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

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

Electronic witness system in IVF—patients perspective

Objective The objective of this study is to evaluate patient concerns about in vitro fertilization (IVF) errors and electronic witness systems (EWS) satisfaction. Design The design of this study is a prospective single-center cohort study. Setting The setting of this study was located in the private IVF center. Patient(s) Four hundred eight infertile patients attending an IVF cycle at a GENERA center in Italy were equipped with an EWS. Intervention(s) Although generally recognized as a very rare event in IVF, biological sample mix-up has been reported in the literature. For this reason, some IVF laboratories have introduced EWS with the aim to further reduce the risk of error during biological samples handling. Participating patients received a questionnaire developed through a Likert scale ranging from 1 to 6. Main outcomes measure(s) Patient concerns about sample mix-up without and with an EWS were assessed. Result(s) 90.4 % of patients expressed significant concerns relating to sample mix-up. The EWS reduced these concerns in 92.1 % of patients, 97.1 % of which were particularly satisfied with the electronic traceability of their gametes and embryos in the IVF laboratory. 97.1 % of patients felt highly comfortable with an IVF center equipped with an EWS. Female patients had a significantly higher appreciation of the EWS when compared to their male partners (p = 0.029). A significant mix-up event occurred in an Italian hospital during the study and patient's satisfaction increased significantly towards the use of the EWS after the event (p = 0.032). Conclusion(s) EWS, by sensibly reducing the risk for sample mix-up in IVF cycles, has been proved to be a trusted strategy from patient's perspective

Meiotic and mitotic aneuploidies drive arrest of in vitro fertilized human preimplantation embryos

Background The high incidence of aneuploidy in early human development, arising either from errors in meiosis or postzygotic mitosis, is the primary cause of pregnancy loss, miscarriage, and stillbirth following natural conception as well as in vitro fertilization (IVF). Preimplantation genetic testing for aneuploidy (PGT-A) has confirmed the prevalence of meiotic and mitotic aneuploidies among blastocyst-stage IVF embryos that are candidates for transfer. However, only about half of normally fertilized embryos develop to the blastocyst stage in vitro, while the others arrest at cleavage to late morula or early blastocyst stages. Methods To achieve a more complete view of the impacts of aneuploidy, we applied low-coverage sequencing-based PGT-A to a large series (n = 909) of arrested embryos and trophectoderm biopsies. We then correlated observed aneuploidies with abnormalities of the first two cleavage divisions using time-lapse imaging (n = 843). Results The combined incidence of meiotic and mitotic aneuploidies was strongly associated with blastocyst morphological grading, with the proportion ranging from 20 to 90% for the highest to lowest grades, respectively. In contrast, the incidence of aneuploidy among arrested embryos was exceptionally high (94%), dominated by mitotic aneuploidies affecting multiple chromosomes. In turn, these mitotic aneuploidies were strongly associated with abnormal cleavage divisions, such that 51% of abnormally dividing embryos possessed mitotic aneuploidies compared to only 23% of normally dividing embryos. Conclusions We conclude that the combination of meiotic and mitotic aneuploidies drives arrest of human embryos in vitro, as development increasingly relies on embryonic gene expression at the blastocyst stage

Meiosis and beyond – understanding the mechanistic and evolutionary processes shaping the germline genome

The separation of germ cell populations from the soma is part of the evolutionary transition to multicellularity. Only genetic information present in the germ cells will be inherited by future generations, and any molecular processes affecting the germline genome are therefore likely to be passed on. Despite its prevalence across taxonomic kingdoms, we are only starting to understand details of the underlying micro‐evolutionary processes occurring at the germline genome level. These include segregation, recombination, mutation and selection and can occur at any stage during germline differentiation and mitotic germline proliferation to meiosis and post‐meiotic gamete maturation. Selection acting on germ cells at any stage from the diploid germ cell to the haploid gametes may cause significant deviations from Mendelian inheritance and may be more widespread than previously assumed. The mechanisms that affect and potentially alter the genomic sequence and allele frequencies in the germline are pivotal to our understanding of heritability. With the rise of new sequencing technologies, we are now able to address some of these unanswered questions. In this review, we comment on the most recent developments in this field and identify current gaps in our knowledge

Investigating the significance of segmental aneuploidy findings in preimplantation embryos

Segmental aneuploidies (SAs) are structural imbalances, namely, gains or losses, involving a chromosomal segment. Most preimplantation genetic testing platforms can detect segmental imbalances greater than 5–10 Mb, either full or mosaic; however, questions remain about clinical significance. An in-depth review was performed to determine the accuracy, frequency, and types of SAs detected in preimplantation embryos. A comprehensive search of the literature revealed an incidence of approximately 8.15% in preimplantation embryos, compared with a prevalence of 3.55% in prenatal diagnosis samples. Several studies have used rebiopsy analysis to validate the accuracy and reproducibility of such findings in blastocyst-stage embryos. A comparison of these studies yielded a mean confirmation rate of SAs slightly higher than 30%. This result could be attributed to their mitotic origin as well as to the technical limitations of preimplantation genetic testing. In addition, the few available studies in which embryos with a segmental finding were transferred in utero are analyzed to discuss the reproductive competence of such embryos. Except for 1 study, all outcomes were described for segmental embryos in a mosaic state. As a result, there is still insufficient evidence to provide accurate information about the effect of segmental imbalances on embryonic reproductive competence and to determine gestational and newborn risks

Artificial oocyte activation with calcium ionophore does not cause a widespread increase in chromosome segregation errors in the second meiotic division of the oocyte

Objective To study the effect of artificial oocyte activation (AOA) on chromosome segregation errors in the meiotic divisions. Design Prospective cohort study with historical control. Setting Private/academic IVF centers. Patient(s) Fifty-six metaphase II oocytes were donated from 12 patients who had undergone IVF between June 2008 and May 2009. Intervention(s) Oocytes were activated by 40 minutes' exposure to 100 ?M calcium-ionophore. The activated oocyte was tubed and analyzed by array comparative genomic hybridization and/or single-nucleotide polymorphism genotyping and maternal haplotyping (meiomapping). A control sample of embryos derived from normally fertilized oocytes was included for comparison. Main Outcome Measure(s) Incidence of chromosome segregation errors in artificially activated and normally fertilized oocytes in relation to pronuclear evaluation. Result(s) Of 49 oocytes that survived the warming procedure, thirty-nine (79.6%) activated. Most activated normally, resulting in extrusion of the second polar body and formation of a single or no pronucleus (2PB1PN: 30 of 39, 76.9%; or 2PB0PN: 5 of 39, 12.8%). Twenty-seven of these were analyzed, and 16 (59.3%) were euploid, showing no effect of AOA on meiotic segregation. Single-nucleotide polymorphism analysis of normally activated oocytes confirmed normal segregation of maternal chromosomes. No difference in the proportion of meiosis II type errors was observed between artificially activated oocytes (28.6%; 95% confidence interval 3.7%–71.0%) compared with embryos obtained from normally fertilized oocytes (44.4%; 95% confidence interval 13.7%–78.8%). The abnormally activated oocytes, with ?2PN (4 of 39, 10.3%) were diploid, indicating a failure to coordinate telophase of meiosis II with polar body extrusion. Conclusion(s) From this preliminary dataset, there is no evidence that AOA causes a widespread increase in chromosome segregation errors in meiosis II. However, we recommend that it be applied selectively to patients with specific indications