Developmental, cytogenetic and epigenetic consequences of removing complex proteins and adding melatonin during in vitro maturation of bovine oocytes.

Background: In vitro maturation (IVM) of germinal vesicle intact oocytes prior to in vitro fertilization (IVF) is practiced widely in animals. In human assisted reproduction it is generally reserved for fertility preservation or where ovarian stimulation is contraindicated. Standard practice incorporates complex proteins (CP), in the form of serum and/or albumin, into IVM media to mimic the ovarian follicle environment. However, the undefined nature of CP, together with batch variation and ethical concerns regarding their origin, necessitate the development of more defined formulations. A known component of follicular fluid, melatonin, has multifaceted roles including that of a metabolic regulator and antioxidant. In certain circumstances it can enhance oocyte maturation. At this stage in development, the germinal-vesicle intact oocyte is prone to aneuploidy and epigenetic dysregulation. Objectives: To determine the developmental, cytogenetic and epigenetic consequences of removing CP and including melatonin during bovine IVM. Materials and methods: The study comprised a 2 x 2 factorial arrangement comparing (i) the inclusion or exclusion of CP, and (ii) the addition (100 nM) or omission of melatonin, during IVM. Cumulus-oocyte complexes (COCs) were retrieved from stimulated cycles. Following IVM and IVF, putative zygotes were cultured to Day 8 in standard media. RNAseq was performed on isolated cumulus cells, cytogenetic analyses (SNP-based algorithms) on isolated trophectoderm cells, and DNA methylation analysis (reduced representation bisulfite sequencing) on isolated cells of the inner-cell mass. Results: Removal of CP during IVM led to modest reductions in blastocyst development, whilst added melatonin was beneficial in the presence but detrimental in the absence of CP. The composition of IVM media did not affect the nature or incidence of chromosomal abnormalities but cumulus-cell transcript expression indicated altered metabolism (primarily lipid) in COCs. These effects preceded the establishment of distinct metabolic and epigenetic signatures several days later in expanded and hatching blastocysts. Conclusions: These findings highlight the importance of lipid, particularly sterol, metabolism by the COC during IVM. They lay the foundation for future studies that seek to develop chemically defined systems of IVM for the generation of transferrable embryos that are both cytogenetically and epigenetically normal.

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.

Preimplantation Genetic Testing for Aneuploidy (PGT-A) Reveals High Levels of Chromosomal Errors in In Vivo-Derived Pig Embryos, with an Increased Incidence When Produced In Vitro.

Preimplantation genetic testing for aneuploidy (PGT-A) is widespread, but controversial, in humans and improves pregnancy and live birth rates in cattle. In pigs, it presents a possible solution to improve in vitro embryo production (IVP), however, the incidence and origin of chromosomal errors remains under-explored. To address this, we used single nucleotide polymorphism (SNP)-based PGT-A algorithms in 101 in vivo-derived (IVD) and 64 IVP porcine embryos. More errors were observed in IVP vs. IVD blastocysts (79.7% vs. 13.6% p < 0.001). In IVD embryos, fewer errors were found at blastocyst stage compared to cleavage (4-cell) stage (13.6% vs. 40%, p = 0.056). One androgenetic and two parthenogenetic embryos were also identified. Triploidy was the most common error in IVD embryos (15.8%), but only observed at cleavage, not blastocyst stage, followed by whole chromosome aneuploidy (9.9%). In IVP blastocysts, 32.8% were parthenogenetic, 25.0% (hypo-)triploid, 12.5% aneuploid, and 9.4% haploid. Parthenogenetic blastocysts arose from just three out of ten sows, suggesting a possible donor effect. The high incidence of chromosomal abnormalities in general, but in IVP embryos in particular, suggests an explanation for the low success of porcine IVP. The approaches described provide a means of monitoring technical improvements and suggest future application of PGT-A might improve embryo transfer success.

Preimplantation Genetic Testing for Aneuploidy Improves Live Birth Rates with In Vitro Produced Bovine Embryos: A Blind Retrospective Study.

Approximately one million in vitro produced (IVP) cattle embryos are transferred worldwide each year as a way to improve the rates of genetic gain. The most advanced programmes also apply genomic selection at the embryonic stage by SNP genotyping and the calculation of genomic estimated breeding values (GEBVs). However, a high proportion of cattle embryos fail to establish a pregnancy. Here, we demonstrate that further interrogation of the SNP data collected for GEBVs can effectively remove aneuploid embryos from the pool, improving live births per embryo transfer (ET). Using three preimplantation genetic testing for aneuploidy (PGT-A) approaches, we assessed 1713 cattle blastocysts in a blind, retrospective analysis. Our findings indicate aneuploid embryos have a 5.8% chance of establishing a pregnancy and a 5.0% chance of given rise to a live birth. This compares to 59.6% and 46.7% for euploid embryos (p < 0.0001). PGT-A improved overall pregnancy and live birth rates by 7.5% and 5.8%, respectively (p < 0.0001). More detailed analyses revealed donor, chromosome, stage, grade, and sex-specific rates of error. Notably, we discovered a significantly higher incidence of aneuploidy in XY embryos and, as in humans, detected a preponderance of maternal meiosis I errors. Our data strongly support the use of PGT-A in cattle IVP programmes.

Copy number analysis of meiotic and postzygotic mitotic aneuploidies in trophectoderm cells biopsied at the blastocyst stage and arrested embryos.

Preimplantation genetic testing for aneuploidy (PGT-A) by copy number analysis is now widely used to select euploid embryos for transfer. Whole or partial chromosome aneuploidy can arise in meiosis, predominantly female meiosis, or in the postzygotic, mitotic divisions during cleavage and blastocyst formation, resulting in chromosome mosaicism. Meiotic aneuploidies are almost always lethal, however, the clinical significance of mitotic aneuploidies detected by PGT-A is not fully understood and healthy live births have been reported following transfer of mosaic embryos. Here, we used single nucleotide polymorphism genotyping of both polar bodies and embryo samples to identify meiotic aneuploidies and compared copy number changes for meiotic and presumed mitotic aneuploidies in trophectoderm cells biopsied at the blastocyst stage and arrested embryos. PGT-A detected corresponding full copy number changes (≥70%) for 36/37 (97%) maternal meiotic aneuploidies. The number of presumed mitotic copy number changes detected exceeded those of meiotic origin. Although mainly in the mosaic range, some of these mitotic aneuploidies had copy number changes ≥70% and would have been identified as full aneuploidies. Interestingly, many arrested embryos had multiple mitotic aneuploidies across a broad range of copy number changes, which may have arisen through tripolar spindle and other mitotic abnormalities.

Abnormal cleavage and developmental arrest of human preimplantation embryos in vitro.

Despite improvements in culture conditions and laboratory techniques still only about 50% of human embryos reach the blastocyst stage of development in vitro. While many factors influence embryo development, aberrant cleavage divisions have only recently been shown to directly affect the genome in individual cells of human embryos resulting in chromosome loss, mosaicism and cell arrest. In this article we review the current literature in the area of aberrant cleavage in human embryos and its effect on blastocyst development. Further to this, we propose a series of common abnormal cleavage events, with particular attention to timing and frequency, and illustrate how these might influence a number of different embryo fates.

Preimplantation genetic testing for aneuploidy versus morphology as selection criteria for single frozen-thawed embryo transfer in good-prognosis patients: a multicenter randomized clinical trial.

OBJECTIVE: To evaluate the benefit of next-generation sequencing (NGS)-based preimplantation genetic testing for aneuploidy (PGT-A) for embryo selection in frozen-thawed embryo transfer. DESIGN: Randomized controlled trial. SETTING: Not applicable. PATIENT(S): Women aged 25-40 years undergoing IVF with at least two blastocysts that could be biopsied. INTERVENTION(S): Randomization for single frozen-thawed embryo transfer with embryo selection based on PGT-A euploid status versus morphology. MAIN OUTCOME MEASURE(S): Ongoing pregnancy rate (OPR) at 20 weeks' gestation per embryo transfer. RESULT(S): A total of 661 women (average age 33.7 ± 3.6 years) were randomized to PGT-A (n = 330) or morphology alone (n = 331). The OPR was equivalent between the two arms, with no significant difference per embryo transfer (50% [137/274] vs. 46% [143/313]) or per intention to treat (ITT) at randomization (41.8% [138/330] vs. 43.5% [144/331]). Post hoc analysis of women aged 35-40 years showed a significant increase in OPR per embryo transfer (51% [62/122] vs. 37% [54/145]) but not per ITT. CONCLUSION(S): PGT-A did not improve overall pregnancy outcomes in all women, as analyzed per embryo transfer or per ITT. There was a significant increase in OPR per embryo transfer with the use of PGT-A in the subgroup of women aged 35-40 years who had two or more embryos that could be biopsied, but this was not significant when analyzed by ITT. CLINICAL TRIAL REGISTRATION NUMBER: NCT02268786.

Karyomapping for simultaneous genomic evaluation and aneuploidy screening of preimplantation bovine embryos: The first live-born calves.

In cattle breeding, the development of genomic selection strategies based on single nucleotide polymorphism (SNP) interrogation has led to improved rates of genetic gain. Additionally, the application of genomic selection to in-vitro produced (IVP) embryos is expected to bring further benefits thanks to the ability to test a greater number of individuals before establishing a pregnancy and to ensure only carriers of desirable traits are born. However, aneuploidy, a leading cause of developmental arrest, is known to be common in IVP embryos. Karyomapping is a comprehensive screening test based on SNP typing that can be used for simultaneous genomic selection and aneuploidy detection, offering the potential to maximize pregnancy rates. Moreover, Karyomapping can be used to characterize the frequency and parental origin of aneuploidy in bovine IVP embryos, which have remained underexplored to date. Here, we report the use of Karyomapping to characterize the frequency and parental origin of aneuploidy in IVP bovine embryos in order to establish an estimate of total aneuploidy rates in each parental germline. We report an estimate of genome wide recombination rate in cattle and demonstrate, for the first time, a proof of principle for the application of Karyomapping to cattle breeding, with the birth of five calves after screening. This combined genomic selection and aneuploidy screening approach was highly reliable, with calves showing 98% concordance with their respective embryo biopsies for SNP typing and 100% concordance with their respective biopsies for aneuploidy screening. This approach has the potential to simultaneously improve pregnancy rates following embryo transfer and the rate of genetic gain in cattle breeding, and is applicable to basic research to investigate meiosis and aneuploidy.

Forty years of IVF.

This monograph, written by the pioneers of IVF and reproductive medicine, celebrates the history, achievements, and medical advancements made over the last 40 years in this rapidly growing field.

'Designer babies' almost thirty years on.

The first pregnancies and live births following in vitro fertilisation (IVF) and preimplantation genetic testing (PGT), formerly known as preimplantation genetic diagnosis, were reported in 1990, almost 30 years ago, in several couples at risk of X-linked inherited conditions, which typically only affect boys inheriting the X chromosome with the affected gene from their carrier mothers. At that time, it was only possible to identify the sex of the embryo by amplifying a Y-linked repeat sequence in single cells biopsied at cleavage stages and avoid the transfer of males, half of which would be affected. The extensive publicity surrounding these cases and the perceived risk of using IVF and PGT for desirable characteristics not related to health, such as sex selection, led to the epithet of 'designer babies' which continues to resonate to this day. Here, I briefly reflect on how the technology of PGT has evolved over the decades and whether it deserves this reputation. With efficient methods for whole genome amplification and the genomic revolution, we now have highly accurate universal tests that combine marker-based diagnosis of almost any monogenic disorder with the detection of aneuploidy. PGT is now clinically well established and is likely to remain a valuable alternative for couples at risk of having affected children.

Tripolar chromosome segregation drives the association between maternal genotype at variants spanning PLK4 and aneuploidy in human preimplantation embryos.

Aneuploidy is prevalent in human embryos and is the leading cause of pregnancy loss. Many aneuploidies arise during oogenesis, increasing with maternal age. Superimposed on these meiotic aneuploidies are frequent errors occurring during early mitotic divisions, contributing to widespread chromosomal mosaicism. Here we reanalyzed a published dataset comprising preimplantation genetic testing for aneuploidy in 24 653 blastomere biopsies from day-3 cleavage-stage embryos, as well as 17 051 trophectoderm biopsies from day-5 blastocysts. We focused on complex abnormalities that affected multiple chromosomes simultaneously, seeking insights into their formation. In addition to well-described patterns such as triploidy and haploidy, we identified 4.7% of blastomeres possessing characteristic hypodiploid karyotypes. We inferred this signature to have arisen from tripolar chromosome segregation in normally fertilized diploid zygotes or their descendant diploid cells. This could occur via segregation on a tripolar mitotic spindle or by rapid sequential bipolar mitoses without an intervening S-phase. Both models are consistent with time-lapse data from an intersecting set of 77 cleavage-stage embryos, which were enriched for the tripolar signature among embryos exhibiting abnormal cleavage. The tripolar signature was strongly associated with common maternal genetic variants spanning the centrosomal regulator PLK4, driving the association we previously reported with overall mitotic errors. Our findings are consistent with the known capacity of PLK4 to induce tripolar mitosis or precocious M-phase upon dysregulation. Together, our data support tripolar chromosome segregation as a key mechanism generating complex aneuploidy in cleavage-stage embryos and implicate maternal genotype at a quantitative trait locus spanning PLK4 as a factor influencing its occurrence.

Tripolar mitosis and partitioning of the genome arrests human preimplantation development in vitro.

Following in vitro fertilisation (IVF), only about half of normally fertilised human embryos develop beyond cleavage and morula stages to form a blastocyst in vitro. Although many human embryos are aneuploid and genomically imbalanced, often as a result of meiotic errors inherited in the oocyte, these aneuploidies persist at the blastocyst stage and the reasons for the high incidence of developmental arrest remain unknown. Here we use genome-wide SNP genotyping and meiomapping of both polar bodies to identify maternal meiotic errors and karyomapping to fingerprint the parental chromosomes in single cells from disaggregated arrested embryos and excluded cells from blastocysts. Combined with time lapse imaging of development in culture, we demonstrate that tripolar mitoses in early cleavage cause chromosome dispersal to clones of cells with identical or closely related sub-diploid chromosome profiles resulting in intercellular partitioning of the genome. We hypothesise that following zygotic genome activation (ZGA), the combination of genomic imbalance and partial genome loss disrupts the normal pattern of embryonic gene expression blocking development at the morula-blastocyst transition. Failure to coordinate the cell cycle in early cleavage and regulate centrosome duplication is therefore a major cause of human preimplantation developmental arrest in vitro.

Generation of meiomaps of genome-wide recombination and chromosome segregation in human oocytes.

We have developed a protocol for the generation of genome-wide maps (meiomaps) of recombination and chromosome segregation for the three products of human female meiosis: the first and second polar bodies (PB1 and PB2) and the corresponding oocyte. PB1 is biopsied and the oocyte is artificially activated by exposure to calcium ionophore, after which PB2 is biopsied and collected with the corresponding oocyte. The whole genomes of the polar bodies and oocytes are amplified by multiple displacement amplification and, together with maternal genomic DNA, genotyped for ∼300,000 single-nucleotide polymorphisms (SNPs) genome-wide by microarray. Informative maternal heterozygous SNPs are phased using a haploid PB2 or oocyte as a reference. A simple algorithm is then used to identify the maternal haplotypes for each chromosome, in all of the products of meiosis for each oocyte. This allows mapping of crossovers and analysis of chromosome segregation patterns. The protocol takes a minimum of 3-5 d and requires a clinical embryologist with micromanipulation experience and a molecular biologist with basic bioinformatic skills. It has several advantages over previous methods; importantly, the use of artificial oocyte activation avoids the creation of embryos for research purposes. In addition, compared with next-generation sequencing, targeted SNP genotyping is cost-effective and it simplifies the bioinformatic analysis, as only one haploid reference sample is required to establish phase for maternal haplotyping. Finally, meiomapping is more informative than copy-number analysis alone for analysis of chromosome segregation patterns. Using this protocol, we have provided new insights that may lead to improvements in assisted reproduction for the treatment of infertility.

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 µM 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.

Karyomapping identifies second polar body DNA persisting to the blastocyst stage: implications for embryo biopsy.

Blastocyst biopsy is now widely used for both preimplantation genetic screening (PGS) and preimplantation genetic diagnosis (PGD). Although this approach yields good results, variable embryo quality and rates of development remain a challenge. Here, a case is reported in which a blastocyst was biopsied for PGS by array comparative genomic hybridization on day 6 after insemination, having hatched completely. In addition to a small trophectoderm sample, excluded cell fragments from the subzonal space from this embryo were also sampled. Unexpectedly, the array comparative genomic hybridization results from the fragments and trophectoderm sample were non-concordant: 47,XX,+19 and 46,XY, respectively. DNA fingerprinting by short tandem repeat and amelogenin analysis confirmed the sex chromosome difference but seemed to show that the two samples were related but non-identical. Genome-wide single nucleotide polymorphism genotyping and karyomapping identified that the origin of the DNA amplified from the fragments was that of the second polar body corresponding to the oocyte from which the biopsied embryo developed. The fact that polar body DNA can persist to the blastocyst stage provides evidence that excluded cell fragments should not be used for diagnostic purposes and should be avoided when performing embryo biopsies as there is a risk of diagnostic errors.

Genome-wide maps of recombination and chromosome segregation in human oocytes and embryos show selection for maternal recombination rates.

Crossover recombination reshuffles genes and prevents errors in segregation that lead to extra or missing chromosomes (aneuploidy) in human eggs, a major cause of pregnancy failure and congenital disorders. Here we generate genome-wide maps of crossovers and chromosome segregation patterns by recovering all three products of single female meioses. Genotyping >4 million informative SNPs from 23 complete meioses allowed us to map 2,032 maternal and 1,342 paternal crossovers and to infer the segregation patterns of 529 chromosome pairs. We uncover a new reverse chromosome segregation pattern in which both homologs separate their sister chromatids at meiosis I; detect selection for higher recombination rates in the female germ line by the elimination of aneuploid embryos; and report chromosomal drive against non-recombinant chromatids at meiosis II. Collectively, our findings show that recombination not only affects homolog segregation at meiosis I but also the fate of sister chromatids at meiosis II.