridization CNV copy number variation CPM confined placental mosaicism CVS chorionic villi sampling DAPI 4’,6-diamidino-2’-phenylindole DET double embryo transfer DMR differentially methylated region DNA deoxyribonucleic acid DOHaD developmental origins of health and disease FISH fluorescence in-situ hybridization FITC fluorescein-12-dUTP FSH follicle-stimulating hormone GnRH gonadotropin releasing hormone GWAS genome-wide association study HBSS Hank’s buffered saline solution xiii hCG human chorionic gonadotropin ICM inner cell mass ICR imprinting control region ICSI intracytoplasmic sperm injection IUGR intrauterine growth restriction IUI intrauterine insemination IVF in vitro fertilization LBW low birthweight LGA large for gestational age LH luteinizing hormone MA maternal age MI meiosis I MII meiosis II MLPA multiplex ligation-dependent probe amplification NGS next generation sequencing NIPT non-invasive prenatal testing OHSS ovarian hyperstimulation syndrome PGD preimplantation genetic diagnosis PGT-A preimplantation genetic testing for aneuploidy PID pelvic inflammatory disease PZN post-zygotic nondisjunction QF-PCR quantitative fluorescence polymerase chain reaction RNA ribonucleic acid xiv SET single embryo transfer SGA small for gestational age SNP single nucleotide polymorphism SRS Silver-Russell syndrome SSC saline sodium citrate TRITC tetramethylrhodamine-5-dUTP UPD uniparental disomy ZGA zygotic gene activation xv Acknowledgements Completion of my dissertation would not have been possible without the ongoing support and guidance of my supervisors, Drs. Alexander Beristain and Wendy Robinson, who agreed to take on the supervision of my project starting in Spring of 2018. Their comments, feedback and general support and encouragement have been instrumental to my progress and development as a graduate student. Additional gratitude is due to members of my supervisory committee, external examiners for my comprehensive exam, and the external examiner for my dissertation and defence, including Drs. Carolyn Brown, Patrice Eydoux, Tim Rowe, Peter Lansdorp, Sylvie Langlois and Evica Separovic. These individuals have each dedicated significant time and input to the completion of my studies and dissertation, as well as the coordination of committee meetings and examinations required for the completion of my program. Case recruitment, sample collection, specimen processing and data analysis were all possible thanks to both research and operational grants from the CIHR and BCCHRI (formerly CFRI). I owe a great deal of thanks to previous members of the Ma lab who have assisted in the recruitment, collection and processing of cord blood and placental samples for this study. As there is no accounting for weekends or holidays in the realm of perinatal sample collection, these students, laboratory assistants and volunteers have been instrumental in contributing to a seamless collection process. Very little of the research comprising this dissertation could have been accomplished without their dedicated help. Instrumental members both during and prior to my time in the lab have been Samuel Schafer, Luke Gooding, Kenny Louie, Richard Ng, Noha Bozreiba, Maryam Rajaee, Rowena Ho, Alyssa Thurston, Steven Garcia, Kevin Dong, Annie Ren, Elizabeth Wu, Chantelle Chand, Edgar Chan Wong, Andrew Wilson, Chiho Hatakeyama, Gordon Kirkpatrick and Agata Minor. xvi Finally, none of this would have been possible without the prior and ongoing support of my parents, Sharon and Casey Martelli; my family and friends, specifically brothers Kenny and Chris Martelli; and friends Drew MacDonald, Jolene Kendzel, Rachelle Anchikoski, Lesley Morgan and Chrissy Avila. All of you have provided me with both financial and emotional support consisting of everything ranging from encouraging text messages, to subsidized tuition and living expenses. I would not have made it through this last decade of education without all of your help, and I am grateful for your ongoing support of my personal and professional development. First and foremost, I would like to thank my spouse and partner, Stephen Johns Watt. You have provided me not only with financial support, but you have been my emotional rock since 2011. I would have never embarked on the tumultuous journey of my Master’s, then PhD, then Master’s (again), without your encouragement, patience and steadfast belief in my potential. I am both grateful and apologetic for all you have had to endure with me through these years, and hope that this will all pay off in our future together in Australia. 1 Chapter 1: Introduction 1.1 Thesis Context and Overview The age at which women have been opting to have their first child has been steadily rising over the past several decades, specifically in developed countries where access to contraception as well as high levels of female education and employment make delayed conception the preferred option (Mills et al., 2011). While postponing pregnancy can be beneficial with regards to increasing financial stability and providing a more favourable environment for childrearing, it comes with the added risks of decreased maternal fertility, and increased chances of miscarriage and pregnancy complications (Nagaoka et al., 2012; Schmidt et al., 2012). To overcome these challenges, the 8-12% of couples worldwide who struggle with infertility have become increasingly reliant on the use of Assisted Reproductive Technology (ART), now to the point where over 6 million pregnancies have been achieved using these techniques, a number which continues to grow each year (Weinerman, 2018). While little was known about the risks of these procedures when they were first introduced in 1978, decades of research in intervening years has indicated that pregnancies conceived using ART may be at a higher risk for chromosomal abnormalities, premature loss, as well as maternal and perinatal complications including preterm birth and restricted fetal growth (Coates et al., 2015; D’Angelo et al., 2011; Farr et al., 2007; Ombelet et al., 2016). The availability of preimplantation testing for ART has revealed that mosaicism – the presence of more than one chromosomally distinct population of cells – is quite common in preimplantation embryos, though whether this phenomenon is influenced by stressors unique to the ART environment, or associated with underlying causes of infertility, is difficult to investigate due to technical and ethical constraints of testing embryos conceived in vivo (van Echten-Arends et al., 2011). It is conceivable that if 2 ART processes contribute to embryonic mosaicism, pregnancies achieved by these may be at a higher than normal risk of confined placental mosaicism (CPM), a condition wherein an aneuploid cell population is restricted to the placenta, placing a variable risk on the fetus for restricted growth, lower placental weight, and stillbirth (Kalousek and Barrett, 1994; Wilkins-Haug et al., 1995; Yong et al., 2009). The availability of cord blood and placenta following childbirth provides a means for non-invasive investigation into whether pregnancies achieved using ART techniques such as in-vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI) and intrauterine insemination (IUI) are more likely than naturally conceived pregnancies to exhibit aneuploidy or mosaicism. While studies abound on mosaicism within the embryo and CPM in natural conception, few studies have fully explored the persistence of aneuploid mosaicism in term ART placentas (Chan Wong et al., 2012; Minor et al., 2006; van Echten-Arends et al., 2011). For my dissertation, I have examined cohorts of ART and naturally conceived pregnancies spanning nearly two decades in order to quantify whether there is an increased risk of aneuploidy and mosaicism in either the cord blood of infants or the chorionic villi of placentas from ART pregnancies. This study serves to expand upon the limited research on the risks of aneuploidy in ART conceptions, specifically with regards to mosaicism in the term placenta, with the goal of contributing to the continued evolution of reproductive technology and its ability to assist infertile couples achieve successful, low-risk pregnancies. 3 1.2 Assisted reproductive technology (ART) The risk of aneuploidy, CPM, and hosts of other pre- and perinatal complications are an ongoing concern for any family making the decision to have children, but for the growing number of couples worldwide who struggle with infertility, there are many additional impediments to take into consideration when it comes to family planning (Ombelet et al., 2008). Fortunately, in the last several decades since its conception, Assisted Reproductive Technology (ART) has grown to where it now assists over 1 million infertile couples to achieve pregnancy each year, and continues to evolve and improve to make the process as safe and efficient as possible (Mansour et al., 2013). ART treatment begins with an effort to diagnose the cause of infertility, in order to select the best options available to the couple for achieving a pregnancy. Testing is offered at many stages of this process to reduce the chances of an aneuploid or mosaic pregnancy, but due to underlying infertility, advanced maternal age, and factors associated with ART techniques themselves, the risk of a failed ART cycle or an abnormality in the pregnancy is never fully eliminated. All these considerations will be explored in the following sections, which will serve to provide an overview of ART techniques, beginning with the condition this technology serves to overcome: infertility. 1.2.1 Infertility Any couple who fails to achieve a pregnancy after 12 months of regular, unprotected intercourse is defined as infertile (Zegers-Hochschild et al., 2009). For these individuals, counselling, diagnostic workup and appropriate treatment are beneficial in achieving a pregnancy in up to 75% of cases, though the chances of success are highly dependent on the source of infertility and the options available to the individuals seeking treatment (Marshburn, 2015). Infertility targets neither sex preferentially, with female infertility accounting for 15-30% of diagnosed cases, and male infertility accounting for roughly 20-30% worldwide (Vander Borght 4 and Wyns, 2018). For the remainder of cases, either both sexes contribute in part, or the source of infertility is unknown. 1.2.1.1 Female factor infertility Female infertility can stem from a variety of factors including maternal age, hormonal imbalances, chronic diseases such as endometriosis, and structural impediments to fertilization or implantation. Advanced maternal age is the most recognized cause of infertility, and it has long been observed that oocyte reserve and quality decline with age. Aneuploid pregnancies are relatively rare for younger females, with around 2% of clinical pregnancies for women 25 years or younger being trisomic, compared to nearly 35% of pregnancies for women over the age of 40 (Hassold and Hunt, 2001). A more recent study found euploidy rates to be fairly consistent in embryos from women between the ages of 24 and 35, after which they drop drastically as maternal age increases (Demko et al., 2016). The majority of hypotheses attempting to explain these large discrepancies hinge on the protracted period at which the oocyte is suspended in meiosis I (MI) – from early fetal development to ovulation – which depending on the age at which the oocyte is ovulated can range from 10 to upwards of 50 years (Nagaoka et al., 2012). Overwhelming evidence suggests that not one but several factors may act in concert during a female’s reproductive lifetime to decrease the reproductive potential of oocytes over time. Oocytes are much more likely than sperm to contain bivalent chromosomes for which no crossovers have occurred, and these “crossover-less” chromosomes are overrepresented in aneuploid gametes (Ottolini et al., 2015). Meiotic checkpoints, such as those used to ensure chromosomal integrity, spindle assembly and proper synapsis between homologues, are also much more permissive in female meiosis, and as a result meiotic missteps that would be detrimental 5 during spermatogenesis are ignored for the meiotic oocyte (Capalbo et al., 2017). Finally, loss of cohesion between sister chromatids over a female’s reproductive lifetime appears to increase nondisjunction events, resulting in aneuploid oocytes (Duncan et al., 2012). All these factors exacerbate one another with the result being a profoundly negative effect on oocyte and embryo quality (Nagaoka et al., 2012) Hormonal conditions, such as Polycystic Ovary Syndrome (PCOS) are another source of female-factor infertility. PCOS is typically diagnosed in women who are overweight, obese and insulin resistant, and is characterized by hyperandogenism and ovarian dysfunction (Rotterdam ESHRE/ASRM-sponsored PCOS consensus workshop, 2004; Vannuccini et al., 2015). Women with PCOS who succeed in getting pregnant are at a higher risk for premature birth, perinatal complications and perinatal mortality (Vannuccini et al., 2015). In many cases of hormonal infertility, imbalances in hormones of the reproductive axis may be the result of a more serious underlying condition, such as thyroid or pituitary dysfunction, therefore standard blood work to investigate hormone levels should be the first line of investigation for otherwise healthy reproductive-age women (Luciano et al., 2013). Endometriosis is a chronic inflammatory disease of the pelvis afflicting up to 10% of women of reproductive age and 35-50% of women with infertility (Burney and Giudice, 2012). The mechanisms by which endometriosis leads to infertility range from physical distortion caused by lesions and fibrosis, to immunological and hormonal disruptions of the female reproductive system (Vander Borght and Wyns, 2018). Women with endometriosis experience higher rates of miscarriage, preterm birth and placental complications than unaffected women, and the effectiveness of hormonal and surgical treatment varies depending on the severity of the disease (Tanbo and Fedorcsak, 2017). 6 Disorders affecting the integrity of the fallopian tubes can lead to infertility by impeding transport of the sperm or fertilized zygote. Tubal factors include pelvic inflammatory disease (PID), scarring from pelvic surgery, effects of endometriosis and pelvic tumours. Of these, PID is the most common tubal factor, triggered by infections such as chlamydia and gonorrhoea which cause inflammation and scarring of the fallopian tubes, and can result in infertility or ectopic pregnancy (Dun and Nezhat, 2012). Both tubal surgery and ART techniques such as in-vitro fertilization (IVF) are offered as infertility treatments for tubal pregnancy (Daniilidis et al., 2017). 1.2.1.2 Male factor infertility Healthy males retain the ability to produce functional sperm throughout their adult life, and age-related fertility decline in males is much more gradual and results in markedly fewer genetic risks compared to advanced maternal age (Eisenberg and Meldrum, 2017). Diagnosis of male infertility can be elusive, with 40% of cases having no discernible cause (Krausz and Riera-Escamilla, 2018). Males presenting with infertility are typically diagnosed following an assessment of medical history, physical examination, blood analysis for hormonal, metabolic or genetic factors, and semen analysis (Marshburn, 2015). When the cause can be determined, male infertility can be attributed to quantitative or qualitative semen parameters, ductal structure or dysfunction, and hormonal imbalances. A semen analysis can quickly diagnose whether infertility stems from low or no sperm count, conditions known as oligozoospermia and azoospermia, respectively. Oligozoospermia is defined as having fewer than 39 million spermatozoa per ejaculate, while azoospermia is the absence of sperm in ejaculate (World Health Organization, 2010). Oligozoospermia and azoospermia can be broadly divided into categories of obstructive, and non-obstructive. In the case of obstructive, the patient tends to be within the normal hormonal limits, and further investigation 7 can reveal the source of obstruction. Non-obstructive oligozoospermia and azoospermia are generally diagnosed based on abnormal hormone results, and can be the result of genetic abnormalities or hormone imbalances (Pan et al., 2018). Cytogenetic analysis and Y-microdeletion testing can be used to diagnose the most common genetic disorders associated with low sperm count, such as Klinefelter syndrome (47,XXY) or its mosaic variants (47,XXY/46,XY), autosomal translocations that interfere with synapsis during meiosis, and deletions of the AZF regions on the Y chromosome (Krausz and Riera-Escamilla, 2018). Human sperm morphology is extremely heterogeneous compared to other species, and the current guidelines for “normal” sperm are quite strict, requiring that the acrosome, head, midpiece and tail all fall under expected ranges for a sperm to be considered normal (Menkveld, 2010). As such, the current threshold for normal sperm morphology requires at least 4% morphologically normal sperm in the ejaculate, and patients with fewer than this are diagnosed as teratozoospermic (World Health Organization, 2010). When gene mutations are identified as the cause, as for large-headed multiflagellar and globozoospermic syndromes, patients present with primary infertility, with nearly 100% of sperm exhibiting gross abnormalities that affect motility and prevent normal fertilization of the oocyte (Coutton et al., 2014). Gene mutations resulting in abnormal sperm flagella are a common cause of asthenozoospermia, wherein fertility is affected by poor sperm motility, and fertility for these patients can be variable depending on the mutations detected (Krausz and Riera-Escamilla, 2018). A simple blood test to detect levels of testosterone, FSH and LH can determine whether a hormonal imbalance is affecting spermatogenesis. Low testosterone in the presence of high FSH and LH suggests primary testicular failure, while high FSH and normal testosterone suggest impaired spermatogenesis. If all three hormones are low, this may suggest an abnormality of the 8 hypothalamus or pituitary, whilst high testosterone and low FSH and LH could indicate exogenous steroid use (Pan et al., 2018) When a semen analysis reveals little or no sperm in the ejaculate, and hormone levels and testicular volume appear normal, follow-up tests can serve to diagnose obstructive oligo- or azoospermia by determining the source of obstruction in the male reproductive tract. Possible sources include physical trauma from previous injury, surgery or genitourinary infections, or a genetic cause, such as congenital bilateral absence of vas deferens (CBAVD), a symptom of homo or heterozygous mutations in the cystic fibrosis transmembrane receptor (CFTR) gene (Wosnitzer and Goldstein, 2014). 1.2.1.3 Idiopathic infertility For 30 to 40% of couples struggling with infertility, no discernible diagnosis will be forthcoming following routine tests of ovulation, tubal patency and semen analysis (Ray et al., 2012). Suggested causes for these cases include impaired bidirectional movement of the tubal cilia, impaired acrosome reaction, exposure to endocrine disrupting chemicals as well as unknown genetic factors that could impair one or several key factors of male or female reproductive biology (Krausz and Riera-Escamilla, 2018; Ray et al., 2012; Vander Borght and Wyns, 2018). There is disagreement in the literature on how best to treat couples with idiopathic infertility, with some studies finding these patients have similar odds of conception using expectant management as with ART techniques, whilst others find significantly improved results when ART is employed (Farquhar et al., 2016; Steures et al., 2006). Patients’ decisions on which methods to use should take into account factors such as age of the female partner, total years of infertility as well the financial and emotional costs of treatment (Ray et al., 2012). 9 1.2.2 ART conception methods For couples who struggle with both explained and unexplained infertility, numerous ART treatment options have been available over the past several decades to increase the chances of conception and live birth. The following sections will describe the current most common techniques employed for ART, as well as the types of infertility each is best suited to treat. 1.2.2.1 Intrauterine Insemination (IUI) IUI is often the first attempted ART treatment for male subfertility and unexplained infertility as it is a simple, minimally invasive procedure, and is relatively low cost in comparison to other ART techniques. However, since the efficacy of IUI is dependent on tubal patency of the female partner and the presence of a minimal number of motile sperm from the male partner, not all infertile couples will benefit from this therapy (Kim et al., 2014). IUI is suggested as a first-line therapy for infertility related to ejaculatory failure, mild endometriosis, immunological incompatibility due to antibodies in the semen or cervical mucus, male subfertility including oligozoospermia, asthenozoospermia and teratozoospermia, and idiopathic infertility (Brinsden and Dickey, 2010). The procedure begins with collection of sperm from the subfertile male or sperm donor, which is washed to remove prostaglandins, infectious agents, antigenic proteins, leukocytes and non-motile spermatozoa, and with the intention of concentrating the sperm sample. The washed, motile spermatozoa are transferred into the uterus using a syringe-drawn insemination catheter which is guided four to six centimetres into the uterus (Dickey et al., 2010). IUI is often performed in conjunction with ovulation induction, which increases the number of oocytes available for fertilization and corrects for any ovulatory irregularities which may be impeding fertilization during a regular cycle (Dickey and Brinsden, 2010). Clomiphene citrate, 10 gonadotropin or letrozole are used to stimulate follicle maturation in the ovaries, with the goal of generating two or fewer stimulated follicles per cycle in order to prevent multiple pregnancy without compromising the odds of conception (Diamond et al., 2015; Van Rumste et al., 2008). It has been estimated that one cycle of IVF has similar odds of success compared to three cycles of IUI for couples with unexplained infertility (Woodward et al., 2016). For this reason, if a couple has failed to achieve a pregnancy after several rounds of IUI, it may be to their benefit to consider a more effective method of ART. 1.2.2.2 In vitro fertilization (IVF) Since the birth of the first IVF baby over four decades ago, in-vitro fertilization techniques have been used as a panacea for numerous causes of infertility, from their origins in the treatment of tubal infertility, to ovulatory dysfunction, endometrial factor, male factor as well as cases for which preimplantation testing is required to rule out inherited disease (L. N. Huang et al., 2015). In contrast to milder forms of ART such as IUI, IVF has proven to have similar success rates across all forms of infertility, with its main limitations associated with advanced maternal age, including diminished ovarian reserve and oocyte quality (Macklon et al., 2009; Wiser et al., 2012). IVF can be used to treat the same reproductive disorders as IUI, with an improvement in the number of cycles to success, as well as the added burden of a higher cost per treatment (Woodward et al., 2016). IVF is recommended for moderate to severe cases of endometriosis, tubal occlusion or dysfunction, severe male factor infertility, and unexplained infertility after first-line treatments such as IUI have proven unsuccessful (L. N. Huang et al., 2015). Whereas for IUI fertilization occurs in vivo, and only the sperm require in vitro manipulation, IVF requires the in vitro manipulation of both oocytes and sperm, which requires oocyte retrieval from the mother or oocyte donor as well as retrieval and preparation of sperm. 11 Both timing of ovulation and optimization of the total number of retrieved oocytes are achieved using superovulation protocols, during which FSH and gonadotropin releasing hormone (GnRH) agonists or antagonists are administered to coordinate maturation of several ovarian follicles simultaneously (Cheong et al., 2011). Meta-analyses have found that the use of the antagonist protocol results in a slight reduction in cumulative live birth rate over agonist protocols, but significantly reduces the risk of ovarian hyperstimulation syndrome (OHSS) (Lambalk et al., 2017; Xiao et al., 2014). Following follicular maturation, oocytes are extracted from the ovaries by aspiration, guided by trans-vaginal ultrasonography (Rienzi and Ubaldi, 2009). Typically 15 oocytes are collected to maximize the odds of a successful pregnancy and live birth, and mature oocytes in meiosis II (MII) are selected for fertilization based on morphological factors (Rienzi and Ubaldi, 2009; Sunkara et al., 2011). Three to four hours after retrieval, mature oocyte-corona-cumulus complexes are placed in drops of media and co-incubated with washed sperm (Karagouga and Morbeck, 2017). The following day, the oocyte is denuded of cumulus cells and fertilization can be confirmed based on the presence of both maternal and paternal pronuclei and two polar bodies (Berger and Hoff, 2017; Karagouga and Morbeck, 2017). Unfertilized oocytes can be fertilized using ICSI and abnormally fertilized zygotes are discarded, while fertilized zygotes are transferred to fresh culture medium and continue to grow (Berger and Hoff, 2017). Embryos are typically assessed by a three-point grading system developed by the Society of Assisted Reproductive Technology (SART) Embryo Morphology Subcommittee, which grades embryos as good, fair or poor at various stages of development (Wang and Chang, 2017) Recently, implementation of time-lapse imaging during embryo incubation has allowed for novel embryo scoring algorithms to be developed, as well as eliminating the need to periodically remove embryos 12 from an incubator for observation, introducing stress due to changes in temperature (Chen et al., 2017). Grading allows the best quality embryos to be prioritized for transfer, while poorer quality embryos can be cryopreserved by vitrification and used for subsequent transfers, if required. Preimplantation genetic testing for aneuploidy (PGT-A) is another optional means for embryo prioritization, ensuring only embryos with euploid results are selected for transfer. The ability for PGT-A to improve pregnancy outcome is controversial, for while it can be argued that transferring only euploid embryos can increase pregnancy rates per number of transfers performed and reduce the number of miscarriages per pregnancy, detection of a large number of aneuploid or mosaic embryos for patients with few viable embryos can further limit their options (Brezina and Kutteh, 2015). Implantation of embryos diagnosed as mosaic by PGT-A can result in healthy live births, though there are currently few fertility clinics willing to provide this option for cases where no euploid embryos are detected (Gleicher et al., 2015; Greco et al., 2015). PGT-A was originally performed on one or two blastomeres of the day 3 cleavage stage embryo, but current practice is to perform a laser biopsy of 5-10 cells from the trophectoderm of blastocyst-stage embryos at day 5, as this technique has shown to be less harmful to the embryo (Scott et al., 2013). PGT-A will be discussed in further detail in Section 1.3.2. Following culture up to day 3 or day 5, one or several embryos will be selected for transfer to the uterus with the goal of implantation. In Canada, it is recommended that fertility clinics opt for single embryo transfer (SET) or double embryo transfer (DET) whenever possible, as this reduces the chances of multiple pregnancy while maintaining acceptable pregnancy rates, though for female patients over 35 and patients with poor quality embryos, transfer of more than two embryos per cycle may be considered (Min et al., 2008). SET in combination with PGT-A has 13 been demonstrated to be effective in both minimizing the risk of multiples and optimizing the chances of a successful pregnancy (Forman et al., 2014). Although meta-analyses have found little to no difference between transfer of day 3 cleavage stage embryos and day 5 blastocysts with regards to cumulative live birth rates and ongoing pregnancy, most clinics now prefer to transfer embryos at the blastocyst stage, especially for patients with a good number of high quality embryos at day 3 (Chang and Wang, 2017; Glujovsky et al., 2016; Nardelli et al., 2014). Culture up to day 5 allows for selection against aneuploid embryos, likely through activation of zygotic gene activation (ZGA), which coincides with the initiation of cell arrest and may function as a mechanism to prevent proliferation of aneuploid cells and survival of aneuploid embryos (Mantikou et al., 2012; McCoy et al., 2015). In addition, culture up to day 5 permits trophectoderm biopsy and subsequent testing by PGT-A and prenatal genetic diagnosis (PGD) for patients who wish to screen embryos for aneuploidy or inherited disease, respectively, before selecting an embryo for implantation (De Vos, 2017). Day 3 or day 5 embryos can be transferred fresh, during the same cycle they were extracted, or if the patient opts for PGT-A or PGD before implantation, biopsied embryos can be vitrified until testing is complete, and healthy, euploid embryos can be thawed and transferred in subsequent cycles (Coll et al., 2018). Transfer of vitrified embryos has proven to be not only as effective as fresh transfer, but also demonstrably safer, due to the adverse effects of controlled ovarian hyperstimulation on pregnancy outcome in concurrent cycles (Aflatoonian et al., 2010; Roque et al., 2013). 1.2.2.3 Intracytoplasmic sperm injection (ICSI) The majority of the procedures used for IVF are identical for ICSI, with the pivotal difference being in techniques used to fertilize the oocyte. ICSI is recommended for patients with 14 fewer than 500,000 motile sperm or very low numbers of morphologically normal sperm in the ejaculate, as well as for cases where conventional IVF has failed (Palermo et al., 2017). Testicular spermatozoa can be used for ICSI when no vital spermatozoa are present in the ejaculate, and ICSI is also compatible with acrosomeless spermatozoa, though the latter requires that oocytes be activated by chemical or electrical stimulation (Neri et al., 2014). In contrast with IVF, oocytes retrieved after superovulation need to be denuded of cumulus cells prior to fertilization by ICSI so they can be assessed for integrity and maturity up to the MII stage, as indicated by extrusion of the first polar body. The denuded oocyte is immobilized using the holding pipette, while the injection pipette is used to immobilize and aspirate a spermatozoon tail-first, and inject it into the ooplasm, avoiding the mitotic spindle proximal to the polar body in the process (Palermo et al., 2017). The fertilization rate of ICSI compared to IVF is very high, with failure being attributed to non-viable injected sperm or asynchronous oocyte maturity, thus ICSI may also be used in cases where few oocytes are available for fertilization after retrieval (Palermo et al., 2012). After the ICSI procedure, fertilized zygotes are incubated for 16 to 20 hours before being examined for the presence of two pronuclei and a second extruded polar body, confirming successful fertilization (Berger and Hoff, 2017). The procedures which follow, including observation and grading of day 3 and day 5 embryos, optional PGT-A, PGD and cryopreservation, and implantation, are identical to those performed for IVF. Since ICSI was first implemented in the early 1990s, numerous studies have concluded that conception rates, cumulative live birth rates and perinatal outcomes are similar between IVF and ICSI conception techniques (Bonduelle et al., 2002; Buckett et al., 2008; Li et al., 2018). Comparing the safety and efficacy of these ART techniques to that of spontaneous 15 conception is slightly more nuanced, as it is frequently difficult to infer which risks arise from the techniques and which are associated with infertility itself. 1.2.3 ART-associated risks Little was known about the risks of IVF and ICSI procedures at their inceptions in 1978 and 1992 respectively, but now that the first generation of children conceived by ART have reached reproductive age, opportunities abound for the investigation of maternal, prenatal, perinatal and long-term risks attributable to the widespread use of these technologies (Esteves et al., 2018; Weinerman, 2018). An important caveat in examining the risks of ART technologies is controlling for underlying infertility in patients who use them to conceive. The financial and emotional burden implicit in ART would make studies with fertile controls expensive and unethical, but the availability of subfertile patients who become pregnant has shed light on which of these risks may be attributable to ART procedures themselves. Complications such as preterm birth and low birth weight (LBW) have been found to be just as likely in ART pregnancies as for pregnancies for women with indications of subfertility compared to matched fertile controls, whereas perinatal death was higher for subfertile women with both single and twin pregnancies, when compared respectively with singleton and twin pregnancies conceived by ART (Declercq et al., 2015). Another study found that relative to fertile individuals, women with ovulation disorders were at a higher risk for gestational diabetes, hospital admissions, preterm birth and LBW regardless of whether they conceived naturally or with ART (Stern et al., 2015). Other causes of subfertility including PID, PCOS and endometriosis are associated with higher risks of fetal growth restriction, preterm birth and adverse pregnancy outcomes, whether ART is used to conceive or not (Vannuccini et al., 2015). The ART-specific variables that have been most highly correlated with 16 excess perinatal morbidity are multiple birth, multiple conception and greater numbers of embryos transferred, suggesting the greatest risk introduced by ART procedures may be the increased likelihood of higher order pregnancies (Luke et al., 2016). Nevertheless, the potential for in-vitro gamete and embryo manipulation and ovarian hyperstimulation to adversely affect ART pregnancies has led to decades of research into exactly what risks are implicit to the use of these technologies. Findings from these studies have served to catalyze improvements in ART treatment regimens, leading to reductions in multiple births and reducing the time to conception in the decades since ART was first introduced. These risks, as well as evidence of association with ART procedures, are presented below. Aneuploidy The incidence of aneuploidy – the presence of fewer or greater than 46 chromosomes – in spontaneous abortions and the pregnancies of women of advanced maternal age has been well documented (Nussbaum et al., 2007), and data collected from PGT-A suggests aneuploidy is very common in cleavage stage embryos. However the lack of feasibility of studying embryos in-vivo makes it difficult to attribute a direct influence of ART to this phenomenon. The primary mechanism by which use of ART may lead to an increased risk of aneuploidy is through bypassing selection, which is more an effect of infertility than of ART procedures themselves. Blastocysts from women of advanced maternal age using IVF have high proportions of aneuploidy and aneuploid-mosaicism than those from women 37 years or younger (Liu et al., 2012). Increased risk of aneuploidy is also observed when ICSI is used to treat severe oligozoospermia versus normospermia, when maternal effect is controlled by use of donor eggs (Coates et al., 2015). 17 For pregnancies persisting to late first trimester, studies using chorionic villus sampling (CVS) found ART pregnancies are not at any higher risk than natural conceptions for aneuploidy or mosaicism, though studies examining multiple sites in term placentas from ICSI pregnancies report higher levels of mosaic polyploidy than similar studies using natural conceptions (Conway et al., 2011; Minor et al., 2006). In spontaneous abortions from all ART pregnancies, including IVF and ICSI, as well as natural conceptions, the incidence of aneuploidy has been found to be similarly high (Ma et al., 2006; Nayak et al., 2011; Qin et al., 2013). Nevertheless, ART pregnancies appear to be more prone to reproductive loss than naturally conceived pregnancies. Early pregnancy loss Spontaneous pregnancies amongst women aged 35 or younger have a 14% to 16% risk of spontaneous failure, as compared to the 21% to 29% risk attributed to pregnancies achieved by ART (Farr et al., 2007; Wang et al., 2004). Pregnancies in the first and second trimesters appear to be at the highest risk of failure, with factors such as advanced maternal age at oocyte retrieval, oocyte stimulation protocols and use of frozen oocytes for transfer influencing higher risks, and factors such as maternal age at conception (ie. when using donor oocytes) and aetiology of infertility not contributing significantly to any increased risk (Farr et al., 2007; Hu et al., 2018; Wang et al., 2004). Amongst women who conceive using ART, early pregnancy loss is influenced by smoking and quality of embryos transferred, but not by obesity or PCOS (Winter et al., 2002). IUGR, SGA, LBW and preterm birth Singletons conceived using ART techniques such as IVF and IUI are at a higher risk of small for gestational age (SGA) and LBW than naturally conceived children, and use of ovulation 18 stimulation protocols with milder ART techniques such as IUI introduces an increased risk of preterm birth and SGA (D’Angelo et al., 2011; Ombelet et al., 2016). Multiple factors associated with ART and infertility increase the risk of intrauterine growth restriction (IUGR), SGA and LBW. As discussed above, infertility itself has been associated with adverse pregnancy outcomes, as observed by comparing pregnancy outcomes from spontaneous pregnancy in subfertile women versus women who conceive using ART, and sibling pregnancies from women who had previously conceived spontaneously then subsequently using ART, wherein both types of pregnancies had similar risks of preterm birth, LBW and SGA (Pinborg et al., 2013). The stage at which the embryo is transferred can also affect perinatal outcome. Most reproductive centres prefer to transfer blastocyst stage embryos, and while transfer at this stage is associated with a reduced risk of SGA compared to transfer at cleavage stage, it is associated with a higher risk of preterm birth, very preterm birth and large for gestational age (LGA) (Alviggi et al., 2018). The use of fresh versus frozen embryo transfer also appears to increase the risk of preterm birth and SGA, suggesting that the endometrial environment may be adversely affected by the use of drugs required for ovarian hyperstimulation, and that cryopreservation of embryos and implantation during subsequent cycles may improve perinatal outcome (Alviggi et al., 2018; Zhao et al., 2016). Congenital anomalies In addition to higher risks of other perinatal defects, meta-analyses of cohort studies examining the outcomes of ART and spontaneously conceived singleton pregnancies have identified the former to have significantly greater risks of congenital defects. As congenital defects occur less frequently than poor perinatal outcomes related to birth weight and prematurity, meta-analyses are useful for resolving differences between these two populations with adequate power. 19 A meta-analysis of IVF and ICSI pregnancies found a 37% increased risk of for congenital abnormalities in singleton ART pregnancies relative to spontaneous pregnancies from women with no history of infertility (Qin et al., 2016). A subsequent meta-analysis by the same group examined the risk of specific congenital anomalies between these two groups and found again that ART singletons were at a significantly increased risk of general congenital defects, with increased risks in most subcategories including cleft lip and palate, malformations of the eyes, ears, mouth, neck and face, chromosomal defects and malformations of respiratory, urogenital, musculoskeletal, circulatory and digestive systems (Chen et al., 2018). A meta-analysis performed by another group found similar results, with an increased risk of 36% for congenital malformations in singletons conceived by IVF and ICSI (Hansen et al., 2013). Both groups found an increased risk of congenital malformations in singletons conceived using ICSI versus IVF, though neither included pregnancies achieved using IUI or ovulation-inducing drugs alone, and neither included spontaneous pregnancies in subfertile patients, making it difficult to conclude whether ART treatments or infertility itself contribute to increased risks of congenital defects (Chen et al., 2018; Hansen et al., 2013). Retrospective cohort studies have indeed found increased risks of congenital malformations in conceptions wherein the patient has a history of subfertility, although the risk is smaller compared to ART conceptions, and again, the use of ICSI is always associated with a significantly increased risk over IVF and spontaneous conception, when the methods are compared (Davies et al., 2012; Jaques et al., 2010; Zhu et al., 2006). These findings suggest there may be some aspect of ICSI, and perhaps IVF techniques themselves that contribute to an increased risk of congenital malformations, although in the case of ICSI it is difficult to rule out biological contributions of severe male infertility. 20 Imprinting defects Imprinting is an epigenetic modification of certain regions of DNA that results in monoallelic silencing of genes in that region, dependent on whether the chromosome involved was paternally or maternally inherited. These imprinted differentially methylated regions (DMRs) are established in the germ cells, as parental methylation is erased and replaced with patterns consistent with the sex of the germ line, assuring that sperm and oocyte will each contribute complementarily imprinted chromosomes to the zygote (Peters, 2014). Mammalian models suggest germ cell imprinting occurs at different points during male and female gonadogenesis: in males, this occurs before meiosis in the mitotic germ cells of the fetus, whereas in females, imprints are likely established in the post-natal ovaries, as germ cells are suspended in meiosis (Hanna and Kelsey, 2014). In contrast, somatic cells of the developing embryo retain these imprints throughout their lifetime, assuring that the genes linked to these DMRs are expressed monoallelically (Barlow and Bartolomei, 2014). Monoallelic expression of imprinted genes can be compromised by several mechanisms. One mechanism is uniparental disomy (UPD), wherein inheritance of both homologs from only one parent results in either double or zero expression of genes regulated by imprinted epigenetic silencing. UPD can occur as a result of trisomy rescue in the embryo, wherein fertilization of an aneuploid gamete results in a trisomic zygote, and euploidy is restored by a subsequent mitotic error involving loss of one of the supernumerary chromosomes (Yamazawa et al., 2010). If the lost chromosome represents the single homolog from one parent, the two remaining chromosomes will be from the other parent, and will have similar methylation patterns. Depending on which chromosome is affected by UPD, and whether affected cells are present in the fetus or restricted to the placenta as in CPM, the results can be variable. UPD in fetal tissues 21 can result in a number of genetic disorders if the chromosome involved contains imprinted genes. For example, Prader-Willi and Angelman syndromes can present as a result of maternal or paternal UPD 15, respectively, whilst Beckwith-Wiedemann syndrome (BWS) occurs as a result of paternal UPD 11 (Nussbaum et al., 2007). Chromosomes 6, 7, 14, 16 and 20 also contain imprinted genes associated with pathologies for UPD, summarized in Table 1.1 (Kotzot, 2008). Other mechanisms interfering with monoallelic expression of imprinted genes include deletion, duplication or mutations in imprinting control regions (ICRs), which modulate imprinting of nearby gene(s), resulting in aberrant imprinting patterns (Amor and Halliday, 2008). These mechanisms can contribute to the phenotypes and growth syndromes presented in Table 1.1. A growing body of work supports that abnormal imprinting, including patterns associated with pathogenicity, may be more frequent in ART conceptions compared to spontaneously conceptions (Choufani et al., 2019; Whitelaw et al., 2014). While more common imprinting disorders, such as Prader-Willi and Angelman syndromes, occur in ART children at similar frequencies to children conceived without reproductive assistance, other syndromes such as BWS and Silver-Russell syndrome (SRS) have been reported to be ten times more frequent in ART children than the general population (Hiura et al., 2012). It has been argued that since ART techniques are employed during critical periods of embryogenesis when the epigenetic marks at DMRs are potentially vulnerable to environmental stress, they may confer an increased risk of epigenetic dysregulation to children conceived in vitro, potentially resulting in imprinting disorders such as BWS and SRS (Hiura et al., 2014). The molecular etiology of disease in children affected with BWS has been shown to be different depending on conception method, with almost all BWS-affected ART children presenting largely as a result of hypomethylation at the 22 KCNQ1OT1 region compared with less than half of affected children conceived without ART (Tenorio et al., 2016). Table 1.1: Examples of chromosomal regions associated with imprinting disorders Chromosome/region Genes Etiology Pathology 6q24 ZAC, HYMAI patUPD6, paternal duplication, maternal hypomethylation Transient neonatal diabetes mellitus (Amor and Halliday, 2008) 7p12.2 7q32.2 GRB10 MEST matUPD7 Silver-Russell syndrome (Amor and Halliday, 2008) 11p15 H19, IGF-2, CDKN1C, KCNQ1 patUPD11, maternal hypomethylation, 11p imprinting defects Beckwith-Weidemann syndrome, Silver-Russell syndrome (Amor and Halliday, 2008; Ishida and Moore, 2013) 14q32 DLK1, MEG3 matUPD14 patUPD14 Maternal UPD 14 syndrome Paternal UPD 14 syndrome (Ishida and Moore, 2013) 15q11.2 -15q13 SNRPN, UBE3A matUPD15, paternal deletion, aberrant methylation patUPD15, maternal deletion, UBE3A mutation, aberrant methylation Prader-Willi syndrome Angelman syndrome (Ishida and Moore, 2013) Chromosome 16 matUPD16 IUGR, detected prenatally (Kotzot, 2008) 20q13 GNAS cluster patUPD20, maternal hypomethylation, maternal deletion Pseudo-hypothyroidism 1B (Ishida and Moore, 2013) Some studies suggest this increased incidence of abnormal imprinting in ART conceptions may originate in the germ cells of infertile parents (Kobayashi et al., 2009; Poplinski et al., 2010). Men with oligozoospermia, who are often referred for ICSI, are more likely to have pre-existing imprinting errors in their sperm, which may contribute to this increased incidence in their offspring (Marques et al., 2008; Tang et al., 2018). ART-associated factors such as ovulation induction and in-vitro oocyte maturation have also been implicated in contributing to aberrant methylation in some controlled studies (Khoureiry et al., 2008; Ludwig et al., 2005), although others have failed to attribute such consequences to these procedures (Rancourt et al., 2012). The increased incidence of imprinting disorders observed in ART children may originate from a combination of aberrant methylation in the germlines of infertile parents, as well as from any number of ART techniques 23 including superovulation, in-vitro maturation and fertilization, embryo culture conditions and other environmental stressors unique to the ART lab setting (Hiura et al., 2014). Long-term effects The increased risk of abnormal epigenetic marks reported for children born after ART may result in imprinting disorders in rare occasions, but a much more likely concern is the potential for predisposition to childhood or adult onset disease. The epigenetic effects of poor maternal nutrition during pregnancy on long-term metabolic disorders in LBW offspring were first proposed by Dr. David Barker, forming the main pillar of the Developmental Origins of Health and Disease (DOHaD) hypothesis (Eriksson, 2016). A growing body of DOHaD research suggests that ART techniques may interfere with epigenetic reprogramming at the critical time during embryo development within which they are employed, predisposing ART children not only to imprinting disorders but also to childhood cancers and adult-onset cardiometabolic syndromes such as diabetes and heart disease (Roy et al., 2017). When controlling for gestational age, birth weight and parity, ART children up to the first five years of life incur significant residual costs of childhood hospitalization over non-ART children, and these costs can be up to ten times higher for children born premature, LBW or with other adverse perinatal complications (Chambers et al., 2014). Some studies found children conceived using IVF and ovulation induction have a significantly increased risk of developing neoplasms within the first ten years of childhood than spontaneously conceived children, suggesting some aspect of drug or hormone-induced ovulation may predispose children conceived using stimulated oocytes to malignancies by some epigenetic mechanism (Wainstock et al., 2017). Nevertheless, childhood neoplasms are relatively rare, and studies finding milder non-significant effects of ART on cancers caution overinterpreting such results (Spaan et al., 2018). The higher 24 risk of ART children to be LBW or SGA could also predispose them to cardiometabolic syndromes in adult life, although since the oldest ART “babies” are currently in their late thirties and represent the earliest and least advanced ART treatments provided to date, it may be several decades before adequate long-term data is available on the risks of modern-day ART techniques for adult-onset disease (Feuer and Rinaudo, 2016). On the upside, recent literature suggests that the adverse perinatal outcomes, such as PTB, SGA and LBW, that have been historically observed for ART pregnancies are occurring less frequently and with milder severity than reported in the past, pointing to a healthier future for children conceived using these technologies (Henningsen and Pinborg, 2014). It is even more encouraging that ART children do not display any growth deficiencies by 3-year follow-up compared to naturally conceived children, suggesting that LBW and SGA ART children are able to catch up with their naturally conceived counterparts given enough time. The improved outcomes observed for ART children in recent studies have been attributed to the evolution of safer ART practices, including a shift from fresh to frozen embryo transfer, and reductions in the number of embryos transferred per cycle (Berntsen et al., 2019). 1.3 Aneuploidy and Chromosomal Rearrangements Aneuploidy occurs in nearly 5% of clinically recognized pregnancies and is the most common cause of spontaneous abortion (Nussbaum et al., 2007). Autosomal aneuploidy generally has more dire consequences than sex chromosome aneuploidy, and trisomies, specifically for chromosomes 21, 13 and 18, tend to be tolerated better than monosomy, as only monosomy X is compatible with life (Ioannou and Tempest, 2015). Aneuploidy can also be present in a mosaic state, in which case the severity of the phenotype will be variable, influenced by the proportion of aneuploid cells and which organs and tissues are affected (Daber et al., 2012; Neiswanger et al., 25 2006). Chromosomal rearrangements, such as inversions and translocations, often do not present with dire phenotypic effects since copy number is not affected, but carriers of rearrangements often present with reproductive difficulty, producing genetically unbalanced gametes when meiotic segregation yields combinations of normal and rearranged chromosomes. Aneuploidy and chromosomal rearrangements can be tested for at various stages of preconception and pregnancy. Genetic testing for aneuploidy and other chromosomal imbalances and rearrangements is usually offered to patients with previous failed or aneuploid pregnancy, advanced maternal age, known translocations or a family history of genetic abnormalities, and couples who are using ART. The following sections review the origins and mechanisms of aneuploidy and rearrangements, their impacts on fertility, as well as the stages at which diagnostic testing is offered and the various methods of testing employed. 1.3.1 Origins of aneuploidy and rearrangements When erroneous cell division occurs during mitosis of somatic cells, resulting aneuploid cells can be at a proliferative disadvantage compared to existing euploid cells (Victor et al., 2019), and are likely to be outcompeted by their more competent neighbours. When aneuploidy is the result of erroneous cell division during oogenesis or spermatogenesis, however, the resulting aneuploid gametes could contribute to aneuploidy in a fertilized zygote, and the consequences for these errors can be far more impactful. The three main mechanisms by which errors during cell division can lead to aneuploid daughter cells are nondisjunction, anaphase lag and endoreplication (Taylor et al., 2014). In nondisjunction, the failure of chromosomes to segregate correctly results in two aneuploid daughter cells. Nondisjunction can occur during either MI, resulting in missing or additional chromosomes in affected daughter cells, or MII, resulting in missing or additional sister chromatids 26 (Nagaoka et al., 2012). Anaphase lag occurs when a chromosome fails to attach to the spindle pole and is not incorporated into the nucleus of one of the daughter cells, resulting in a monosomy (Taylor et al., 2014). Lastly, endoreplication occurs when the cell cycle malfunctions such that chromosome replication proceeds without subsequent cytokinesis. This results in a cell with an additional copy of all chromosomes, leading to tetraploidy (Mantikou et al., 2012). The vast majority of aneuploidies detected in human conceptions originate from maternal MI, with the exception of Klinefelter syndrome (47,XXY) which is slightly more likely to originate from errors in paternal MI (Hassold and Hunt, 2001). Recognized as the “maternal age effect”, the increased incidence of aneuploidy in oocytes from older mothers is suspected to arise due to the extensive window of time between which female MI is initiated and suspended – commencing in the oocytes of the developing fetus, and concluding decades later when the oocyte is ovulated –during which the cellular machinery ensuring the integrity of synapsis between chromosomes may become compromised (Nagaoka et al., 2012). Crossover frequency has been demonstrated to influence synaptic integrity: global recombination levels are positively correlated with euploidy in both young women and women of advanced maternal age, and chromosomes with no crossovers are at an increased risk of premature sister chromatid separation (Hou et al., 2013; Ottolini et al., 2015). In addition, the unique asymmetric meiotic divisions of the oocyte require atypical spindle morphogenesis, facilitated by less stringent MI spindle assembly checkpoints which are consequently more permissive of meiotic segregation errors (Bennabi et al., 2016). Male spermatogenesis, on the other hand, is drastically less error-prone. The majority of paternal meiotic errors involve sex chromosomes, as the X and Y chromosomes share much less homology than two X chromosomes, and this lack of similarity makes proper sex chromosome segregation more challenging in male versus female MI (Touati and Wassmann, 2016). This is 27 particularly evident in the sperm of oligospermic males, which features an increased incidence of sex chromosome aneuploidy compared to that of males with normospermia (Sbracia et al., 2002). For the remainder of the autosomes, however, aneuploidy is markedly less frequent in spermatogenesis versus oogenesis, likely due to the observation that crossover maturation in oocytes is less efficient than in spermatocytes, permitting the formation of crossovers in regions that reduce synaptic stability and compromise proper segregation (Wang et al., 2017). Events leading to aneuploidy can also occur during normal mitotic cell division, a particular risk for rapidly dividing cells such as those of the post-zygotic embryo, wherein cell cycle checkpoints which usually ensure mitotic fidelity behave less stringently in favour of rapid cell division (Kiessling et al., 2010). Overall, mitotic errors do not account for aneuploidy in post-zygotic embryos as frequently as maternal meiotic errors, however their contribution is significant for certain aneuploidies, notably 45,X, which almost always leads to spontaneous abortion when it occurs in a non-mosaic state, as it would for a meiotic error (Hook and Warburton, 2014). Aneuploidy can also result from chromosomal rearrangements, which can take a variety of forms. The most common rearrangements are reciprocal translocations which occur in 0.092% of the population, and involve the mutual exchange of chromosome regions between two chromosomes (Figure 1.1 A) (De Braekeleer and Dao, 1991). Robertsonian translocations, occurring in 0.086% of the population, involve the fusion of two acrocentric chromosomes (De Braekeleer and Dao, 1991). Inversions in which the centromere is within the inverted region are known as pericentric inversions, and occur in 1-2% of individuals, while paracentric inversions, in which the inverted region does not include the centromere, occur in 0.002-0.049% (Anton et al., 2005). Complex chromosomal rearrangements are extremely rare rearrangements involving three or more chromosomes and three or more breakpoints (Morin et al., 2017). 28 Figure 1.1: Balanced translocations and meiotic segregation patterns. A) Ideogram of a balanced translocation between two non-homologous chromosomes B) Cruciform quadrivalent structure formed by synapsis of normal and translocation chromosomes at meiosis I C) Alternate segregation patterns result in balanced gametes D) Adjacent segregation patterns result in unbalanced gametes The incidence of chromosomal rearrangements in the infertile population is ten times that of the general population (Egozcue et al., 2000). Infertility in male carriers manifests as reduced sperm parameters, and in both sexes rearrangements result in an increased risk for the production of chromosomally abnormal gametes with additional or missing regions of the chromosomes involved in the rearrangement, resulting from meiotic mis-segregation (Nussbaum et al., 2007). During MI, the rearranged chromosomes and their normal homologous chromosomes must adopt unique structures in order for homologous recombination to occur: reciprocal translocations form a cruciform quadrivalent structure between the two rearranged chromosomes and their normal homologs (Figure 1.1 B), while Robertsonian translocations form a trivalent structure between the fusion product and the two normal homologous chromosomes (Nussbaum et al., 2007). When inverted chromosomes pair with their homologs, an inversion loop forms, wherein one chromosome loops back along the inverted region, allowing the entire length of the chromosome 29 to pair. Complex chromosomal rearrangements form structures similar to quadrivalents, though involving more chromosomes (Pellestor et al., 2011). While these structures allow the rearranged and normal chromosomes to pair and presumably escape meiotic arrest, they permit both balanced and unbalanced segregation products, dependent on whether alternate or adjacent segregation occurs (Figure 1.1 C & D). In inversion carriers, unbalanced products are generated only if an odd number of recombination events occur within the inversion loop, resulting in a chromosome with segmental aneuploidy (Nussbaum et al., 2007). Complex chromosomal rearrangements have a number of possible segregation patterns, in proportion to the complexity of the rearrangement, and the vast majority of these outcomes are unbalanced (Morin et al., 2017). 1.3.2 Preimplantation testing For patients attempting to conceive using ART, preimplantation testing such as PGD and PGT-A can be offered if there is a significant risk of pathogenic mutations or aneuploidy in the embryos produced (Dahdouh et al., 2015). While preimplantation testing is strictly diagnostic and offers no solutions for affected or aneuploid embryos, it can provide peace of mind to patients knowing they have selected the best candidate embryos for implantation. Preimplantation testing involves the biopsy of cells from the preimplantation embryo and subsequent testing of DNA from these cells to diagnose monogenic disorders or aneuploidy (Dahdouh et al., 2015). The initial biopsy can be performed at one of three stages: polar body biopsy requires the removal of one or both polar bodies either before or after fertilization, cleavage stage biopsy is performed at the 8-cell stage around day 3 post-fertilization and involves removal of one or two blastomeres, and blastocyst biopsy is performed around day 5 or 6 post-fertilization and involves laser-assisted removal of several cells from the trophectoderm of the blastocyst 30 (Palini et al., 2015). Polar body biopsy is the least popular method, as only the maternally-inherited complement is tested, and very few fertility clinics currently offer this option (Sermon et al., 2016). Cleavage and blastocyst stage biopsies are more frequently practiced, with blastocyst stage recently emerging as the preferred method at most clinics, due to several advantages it offers over cleavage stage biopsy, to be discussed below (Sermon et al., 2016). Cleavage stage biopsy involves “drilling” a hole in the zona pellucida and removing one or two blastomeres for subsequent aneuploidy testing (Wilton, 2002). Zona drilling was initially performed using acid Tyrode’s solution, but has since been replaced by infrared laser drilling, which is up to 3 times faster and reduces exposure of embryos to acid as well as to prolonged periods outside the incubator (Geber et al., 2011; Harper et al., 2012). Aneuploidy testing, while first achieved using fluorescence in-situ hybridization (FISH) for common aneuploidies, was quickly replaced by comprehensive chromosome screening techniques such as array comparative genome hybridization (aCGH), single nucleotide polymorphism (SNP) array, quantitative fluorescence polymerase chain reaction (QF-PCR) and next generation sequencing (NGS), which allow examination of all chromosomes for copy number variation (CNV), UPD and inheritance of pathogenic alleles (Palini et al., 2015). In the past decade, randomized and paired clinical trials have revealed that biopsy at the cleavage stage significantly delays subsequent development of biopsied embryos and reduces implantation potential compared to blastocyst-stage biopsy, fueling a shift toward performing biopsies at the blastocyst stage in most fertility clinics (Bar-El et al., 2016; Scott et al., 2013; Sermon et al., 2016). Blastocyst biopsy for preimplantation testing is performed on hatched blastocysts at day 5 or 6 post-fertilization, and uses infrared laser pulses at the cell junctions of trophectoderm cells to remove 5 to 10 trophoblasts for molecular testing (Oliana et al., 2015). The blastocyst is secured 31 with a holding pipette with the ICM distal from the site of the biopsy, and since the trophoblasts removed are destined to form the placenta while the fetus-forming cells of the ICM are left undisturbed, this technique is considered to be less invasive and harmful compared to removal of one or two totipotent blastomeres at the cleavage stage (Oliana et al., 2015; Scott et al., 2013). After the biopsy, the blastocyst is typically vitrified until the biopsied cells can be diagnosed for aneuploidy or the presence of pathogenic alleles, and a suitable candidate embryo or embryos are thawed and transferred in subsequent cycles (Cimadomo et al., 2016). This strategy has the benefit of not only reducing multiple pregnancies by allowing selection of a single embryo with good implantation potential, but it also offers the added benefit of avoiding the hormonal side-effects of superovulation in fresh-cycle transfer (Cimadomo et al., 2016). Another benefit of performing biopsy and preimplantation testing at the blastocyst stage is that by day 5 or 6, most genetically incompatible or aneuploid embryos are selected against and fail to become blastocysts, reducing the number of embryos that need to be biopsied or cryopreserved and enriching the surviving population for euploidy (Adler et al., 2014). Despite the purported benefits of testing embryos prior to implantation, there remains a risk for aneuploidy or mosaicism to be detected in most or all embryos, limiting the number of embryos available for transfer. This is especially significant for poor prognosis patients, as surviving embryos that could have been transferred in the absence of PGT-A must now be discarded, resulting in subsequent superovulation cycles if useable embryos are desired (Gleicher et al., 2015). Healthy pregnancies and live births have resulted from mosaic embryo transfer for women with no euploid embryos, however not all laboratories permit the transfer of these embryos (Greco et al., 2015). Whenever the option of mosaic embryo transfer is offered, clinicians suggest prioritization of embryos with aneuploidies incompatible with ongoing pregnancy, so as to avoid 32 aneuploid live births and promote sequestering the aneuploidy to the placenta, as CPM can usually be tolerated with minimal risk to the fetus (Esfandiari et al., 2016). 1.3.3 Prenatal testing An estimated 30% of pregnancies are lost within the first trimester, and of these losses 70% are biochemical pregnancies while the remaining 30% are clinically diagnosed (Alberman, 1992). Aneuploidy is the leading cause of early pregnancy loss, with an estimated 50% of products of conception being diagnosed with chromosomal abnormality by cytogenetic analysis (Hardy and Hardy, 2015). In Canada, prenatal screening tests are offered to all pregnant women with the goal of reducing the risk of aneuploid pregnancies while avoiding the use of invasive testing unless absolutely necessary (Nshimyumukiza et al., 2018). The risk of aneuploidy is markedly reduced as pregnancy progresses, however for certain high-risk groups of patients – such as women of advanced maternal age, patients with known chromosomal or monogenic disorders or a family history of such disorders, and patients with a history of recurrent spontaneous abortion – prenatal screening will be necessary to rule out aneuploidy or monogenic disorders in ongoing pregnancies, and invasive testing may be required to confirm any abnormalities detected by these techniques. Maternal serum screening can be performed in either the first or second trimester. The purpose of this screen is to compare detected levels of biomarkers in maternal blood with expected levels for normal pregnancies, and determine the risk for aneuploidy based on deviation between detected and expected (Harris et al., 2018). In combination with nuchal translucency (NT) measurement by ultrasound, these blood screening tests have an aneuploidy detection rate of up to 87% for common trisomies including 21 (Down syndrome), 13 (Patau syndrome), 18 (Edwards syndrome) and monosomy X (Turner syndrome) (Harris et al., 2018). If a non-invasive screen for 33 these markers suggests a higher than normal risk of any of these aneuploidies, follow-up invasive testing is usually recommended to confirm if the aneuploidy is real (Nussbaum et al., 2007). Cell-free fetal DNA (cffDNA) Screening, commonly referred to as NIPT, is a recently-validated method of non-invasive prenatal screening with significant improvements to aneuploidy detection rates compared with traditional maternal serum screening (Tamminga et al., 2016). cffDNA screening works based on the observation that maternal plasma contains fragments of DNA from lysed cells, largely of maternal haematopoetic lineage, but to a smaller degree from apoptosed cells of the placental trophoblasts (Harris et al., 2018). cffDNA screening technology analyzes the ratios of sequenced DNA fragments from each chromosome to detect aneuploidy across the entire genome using massively parallel sequencing (MPS), or can focus on more commonly detected aneuploidies, such as 13, 18, 21 and X, using targeted sequencing, wherein only DNA from these fragments is sequenced (Allyse et al., 2015). cffDNA screening is performed around 10 weeks into pregnancy, when the fetal fraction—the proportion of cffDNA versus maternal cell-free DNA—is optimally near 10% (Allyse et al., 2015). With a positive predictive value ranging from 92-99%, and a false positive rate as low as 1-3%, cffDNA screening is remarkably more accurate than maternal serum screening, reducing the need for further invasive testing in many cases. Nevertheless, cffDNA screening is not recommended for twin pregnancies, cases where vanishing twin syndrome is suspected, pregnancies conceived by donor oocyte or if the mother is a transplant recipient, due to the need to directly compare DNA from mother and fetus (Harris et al., 2018). In addition, cffDNA screening programs are estimated to detect only 85% of chromosomal abnormalities, mainly common trisomies, while the remainder of abnormalities, including low-level mosaicism, balanced 34 translocations and polyploidy, remain undetectable without invasive procedures and traditional cytogenetics (Norton et al., 2014). Chorionic villus sampling (CVS) is the earliest invasive prenatal test available for confirmation of abnormal results as detected by NIPT, and is offered 10-13 weeks into the pregnancy (Monni et al., 2010). While both fibroblast (mesenchymal) and cytotrophoblast cells can be obtained from CVS, it is the former that is more likely to represent the chromosomal constitution of the fetus, as both mesenchymal and fetal cells are derived from the ICM (Monni et al., 2010). If aneuploidy or mosaicism is restricted to trophoblast cells, the fetal cells are likely not affected, and these results would suggest CPM (Nussbaum et al., 2007). When a specific aneuploidy is suspected based on NIPT and the patient desires rapid results, uncultured cells from CVS can be analyzed by FISH using probes specific for the aneuploidy, generating results in a matter of hours, compared to the 7-10 days minimum required for cultured CVS to be analyzed by cytogenetics (Monni et al., 2010). Elevated risks of pregnancy loss as well as limb deformities have been previously cited as side-effects of CVS procedures, although follow-up investigation has largely attributed the latter to poor procedural technique and CVS being performed earlier than 9 weeks into pregnancy (Monni et al., 2010). Amniocentesis, like CVS, is an invasive procedure used to confirm NIPT results that suggest aneuploidy or mosaicism. Amniocentesis involves sampling of 20-30mL of amniotic fluid for the purpose of culturing epithelial fetal cells present in the fluid for cytogenetic or molecular analysis (Elias, 2010). The procedure is most effective 15 to 16 weeks into pregnancy, when the uterus can be accessed via the abdomen, there is sufficient amniotic fluid for sampling, and fetal cells are rapidly dividing (Elias, 2010; Randolph, 2005). In previous decades, amniocentesis procedures were reported to result in a 0.5% to 2.5% increased risk of fetal demise; a risk which 35 had to be weighed against the risk of aneuploidy for patients confronted with the choice of whether or not to pursue invasive testing (Randolph, 2005). A more recent meta-analysis examining the added risk of amniocentesis and CVS procedures for fetal loss found an additional weighted risk of 0.1% and 0.2% respectively over the background risk of spontaneous loss for pregnancies at this point in gestation, which is significantly reduced compared to that reported in earlier studies, as well as to what is frequently quoted to patients by healthcare providers (Akolekar et al., 2015). 1.3.4 Cytogenetic and molecular testing Numerical and structural abnormalities including whole chromosome aneuploidy, rearrangements and microdeletion/duplication syndromes can be tested by a vast array of technologies, from cultured cell preparations for cytogenetics, to commercially available testing kits and next-generation sequencing. Fetal or placental DNA, collected from maternal plasma or invasive techniques (CVS and amniocentesis), can be tested either directly or following culture or amplification. Cytogenetic analysis, such as counting, sexing and karyotyping, can be performed when live cells are collected by invasive testing techniques. Culturing and harvesting mitotically active cells allows their condensed chromosomes to be fixed to slides, banded, and analyzed using Giemsa banding (G-banding) which preferentially stains heterochromatic regions of chromosomes with a methanol-based dye preparation of Giemsa and Leishmann stains, creating “bands” which allow for detection of aneuploidy as well as invertions, deletions, duplications and translocations larger than 5 Mb (Benn et al., 2013). Other banding techniques, such as C-banding and NOR-staining, are used to preferentially stain centromeres and heterochromatic pericentromeric regions of chromosomes 1, 9 and 16, and the distal q arm of Y, as well as the satellite stalks of acrocentric chromosomes, respectively (Craig-holmes and Shaw, 1971; Goodpasture and Bloom, 1975). Prior 36 to the implementation of more precise molecular techniques such as sequencing and array CGH, C-banding and NOR-staining were useful for identifying whether marker chromosomes – supernumerary chromosomes of unknown origin – contained additional gene-rich material, contributing to partial trisomy and potential pathogenesis if detected in prenatal samples. Cytogenetic analysis is considered the gold standard for detection of aneuploidy and chromosomal rearrangements, as it allows for the identification of all chromosomal components, including gene-poor heterochromatic regions such as centromeres and satellites, which are largely composed of repetitive elements which molecular techniques, such as microarray and sequencing, are ill-equipped to identify and quantify (Miklos and John, 1979; Treangen and Salzberg, 2013). While molecular techniques are much more effective in picking up CNV than cytogenetic techniques, their limitations for detection of balanced rearrangements and low-level mosaicism have thus far safeguarded the role of cytogenetics in prenatal diagnosis (Sanlaville et al., 2005; Shah et al., 2017). Rapid cytogenetic techniques such as FISH, QF-PCR and MLPA are commonly employed when patients desire results more quickly than can be achieved using traditional cytogenetic techniques, which can have turnaround times as long as several weeks. These molecular karyotyping technologies can be used to fluorescently probe or amplify portions of potentially aneuploid chromosomes, yielding confirmation in as short as 24 hours (Benn et al. 2013). Unfortunately, since the results of rapid cytogenetic techniques are less informative than traditional cytogenetics, they should be interpreted with consideration of their limitations, and followed up with conventional cytogenetics for confirmation. Chromosomal microarray analysis, including aCGH and SNP arrays, involves the hybridization of labelled DNA to slides containing thousands of probes in order to detect CNV in 37 test DNA, which can be applicable for investigation of aneuploidy as well as duplications and deletions too small to detect using conventional cytogenetics (Van den Veyver, 2016). As DNA for arrays can be extracted from cells or tissue obtained by invasive techniques and tested directly, cell culture is not required, avoiding extended turnaround times, the risk of cell culture failure and the risks of maternal cell contamination. Some limitations to the applicability of microarray are its inability to detect polyploidy or balanced chromosomal rearrangements, as it measures relative copy numbers of total DNA. Since large amounts of purified fetal DNA are required for probe preparation, array techniques have largely been replaced by cffDNA sequencing for prenatal screening, however microarray analysis remains the most comprehensive method for detecting CNV in follow-up prenatal testing for abnormal screens, using DNA from CVS or amniocentesis. Sequencing technologies feature prominently in cffDNA testing, as both maternal and fetal DNA fractions present in maternal plasma must be quantified by massively parallel sequencing to extrapolate fetal DNA copy number (Allyse et al., 2015). cffDNA can also be used for targeted sequencing, not only reducing total costs in terms of reagents and machine processing time, but also reducing the amount of data, specifically with regards to unknown variants, which must be investigated for any associated pathologies (Tamminga et al., 2016). Targeted sequencing can focus on specific chromosomes, typically those involved in the most frequent aneuploidies such as 13, 18, 21, X and Y, or focus exclusively on exomes, as in targeted exome sequencing – a method effective for investigating potential genetic causes when adverse fetal morphology and growth are detected by ultrasonography (Best et al., 2018; Simón and Rubio, 2018). 1.4 Confined Placental Mosaicism (CPM) CPM is an additional risk that could be attributed to ART procedures, but its prevalence in term pregnancies has thus far primarily been investigated in naturally conceived and IUGR 38 pregnancies (Lestou and Kalousek, 1998; Minor et al., 2006; Robinson et al., 2010). Other than two small studies by former members of my lab, most investigations into CPM in ART pregnancies have relied on single site chorionic villus sampling from late first trimester pregnancies, which could possibly miss low-level mosaicism detected when several sites of a term placenta are examined for aneuploidy (Chan Wong et al., 2012; Henderson et al., 1996; Minor et al., 2006). The reported incidence of CPM detected in these studies ranges from ~1.3% which falls within the 1-2% risk commonly cited for all pregnancies, up to 6.25%, as reported in the earliest study investigating CPM in ART pregnancies (Huang et al., 2009; In’t Veld et al., 1995; Jacod et al., 2008; Margot et al., 2018). The significant incidence of mosaicism as detected by preimplantation testing of ART embryos would be nearly impossible to examine in embryos in vivo, but if it could be approximated by investigating CPM in term placentas from ART and non-ART pregnancies, we could elucidate whether conception method might influence embryonic mosaicism. The following section will cover the meiotic and mitotic origins of chromosomal errors resulting in embryonic mosaicism, and how they might lead to CPM. 1.4.1 Origins of CPM The study of human embryos as they develop in vitro has led to observations that meiotic and mitotic errors in the early dividing embryo are surprisingly common, even in young and healthy patients, with observed rates of mosaicism in cleavage-stage embryos up to 73% and between 23% and 33% in blastocysts (Fragouli et al., 2019; van Echten-Arends et al., 2011). The dividing embryo in the days following fertilization is especially prone to mitotic errors, as cell-cycle drivers are overexpressed and cell-cycle checkpoints under-expressed in an effort to promote rapid division of the embryo at the cost of genetic accuracy (Kiessling et al., 2010). 39 The early embryo may recover somewhat from a mitotic error leading to mosaic aneuploidy, with embryos biopsied at both cleavage and blastocyst stages exhibiting reduced incidence of mosaicism at the latter stage (J. Huang et al., 2015; Li et al., 2005). This may be due to the ability of normal, diploid cells to outcompete their less fit aneuploid counterparts, accounting for a greater proportion of cells by the time the embryo is ready to implant (Victor et al., 2019). Roughly 10% of the 107 cells comprising the 5-day blastocyst form the ICM, and even fewer of these cells go on to form the embryo; the remainder form the extraembryonic structures (Markert and Petters, 1978). Therefore, there is a greater likelihood that a mosaic aneuploidy persisting to the blastocyst stage will be present in placental precursor cells than in those destined to form the fetus. The precise location and timing of the error leading to aneuploidy will affect the type of CPM that develops, and will be further discussed below. A portion of the tissues forming the placenta – including the chorion – originate from the trophectoderm of the blastocyst, while the fetus and the remainder of the extraembryonic tissues — including the amnion, yolk sac, allantois and chorionic mesenchyme — are derived from the ICM (Boss et al., 2018). The chorionic villi – the functional units of placental nutrient exchange between mother and fetus – branch from the fetal-facing chorionic plate, and are bathed directly in maternal blood which flows into the intervillous space via maternal spiral arteries and veins in the basal plate (Figure 1.2). 40 Figure 1.2: Chorionic villi - the functional unit of the placenta. A) The chorionic villi extend from the chorionic plate into the intervillous space where it is bathed in the maternal blood which enters and exits the space via the maternal spiral arteries and veins, respectively. Cytotrophoblast cells from the villus invade the maternal tissues via anchoring villi, which connect the villi to the basal plate. B) A cross-sectional view of the villus: the outer layer is composed of the multinucleated syncytiotrophoblast, supported by the multicellular cytotrophoblast layer which surrounds the mesenchymal core. Gas exchange occurs between fetal and maternal circulation via fetal capillaries which run through the mesenchymal core of the villi. Hofbauer cells are fetal monocytes which reside in the mesenchymal core. The villi are formed from projections of the trophectoderm from the original implanting embryo, which differentiate into an external layer of syncytiotrophoblast — a multinucleated continuous cellular covering which is in direct contact with the basal plate via anchoring villi – and an inner bilayer of mononucleated cytotrophoblast cells (Boss et al., 2018). The cytotrophoblast cells both support and contribute material to the expanding syncytiotrophoblast as well as differentiate into extravillous trophoblasts, which invade the maternal decidua via the anchoring villi, remodelling the maternal spiral arteries and ensuring adequate placental perfusion throughout pregnancy. If mitotic errors occur in the trophectoderm, mosaicism will be restricted to cells in the chorionic villi derived from this lineage (cytotrophoblasts, syncytiotrophoblasts, and extravillous trophoblasts), resulting in Type I CPM. If the error occurs in an extraembryonic precursor in the 41 ICM, mosaicism will be mainly restricted to cells within the mesenchymal core (including fibroblasts, pericytes, vascular smooth muscle cells and endothelial cells) that comprise the structural core of the villi, resulting in Type II CPM. Type III CPM occurs when mosaicism is present in both cytotrophoblast and mesenchymal cells (Kalousek and Vekemans, 1996). The earlier the trisomy arises, the greater the number of cells which are aneuploid and the more widespread the mosaicism. Overall, the effect of mosaic aneuploidy on the developing fetus can be highly variable, depending on both the nature of the aneuploidy and the proportion of placental (or fetal) cells involved (Lestou and Kalousek, 1998). Trisomy rescue, briefly mentioned in Section 1.2.3, arises after an aneuploid gamete contributes an extra copy of a chromosome to the zygote, usually as a consequence of nondisjunction or anaphase lag during meiosis (Taylor et al., 2014). A subsequent mitotic error in the early embryo in which a trisomic chromosome is lost “rescues” any downstream cells from the trisomy. CPM that arises after trisomy rescue – typically Type III CPM– results in a far greater proportion of aneuploid cells in the placenta as only the lineage proceeding post-rescue will be euploid, and these pregnancies are at an increased risk of IUGR (Robinson et al., 1997). Euploid cells that persist in the ICM after trisomy rescue are able to selectively outcompete neighbouring trisomic cells to some extent, inferred from extreme X-chromosome skewing (Lau et al., 1997) and the primarily euploid constitution of tissues originating from cells of the embryonic mesoderm (Robinson et al., 2002), for conceptions involving meiotic-origin trisomy. A significant risk for CPM originating from trisomy rescue is UPD, for which consequences were discussed in Section 1.2.3, and include any of the disorders presented in Table 1.1 when UPD occurs in the cells that form the fetus. Generally, mosaic aneuploidy and UPD that are highly pathogenic in the fetus may be tolerated when confined to the placenta, although certain 42 aneuploidies have more severe consequences than others, even when restricted to placental tissues (Wolstenholme, 1996). As the main purpose of the placenta is to support the nutrition and development of the fetus, the majority of pathologies associated with CPM, to be discussed in the following section, involve impaired fetal growth. 1.4.2 Pathology of CPM CPM is detected in approximately 2% of cases where chorionic villi sampling (CVS) is performed at the 9th to 11th week of gestation (Kalousek and Vekemans, 1996). CVS is recommended to patients when an early non-invasive prenatal test (NIPT) indicates a risk for an abnormal result which would need to be confirmed by invasive testing, such as CVS or amniocentesis, in order to establish the likelihood of the fetus being affected (Allyse et al., 2015). The risk of mosaic or non-mosaic aneuploidy in the fetus is difficult to completely rule out if aneuploidy is detected by CVS, but a follow-up with amniocentesis can help to clarify the actual risk of a trisomic or mosaic fetus (Nussbaum et al., 2007). Even in the absence of aneuploidy in the fetus proper, CPM is associated with higher risks of IUGR, SGA, lower placental weight, and stillbirth, though CPM itself does not seem to have any long-term effects on the health of live born children from these pregnancies (Amor et al., 2006; Kalousek and Barrett, 1994; Lestou and Kalousek, 1998; Wilkins-Haug et al., 1995; Yong et al., 2009). To further complicate matters, not every type of CPM tends to carry the same risks for the pregnancy, and many studies have conflicting results as to the precise risks for each type of CPM. For type I CPM, which refers to mosaicism in the trophoblast cells, studies have reported risks of LBW, SGA, spontaneous abortion, IUGR and perinatal morbidity (Baffero et al., 2012; Johnson et al., 1990). For type II CPM, where mosaicism is detected in the mesenchyme of the villi (usually by analysis of the fibroblasts), increased incidence of pregnancy-induced maternal 43 hypertension has been observed, but this type of CPM generally results in fewer pathologies than the other two types of CPM (Baffero et al., 2012; Johnson et al., 1990; Toutain et al., 2018). Type III CPM, where mosaicism is detected in both the trophoblasts and the mesenchyme of the villi, has been associated with the greatest number of complications of the three types of CPM, with reports of preterm birth, SGA, adverse pregnancy outcomes, IUGR and early pregnancy loss (Johnson et al., 1990; Kalousek et al., 1991; Toutain et al., 2018). The higher risk for Type III CPM may be attributable to these cases being more likely to arise from trisomy rescue, wherein they would have both a higher proportion of aneuploid cells than Types I and II as well as a 1 in 3 risk of UPD (Robinson et al., 1997; Wolstenholme, 1996). In addition to the risk of UPD, the identity of the aneuploid chromosome in CPM can have variable phenotypic effects on the pregnancy. Trisomy 8 CPM tends to be tolerated when it arises from a mitotic error, but meiotic-origin trisomy 8 is ascertained almost exclusively from spontaneous abortion, suggesting this trisomy may not be tolerated before trophoblast differentiation (Robinson et al., 1999). Trisomy 16 is almost always confined to the placenta, but is associated with the most severe risks to the pregnancy, including premature birth, SGA and increased risk of congenital abnormalities (Langlois et al., 2006). Trisomy 22 in CPM is frequently associated with recurrent pregnancy loss, trisomy 2 CPM with fetal growth restriction and trisomy 13 CPM with poor fetal outcome (Farra et al., 2000; Wolstenholme, 1996). Trisomy 7 is the most commonly observed aneuploidy in CPM but confers a very small risk of restricted growth during pregnancy, while CPM for trisomy 3 is not commonly associated with any adverse outcomes (Farra et al., 2000; Kalousek et al., 1996; Wolstenholme, 1996). Nearly one hundred additional maternally-imprinted DMRs unique to the human placenta and extraembryonic tissues have been identified, and methylation at these regions has been 44 observed to be highly polymorphic (Hanna et al., 2016; Sanchez-Delgado et al., 2016). The specific function of this phenomenon in human development remains unclear, however it is possible that CPM for genomic regions encompassing these imprints could influence placental function, accounting for some of the risks observed with CPM. 1.4.3 Diagnosis of CPM With the growth in popularity and validation of cffDNA screening as an accurate preliminary form of NIPT over the past decade, the toolkit available for clinicians to detect and confirm CPM has become much more streamlined, reducing the need for invasive testing such as CVS to only extreme cases where there is a high risk of an abnormality (Langlois et al., 2013). For cases where invasive testing fails to confirm abnormal NIPT results, subsequent testing of the term placenta might uncover CPM after parturition. Chorionic Villus Sampling (CVS) CVS is an invasive procedure wherein a biopsy of tertiary chorionic villi, including both cytotrophoblast and syncytiotrophoblast layers as well the mesenchymal core, is extracted either transcervically or transabdominally from the placenta (Nussbaum et al., 2007). CVS is used to confirm abnormal results from NIPT in the first trimester of pregnancy, as previously discussed in Section 1.3.3, often allowing a decision to be made pending confirmation of aneuploid results to terminate the pregnancy before it has progressed much further (Monni et al., 2016). Cells from CVS are cultured both short-term and long-term, promoting growth of cytotrophoblast and mesenchymal cells respectively (Monni et al., 2010). Long-term mesenchymal cell culture is more likely to represent the fetal constitution, as both tissues are derived from cells of the ICM, whereas the short-term cytotrophoblast cells largely represent the trophoblast lineage, however, CPM can only be ruled out with follow-up amniocentesis or 45 cordocentesis (Nussbaum et al., 2007). The ability to “separate” the extraembryonic and trophoblastic cell lineages by culture length enables detection of mosaicism in either or both placental tissues, in order to diagnose either Type I, II or III CPM, which as previously discussed can have an impact on pregnancy outcome (Monni et al., 2010). Successful short-term and long-term cultures of CVS cells are analyzed using cytogenetic techniques to detect presence of aneuploidy and/or mosaicism in both placental lineages (Nussbaum et al., 2007). Analysis of the Term Placenta Dissection of the term placenta and extraction of cord blood post-pregnancy are effective and non-invasive methods of obtaining cells to confirm the extent of CPM after pregnancy, as the placenta is no longer required at this point. Cytotrophoblasts, fibroblasts from the mesenchyme of the villi, as well as lymphocytes from cord blood can be cultured to produce dividing cells for cytogenetic analysis, or DNA from any of these cell types and tissues can be extracted and examined using molecular techniques such as CGH, MLPA and flow cytometry (Barrett et al., 2001; Kooper et al., 2009). Culturing and harvesting cells from the placenta or cord blood for cytogenetic analysis is the gold standard for detecting abnormalities such and aneuploidy, polyploidy and mosaicism in placental tissue. However, downsides to cytogenetic analysis include the risk of culture failure due to reduced viability of tissues from term placentas, and the proliferation of contaminating maternal cells, resulting in 46,XX metaphases that do not represent fetal cells. In these cases, intact DNA can be extracted from placental cells for molecular analysis. CGH is a molecular technique which detects whole or partial chromosome aneuploidy in DNA extracted from blood or tissue, by hybridizing differentially labelled reference and patient (test) DNA to intact, male metaphase chromosomes (Barrett et al., 2001). The fluorescence ratios 46 between the competitively bound reference and test DNA allow for detection of losses or gains in the patient. The disadvantage of CGH over traditional cytogenetics is the inability to detect balanced translocations or polyploidy, as neither of these abnormalities will introduce changes in the total ratio of reference to patient DNA. MLPA is another molecular technique used to detect whole chromosome gains or losses for the purpose of testing placental tissue for CPM (Kooper et al., 2009). This technique uses pairs of probes which bind specifically to the telomeres of each chromosome arm, and are ligated together once hybridized to their target (Massalska et al., 2013). Each ligated pair of probes is a different length due to a “stuffer sequence”, but all use the same universal primers to amplify any ligated probes that have bound to their respective targets. Gains and losses of whole chromosome arms are quantified by running the amplified probes on a capillary electrophoresis bioanalyzer and comparing the height of each peak. Like CGH, this technique is unable to detect balanced translocations or polyploidy, but since the probes are present at the telomeres, MLPA is unable to detect interstitial gains or losses, nor is it able to determine how much of a chromosome arm is missing for any terminal deletion (Saxena et al., 2016). For this reason, any gain or loss detected by MLPA should be followed up using a more comprehensive molecular technique such as microsatellite PCR, microarray or targeted sequencing. As the majority of molecular technologies for DNA analysis are unable to detect polyploidy, CGH and MLPA should be complemented with flow cytometry for fully comprehensive analysis of CPM (Barrett et al., 2001). Polyploidy is usually not compatible with life when present in fetal tissues, but polyploid cells have been observed in placental tissues in certain cases of CPM (Minor et al., 2006). DNA in intact placental cells can be stained with propidium iodide and analyzed by flow cytometry to detect single cells with twice the normal 47 amount of DNA, suggesting polyploidy (Rua et al., 1995). When polyploidy is suspected after flow cytometry analysis, leftover cells can be analyzed by FISH to confirm tetraploidy as well as quantify the degree of mosaicism in the sample. 1.5 Hypothesis and Objectives I hypothesize that there will be an increased incidence of CPM and fetal aneuploidy – mosaic or otherwise – in ART conceptions compared to natural conceptions, as a reflection of the increase in aneuploid mosaicism observed in ART embryos. To address this hypothesis for my dissertation, I had two objectives: 1) I sought to investigate CPM in a minimum of 105 term IVF and 105 term IUI placentas using CGH, MLPA and flow cytometry to detect numerical abnormalities at a minimum of three isolated placental sites per case. I was able to achieve this for 82 IVF and 15 IUI placentas. My selection of analytical techniques was influenced by both a need to prioritize detection of structural and numerical abnormalities, as well as maximize the number of analyzable samples by avoiding potential cell culture failure. 2) I sought to investigate aneuploidy in infants from ART pregnancies using a combination of cytogenetic and molecular techniques. The availability of cord blood from term pregnancies permitted full karyotyping for 424 ART cases (188 IVF and 220 ICSI) and 288 natural conception pregnancies. Extracted DNA was used for molecular analysis in cases (n=118) that could not be karyotyped due to culture failure, or if follow-up by FISH, MLPA or array techniques was required to confirm ambiguous results. 48 The frequent detection of aneuploidy and mosaicism in preimplantation embryos used for ART procedures suggests these pregnancies could be at a greater risk of CPM, however the limited number of existing studies that have investigated this phenomenon have found conflicting results as to whether or not this number differs significantly from what is seen in natural conceptions. My intention was to supplement these existing studies by using several sites from term placentas for both aneuploidy and polyploidy, in an effort to detect mosaicism which may have been missed in similar studies using only a single site from first trimester CVS for CPM detection. By examining the constitutional karyotypes of children born of these pregnancies by cytogenetic analysis of cord blood, I was also able to rule out aneuploidy or mosaicism in the fetus proper for cases of CPM, as well as investigate whether infants born from ART pregnancies are at a greater risk than naturally conceived children for constitutional abnormalities. 49 Chapter 2: Materials and Methods 2.1 Patient Recruitment Patients who had conceived via IVF, ICSI or IUI were recruited from several ART centres across Canada, including the UBC Centre for Reproductive Health (Vancouver, BC), Olive Fertility Centre (Vancouver, BC), Pacific Centre for Reproductive Medicine (Burnaby, BC), Regional Fertility Program (Calgary, AB), Genesis Fertility Centre (Vancouver, BC) and Victoria Fertility Centre (Victoria, BC). In addition, patients who had conceived via natural conception or ART were recruited from hospitals in the Greater Vancouver Regional District including BC Children and Women’s Hospital (Vancouver, BC), Lions Gate Hospital (North Vancouver, BC), Surrey Memorial Hospital (Surrey, BC) and Royal Columbian Hospital (New Westminster, BC). At delivery, the attending nurse collected data including time, date and location of birth; details of delivery including sex of the infant, delivery method, gestational age, infant’s weight and measurements, and Apgar scores; as well as any antenatal complications. The patient was asked to complete a voluntary questionnaire to collect parental information including height and weight, ethnicity, medical history, previous pregnancies or infertility, and use of cigarettes or drugs. This information was used to determine suitability for studies, as well as for categorizing samples. After delivery, the attending nurse collected placenta and two 5mL vacutainers (1x heparin, 1x EDTA) of cord blood, which were either picked up in the hours following by a member of the lab, or delivered on ice via overnight shipping. 50 2.2 Placenta Biopsy Upon collection, placentas were weighed and measured, and any notable physical characteristics were recorded. Chorionic villi biopsies of approximately 2cm3 were excised from the fetal facing side of the placenta under sterile conditions in a biosafety cabinet. A total of ten biopsies were collected per placenta, as illustrated in Figure 2.1. The first three biopsy sites were proximal to the cord (1), between the cord and the edge of the placenta (2), and at the edge of the placenta (3), and the remaining biopsies (4-10) were taken at sites moving clockwise around the cord from the first three biopsy sites. Figure 2.1: Illustration depicting typical site selection for placenta biopsy. Sites 1-3 were selected near, proximal and distal from the umbilical cord, and the remainder of sites were taken moving clockwise around the cord from the initial 3 biopsy sites. An adjacent piece of amnion was collected for each biopsy, and the villi sample was excised from beneath the chorionic plate. A sample of umbilical cord and maternal decidua from the basal plate were also collected for each case. All biopsied samples were immediately frozen on dry ice and stored at -80°C until they could be processed for further analysis. 51 2.3 Blood Culture and Harvest 1mL of heparinized blood was used to set up paired flasks of stimulated blood culture, each with 0.5mL cord blood and 8.5mL RPMI medium (Gibco, Thermo Scientific, Waltham MA) supplemented with 1.75mL bovine serum albumin (Gibco), 250µL phytohaemagglutinin (Gibco), 150µL penicillin/streptomycin (Gibco), 150µL L-Glutamine (Gibco) and 25µL heparin (Sigma-Aldrich, St. Louis, MO). Flasks were cultured at 37°C for 72-96 hours before harvesting. 24.5 hours before harvest, cultures were synchronized using 5-fluoro-2’-deoxyuridine (Sigma) for cell cycle suspension, and excess thymidine (Sigma) was added 3.5 hours before harvest for cell cycle release. Harvest was initiated by addition of KaryoMAX colcemid (Gibco) and 10 minutes incubation, followed by hypotonic treatment with potassium chloride (0.075M) (Fisher Scientific, Hampton NH) for 10 minutes, and fixation and red blood cell lysis with four washes of 3:1 methanol:acetic acid fixative (Fisher). Fixed cells were dropped onto washed glass slides and aged at room temperature for two weeks before G-banding using 0.05% trypsin in Hank’s buffered saline solution (HBSS) (Gibco) for cytoplasmic digestion and methanol-based Giemsa and Leishmann stains (Sigma) for banding. 2.4 Karyotype, Counting and Sexing Twenty-five banded metaphases were analyzed for each patient – five metaphases were karyotyped using a Zeiss Axio Imager M2 microscope (Carl Zeiss Light Microscopy, Gottingen, Germany) and Cytovision v7.2 software (Leica Biosystems, Nussloch, Germany), and twenty additional metaphases were counted and sexed. If mosaicism was suspected after analysis of the first twenty-five cells, a minimum of twenty-five additional cells were analyzed. 52 2.5 DNA Extraction DNA was extracted from EDTA vacutainers of cord or peripheral blood, and chorionic villi using the Gentra Puregene kit (QIAGEN, Venlo, Netherlands) following manufacturer’s instructions for blood and tissue respectively, with slight modifications as described below. Whole Blood (Cord or Peripheral) 3mL whole blood was lysed with 9mL RBC lysis buffer (QIAGEN; Cat# 1045722) for 20 minutes, spun down for 10 minutes at 3000rpm, and supernatant was discarded. 3mL cell lysis solution (QIAGEN; Cat# 1045723) was added to pellet, and resuspended by vortexing. 2mL protein precipitation solution (QIAGEN; Cat# 1045701) was added, vortexed for 20 seconds, and centrifuged for 10 minutes at 3000rpm. Supernatant was decanted and added to a fresh tube containing 3mL 100% isopropanol, and inverted 50 times to precipitate DNA. DNA pellet was retrieved, washed in 70% ethanol, and dried in a 1.5mL microcentrifuge tube. DNA was eluted with 200-300µL DNA hydration solution (QIAGEN; Cat# 1045702) and incubated at 65°C for 1 hour, then room temperature overnight. Chorionic villi A small 10-20mg sample of chorionic villi was thawed and washed in PBS to remove all traces of blood. The sample was placed in 300µL cell lysis solution, minced using surgical shears, and 1.5µL proteinase K (QIAGEN) was added. The sample was incubated at 65°C for 1 hour or until tissue was completely dissolved. 1.5µL RNase A solution (QIAGEN) was added to the sample after tissue dissolution, followed by incubation at 37°C for 15 minutes. Sample was cooled on ice for 60 seconds, and 120µL protein precipitation solution was added followed by 20 seconds of vigorous vortexing. Sample was spun down at 13,000rpm at 4°C for 10 minutes. Supernatant 53 was decanted and added to a fresh tube containing 300µL 100% isopropanol. Tube was inverted 50 times to precipitate DNA, then spun at 13,000rpm for 2 minutes. Supernatant was discarded and DNA pellet was washed with 300µL ice cold 70% ethanol and spun down for 1 minute at 13,000rpm. Supernatant was discarded and pellet was dried then eluted with 50µL DNA hydration solution and incubated for 1 hour at 65°C, then room temperature overnight. Eluted DNA from whole blood and chorionic villi was quantified using a NanoDrop™ (Thermo Fisher, Waltham MA) for subsequent molecular analysis by chromosomal CGH and MLPA, and Quantus™ Fluorometer and QuantiFluor™ ONE dsDNA system (Promega, Madison WI) for subsequent molecular analysis by SNP array. 2.6 Chromosomal CGH For cord blood and peripheral blood cultures that failed to generate adequate metaphases for karyotype analysis, DNA extracted from EDTA blood was used for chromosomal CGH analysis. Chromosomal CGH, as described in Section 1.4.3, is able to detect partial- or whole-chromosome aneuploidy by hybridization of differentially labeled patient (test) DNA and reference DNA to metaphase chromosomes and comparison of signals using fluorescence microscopy. Since chromosomal CGH can only detect gains or losses of DNA, it is unable to detect balanced rearrangements and polyploidy. CGH was performed according to a previously established lab protocol (Minor et al., 2006). 2μg of test DNA from whole chorionic villi was labelled by nick translation using 4μL prepared mix of dNTPs (Invitrogen, Carlsbad CA)/fluorescein-12-dUTP (FITC) (Roche Diagnostics, Penzberg, Germany), 2.0μL of nick translation enzyme mix (Roche Diagnostics), and 1.0μL of DNA polymerase I (New England Biolabs, Ipswich, Germany). 3μg reference DNA from whole chorionic villi of a normal female pregnancy was labelled by nick translation using 8μL 54 prepared mix of dNTPs/tetramethylrhodamine-5-dUTP (TRITC) (Roche Diagnostics), 2.5μL of nick translation enzyme mix, and 1.5μL of DNA polymerase I. Nick translation was carried out at 15°C for 1-3 hours to achieve fragment lengths of 500bp-2kb. The size of the fragment lengths was verified by electrophoresis in a 1% agarose gel using a lambda-HindIII as a size standard. Equal amounts of test and reference fluorescent nick translated products, as estimated by band intensities, were precipitated with 20μL of human Cot-1 DNA (Invitrogen) using 200μL of 100% ethanol and 5μL of 4M LiCl overnight at -20°C. The following day, precipitated DNA was spun down at 20,000rpm in a 4°C temperature-controlled centrifuge and resuspended in hybridization buffer (50% formamide/10% dextran sulfate / 2x saline sodium citrate (SSC)). Resuspended probes were incubated at 37°C for 1 hour, denatured at 72°C for 5 minutes, and incubated for another hour to allow Cot-1 DNA to anneal to subtelomeric regions of chromosomal DNA. Meanwhile, fixed suspensions of normal male metaphase chromosomes were dropped fresh onto glass slides and pre-treated with RNAse A (0.125mg/mL; Sigma) in 200μL 2xSSC solution for 1 hour in a 37°C humid chamber. Slides were subsequently washed twice for 5 minutes in PBS, fixed with 10% buffered formalin (Fisher) for 15 minutes, washed twice for 5 minutes in PBS and dehydrated in a 70%, 85% and 100% ethanol series for 3 minutes each. Dehydrated slides were denatured with 70% formamide/2xSSC at 72°C for 5 minutes, followed by an ice-cold 70%, 85% and 100% ethanol series for 2 minutes each. Treated slides were hybridized with the denatured probe suspensions after evaporation of the final ethanol wash on a 42°C slide warmer for 30 seconds. After applying denatured probe, slides were sealed with 22mm square glass coverslips (Fisher) and rubber cement, and hybridized at 37°C in a humid chamber for 72 hours. 55 Following hybridization, the coverslips were removed and slides were washed in 0.4xSSC/0.3% NP-40 at 72°C for 2 minutes, 2xSSC/0.1% NP-40 at room temperature for 30 seconds, and counterstained with 0.2μg/mL 4’,6-diamidino-2’-phenylindole (DAPI) for 1 minute. Each slide was mounted with Vectashield anti-fade solution (Vector Laboratories, Burlington, CA) and a 22mm square glass coverslip. The slides and fluorophores were initially stabilized and stored at -20°C for several hours before analysis by fluorescence microscopy. Metaphase chromosomes were captured under a Zeiss Axio Imager M2 microscope (Carl Zeiss Light Microscopy) using DAPI, FITC and TRITC filters, and probe hybridization was considered successful when a minimum of 10 captured metaphases passed the QC requirements for the Cytovision v7.2 software, CGH package (Leica Biosystems). Heterochromatin-rich regions such as the centromeric regions of chromosomes 1, 9, 16 and the distal q arm of chromosome Y, as well as the satellite regions of the acrocentric chromosomes were excluded from analysis, as they are suppressed by Cot-1 DNA and produce variable CGH profiles (Lestou et al., 1999) . Average signal intensities for test and reference fluorophores were used to detect aneuploidy via gains or losses in patient DNA in relation to test DNA for autosomes, as indicated by shifts from the expected 1:1 profile for euploid samples. Shift in the ratios of X and Y signals indicating loss of X and gain of Y signals in relation to the female reference DNA was used to identify male samples, as well as sex chromosome aneuploidy. 2.7 MLPA In addition to CGH, MLPA was employed when detection of aneuploidy by traditional cytogenetic analysis was not possible due to blood culture failure. As discussed in Section 1.4.3, MLPA relies on the amplification of probes that hybridize to the subtelomeric regions of each chromosome in copy-dependent proportions, detecting aneuploidy by using subtelomeric regions 56 as proxies for whole chromosome arms. Like CGH, MLPA is unable to detect balanced rearrangements and polyploidies. In addition to this, MLPA is unable to detect gains and losses that do not encompass telomeres, such as segmental deletions and duplications. The application of MLPA is most suitable for detection of whole chromosome aneuploidy. Two subtelomeric probemix kits were used to detect CNV in DNA samples extracted from EDTA blood or chorionic villi: SALSA MLPA P070 Subtelomeres mix 2B and SALSA MLPA P036 Subtelomeres mix 1 (MRC-Holland, Amsterdam, Netherlands). The former was used for all samples initially to detect aneuploidy, and if detected, the latter was used to confirm results from the first kit. DNA extracted from blood or chorionic villi was prepared for MLPA using manufacturer’s instructions, as previously described (Schouten et al., 2002). Sample DNA was diluted to 50ng/μL with TE, denatured for 5 minutes at 98°C, and cooled to 25°C. 3μL hybridization mastermix (1.5μL probemix, 1.5μL MLPA buffer) was added to each cooled sample, mixed, and incubated at 95°C for 1 minute, followed by a reduction to 60°C for 16-20 hours overnight to allow hybridization of the pairs of probes to subtelomeric regions of genomic DNA. The following day, samples were cooled to 54°C and held while 32μL Ligase-65 mastermix (25μL Ultrapure water, 3μL Ligase Buffer A, 3μL Ligase Buffer B) was added to each tube and mixed by pipetting. Ligation of adjacent hybridized probe pairs occurred at 54°C for 15 minutes, after which the ligase was heat-deactivated by 5 minutes of incubation at 98°C, and cooled to 20°C. Next, the ligated probes were amplified by 35 cycles of PCR following addition of 10μL Polymerase mastermix (7.5μL Ultrapure water, 2μL SALSA PCR primer mix, 0.5μL SALSA 57 Polymerase). Each cycle consisted of 30 seconds at 95°C, 30 seconds at 60°C, and 60 seconds at 72°C, and the final cycle was followed with 20 minutes of incubation at 72°C then held at 15°C. Following probe amplification, 7.5μL of the product was diluted with 7.5μL TE, mixed and spun down. 0.7μL of diluted probe mix was combined with 0.3μL ROX size standard (Thermo Fisher) and 9μL HiDi formamide (Thermo Fisher), heated to 86°C for three minutes and cooled to 4°C for two minutes in a sealed injection plate prior to being resolved on an ABI-3130 bioanalyzer (Applied Biosystems, Foster City, CA). Quality control and peak analysis of capillary-resolved probes was performed using Coffalyser.net software (MRC-Holland). The software normalizes test and reference probes to calculate a Dosage Quotient (DQ) – the peak ratio between test and reference samples. For normal samples with no aneuploidy, DQ for all probes will be ~1.0. Heterozygous deletions will result in a DQ of ~0.5 for the corresponding probe, whereas heterozygous duplications will result in a DQ of ~1.5. The expected DQ ranges for normal and aneuploid samples as supplied by the manufacturer are presented in Table 2.1. Table 2.1: Expected copy number for Dosage Quotient ranges generated by MLPA Dosage Quotient Range Copy number DQ = 0 0 copies (homozygous deletion) 0.40 < DQ < 0.65 1 copy (heterozygous deletion) 0.80 < DQ < 1.20 NORMAL (identical to reference samples) 1.30 < DQ < 1.65 3 copies (heterozygous duplication) 1.75 < DQ < 2.15 4 copies (homozygous duplication) All other values Ambiguous result Table adapted from manufacturer’s protocol (MRC-Holland, 2018) 2.8 Flow Cytometry In order to detect polyploidy in the cytotrophoblasts and mesenchyme cells of the chorionic villi, flow cytometry for cell cycle analysis was performed on isolated cells. Cytotrophoblast cells were isolated from thawed chorionic villi by digestion with 1mg/mL collagenase 1A (Sigma) in 58 HBSS (Gibco) at 37°C for 10 minutes. Mesenchyme cells were subsequently isolated by mincing villi and digestion with 0.5% pepsin (Sigma) in deionized water at 37°C for 15 minutes. Cytotrophoblast and mesenchyme cells were strained through a 40nm sieve (Fisher) to obtain a single cell suspension, and fixed in 70% ethanol overnight at -20°C before staining for flow cytometry. For each sample, approximately 2.5 x 105 fixed cells were washed with PBS and stained with 250μL of 40μg/mL propidium iodide (Sigma) in PBS with 0.1% v/v Triton X-100 (Sigma) and 200μg/mL RNase A (QIAGEN) for 30 minutes at room temperature, then incubated on ice. 20,000 cells were assayed per sample with a CytoFlex flow cytometer (Beckman Coulter, Indianapolis, Indiana) using the 690/50 BP PerCP fluorescence channel, at the BCCHRI Flow Cytometry Core Facility (Vancouver, BC, Canada). The instrument was initially calibrated and gated using a diploid negative control sample and a tetraploid positive control sample. Data from each run was exported and analyzed using FlowJo version 10.0.8 software (FlowJo, Ashland, Oregon). Diploidy was confirmed if the diploid peak did not shift more than 10% from that of the negative control, and the proportion of tetraploid cells (G2) was less than 30% of the diploid peak. For any samples where tetraploidy was suspected based on flow cytometry results, FISH was performed on the same sample of fixed cells to confirm results. 2.9 FISH FISH was used to confirm aneuploidy or polyploidy detected using CGH, MLPA or flow cytometry. Single cell suspensions from query samples were isolated and fixed to slides as previously described in the literature (Henderson et al., 1996; Klever et al., 1992), and hybridized with FISH probes for the aneuploidy of interest, or centromeric enumeration probes (CEP) X/Y/18 59 (Vysis, Abbott Molecular, Abbott Park, Illinois) for suspected polyploidy. FISH probes used for aneuploidy confirmation for chromosome 21 were alpha satellite (LSI) 13/21 (Vysis). Cytotrophoblasts: Suspensions of single cytotrophoblast cells were obtained as described for flow cytometry in Section 2.8, and ethanol-fixed cells were dropped onto clean silanized glass slides. Cells were prepared for FISH by pre-treatment with 0.03% trypsin (Sigma) in PBS for 10 seconds, and fixed with 10% buffered formalin for 10 minutes, and washed three times in PBS for 5 minutes each. Mesenchymal cells: Suspensions of fibroblasts from the mesenchyme of the villi were obtained as described for flow cytometry in Section 2.8, and ethanol-fixed cells were dropped onto clean silanized glass slides. Cells were fixed in 10% buffered formalin for 2 hours 15 minutes, washed in PBS for 1 minute, and dehydrated in a 70%, 85% and 100% ethanol series for 2 minutes each. Cord blood: Fixed suspensions of interphase and metaphase lymphocytes were obtained by methods described for blood culture and harvest in Section 2.3. Fixed cells were dropped onto wet silanized glass slides, air dried, and dehydrated in a 70%, 85% and 100% ethanol series for 2 minutes each. Slides were subsequently pre-treated by incubation in 2xSSC for 15 minutes at 37°C, and dehydrated again in an ethanol series. Slide denaturation & FISH probe hybridization Following fixation and pre-treatment, slides were denatured and FISH probes were prepared according to manufacturer’s instructions. Slides were heat denatured in 70% formamide/2xSSC (pH 7.0) for 5 minutes at 73°C, and dehydrated in an ice cold 70%, 85% and 100% ethanol series for 2 minutes each, then air dried briefly on a 45°C slide warmer before probe application. Prepared aliquots of FISH probe were denatured for 5 minutes at 73°C and either held 60 on a 45°C slide warmer or applied to prepared slides immediately. Slides were sealed with a 22mm square glass coverslip and rubber cement, then placed in a humid chamber at 37°C overnight. The following day, coverslips were removed followed by post-hybridization washes in 0.4xSSC/0.3% NP-40 for 2 minutes at 72°C, then 2xSSC/0.1% NP-40 at room temperature for 30 seconds. Slides were air dried then counterstained with 10μL DAPI II (Vysis), sealed with a 22mm square glass coverslip, and incubated for 1-2 hours minimum at -20°C before counting and capturing cells on a Zeiss Axio Imager M2 microscope (Carl Zeiss Light Microscopy) using Cytovision v7.2 software, (Leica Biosystems). Signals from at least 200 interphase nuclei were examined for each sample, and up to 500 cells were examined when mosaicism was suspected. Each FISH assay or set of assays was performed in parallel with the corresponding cells of a normal male control for statistical comparison of abnormal signal frequency. 2.10 SNP array SNP array is a DNA microarray technology applicable for detection of CNV, UPD and mosaicism. Samples were sent for analysis on the Illumina Omni 2.5 SNP array when results from CGH, MLPA or karyotyping were inconclusive. These samples as well as the abnormalities needing confirmation by SNP array are presented in Table 2.2. The Illumina Omni2.5-8 BeadChip v1.3 contains over 2.5 million markers, with a median spacing of one marker per every 560bp. Markers include both rare and common SNPs, covering phenotypically relevant regions of CNV and regions commonly screened for cytogenetic anomalies and segmental duplications and deletions. SNP-poor regions are covered by intensity-only markers used to quantify copy number in the absence of a SNP. 61 Table 2.2: Samples submitted for SNP array Patient Sample Tissue Suspected abnormality/CNV (assay used for detection) SM02-8 site 3 Placental villi Gain of distal 7q, loss of distal Xp (CGH) SM12-1081 site 1 Placental villi Query XXY (CGH) SM12-1081 site 3 Placental villi Query XXY (CGH) SM12-1081 site 9 Placental villi Query XXY (CGH) SM07-271 Cord blood Rearrangement of terminal 1p (karyotype) SM14-1246 Peripheral blood Mosaic for supernumerary chromosome (karyotype) 50ng of genomic DNA was submitted to TCAG Microarray Facility at Sick Kids Hospital (Toronto, ON), where it was prepared according to manufacturer’s instructions for the Infinium LCG Platform (Illumina, San Diego, CA) using Tecan automated liquid handling robots (Männendorf, Switzerland) for sample preparation. In this process, the submitted genomic DNA samples were amplified, fragmented, precipitated and resuspended before being dispensed onto an Infinium Omni2.5-8 BeadChip v1.3 (Illumina). The raw data, GenomeStudio project and exported calls were submitted to our lab by TCAG for subsequent analysis. Output for the array consists of a B allele frequency and log R ratio for each SNP marker. B allele frequency is the measure of how many copies of each allele is detected – AA, AB, BB in the case of diploidy – allowing the detection of loss of heterozygosity (ie. AB is absent) when UPD or deletions are present, or additional ratios of heterozygosity (ie. AAB, ABB are present) for duplications. The log R ratio is a measure of signal intensity – given as the log2 of the observed over expected signal intensity per sample – which allows the source of abnormal B allele frequency profiles to be discerned between deletion (log R ratio ~ 0.5), UPD (log R ratio ~ 1.0) or duplication (log R ratio ~ 1.5). In the case of mosaicism, the log R ratio will fall in between these values relative to the ratio of aneuploid to euploid DNA in the sample. 62 Analysis of B allele frequency and log R ratio for submitted samples was performed using GenomeStudio software version 2.0.4 (Illumina) using the cnvPartition plugin version 3.2.0 (Illumina) for detection and quantification of CNV, UPD and mosaicism. CNVs within 200kb of the centromere were filtered, as well as gains < 500kb and losses < 200kb in intergenic regions. Minimum cut-offs for CNVs were defined at 100 kb for any consecutive gains and 50 kb for any consecutive losses. 2.11 Validation of MLPA for aneuploidy detection At the outset of my investigation, chromosomal CGH was employed to detect aneuploidy in the chorionic villus DNA of the placenta, in keeping with the methodology of previous CPM studies conducted in my lab (Chan Wong et al., 2012; Minor et al., 2006). I experienced variable success in the hybridization of nick-translation-labelled placenta DNA for CGH, likely due to sample degradation after prolonged storage of tissue at -80°C. Labeled fragments of DNA from older tissues were observably shorter (100-300bp) compared to the optimal fragment lengths achievable with fresher samples (500-2000bp) under the same digestion conditions, and CGH for these samples frequently failed due to poor hybridization of test probe to metaphase chromosomes. This prompted a switch in aneuploidy detection methodologies to MLPA, which has been demonstrated to be comparable to traditional cytogenetic methods with regards to whole chromosome aneuploidy detection (Saxena et al., 2016). Compared to CGH, MLPA is at a disadvantage for detection of interstitial gains and losses, but its lower cost, reduced turnaround time and ability to process more than double the number of samples simultaneously would arguably make up for that disadvantage by allowing the investigation of a greater number of cases with a potentially reduced incidence of sample failure. In addition, a single MLPA reaction requires only 50ng of DNA compared to the 2µg required for 63 CGH, so there is a reduced risk of having to re-extract DNA from frozen placental biopsies and running out of patient sample. To validate the MLPA methodology (Section 2.7) against our existing CGH methodology (Section 2.6) for the detection of aneuploidy in chorionic villus DNA, we compared the results of 93 samples from 31 cases that had previously been diagnosed via CGH with the results obtained for the same DNA samples using MLPA. These results are presented in Table 2.3. The sample set consisted of 10 cases each from ICSI, IVF and IUI conceptions, and one natural conception case, for a total of 31 cases; DNA from three chorionic villus samples was investigated from each case for a total of 93 samples. We included cases that had previously been found to have either constitutional aneuploidy or CPM as positive controls, including an ICSI case previously reported by CGH to have CPM for a gain of 7q and loss of Xp (Minor et al., 2006), an ICSI case with monosomy X CPM, an ICSI case with non-mosaic trisomy 21 in both cord blood and placenta, an IVF case with 47,XXY in both cord blood and placenta, an IVF case with constitutional mosaicism for monosomy X and loss of X in all placental sites investigated by CGH and FISH, an IVF and IUI case with suspected CPM for whole and partial monosomies, and a natural conception case for which mosaicism for a supernumerary chromosome was observed in karyotyped lymphocytes but no abnormalities were detected by CGH. 64 Table 2.3: Results from CGH and MLPA for methodology validation. Results in black text indicate samples were detected as normal for both CGH and MLPA. Green text indicates abnormal results were congruent between both methods. Purple text indicates non-congruent results between both methods. Red text indicates sample failure. 26/31 (83.9%) cases had congruent results at all three sites for both CGH and MLPA. 1/31 cases had a site that failed to generate results by MLPA after two attempts, and 5/31 (16.1%) cases had non-congruent results for at least one site between MLPA and CGH testing methodologies. All three sites from SM10-523 showed a loss of X by CGH but a balanced female profile by MLPA. FISH on cytotrophoblasts from each of these sites found 30.3%, 19.7% and 11.4% of cells from each respective site to have only one signal for chromosome X and cytogenetic analysis of cord blood lymphocytes found monosomy X in 7/50 (14%) of cells examined, suggesting that this low level of mosaicism was undetectable by MLPA. It is surprising, however, that such low levels of mosaicism were picked up by CGH using the same DNA sample, as this method has been 65 reported to be unable to detect mosaicism lower than 32% (Lomax et al., 2000). These findings are presented in Chapter 3: Results (Section 3.5) in Table 3.3 and Table 3.6. For one non-congruent case (SM10-620), one sample (site 3) suggested a loss of 21 by CGH but a balanced male profile by MLPA (Figure 2.2). Uncultured fibroblasts from the mesenchymal core of the same biopsied site were used for follow-up by FISH, and no loss of 21 was observed in 200/200 (100%) cells analyzed, suggesting the MLPA results reflect the true genotype for this sample. Figure 2.2: Non-congruent results for SM10-620 S3. A) CGH results suggest a male with a loss of chromosome 21 B) MLPA results suggest a balanced male profile. One case (SM12-841) showed non-congruence at one of three sites examined, suggesting possible mosaicism for interstitial loss by CGH, which was not detected by MLPA using the same DNA sample. CGH on this sample (site 4) was a query loss of proximal Xq that did not extend to the distal q-arm, and as expected, no aneuploidy was detected by MLPA. The signal on the CGH profile was not shifted completely to the left, so it is unclear whether this represents an actual loss in mosaic form, or an artifact from inconsistent hybridization of CGH probes. A real imbalance in this region would be undetectable by MLPA, as the MLPA probes hybridize to the subtelomeric region, which would be present in two copies regardless of any interstitial gains or losses present. 66 Another case (SM12-842) had incongruent results for one of three samples (site 9). CGH on this sample suggested a gain of proximal 11q by CGH, but MLPA using the same DNA sample showed a balanced female profile. As with the previous case, the 11q gain detected by CGH was not completely right-shifted, so it is unclear whether it represents an actual gain in mosaic form, or an artifact from inconsistent CGH probe hybridization. Again, this imbalance, being interstitial, would not be detectable by MLPA even if it were real, so we were unable to confirm it with the second methodology. Figure 2.3 Analysis of mosaic aneuploidy for SM02-8 site 3. A) CGH results suggest a gain of distal 7q and a loss of distal Xp. B) MLPA results suggested reduced copies of Xp may be present in the sample, but no significant increase in copies of 7q was observed. C) SNP array confirmed the mosaic gains in distal 7q and mosaic losses in Xp for this sample. The final non-congruent case was originally analyzed by a previous member of my lab and was found to have a gain of 7q31 to 7qter and a loss of Xp21.3 to Xpter by CGH (Figure 2.3 A) (Minor et al., 2006). The MLPA results for this sample suggested a mosaic loss of Xp but no notable gain of 7q (Figure 2.3 B). To resolve the discrepancy between the two methodologies, the sample was analyzed by SNP array. Array results were consistent with the original CGH results, 67 demonstrating a 9.87 Mb segmental gain of the terminal q arm of Chromosome 7 and a 39.20 Mb segmental loss of the terminal p arm of Chromosome X. The log R ratio shift for these two imbalances was small, suggesting mosaic gains and losses (Figure 2.3 C). This is consistent with the observation that this imbalance was only detected for one biopsied site of the placenta, suggesting mosaicism. Overall, congruent results for CGH and MLPA were observed for 26/31 (83.9%) cases, or 85/93 (91.3%) samples. 1/93 (1.1%) samples failed to generate results using MLPA, and 5/31 (16.1%) cases – 7/93 (7.5%) samples – had non-congruent results between MLPA and CGH testing methodologies. 68 Chapter 3: Confined Placental Mosaicism in IVF and IUI term pregnancies 3.1 Introduction The goal of my first objective was to investigate the incidence of CPM in IVF and IUI term placentas. By examining the genetic constitution of villi isolated from three distinct sites in each term placenta, it was my intention to maximize the odds of detecting mosaicism, should it be present. My rationale for this investigation was that ART pregnancies, including IVF pregnancies, are at an increased risk of preterm birth, IUGR and SGA (Ombelet et al., 2016) – perinatal complications that are also associated with the presence of CPM (Toutain et al., 2018). Mosaicism is frequently observed in preimplantation embryos generated by ART (van Echten-Arends et al., 2011). I predicted that if some variable of the IVF procedure is influencing mosaicism, this should be reflected as an increased incidence of CPM for IVF embryos. I included a small sample of IUI term placentas in order to control for infertility in the absence of in vitro embryo culture. 3.2 Case Ascertainment Inclusion criteria were pregnancies resulting in live birth, conceived by either IVF or IUI techniques, wherein placenta was collected after delivery. Previous studies examining mosaicism in term placentas from healthy, natural conception pregnancies were used for comparison, as well as term placentas from naturally conceived IUGR pregnancies. These studies are summarized in Table 3.1. CPM has been observed to occur more frequently in pregnancies affected by IUGR (Amiel et al., 2002; Kalousek et al., 1991; Robinson et al., 2010; Stipoljev et al., 2001; Wilkins-Haug et al., 1995), and since my ART cohorts included pregnancies with normal outcomes as well as IUGR, both outcomes were considered separately for natural conception controls as well. Table 3.1: Studies on the incidence of CPM in normal and IUGR natural conception term pregnancies Study Normal outcome Abnormal (%) IUGR Abnormal (%) # of sites examined Analysis method CPM abnormality detected (# cases) Kalousek & Dill (1983) 0/9 (0) 2/9 (22.2) “several” Karyotype 47,XXX(1); 47,XY,+22(1) Artan et al. (1995) 0/115 (0) 6/10 (60.0) 10 Karyotype 47,XX,+14(1); 92,XXXX(1); 47,XX,+21(1); 47,XY,+21(1); 45,X(1), 47,XY,+18(1) Wilkins-Haug et al. (1995) 2/24 (8.3) 3/12 (25) not specified Karyotype, FISH Control: 47,XY,+10(1); 92,XXXX(1) IUGR: 48,XX,+17,+21(1); 92,XXXX(1); 92,XXYY(1) Verp & Unger (1990) 0/2 (0) 0/11 (0) 1 Karyotype None Krishnamoorthy et al. (1995) 0/30 (0) 4/26 (15.4) 1 Karyotype, FISH monosomy 21(2); monosomy 3(1); multiple aneuploidies(1) Kennerknecht et al. (1993) 0/24 (0) 0/71 (0) 2 Karyotype None Barrett et al. (2001) 5/219 (2.3) None not specified CGH, FISH 46,XXX(1); 47,XXY(1); trisomy(3) Stipoljev et al. (2001) 0/20 (0) 2/20 (10) not specified Karyotype 47,XY,+15(1), 16p deletion(1) Masuzaki et al. (2004) None 3/50 (6) 2 Karyotype 47,XX,+22(1), 47,XX,+7(1), 47,XY,+2(1) Robinson et al. (2010) 1/85 (1.2) 4/43 (9.3) 2 CGH, microsatellite PCR Control: 47,XXX(1) IUGR: 47,XY,+7(1); 47,XX,+7(1); 47,XX,+13(1); 47,XX,+2(1) TOTAL 8/528 (1.5) 24/252 (9.5) 3.3 Case Sampling To maximize the chances of detecting mosaicism, chorionic villi from three biopsy sites were used to investigate aneuploidy for each case: one site proximal to the cord, one site near the edge of the placenta, and one site distal from these two sites. Typically, these would be biopsies 1,3 and 9 in Figure 2.1 of Section 2.2. Each of the villus samples were processed in order to a) isolate villus DNA for analysis by chromosomal CGH and/or MLPA and b) separate cytotrophoblast cells from cells of the mesenchyme for detection of polyploidy using flow cytometry. If any of the three sites were found to have a different chromosomal constitution than fetal lymphocytes (or amnion), villi from an additional seven placental sites (biopsies 2,4,5,6,7,8,10 from Figure 2.1) were to be examined to establish the extent of CPM for the case. Ideally, fresh cord blood was collected concurrently with placenta, as this allowed any abnormalities detected in placental tissue to be ruled out or confirmed in the child by harvesting and karyotyping lymphocytes from cord blood. In cases of lymphocyte culture failure, DNA extracted from cord blood lymphocytes was examined by chromosomal CGH or MLPA. For the few cases where cord blood could not be collected, DNA was extracted from a sample of amnion, the placental tissue most closely related to fetal tissues, as both amniotic and fetal cell lineages are derived from the epiblast of the ICM (Boss et al., 2018). Cases were considered positive for CPM if aneuploidy was detected for one or more placental sites, but not in the cord blood or amnion. If the same aneuploidy was also present in cord blood or amnion, these were cases of aneuploidy, not CPM. 3.4 Statistical Analysis An a priori power analysis conducted using G*Power software version 3.1.9.2 (Dusseldorf, Germany) concluded that in order to determine that IVF or IUI pregnancies are at significantly 71 increased risk (p<0.05) than the 1.5% risk of CPM in normal, natural conception pregnancies – using a one-tailed linear bivariate regression model –, we would need to examine a minimum of 100 cases for a power of 80% (Faul et al., 2007). 3.5 Results A total of 82 IVF and 15 IUI singleton pregnancies were investigated for CPM. Of these, the results from 22 IVF cases were previously published as a pilot study by a former member of the lab (Chan Wong et al., 2012). Maternal age, gestational age and birth weight were not available for every case, but those for which records were available are summarized in Table 3.2. Any abnormalities detected among the subjects of each cohort are summarized in Table 3.3. Table 3.2: Maternal Clinical Information for CPM study cohorts Cohort Maternal age (yrs) (mean ± 95% CI) Gestational age (days) (mean ± 95% CI) Birth weight (grams) (mean ± 95% CI) IVF (n=82) 36.25 ± 1.42 (n=36) 270.93 ± 4.44 (n=43) 3303.12 ± 184.87 IUI (n=15) 35.25 ± 8.84 (n=4) 279.60 ± 3.44 (n=15) 3479.07 ± 352.68 Of these, a male IVF baby (SM09-478) was found to be 47,XXY by both karyotype and CGH for cord blood (Figure 3.1). CGH on five placental sites found two sites with an additional trisomy 13 abnormality. This case was investigated by a previous member of the Ma lab as part of his thesis, and this data was published in the aforementioned pilot study on CPM in IVF pregnancies (Chan Wong et al., 2012). Table 3.3: Abnormalities detected in cases examined for CPM Sample ID Cohort Maternal Age Perinatal outcome Karyotype/CGH Abnormal Sites Placenta abnormality [method] SM09-447 IVF unknown normal 46,XX 2/3 villi ish cgh enh(13)(q31q33) [CGH] SM09-478 IVF unknown normal 47,XXY 2/5 villi ish cgh enh(X),enh(13) [CGH] SM10-523 IVF unknown normal 45,X[7]/46,XX[43] 3/3 villi 45,X/46,XX (mosaic) [CGH, FISH] SM15-1386 IVF 37 normal 46,XY.ish cgh 1/3 villi tetraploidy (mosaic) [flow cytometry, FISH] SM15-1390 IVF 39 normal 46,XX,inv(9)(p13.3q13) N/A SM17-1476 IVF 32 normal 46,XX 1/3 villi tetraploidy (mosaic) [flow cytometry, FISH] SM12-841 IUI unknown low HR/nuchal cord 46,XX 1/3 villi ish cgh dim(X)(q12q21.3) [CGH] SM12-842 IUI unknown SGA 46,XX 1/3 villi ish cgh enh(11)(q13.4q22.3) [CGH] SM13-1104 IUI 42 preterm birth 46,XY,inv(9)(p12q21) N/A Figure 3.1: Confined Placental Mosaicism for male IVF baby SM09-478. A) 47,XXY karyotype B) CGH results for placental villi show XXY with additional trisomy 13 (indicated by red box) in 2/5 sites. One female IVF baby (SM10-523) was found to be mosaic for monosomy X in 7 of 50 cells examined by cytogenetic analysis of cord blood lymphocytes. Mosaicism for monosomy X in the chorionic villi was suspected based on CGH, but these results were not corroborated by MLPA (Figure 3.2). Isolated cytotrophoblast cells from placental villi were analyzed by FISH using CEP X/Y/18 probes (Vysis) to quantify monosomy X mosaicism (Table 3.4, Figure 3.3). Since monosomy X mosaicism was observed in cord blood as well as placenta, this case more than likely represents generalized mosaicism versus CPM. Figure 3.2: Monosomy X mosaicism for villi of SM10-523. A) CGH results reveal monosomy X mosaicism (indicated by red box) in 3 of 3 placental villi sites B) MLPA did not detect monosomy X in the same villi sites used for CGH. 74 Table 3.4: FISH confirmation of monosomy X in cytotrophoblast cells of SM10-523 biopsies Case n Disomy* (%) Monosomy (%) p** control male 300 300 (100) 0 SM09-523 site 1 300 208 (69.3) 91 (30.3) <.0001 SM09-523 site 2 300 240 (80.0) 59 (19.7) <.0001 SM09-523 site 3 305 270 (88.5) 35 (11.4) <.0001 * for control male, “disomy” refers to sex chromosome disomy in which one X and one Y signal were observed. ** by comparison with control using chi square test Figure 3.3: FISH confirmation of monosomy X in cytotrophoblast cells of SM10-523 biopsies. FISH was carried out using probes for the centromeric regions of chromosomes X (green), Y (red) and 18 (aqua). A) Diploid cells from a normal, male control mesenchyme sample showing one signal for each of X and Y, and two signals for 18. B) Diploid cells from SM10-523 showing two signals for each of X and 18. C) Monosomic cells from female sample SM10-523 showing one signal for X and two signals for 18. One female IVF baby (SM09-447) was found by CGH to have a positive shift in the distal 13q region for two of three placental sites (1 & 3), suggesting partial trisomy, although the region identified as trisomic by CGH is not identical for both samples (Figure 3.4). These samples were not included in the validation study for MLPA. It would be valuable to follow up these results with either MLPA or microsatellite analysis, as the region identified as trisomic by CGH may extend to the telomere. 75 Figure 3.4: CGH results for SM09-447 suggest partial trisomy 13. Chromosome 13 CGH profiles for A) site 1 and B) site 3 The flow cytometry results for two IVF cases – one male (SM15-1386, site 3) and one female (SM17-1476, site 1) – suggested tetraploidy in the mesenchyme cells from one out of three placental villi sites investigated for each patient (Figure 3.5). FISH on 500 mesenchymal cells confirmed low-level mosaicism for tetraploidy in each of these samples, by comparison with 300 mesenchymal cells from a normal male pregnancy (Table 3.5, Figure 3.6) Figure 3.5: Flow cytometry results for two IVF cases suggesting tetraploidy. SM15-1385 site 3 and SM17-1476 site 1 had cell populations comparable with a positive control for tetraploidy. Table 3.5: FISH confirmation of tetraploidy detected by flow cytometry in mesenchymal cells of two IVF pregnancies Case n Diploid (%) Tetraploid (%) p* control male 300 298 (99.3) 0 SM15-1386 site 3 500 460 (92.0) 38 (7.6) <.0001 SM17-1476 site 1 500 465 (93.0) 34 (6.8) <.0001 * by comparison with control using chi square test 76 Figure 3.6: FISH confirmation of tetraploidy detected by flow cytometry in mesenchymal cells of two IVF pregnancies. FISH was carried out using probes for the centromeric regions of chromosomes X (green), Y (red) and 18 (aqua). A) Diploid cells from a normal, male control mesenchyme sample showing one signal for each of X and Y, and two signals for 18. B) A tetraploid cell from male sample SM15-1386 site 3, showing two signals for each of X and Y, and four signals for 18. C) A tetraploid cell from female sample SM17-1476 site 1, showing four signals for each of X and 18. The CGH results of one female IUI case (SM12-841, site 4) suggested monosomy for the proximal q arm of chromosome X in one of three placental sites examined. (Figure 3.7 A) As the signal on the CGH profile was only partially shifted to the left, the loss, if real, is likely present in mosaic form. The MLPA results for this sample suggested a normal female profile (Figure 3.7 B), which was expected considering the loss (as suggested by the CGH results) does not encompass the subtelomeric region of the q arm to which the MLPA probes hybridize. Figure 3.7: Results suggesting partial mosaic loss for SM12-841 site 4. A) CGH results show a partial leftward shift of the signal from q14 to q22 on Chromosome X site 4, suggesting a mosaic loss for this region. B) MLPA results using the same DNA sample as for CGH could not detect a loss at Xq proximal, and show a normal female profile. 77 One of the placental sites from another female IUI baby (SM12-842, site 9) showed possible trisomy of the proximal q arm (q14 to q22) of chromosome 11 when examined by CGH (Figure 3.8 A). As with the sample mentioned above, this gain was not completely right-shifted on the CGH profile, so it likely indicates a mosaic gain, if real. As the alleged gain would be in the interstitial region of the q arm, it could not be detected by MLPA, and MLPA results indicated a balanced female profile (Figure 3.8 B). Figure 3.8: Results suggesting partial mosaic gain for SM12-842 site 9. A) CGH results show a partial rightward shift of the signal from q14 to q22 on Chromosome 11 site 9, suggesting a mosaic gain for this region. B) MLPA results using the same DNA sample as for CGH could not detect a gain at 11q proximal, and show a normal female profile. One male IVF baby (SM15-1390) and one female IUI baby (SM13-1104) were found to possess a pericentric inversion of chromosome 9. Rearrangements of chromosome 9qh, the heteromorphic region of the q arm proximal to the centromere, are considered to be polymorphic and are not associated with any demonstrable phenotypic effects in carriers (Kosyakova et al., 2013). These abnormalities were also not included in the total count for CGH cases. The totals for CPM detected in IVF and IUI cases are presented in Figure 3.6. 78 of the 82 IVF cases examined were found to be normal – with euploidy and diploidy detected in all three placental sites – after investigation of CPM by CGH and flow cytometry, respectively. Four cases were found to be abnormal, i.e. positive for CPM: the male 47,XXY case where trisomy 13 was 78 detected in 2/5 placental sites, the female case where partial trisomy 13 was detected in 2/3 placental sites, and the two cases where low level mosaicism for tetraploidy was detected in 1/3 placental sites. This is a total of 4/82 (4.9%) CPM cases for IVF pregnancies, which compared to the 8/528 (1.5%) CPM detected in non-IUGR natural conception cases reported in the literature is not significantly increased (P=0.064, Fisher’s Test). However, since the number of cases I analyzed fell short of that required to detect a significant difference between groups (n=100), I would need to analyze more IVF cases to expect to resolve whether there is any real increase in the incidence of CPM for these pregnancies. For the IUI cohort, the sample size was only 15, and of these, 13 cases were determined to be euploid and diploid for all three placental sites. 2 cases were positive for CPM: one case with partial monosomy X for 1/3 placental sites and one with partial trisomy 11 for 1/3 placental sites. This is a total of 2/15 (13.3%) CPM cases in IUI conceptions, significantly greater than the 1.5% detected in natural conceptions (P=0.028, Fisher’s Test). There was not a significant difference between the IVF and IUI cohort (P=0.23, Fisher’s Test), but sample size is small. Table 3.6: Results for CPM detection in IVF and IUI cohorts IVF IUI Normal: All sites euploid, diploid 78 cases 13 cases Abnormal: 2/5 sites gain(13) (CGH, FISH) 1/3 sites loss(X)(q14q22) (CGH) 2/3 sites gain(13)(q31q33) (CGH) 1/3 sites gain(11)(q14q22) (CGH) 1/3 sites LL tetraploid (FACS, FISH) 1/3 sites LL tetraploid (FACS, FISH) TOTAL 82 cases: 78 normal, 4 abnormal 15 cases: 13 normal, 2 abnormal 79 3.6 Discussion For the first objective of my dissertation, I examined the incidence of CPM in term placentas from pregnancies achieved through IVF and IUI by 1) CGH or MLPA analysis of placental DNA, and 2) flow cytometry analysis of isolated cells, from at least 3 placental sites per case. While the incidence of CPM in pregnancies conceived using IVF (4/82, 4.9%) was slightly greater than what is reported for natural conceptions (8/528, 1.5%) this observed difference was not significant. A small pilot cohort of IUI placentas was also examined to control for infertility without in vitro embryo culturing, and while the incidence of CPM was significantly greater than controls in this cohort (2/15, 13.3%), the sample size of 15 is too small to make any conclusions. While the incidence of CPM in both these cohorts trended higher than that observed for natural conception pregnancies, these numbers may have been inflated due to my sampling and testing criteria. For instance, I examined 3 sites per placenta, whereas 1-2 sites were examined for most control cases (Table 3.1), enabling me to detect mosaicism that may have been missed by testing fewer sites. Importantly, I only observed one case of trisomy, which is an abnormality that would have been more reliably diagnosed in other studies. My other CPM-positive cases involved partial losses that required more sensitive methods to detect (ie. CGH), and polyploidy which requires either flow cytometry or cytogenetic analysis of upwards of 50 metaphases. Additionally, the mean maternal age for each cohort – 36.25 years for IVF and 35.25 years for IUI – was likely greater than that of the natural conception cohort, however due to both the Ma lab’s failure to record maternal age for many cases prior to 2011 and the absence of this data in most of the studies compiled for the natural conception cohort, I was unable to control for this difference. This preponderance of advanced maternal age for ART cases likely biased my findings toward greater numbers of abnormalities detected. Finally, I included both SGA and IUGR pregnancies in both 80 my cohorts, which may also have inflated the incidence of CPM detected due to the increased incidence of CPM for cases with fetal growth restriction (Table 3.1). Despite these design flaws, my results were consistent with previous studies which, based on invasive testing using CVS, found no increased incidence of CPM in ART versus spontaneous pregnancies (Huang et al., 2009; Jacod et al., 2008). Since CVS is done at a single site in the placenta, I considered that this methodology might miss mosaicism that could be picked up by sampling multiple sites in the term placenta, however my results using this methodology concur with what has previously been reported from investigation of term placentas from ICSI pregnancies —that there is no significant increase in incidence of CPM between ART and natural conception pregnancies (Minor et al., 2006). A pilot study previously published by the Ma research group suggested IVF pregnancies might be at higher risk of CPM (Chan Wong et al., 2012), however by increasing the sample size of the IVF cohort, this difference was less striking. For this objective, half of the cases of CPM detected for IVF pregnancies were for low-level tetraploidy (2/4). Since polyploidy (triploidy, tetraploidy etc.) is undetectable by molecular methods such as CGH and sequencing (used for cffDNA screening/NIPT), these types of abnormalities would not usually be detected by prenatal testing unless an invasive procedure was required and traditional karyotyping was performed. As the Ma research group works with term placental tissue, I was able to use flow cytometry to detect polyploidy, but for most pregnancies the placenta would not be examined for the presence of polyploidy unless there were some pathological indication to do so. Although CPM for tetraploidy has been observed in several IUGR cases (Cowles et al., 1996; Leschot et al., 1996; Wilkins-Haug et al., 1995), both babies born from low-level mosaic tetraploid placentas in this study were of average gestational age and had no complications at birth. The low level of tetraploidy in these two cases may account for the lack of 81 perinatal complications, however healthy deliveries of average gestational age have also been reported for cases with CPM for tetraploidy (Leschot et al., 1996; Minor et al., 2006). The third CPM-affected IVF case was the 47,XXY non-mosaic male baby previously published by a former member of the Ma research group (Chan Wong et al., 2012). All 5 placental sites examined for this case showed a 47,XXY profile by CGH, but 2/5 sites had an additional trisomy 13. This infant was of average gestational age and had no perinatal complications, which has been previously observed for low-grade trisomy 13 CPM (Etoubleau et al., 2011), however trisomy 13 in the placenta has also been reported for cases of IUGR (Farra et al., 2000; Hall et al., 2013; Leschot et al., 1996; Miura et al., 2006), maternal hypertensive disorder (Dotters-Katz et al., 2018) and spontaneous abortion (Farra et al., 2000; Leschot et al., 1996). Whole or partial trisomy 13 CPM has also been detected in IUGR pregnancies in combination with other trisomies or monosomies detected in placental tissues by CGH (Amiel et al., 2002; Robinson et al., 2010). The fourth IVF case for which CPM was detected was a segmental trisomy of chromosome 13 present in 2/3 sites examined, though unfortunately birth weight and perinatal outcome were not collected for this pregnancy. Since this trisomy was detected by CGH alone and could not be confirmed by either MLPA nor FISH as neither the telomere nor centromere were involved in the trisomy, these two samples should be ideally followed up with either microsatellite, array or sequencing to confirm the aneuploidy. I detected two additional segmental aneuploidies amongst the IUI cases. Both a proximal Xq deletion for SM12-841 and a proximal 11q duplication for SM12-842 were detected by CGH in 1/3 placental sites for each case. The infant with the proximal X segmental monosomy displayed fetal heart rate anomalies, but these were likely the result of nuchal cord (umbilical cord around the neck of the fetus). This proximal Xq region contains the XIST gene, transcribing a long non- 82 coding RNA integral for X-chromosome inactivation, which in the human placenta occurs post-fertilization and is random (Sahakyan et al., 2018). Reports of phenotypic evidence for proximal Xq monosomy in CPM are scarce, however monosomy for this region in adult females (due to deletions) is often phenotypically benign, with the most prominent consequence being severe skewing of the inactive X (Mercer et al., 2013). One gene likely involved in the severe intrauterine mortality of most 45,X conceptions, CSF2RA, which is essential for placental function, lies in the pseudoautosomal-one region (PAR-1) of chromosome X (Urbach and Benvenisty, 2009). The phenotype for the case I detected may have been normal because the PAR-1 region would be intact for the segmental mosaic, thus genes in this region, including CSF2RA, which are normally present in two copies for both males and females, would be intact. The IUI infant with CPM for the segmental 11q trisomy in 1/3 sites was SGA, and the attending nurse noted the neonate “looks IUGR”. Trisomy 11 is rarely found in either spontaneous abortions or CPM (Lestou and Kalousek, 1998) and is not often associated with adverse perinatal outcomes (Daniel et al., 2004; Farra et al., 2000; Wang et al., 1993). Since the segmental trisomy was detected at only one placental site and the shift as detected by CGH was small, suggesting mosaicism within the single site, this trisomy likely resulted from a mitotic error. It is possible this segmental trisomy contributed to the SGA phenotype of this infant, however due to the low level of abnormal cells detected for this case and an absence of molecular follow-up to confirm the abnormality, any connection between CGH results and perinatal outcome is uncertain. One of the earliest studies to examine CPM in ART pregnancies using invasive CVS procedures found a five-fold increase in the incidence of CPM in ART pregnancies versus naturally conceived controls (7.5% vs 1.5%) (In’t Veld et al., 1995). Subsequent studies using CVS failed to reproduce these results, finding incidences of aneuploidy in ART placentas ranging 83 from 0.8% to 1.15% (Huang et al., 2009; Jacod et al., 2008). These values were not significantly different from those observed for natural conception controls in the same studies (0.41% to 2.2%) nor from the 1-2% typically cited in the literature (Kalousek and Vekemans, 1996). Controlled studies such as these are now nearly impossible to reproduce, since invasive prenatal screening has largely been replaced by NIPT and patients are now only offered invasive testing to confirm abnormal results from non-invasive screens. On the other hand, since NIPT results are based on cell-free DNA originating from the trophectoderm-derived cells of the chorionic villi, this screening test is uniquely suited for detection of aneuploidy in the placenta (Flori et al., 2004). CPM has been found to account for 10% of false positive NIPT results, likely a conservative estimate since in 67% of cases the source of discrepancy between NIPT and fetal karyotype is unknown, and a small percentage of this could be accounted for by incidences where follow-up with CVS misses the aneuploid portion of the placenta (Hartwig et al., 2017). Amniocentesis can also be performed as an invasive follow-up to abnormal NIPT results, but since the majority of cells examined from amniocentesis are of fetal origin (Gosden, 1983), CPM would not be confirmed using this procedure. Since the DNA tested in NIPT originates from the syncytiotrophoblast (Tjoa et al., 2006) – which ranges across the entire placenta at the maternal interface – the DNA collected for NIPT may more accurately reflect the heteromorphism of placental DNA than the isolated sample obtained by CVS, ostensibly providing a more sensitive method for CPM detection than invasive testing. Future studies could explore the incidence of CPM in ART pregnancies through the use of NIPT, which has the advantage of now being widely offered for both normal as well as higher-risk pregnancies. This would allow for a controlled study at a reduced cost, as term placenta biopsy would likely only be necessary for confirmation of CPM in cases where detection occurred through 84 routine screening and testing. When confounding factors such as vanishing twin syndrome, maternal CNVs/mosaicism and maternal malignancies are ruled out, NIPT has been demonstrated to detect CPM with 90% accuracy using algorithms wherein the Z-score (copy number value) for the query chromosome lies between the expected values for euploidy and trisomy (Brison et al., 2018). No large-scale studies have been published to date on the incidence of CPM in pregnancies using NIPT as a screening method and following up with invasive testing (either CVS or amniocentesis) and confirmation in term placenta. Such a study could serve to confirm or update the commonly cited 1-2% incidence of CPM inferred from studies using single-site CVS, and subsequent studies could examine whether the incidence differs for ART pregnancies. A notable weakness of my study was a failure to account for maternal age in the vast majority of samples, though we may presume from the data available that maternal age is higher for ART than spontaneous pregnancies. Future studies would do well to take this into consideration, as it likely has an effect on the incidence of aneuploidy in ART conceptions, and possibly on the incidence of CPM for these pregnancies. Controlling for maternal age would strengthen any evidence in a study such as mine as to whether factors associated with ART might contribute to an elevated incidence of CPM. 85 Chapter 4: Aneuploidy and Mosaicism in ART pregnancies 4.1 Introduction Most whole chromosome aneuploidies that have serious phenotypic effects (reviewed in Section 1.3) are detected either during routine prenatal testing, or very soon after birth, if they are compatible with life. Balanced rearrangements and aneuploidies with milder phenotypes, such as sex chromosome trisomy, may not be detected until the affected individual reaches reproductive age, if they are even detected at all. The Ma laboratory has been collecting cord blood for cytogenetic analysis from ART conceptions, including IVF and ICSI, as well as natural conception controls, since 2002. Since neonates in Canada are not typically tested for chromosomal abnormalities without a prior indication for doing so, these cases provide a unique opportunity to compare the incidence of abnormalities in neonates conceived spontaneously with those of ART infants. As for my first objective (presented in the previous chapter), the purpose of my second objective was to determine whether some variable of the ART process puts children conceived using these methods at a greater risk of chromosomal abnormalities. 4.2 Case description Cases collected for this objective include term pregnancies conceived using natural conception, IVF and ICSI from 2002 to 2017, recruited as described in Section 2.1. The control cohort were healthy, naturally conceived pregnancies. Inclusion criteria for this cohort were singleton and multiple pregnancies resulting in live birth, conceived spontaneously after less than one year of unprotected intercourse. Inclusion criteria for the test cohorts were any singleton or multiple term pregnancies conceived using ART, including IVF and ICSI. These pregnancies were from couples with either 86 female, male or unknown sources of infertility. Maternal age, BMI and gestational age were available for some but not all participants, and these are summarized in Table 4.1. Table 4.1: Maternal Clinical Information – Range and (Mean) NC (n=336) IVF (n=220) ICSI (n=273) ART (IVF+ICSI) (n=493)* Maternal Age 19-43 (31.7) 22-46 (35.6)∆ 28-44 (35.8)∆ 22-46 (35.8)∆ Maternal BMI 14-49 (25.4) 16-42 (25.5) 17-44 (24.3) 16-44 (25.0) Gestational Age (wks) 29-42 (38.7) 27-42 (37.7)∆ 31-42 (38.8) 27-42 (38.1)∆ Birthweight (g) 853-5050 (3217.3) 820-5280 (2981.1) ∆ 1655-4680 (3262.5) 820-5280 (3101.4) * n is greater than sum of IVF and ICSI because some samples received were designated as “IVF and ICSI”. ∆ significant difference from NC value (P<0.05, two-sample t-Test) Of note, the mean maternal age is significantly higher for both the IVF and ICSI groups and all ART combined, compared to NC, using a two-sample t-test. This is to be expected, as fertility decreases with age, and many women who delay pregnancy until they fall within the advanced maternal age category of 35 years or older may require ART to get pregnant. Mean gestational age was also significantly shorter for both IVF and combined ART pregnancies, compared to NC, but no difference was observed for ICSI. Finally, birth weight for IVF pregnancies was significantly lower compared to NC and ICSI, but combined birth weight for all ART pregnancies was not significantly different from NC. The use of patient questionnaires to collect demographic data, including parental ethnicity and country of origin, was not implemented until later in the study. From the available data, the ethnic breakdown for each cohort is visibly dissimilar (Table 4.2). This is primarily due to the majority of natural conception cases originating from Surrey Memorial Hospital, which serves a primarily South Asian population, and consequently, the proportion of mothers of South Asian ethnicity is distinctly greater for spontaneous conceptions. Comparatively, white mothers represent the vast majority of ICSI cases for which demographic data was collected. 87 Table 4.2: Breakdown of Maternal Ethnicity per Conception Cohort NC IVF ICSI ART Unknown 210 (62.5%) 140 (63.6%) 205 (80.4%) 349 (70.6%) White 44 (13.1%) 34 (15.5%) 43 (16.9%) 85 (17.2%) East Asian 18 (5.3%) 10 (4.5%) 4 (1.6%) 19 (3.8%) South Asian 58 (17.3%) 27 (12.3%) 2 (0.8%) 31 (6.3%) Other* 6 (1.8%) 9 (4.1%) 1 (0.4%) 10 (2.0%) TOTALS 336 220 255 494 Predominating known ethnicity for each cohort is highlighted in bold *includes Aboriginal, Black, Latino, Pacific Islander and mixed race 4.3 Results In total, 830 cases were included in the analysis. Cases analyzed by either traditional karyotyping analysis or CGH were included. CGH was used to detect aneuploidy for cases where culture of cord blood lymphocytes failed. Since CGH is only able to detect copy number changes and not balanced rearrangements, CGH analysis may have missed balanced translocations, rearrangements and structural polymorphisms for these cases. CGH is also limited for the detection of mosaic profiles that occur at levels lower than than 32% (Lomax et al., 2000). Therefore, it is possible low-level mosaicism may have been missed in any of the samples for which traditional karyotyping was not available. The numbers of cases in each conception cohort as well as the numbers of cases for which results were obtained either by karyotyping or CGH are summarized in Table 4.3. Cases with numerical and/or structural abnormalities were detected in each conception group. The results for all fully karyotyped cases are summarized in Table 4.4. No unbalanced genetic profiles were detected in any of the cases for which only CGH results were available. 88 Table 4.3: Summary of cases analyzed – by cohort and analysis method Analysis method NC IVF ICSI ART (IVF+ICSI)* All Karyotyping 288 188 220 424 712 CGH 48 32 35 70 118 Total: 336 220 255 494 830 * n is greater than sum of IVF and ICSI because some samples received were designated as “IVF and ICSI”. Table 4.4: Summary of results from karyotyped cases Group Normal Karyotype (%) Abnormal Karyotype (%) Total (P value*) NC 283 (98.3) 5 (1.7) – 3 (1.0) 47,XYY – 1 (0.3) 47,XX,+21 mosaic – 1 (0.3) 47,XY,+der(15) mosaic 288 IVF 185 (98.4) 3 (1.6) – 2 (1.1) 47,XXY – 1 (0.5) 45,X mosaic 188 (1) ICSI 216 (98.2) 4 (1.8) – 1 (0.3) 46,XY,del(Y) – 1 (0.3) 45,X – 1 (0.3) 47,XX,+21 – 1 (0.3) 47,XXY 220 (1) ART (IVF + ICSI) 417 (98.3) 7 (1.7) 424 (1) * by comparison with NC using Chi-Square test Natural Conception Abnormalities A total of five cases were found to have abnormal chromosomal complements by karyotype analysis in the natural conception cohort, summarized in Table 4.5. Table 4.5: Cytogenetic Abnormalities Detected in Natural Conception Pregnancies Sample ID Karyotype Maternal Age Perinatal outcome Detection Method(s) SM12-905 47,XYY unknown normal karyotype SM12-1019 47,XX,+21[7]/46,XX[93] unknown normal karyotype SM12-1069 47,XYY 29 normal karyotype SM13-1144 47,XY,+mar[18]/46,XY[32] 40 LGA karyotype, SNP array SM13-1221 47,XYY 32 normal karyotype 89 Three male babies (SM12-905, SM12-1069 and SM13-1221) were found to have the same non-mosaic sex chromosome aneuploidy, 47,XYY, by cytogenetic analysis. A female baby (SM12-1019) was found to be mosaic for trisomy 21 in 7/100 metaphases examined, with the remaining 93/100 cells having a normal 46,XX complement. One male natural conception case (SM13-1144) was mosaic for an acrocentric marker chromosome in 18/50 metaphases examined from cord blood lymphocytes (Figure 4.1 A). Based on banding patterns, the marker chromosome was suspected to consist of the p arm, centromere and proximal q arm of chromosome 15. CGH and MLPA were performed in an attempt to confirm the identity of the marker chromosome, but both assays failed to detect any genetic imbalance (Figure 4.2). Peripheral blood from the same child (SM14-1246) was drawn at 6 months of age for follow-up karyotype analysis and cultured peripheral blood lymphocytes revealed the same acrocentric marker chromosome in 52/100 metaphases examined (Figure 4.1 B). Figure 4.1: Karyotypes showing mosaicism for marker chromosome. A) SM13-1144: 18/50 karyotypes from cord blood lymphocytes were 47,XY,+mar. B) SM14-1246: 52/100 karyotypes from peripheral blood lymphocytes were 47,XY,+mar. DNA from peripheral blood was sent to TCAG at The Hospital for Sick Children (Toronto, ON) for SNP array analysis and results confirmed mosaic trisomy for 3.2 Mb of the proximal q arm of chromosome 15 (Figure 4.3). The proximal q arm of Chromosome 15 contains several recombination hotspots (or breakpoints), associated with the formation of rearrangements such as 90 the marker chromosome identified in this child, as well as pathogenic rearrangements including deletions and duplications of the imprinted region of Chromosome 15, associated with Prader-Willi/Angelman Syndromes (Amos-Landgraf et al., 1999; Huang et al., 1997). The SNP array data suggests the marker chromosome for this case terminates at “breakpoint 2”, which is upstream of the imprinted Prader-Willi/Angelman syndrome region of chromosome 15. The marker chromosome contains only one gene listed as potentially pathogenic: NIPA1, non-imprinted gene in Prader-Willi syndrome/Angelman syndrome chromosome region 1. Patients with small marker chromosomes of this conformation rarely present with any of the symptoms associated with longer isochromosomes of chromosome 15, and NIPA1 itself is not associated with dosage sensitivity (Huang et al., 1997; Leana-Cox et al., 1994). Figure 4.2: Molecular testing to identify marker chromosome for SM13-1144 cord blood. A) Chromosomal CGH testing suggests a balanced male profile with no gains for any acrocentric chromosomes. B) MLPA testing did not identify any imbalances. 91 Figure 4.3: SNP array using peripheral blood DNA for SM14-1246 to identify marker chromosome. SNP array identified mosaic trisomy for a 3.2 Mb region of proximal chromosome 15, indicated by an additional track for B Allele Frequency and a slight increase in Log R Ratio for the region highlighted in blue. The probe data presented encompasses the area of chromosome 15 within the red box of the ideogram at the bottom of the figure. IVF Abnormalities For the IVF cohort, a total of three cases were found to have abnormal chromosomal complements, summarized in Table 4.6. Two male babies (SM09-478 and SM12-815) were found by karyotype analysis of cord blood lymphocytes to be 47,XXY. One of these (SM09-478) is the case previously discussed for Chapter 3 which was found to have CPM for an additional trisomy for chromosome 13 in 2 of 5 placental sites examined by CGH. Table 4.6: Cytogenetic Abnormalities Detected in IVF Pregnancies Sample ID Karyotype Maternal Age Perinatal outcome Detection Method(s) SM09-478 47,XXY unknown normal karyotype SM10-523 45,X[7]/46,XX[43] unknown normal karyotype SM12-815 47,XXY unknown normal karyotype The third IVF case with an abnormal karyotype was a female baby (SM10-523) with mosaicism for monosomy X in 7/50 metaphases examined from cord blood lymphocytes. 92 ICSI Abnormalities A total of four ICSI cases were found to have abnormal chromosomal complements, summarized in Table 4.7. One male baby (SM04-74) had a deletion of most of the q arm of chromosome Y in all metaphases examined from cord blood lymphocytes (Figure 4.4). A female baby in a set of twins (SM05-111A) was found to be 45,X in all metaphases examined, while her female twin had a normal 46,XX karyotype. Another female baby (SM10-575) was 47,XX,+21 in all metaphases examined. The last abnormality detected within the ICSI cohort was a male baby (SM16-1439) for whom all metaphases from cord blood lymphocytes were 47,XXY. Table 4.7: Cytogenetic Abnormalities Detected in ICSI Pregnancies Sample ID Karyotype Maternal Age Perinatal outcome Detection Method(s) SM04-74 46,XY,del(Y) unknown normal karyotype SM05-111A 45,X unknown normal karyotype SM10-575 47,XX,+21 unknown normal karyotype SM16-1439 47,XXY 37 normal karyotype In total, abnormal complements were identified in 5/336 (1.49%) of naturally conceived cases examined cytogenetically or by chromosomal CGH using cord blood lymphocytes. By comparison, 3/220 (1.36%) of IVF cases and 4/255 (1.57%) of ICSI cases were found to have abnormal karyotypes. When all IVF and ICSI cases were combined into one group, 7/494 (1.42%) of these cases were cytogenetically abnormal. The proportions for neither IVF, ICSI or both ART groups combined proved to be significantly different from the proportions of abnormal to normal cases from the natural conception control cohort when compared using the Chi-Square statistical test (P=1 for all comparisons). 93 Figure 4.4: Karyotype for male IVF case (SM04-74) with Y deletion. All cord blood lymphocytes examined had a deletion of most of the terminal q arm of chromosome Y, indicated in the red box. The majority of abnormalities detected in each cohort are sex chromosome aneuploidies, while the remainder were autosomal. For 3/5 (60%) of NC, 3/3 (100%) of IVF and 3/4 (75%) of ICSI cases wherein an abnormality was detected, a sex chromosome was involved (Table 4.4). As microsatellite analysis was not performed to examine the parental origin of any abnormalities, I could not verify whether they originated from maternal or paternal errors in meiosis, or from post-zygotic nondisjunction (PZN) in the dividing embryo. 4.4 Discussion For the second objective of my dissertation, I examined the incidence of chromosomal abnormalities in term pregnancies achieved by ART compared to spontaneous pregnancies. Through karyotyping cord or peripheral blood lymphocytes or analyzing CGH profiles of lymphocyte DNA, I failed to find any significant difference in the incidence of constitutional 94 chromosomal aneuploidy between children conceived using ART techniques including IVF (3/220, 1.36%) and ICSI (4/255, 1.57%), and children conceived spontaneously (5/336, 1.49%). The majority of abnormalities detected in each cohort were for sex chromosome aneuploidy – 60% for NC, 100% for IVF and 75% for ICSI. Early detection of trisomies 13, 18 and 21 as well as sex chromosome aneuploidies is possible now that NIPT using cffDNA is now widely offered to pregnant women in North America, whereas prior to NIPT, early non-invasive screening could only detect pregnancies at risk of trisomy 13, 18, 21 and monosomy X, and confirmation required invasive testing by amniocentesis or CVS (Harris et al., 2018). Since my cohorts were recruited between 2002 and 2017, most of the women in the study were pregnant before 2011 when NIPT using cffDNA began being offered, and may not have even been aware of more benign sex chromosome aneuploidies in the fetus, such as Y chromosome deletions, 47,XXY or 47,XYY, except for cases of advanced maternal age (>35 years) wherein invasive testing was pursued. Current estimates of the incidence of chromosomal abnormalities in live-born infants are based on cytogenetic studies conducted up to the 1980s. These studies found one in every 153 newborns (0.65%) had at least one chromosomal abnormality – including either autosomal (0.16%) or sex chromosomal (0.22%) aneuploidy and both balanced (0.20%) and unbalanced (0.06%) structural rearrangements (Benn, 2016). The most comparable modern study published in the interim was by Forabosco et al. (2009), an Italian group which examined the cytogenetic results of 88 965 second trimester amniocenteses conducted in Italy between 1995 and 1996. This two-year period was selected to avoid ascertainment bias introduced once invasive testing became a refractive test for abnormal NIPT results. This group also found an incidence of 0.65% for any chromosomal abnormality, in pregnancies that included both women younger than 35 years as well 95 as women of advanced maternal age. Even though I did not observe any significant difference between natural conception and ART cohorts, the incidence of aneuploidy I detected for all cases (1.44%) is significantly higher than the 0.65% reported from these previous studies. These results could be explained by the observation that mean maternal age has increased in recent years (Mills et al., 2011), and despite advancements in aneuploidy detection over this time period, most abnormalities involving sex chromosomes and/or abnormalities that are phenotypically benign would not warrant pregnancy termination, and we might expect to see more of these types of aneuploidy as the mean age of childbearing mothers increases. Furthermore, the majority of abnormal cases I detected were pre-cffDNA testing, and it is likely these benign aneuploidies and rearrangements were not indicated by maternal serum screening and the patients either had no cause to pursue invasive testing, or, as is frequently the case with ART pregnancies at least, couples declined invasive testing to avoid the risk of spontaneous loss (Gjerris et al., 2008). My sample size of 830, while adequate for my objective, was likely too small to accurately reflect the incidence of rare abnormalities estimated from the aggregated studies from pre-1980s and the Italian amniocentesis cohort. For instance, unbalanced Robertsonian translocations occur roughly once in every 13,400 cases, whereas more common abnormalities, such as 47,XXY, occur once in every 800 cases (Benn, 2016). To illustrate this point, I found one case of a supernumerary chromosome in 830 cases, which would be expected based on the incidence of 1/685 reported by Forabosco et al. (2009). However other than two cases of pericentric inversion of chromosome 9 – considered a polymorphism and not a structural abnormality – I observed no balanced rearrangements in any of the infants karyotyped, despite their reported incidence of 1/285 in second trimester conceptions, 1/500 in the general population, and an even higher frequency among individuals with reproductive difficulty (Forabosco et al., 2009; Morin et al., 2017). These 96 cases represent a small snapshot of reproductive-age Western Canadians, and within this sample, the method of conception does not appear to be associated with an increased risk of any chromosomal rearrangement. As I was unable to perform microsatellite analysis prior to the conclusion of my data collection, I could not determine the origin of abnormality for my aneuploid cases. However, from studies which have determined both the parental (meiotic) and mitotic origins of similar abnormalities, I can estimate that the majority of errors in my natural conception cohort likely arose from paternal meiotic errors (3/5 cases were non-mosaic 47,XYY) while the remaining two cases, being mosaics with a smaller population of abnormal cells compared to normal cells, were likely the result of PZN. In contrast, the IVF and ICSI cohorts were more evenly balanced, with regards to likely origin of abnormality (e.g. maternal or paternal), to be discussed below. The vast majority of errors contributing to autosomal trisomy – as well as 47,XXX – can be traced to maternal MI, with the remainder occurring during maternal MII, post-zygotic mitoses, and very infrequently, during paternal meiosis (Hassold et al., 2007). For the remainder of aneuploidies involving the sex chromosomes, errors in paternal meiosis are as likely as maternal meiotic errors to contribute to 47,XXY (Thomas and Hassold, 2003) and the extra Y is always paternally inherited for 47,XYY conceptions, usually as a result of MII errors (Robinson and Jacobs, 1999). For 45,X conceptions, origin-of-abnormality studies have found that the origin of the single retained X is maternal in 71% to 100% of cases examined (Grande et al., 2019). It has been suggested the increased contribution of paternal errors to sex chromosome aneuploidy stems from the reduced area of crossover between the X and Y chromosomes (Hassold and Hunt, 2001). Pseudoautosomal region 1 (PAR1) is a ~2.7Mb region of homology between the distal P arms of chromosomes X and Y, and behaves as a recombination hotspot, however failure to achieve 97 recombination in PAR1 is the most common instigator for X-Y nondisjunction in paternally-derived 47,XXY (Poriswanish et al., 2018; Thomas and Hassold, 2003). De novo deletions of the Y chromosome are estimated to originate almost exclusively from errors in paternal meiosis, as these deletions are never reported as mosaic (Silber and Repping, 2002). These deletions are observed in infertile men nearly 50 times more frequently than in the general population, and up to 12% of men with azoospermia and oligozoospermia have microdeletions of the AZF regions of the Y chromosome (Gekas et al., 2001) Non-mosaic 47,XXY was the most frequent abnormality detected in the ART cohorts for this study, found in 2/3 of abnormal IVF cases and 1/4 abnormal ICSI cases. None were detected among natural conceptions. Since the additional X chromosome in 47,XXY is as likely to originate from errors in maternal versus paternal meiosis, it would have been valuable to have tested these aneuploidies to determine its parental origin. Though paternal age can have a small effect, advanced maternal age is by far the greatest associated risk factor for the incidence of 47,XXY, especially when the additional X results from nondisjunction at maternal MI (De Souza et al., 2010; Harvey et al., 1991; MacDonald et al., 1994). The average maternal age for both ART cohorts in this study was significantly greater than that of the natural conception controls, therefore this may have contributed to the preponderance of 47,XXY cases for ART conceptions. A 45,X case was detected in each of the IVF and ICSI cohorts, as a mosaic and non-mosaic monosomy, respectively. The mosaic IVF case most likely resulted from PZN, as the monosomy was detected in a small population of cord blood lymphocytes (7/50). The monosomy X detected in the ICSI case is most likely to have arisen due to a paternal meiotic error, as it was detected in all cells examined, and in all origin-of-abnormality studies conducted for non-mosaic monosomy X, the single remaining X is maternal in origin – implying loss of the paternal X – in the vast 98 majority of cases (Grande et al., 2019). Given that the odds of the retained X being maternal should only be twice those of it being paternal (66% vs 33%, since all female gametes should have one X, versus half of male gametes) it has been suggested this disproportionate representation of paternal loss for monosomy X cases may be related to differences in viability based on parental imprinting, as phenotypic effects for adults with monosomy X differ depending on the parental origin of the retained X (Bondy et al., 2012; Grande et al., 2019). Trisomy 21 was detected in two cases: one was a natural conception case in which the aneuploidy was mosaic and, since the 47,XX,+21 cell population was much smaller (7/100) than that of normal, 46,XX cells, this aneuploidy was likely the result of PZN. The other was an ICSI case with non-mosaic trisomy 21, which was most likely of maternal origin. Maternal age for this case was not recorded at collection, however considering maternal age was significantly greater in our ICSI cohort compared with natural conception, advanced maternal age could have been a factor for this case, as it is known to greatly influence the incidence of autosomal trisomy. I observed two additional abnormal cases for this objective. The first was an ICSI case with a large deletion of the heterochromatic region of chromosome Y. This case has previously been published by former members of the Ma research group, and both the infant’s father and paternal grandfather were found to have the same deletion (Minor et al., 2007). The second abnormal case was a natural conception for which a small supernumerary chromosome was detected in 18/50 cord blood lymphocytes and 52/100 peripheral blood lymphocytes at a 6-month follow-up. SNP array investigation of this case confirmed the supernumerary chromosome was the p arm and proximal q of chromosome 15. Markers derived from chromosome 15 are by far the most frequently observed supernumerary chromosomes, and are present as mosaics in roughly 20% of cases (Crolla et al., 2005). Although generally the incidence of de-novo supernumerary 99 chromosomes is not age-dependent, markers derived from chromosome 15 in particular are associated with increased maternal age, reflected by the preponderance of cases which are non-mosaic and likely result from errors in maternal meiosis. This particular natural conception case, possessing the supernumerary chromosome in roughly 50% of cells, could have arisen either through subsequent mitotic loss of a parentally (likely maternally) contributed meiotic error, or could have arisen de-novo due to an error in post-zygotic mitosis. Advanced maternal age (40 years) was likely a contributing factor for the cytogenetic outcome of this pregnancy, however the infant appeared unaffected and suffered from no adverse perinatal outcomes. Dosage sensitivity for the region of Chromosome 15 affected in this case has not been reported, and it is likely this marker chromosome is phenotypically benign (Huang et al., 1997). The preponderance of 47,XYY cases in the natural conception cohort for my study is of particular interest. I detected three 47,XYY cases among 336 newborns: a rate nearly ten times higher than expected based on previous studies (Benn, 2016). The incidence of 47,XYY is not consistent between populations, and ranges from 1/1000 from a study using a Danish cytogenetic registry (Berglund et al., 2019), to 1/3000 in a Japanese study on newborns (Maeda et al., 1991), to 1/3200 in a study from invasive testing in Hong Kong (So et al., 2017) and 1/4000 in a Hungarian study using prenatal results from amniocentesis (Nagy et al., 2015). The increased incidence of 47,XYY in the natural conception cohort (1/113) and even our entire study combined (1/277) as compared to these other studies could be explained either though population variation, or sampling error. While demographic data (such as ethnicity) was not collected for all samples in our study – due to implementation of a questionnaire to gather such data in the final years of the study – the majority of natural conception samples were collected from Surrey Memorial Hospital in the Greater Vancouver Regional District, a hospital which serves a primarily South Asian 100 population. As such, there is an overrepresentation of patients of South Asian ethnicity in the natural conception cohort, whereas for the ART cohort, most of the patients for which data is available identified as White (Table 4.2). Genetic variation between discrete ethnic populations can predispose individuals of certain ethnic backgrounds to disease susceptibility, and genome-wide association studies (GWAS) are continuously uncovering genetic variants that contribute to impaired spermatogenesis, some of which may cluster within certain ethnic populations (Krausz and Riera-Escamilla, 2018). However, in the absence of any evidence thus far of intra-ethnic variation in genes involved in sperm aneuploidy, I conclude that the 10x increased risk incidence of 47,XYY I observed in the natural conception cohort is most likely sampling error, which could be resolved with the inclusion of more cases. Overall, the incidence of constitutional chromosomal abnormalities I observed for my second objective, while marginally greater than what has previously been published for newborns, does not appear to differ at all between term pregnancies achieved either spontaneously, or using ART. 101 Chapter 5: Conclusion ART has been helping couples with reproductive difficulty achieve pregnancy for nearly four decades. The techniques involved in procedures such as IVF, ICSI and IUI have been evolving since these methods were first implemented, making it difficult to ascertain whether factors implicit in these procedures have tangible effects on pregnancy outcome, not to mention long-term effects on the health of children conceived by ART. To further obscure any associations between ART and pregnancy outcome, individuals who seek ART are often affected with at least one of many sources of infertility, which can also contribute to both pre- and perinatal complications, in spite of whether or not the affected individuals use ART to conceive. The aim of my dissertation was to determine whether pregnancies conceived using ART procedures are at an increased risk of chromosomal abnormalities and mosaicism. For Chapter 3, my objective was to examine DNA from term IVF placentas at a minimum of 3 sites to detect large (>10Mb) chromosomal imbalances, as well as polyploidy. This objective also included a pilot cohort of term IUI placentas, to control for the impact of infertility in the absence of in-vitro embryo culture. I found the incidence of CPM in IVF placentas to be increased, though not significantly, compared to what has previously been observed in term placentas from natural conceptions. The IUI placentas I investigated also showed an increased incidence of CPM, though since the total number of placentas examined was very small (n=15), these results should be interpreted with caution. For Chapter 4, I investigated the chromosomal constitution of cord blood (or peripheral blood) lymphocytes from live-born infants conceived using IVF, ICSI or either technique, and compared the results with those from live-born, spontaneously conceived infants. I did not find any differences in the incidence of chromosomal abnormalities – including whole-chromosome 102 aneuploidy, polyploidy, deletions, duplications, rearrangements, or mosaicism for any of these abnormalities – between the ART infants and naturally conceived infants investigated by my research group. Nevertheless, the total incidence of abnormalities detected for these groups was greater than what has previously been reported in similar studies examining both series of live-born infants, as well as the cytogenetic results from invasive prenatal testing with no indication of abnormal pregnancy. For my dissertation, I specifically focused on detecting abnormalities in term pregnancies achieved by ART, and used healthy, naturally conceived term pregnancies as controls. It would have been beneficial to compare the ART groups to infertile controls who had managed conceive (in order to control for infertility in our treatment groups), however such pregnancies are often troublesome to recruit due to the difficulty of diagnosing infertility in individuals who are eventually able to conceive, and the decreased incidence of spontaneous pregnancy for individuals with reproductive difficulty. I included the cohort of IUI placentas in Chapter 3 to mitigate this limitation, however due to time constraints, I fell short of the numbers required in my power analysis to determine significance. Notable limitations to my studies were my inability to control for advanced maternal age between cohorts, and inclusion of SGA and IUGR infants for ART cohorts, which have been discussed in previous chapters. Additional limitations relate to the lack of resolution implicit in both the cytogenetic and molecular techniques I used for aneuploidy detection. Even though traditional, G-banded karyotyping of chromosomes (used in Chapter 4) is unrivalled for the detection of balanced rearrangements and low-level mosaicism, it is less efficient than current CNV detection technologies (ie. whole or targeted sequencing and microarrays) for detecting imbalances smaller than 10Mb. These more sensitive, modern techniques may have detected 103 additional CNVs in both my objectives – however the Ma research group, which began collecting and processing these cases in 2002, did not have such tools at its disposal when the study began, and I only had the resources to use them for several cases to resolve or verify suspected abnormalities. It also would have been optimal to confirm some of the abnormalities detected by chromosomal CGH in Chapter 3 using microsatellite PCR or microarray, however this was not possible prior to the study’s conclusion. Another limitation to studies investigating the safety of ART is the tendency for its associated techniques and protocols to improve over time, making it difficult to conclude that any findings, especially with regards to now-adult “children” conceived using ART decades ago. Even the timeframe of roughly fifteen years during which our cases were compiled presents a large window of time – almost half the time ART has been in practice – over which we may have detected abnormalities which are the consequences of protocols since abandoned. It has been proposed that methods be put in practice to ensure fertility clinics collect data on all variables involved in embryo culture and handling, including but not limited to ovulation induction protocols, disturbance of embryo culture, O2 concentration, brands of culture media used, and use of fresh or previously frozen embryos for transfer; as all of these variables have been observed to impact embryo quality and/or treatment outcome (Kirkegaard et al., 2013; Kleijkers et al., 2016; Lambalk et al., 2017; Qiang Zhang et al., 2010; Zhao et al., 2016). Registries for the collection of data pertaining to ART treatment exist in Europe (European IVF Monitoring of ESHRE), the United States (Society for Assisted Reproductive Technology), Africa (African Network and Registry for Assisted Reproductive Technology) and globally (International Committee Monitoring Assisted Reproductive Technologies), although these databases focus primarily on more broad indicators of ART success, such as number of cycles, rate of success per cycle, embryo 104 transfers per cycle and multiple births (De Geyter et al., 2016; Fauster, 2019). While the collection of such data is useful for monitoring the progression of ART with regards to successful outcomes (e.g. increased chances of singleton births per cycle of treatment), it does little to tease out which treatment-associated variables (e.g. ovulation induction regimens, culture media, micromanipulation techniques, etc.) are associated with the success and safety of ART pregnancies and their long-term impact on the health of resulting children. In this dissertation, I investigated the broad genetic outcomes of ART versus natural conception pregnancies, and found little evidence to suggest these methods are associated with large CNVs or chromosomal rearrangements in either the placenta, or the fetus/infant. These results do not rule out the possibility that smaller CNVs, rearrangements or epigenetic effects may result from the use of these technologies. As discussed in Chapter 3, NIPT using cffDNA may prove a valuable tool for further investigating CNV between natural conception and ART pregnancies, as these methods are moving toward being universally implemented (at least in North America and Europe) to test for CNV as early as 10 weeks into pregnancy, and can provide information on both fetal and placental chromosomal complements (Birko et al., 2019; Hartwig et al., 2017; Rego De Sousa et al., 2018). Investigation of potential epigenetic effects of ART procedures with regards to pre- and post-natal health suggest that environmental variables introduced via in-vitro gamete and embryo manipulation and culture may have both short- and long-term effects on ART children. Meta-analyses on cardiovascular and metabolic health found children conceived using ART were at an increased risk of elevated blood pressure, fasting blood glucose and total body fat composition compared to spontaneously conceived children, however the risk was eliminated for younger cohorts of ART children, suggesting that current ART practices (including milder ovarian 105 stimulation, SET and improved culture protocols) may have milder epigenetic consequences for cultured embryos (Guo et al., 2017). The incidence of imprinting disorders, such as BWS, Prader-Willi Syndrome, Angelman Syndrome and Silver-Russel Syndrome (discussed in Section 1.2.3), has been extensively studied in ART children, although many of these studies suffer from either small sample sizes or failure to control for the impact of parental infertility (Berntsen et al., 2019). Only BWS has consistently been observed at higher than expected frequencies in ART children, specifically for cases originating from hypomethylation at the KCNQ1OT1 DMR (Tenorio et al., 2016; Vermeiden and Bernardus, 2013). Aberrant methylation in the placenta may account for the increased incidence of perinatal complications (such as LBW, IUGR and preterm birth) for ART pregnancies. DMRs such as KCNQ1OT1 and H19/IGF2 as well as LINE-1 and ERVFRD-1 transposon families are hypomethylated in ART placentas compared to natural conception controls, which may lead to lower birth weight via dysregulation of genes involved in the formation of the syncitiotrophoblast, and the progression of maternal immunotolerance toward the fetus (Choux et al., 2018). Such epigenetic differences between ART and non-ART pregnancies have been detected in first trimester CVS for genes involved in cell cycle, transcription regulation and trophoblast outgrowth, all of which could contribute to placental insufficiency for ART pregnancies (Xu et al., 2017). When considering epigenetic effects on children conceived by ART, one must bear in mind the potential for genetic or epigenetic dysregulation in the infertile parents to be passed on to their offspring, and study design should be taken into account when interpreting results from these studies. Comparison of placental methylation and expression patterns between ART, fertile donor-egg and spontaneous conceptions found both ART as well as donor-egg placentas are aberrantly methylated compared to control placentas, suggesting that some part of the ART procedures 106 themselves may have an effect on placental methylation, independent of parental infertility (Song et al., 2015). Modifiable variables introduced by ART, including oxygen concentration and fresh versus frozen embryo transfer, contribute to potentially detrimental global methylation differences observed in ART placentas from LBW pregnancies (Ghosh et al., 2017). These observations of dysregulated methylation for ART conceptions are likely related to overlap between the window of development during which embryos cultured in vitro are exposed to exogenous factors, and the period of embryogenesis wherein two important processes – global demethylation and maintenance of methylation at imprinted regions – are sustained in the dividing embryo (Menezo et al., 2017). If environmental factors introduced by ART can upset the delicate balance during this sensitive period of embryogenesis, the effects may result in the metabolic dysfunction of the placenta, leading to both fetal growth pathologies and long-term epigenetic changes for individuals conceived by ART. Interestingly, the ART-induced epigenetic differences at the H19/IGF-2 locus are influenced by the presence of a common polymorphism in the paternal allele, demonstrating that genetic as well as environmental variables can influence the perinatal outcome of ART pregnancies (Marjonen et al., 2018). It is my hope that my dissertation will add support to the notion that pregnancies achieved through ART are not at an explicit disadvantage compared to spontaneous pregnancies for large imbalances, rearrangements or CPM. Nevertheless, a better understanding of the genetic and environmental factors implicit in infertility and ART regimens, respectively, will lead to both improved outcomes of infertility treatment as well as improved health for children conceived using these practices. 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Thesis/Dissertation
eng
Reproductive and Developmental Sciences
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Investigation of aneuploidy and mosaicism throughout pregnancies conceived through assisted reproductive technology (ART)
Text