Use of artificial intelligence embryo selection based on static images to predict first-trimester pregnancy loss.

RESEARCH QUESTION: Can an artificial intelligence embryo selection assistant predict the incidence of first-trimester spontaneous abortion using static images of IVF embryos? DESIGN: In a blind, retrospective study, a cohort of 172 blastocysts from IVF cases with single embryo transfer and a positive biochemical pregnancy test was ranked retrospectively by the artificial intelligence morphometric algorithm ERICA. Making use of static embryo images from a light microscope, each blastocyst was assigned to one of four possible groups (optimal, good, fair or poor), and linear regression was used to correlate the results with the presence or absence of a normal fetal heart beat as an indicator of ongoing pregnancy or spontaneous abortion, respectively. Additional analyses included modelling for recipient age and chromosomal status established by preimplantation genetic testing for aneuploidy (PGT-A). RESULTS: Embryos classified as optimal/good had a lower incidence of spontaneous abortion (16.1%) compared with embryos classified as fair/poor (25%; OR = 0.46, P = 0.005). The incidence of spontaneous abortion in chromosomally normal embryos (determined by PGT-A) was 13.3% for optimal/good embryos and 20.0% for fair/poor embryos, although the difference was not significant (P = 0.531). There was a significant association between embryo rank and recipient age (P = 0.018), in that the incidence of spontaneous abortion was unexpectedly lower in older recipients (21.3% for age ≤35 years, 17.9% for age 36-38 years, 16.4% for age ≥39 years; OR = 0.354, P = 0.0181). Overall, these results support correlation between risk of spontaneous abortion and embryo rank as determined by artificial intelligence; classification accuracy was calculated to be 67.4%. CONCLUSIONS: This preliminary study suggests that artificial intelligence (ERICA), which was designed as a ranking system to assist with embryo transfer decisions and ploidy prediction, may also be useful to provide information for couples on the risk of spontaneous abortion. Future work will include a larger sample size and karyotyping of miscarried pregnancy tissue.

Understanding microchromosomal organization and evolution in four representative woodpeckers (Picidae, Piciformes) through BAC-FISH analysis.

The genome organization of woodpeckers has several distinctive features e.g., an uncommon accumulation of repetitive sequences, enlarged Z chromosomes, and atypical diploid numbers. Despite the large diversity of species, there is a paucity of detailed cytogenomic studies for this group and we thus aimed to rectify this. Genome organization patterns and hence evolutionary change in the microchromosome formation of four species (Colaptes campestris, Veniliornis spilogaster, Melanerpes candidus, and Picumnus nebulosus) was established through fluorescence in situ hybridization using bacterial artificial chromosomes originally derived from Gallus gallus and Taeniopygia guttata. Findings suggest that P. nebulosus (2n = 110), which was described for the first time, had the most basal karyotype among species of Picidae studied here, and probably arose as a result of fissions of avian ancestral macrochromosomes. We defined a new chromosomal number for V. spilogaster (2n = 88) and demonstrated microchromosomal rearrangements involving C. campestris plus a single, unique hitherto undescribed rearrangement in V. spilogaster. This comprised an inversion after a fusion involving the ancestral microchromosome 12 (homologous to chicken microchromosome 12). We also determined that the low diploid number of M. candidus is related to microchromosome fusions. Woodpeckers thus exhibit significantly rearranged karyotypes compared to the putative ancestral karyotype.

Understanding the chromosomal evolution in cuckoos (Aves, Cuculiformes): a journey through unusual rearrangements.

The Cuculiformes are a family of over 150 species that live in a range of habitats, such as forests, savannas, and deserts. Here, bacterial artificial chromosome (BAC) probes (75 from chicken and 14 from zebra finch macrochromosomes 1-10 +ZW and for microchromosomes 11-28 (except 16)) were used to investigate chromosome homologies between chicken and the squirrel cuckoo (Piaya cayana). In addition, repetitive DNA probes were applied to characterize the chromosome organization and to explore the role of these sequences in the karyotype evolution of P. cayana. We also applied BAC probes for chicken chromosome 17 and Z to the guira cuckoo (Guira guira) to test whether this species has an unusual Robertsonian translocation between a microchromosome and the Z chromosome, recently described in the smooth-billed ani (Crotophaga ani). Our results revealed extensive chromosome reorganization with inter- and intrachromosomal rearrangements in P. cayana, including a conspicuous chromosome size and heterochromatin polymorphism on chromosome pair 20. Furthermore, we confirmed that the Z-autosome Robertsonian translocation found in C. ani is also found in G. guira, not P. cayana. These findings suggest that this translocation occurred prior to the divergence between C. ani and G. guira, but after the divergence with P. cayana.

Integrative comparative analysis of avian chromosome evolution by in-silico mapping of the gene ontology of homologous synteny blocks and evolutionary breakpoint regions.

Avian chromosomes undergo more intra- than interchromosomal rearrangements, which either induce or are associated with genome variations among birds. Evolving from a common ancestor with a karyotype not dissimilar from modern chicken, two evolutionary elements characterize evolutionary change: homologous synteny blocks (HSBs) constitute common conserved parts at the sequence level, while evolutionary breakpoint regions (EBRs) occur between HSBs, defining the points where rearrangement occurred. Understanding the link between the structural organization and functionality of HSBs and EBRs provides insight into the mechanistic basis of chromosomal change. Previously, we identified gene ontology (GO) terms associated with both; however, here we revisit our analyses in light of newly developed bioinformatic algorithms and the chicken genome assembly galGal6. We aligned genomes available for six birds and one lizard species, identifying 630 HSBs and 19 EBRs. We demonstrate that HSBs hold vast functionality expressed by GO terms that have been largely conserved through evolution. Particularly, we found that genes within microchromosomal HSBs had specific functionalities relevant to neurons, RNA, cellular transport and embryonic development, and other associations. Our findings suggest that microchromosomes may have conserved throughout evolution due to the specificity of GO terms within their HSBs. The detected EBRs included those found in the genome of the anole lizard, meaning they were shared by all saurian descendants, with others being unique to avian lineages. Our estimate of gene richness in HSBs supported the fact that microchromosomes contain twice as many genes as macrochromosomes.

Probiotics as an alternative to antibiotics in modulating the intestinal microbiota and performance of broiler chickens.

AIMS: Gut bacteria play an important role in poultry nutrition and the immune defense system. Changes in the intestinal microbiome affect the physiological state, metabolism, and innate immunity of poultry. The present study aimed to characterize age-related changes in the gastrointestinal tract microflora in broiler chickens, depending on supplementation of the diet with the in-feed antibiotic Stafac® 110 and a Bacillus subtilis strain-based probiotic. METHODS AND RESULTS: In this regard, a comprehensive analysis of the taxonomic structure of the microbial community in the gastrointestinal tract (GIT) of broiler chickens was carried out using a molecular genetic technique of the terminal-restriction fragment length polymorphism analysis and taking into account age dynamics and feeding treatment. A beneficial effect on the microbiological composition and body weight of broilers was observed when using the antibiotic and probiotic in compound feeds. Different bacterial communities were revealed in the duodenum and cecum, and their positive impact on broiler growth was established. The results obtained shed light on the formation of GIT microflora of broiler chickens during the growing period and its changes in response to the use of the antibiotic and the probiotic. CONCLUSIONS: We suggest that the implementation of the tested in-feed antibiotic and probiotic can be beneficial in regulating the intestinal microflora microbiological processes in the GIT and improving the feeding efficiency and productivity of broiler chickens.

Successful recovery of motile and viable boar sperm after vitrification with different methods (pearls and mini straws) using sucrose as a cryoprotectant.

Vitrification of sperm by direct contact with liquid nitrogen is increasing in popularity as an alternative to conventional (slow) freezing. Although slow freezing is very challenging in boar sperm cryopreservation, this is currently the standard method used. We compared vitrification in "pearls" and in "mini straws" using the in vitro fertilization media Porcine Gamete Media with 0.3 M sucrose with the standard (slow) method used to preserve boar sperm. Both vitrification methods reduced the viability of the sperm sample more than slow freezing (42.2 ± 4.3% total motility and 71.4 ± 2.3% alive), however, both protocols allowed for the successful recovery of the sperm samples. By comparing two different methods of vitrification and two different methods of post-thaw preparation we were able to determine the optimal vitrification-thaw protocol for boar sperm. When comparing pearls and mini-straws, the smaller liquid volume associated with pearls had a positive effect on the survivability of the samples, reducing sperm DNA damage (1.2 ± 0.2% vs. 5.1 ± 0.1.7%) and preserving motility (26.15 ± 2.8% vs 9.39 ± 0.9%) after thawing. In conclusion, the pearl method was the most suitable of the vitrification techniques for use with boar sperm.

What proportion of embryos should be considered for transfer following a mosaic diagnosis? A study of 115 clinics from a central diagnostic laboratory.

PURPOSE: The aim of this study is to identify what proportion of mosaic embryo diagnoses should be considered for transfer, and thereby assess the impact on patient cases. METHODS: We categorised mosaic embryos into 3 groups; high, medium and low priority for transfer based on the percentage of biopsy sample being aneuploid and the chromosomes involved. The categories were applied to those patients that had no euploid embryo diagnoses but 1 or more mosaic embryos identified as mosaic available after PGT-A. RESULTS: In total, 6614 PGT-A cases from 115 clinics and a single diagnostic laboratory were reviewed. Further, 1384 [20.9%] cases only had aneuploid embryos, 4538 [68.6%] cases had one or more euploid embryos and 692 [10.5%] cases had no euploid and one or more mosaic embryo. The mosaic embryos in the no euploid, one or more mosaic group, when reviewed using priorities, resulted in: 111 [1.7%] of cases having at least one high priority mosaic available. 184 [2.8%] of cases having no high priority but at least one medium priority mosaic available. 397 [6.0%] of cases only having low priority mosaic embryos available. CONCLUSION: Based on this data, embryos identified as mosaic will only be considered for transfer in the first instance for around 4.5% (when taking high and medium priority and excluding low priority cases) of all PGT-A cases.

Why PGT-A, most likely, improves IVF success.

Preimplantation genetic testing for aneuploidies (PGT-A), with its vocal advocates and opponents, is at the epicentre of a perpetual, often heated, debate. The main issues include the following. First, how do we interpret the existing evidence-base? Around 100 retrospective and single-centre studies, two non-selection trials and at least two meta-analyses point to its efficacy in improving live birth rates, although randomized controlled trials are more mixed. Second, what should be done in relation to euploid/aneuploid mosaicism? Recent data suggest that low-level mosaic pregnancies can proceed uneventfully to term, so intelligent interpretation of the diagnostic data is appropriate. Third, what is the stance of the Human Fertilisation and Embryology Authority? The 'traffic light' system is much debated and is perhaps best described as well-intentioned, but misguided in places. Fourth, what is the motivation of people who maintain their point of view despite the evidence? Sadly, the presentation of new empirical evidence polarizes, rather than reconciles, opinion. Too many have made a career out of either promoting or denigrating PGT-A for them to back down easily. Finally, how can we find common ground and move forward? All patients should be counselled in a non-directive manner on whether to embark on PGT-A, summarizing for them the whole evidence base so they can make up their own mind.

Supplementation of porcine in vitro maturation medium with FGF2, LIF, and IGF1 enhances cytoplasmic maturation in prepubertal gilts oocytes and improves embryo quality.

In porcine in vitro production (IVP) systems, the use of oocytes derived from prepubertal gilts, whilst being commercially attractive, remains challenging due to their poor developmental competence following in vitro maturation (IVM). Follicular fluid contains important growth factors and plays a key role during oocyte maturation; therefore, it is a common supplementation for porcine IVM medium. However, follicular fluid contains many poorly characterized components, is batch variable, and its use raises biosecurity concerns. In an effort to design a defined IVM system, growth factors such as cytokines have been previously tested. These include leukaemia inhibitory factor (LIF), fibroblast growth factor 2 (FGF2), and insulin-like growth factor 1 (IGF1), the combination of which is termed 'FLI'. Here, using abattoir-derived oocytes in a well established porcine IVP system, we compared follicular fluid and FLI supplementation during both IVM and embryo culture to test the hypothesis that FLI can substitute for follicular fluid without compromising oocyte nuclear and cytoplasmic maturation. We demonstrate that in oocytes derived from prepubertal gilts, FLI supplementation enhances oocyte meiotic maturation and has a positive effect on the quality and developmental competence of embryos. Moreover, for the first time, we studied the effects of follicular fluid and FLI combined showing no synergistic effects.

Do sex chromosomes of snakes, monitor lizards, and iguanian lizards result from multiple fission of an "ancestral amniote super-sex chromosome"?

Sex chromosomes in some amniotes share linkage homologies with distantly related taxa in regions orthologous to squamate reptile chromosome 2 (SR2) and the snake W sex chromosome. Thus, the SR2 and W chromosomes may formerly have been part of a larger ancestral amniote super-sex chromosome. Comparison of various sex chromosomal linkage homologies in Toxicofera with those in other amniotes offers an excellent model to assess key cytological differences, to understand the mechanisms of amniote sex chromosome evolution in each lineage and the existence of an ancestral amniote super-sex chromosome. Chromosome maps of four species of Toxicofera were constructed using bacterial artificial chromosomes (BACs) derived from chicken and zebra finch libraries containing amniote sex chromosomal linkages. Different macrochromosome linkage homologies were highly conserved among Toxicofera, and at least two BACs (CH261-125F1 and CH261-40D6) showed partial homology with sex chromosomes of amniotes associated with SR2, which supports the hypothesis of an ancestral super-sex chromosome with overlaps of partial linkage homologies. The present data also suggest a possible multiple fission mechanism of an ancestral super-sex chromosome, which resulted in further development of various sex chromosomal linkages of Toxicofera based on particular properties that favored the role of sex chromosomes.

Analysis of IVF live birth outcomes with and without preimplantation genetic testing for aneuploidy (PGT-A): UK Human Fertilisation and Embryology Authority data collection 2016-2018.

PURPOSE: To examine the live birth and other outcomes reported with and without preimplantation genetic testing for aneuploidy (PGT-A) in the United Kingdom (UK) Human Embryology and Fertilization Authority (HFEA) data collection. METHODS: A retrospective cohort analysis was conducted following freedom of information (FoI) requests to the HFEA for the PGT-A and non-PGT-A cycle outcomes for 2016-2018. Statistical analysis of differences between PGT-A and non-PGT-A cycles was performed. Other than grouping by maternal age, no further confounders were controlled for; fresh and frozen transfers were included. RESULTS: Outcomes collected between 2016 and 2018 included total number of cycles, cycles with no embryo transfer, total number of embryos transferred, live birth rate (LBR) per embryo transferred and live birth rate per treatment cycle. Data was available for 2464 PGT-A out of a total 190,010 cycles. LBR per embryo transferred and LBR per treatment cycle (including cycles with no transfer) were significantly higher for all PGT-A vs non-PGT-A age groups (including under 35), with nearly all single embryo transfers (SET) after PGT-A (significantly more in non-PGT-A) and a reduced number of transfers per live birth particularly for cycles with maternal age over 40 years. CONCLUSION: The retrospective study provides strong evidence for the benefits of PGT-A in terms of live births per embryo transferred and per cycle started but is limited in terms of matching PGT-A and non-PGT-A cohorts (e.g. in future studies, other confounders could be controlled for). This data challenges the HFEA "red traffic light" guidance that states there is "no evidence that PGT-A is effective or safe" and hence suggests the statement be revisited in the light of this and other new data.

Forecasting early onset diminished ovarian reserve for young reproductive age women.

PURPOSE: To investigate the biological networks associated with DOR in young women and the subsequent molecular impact on preimplantation embryos. METHODS: Whole peripheral blood was collected from patients: young women presenting with diminished ovarian reserve (DOR) and age-matched young women with normal ovarian reserve. Maternal exome sequencing was performed on the NovaSEQ 6000 and sequencing validation was completed using Taqman® SNP Genotyping Assays. Blastocyst global methylome and transcriptome sequencing were also analyzed. RESULTS: Exome sequencing revealed 730 significant DNA variants observed exclusively in the young DOR patients. Bioinformatic analysis revealed a significant impact to the Glucocorticoid receptor (GR) signaling pathway and each young DOR female had an average of 6.2 deleterious DNA variants within this pathway. Additional stratification based on patient age resulted in a cut-off at 31 years for young DOR discrimination. Embryonic global methylome sequencing resulted in only a very small number of total CpG sites with methylation alterations (1,775; 0.015% of total) in the DOR group. Additionally, there was no co-localization between these limited number of altered CpG sites and significant variants, genes, or pathways. RNA sequencing also resulted in no biologically significant transcription changes between DOR blastocysts and controls. CONCLUSION: GR signaling DNA variants were observed in women with early-onset DOR potentially compromising oocyte production and quality. However, no significant downstream effects on biological processes appear to impact the resulting blastocyst. The ability to forecast premature DOR for young women may allow for earlier identification and clinical intervention for this patient population.

Telomere Distribution in Human Sperm Heads and Its Relation to Sperm Nuclear Morphology: A New Marker for Male Factor Infertility?

Infertility is a problem affecting an increasing number of couples worldwide. Currently, marker tests for male factor infertility are complex, highly technical and relatively subjective. Up to 40% of cases of male factor infertility are currently diagnosed as idiopathic therefore, there is a clear need for further research into better ways of diagnosing it. Changes in sperm telomere length have been associated with infertility and closely linked to DNA damage and fragmentation, which are also known to be related to infertility. However, telomere distribution is a parameter thus far underexplored as an infertility marker. Here, we assessed morphological parameters of sperm nuclei in fertile control and male factor infertile cohorts. In addition, we used 2D and 3D fluorescence in situ hybridization (FISH) to compare telomere distribution between these two groups. Our findings indicate that the infertile cohort sperm nuclei were, on average, 2.9% larger in area and showed subtle differences in sperm head height and width. Telomeres were mainly distributed towards the periphery of the nuclei in the control cohort, with diminishing telomere signals towards the center of the nuclei. Sperm nuclei of infertile males, however, had more telomere signals towards the center of the nuclei, a finding supported by 3D imaging. We conclude that, with further development, both morphology and telomere distribution may prove useful investigative tools in the fertility clinic.

Ultra-Structural Imaging Provides 3D Organization of 46 Chromosomes of a Human Lymphocyte Prophase Nucleus.

Three dimensional (3D) ultra-structural imaging is an important tool for unraveling the organizational structure of individual chromosomes at various stages of the cell cycle. Performing hitherto uninvestigated ultra-structural analysis of the human genome at prophase, we used serial block-face scanning electron microscopy (SBFSEM) to understand chromosomal architectural organization within 3D nuclear space. Acquired images allowed us to segment, reconstruct, and extract quantitative 3D structural information about the prophase nucleus and the preserved, intact individual chromosomes within it. Our data demonstrate that each chromosome can be identified with its homolog and classified into respective cytogenetic groups. Thereby, we present the first 3D karyotype built from the compact axial structure seen on the core of all prophase chromosomes. The chromosomes display parallel-aligned sister chromatids with familiar chromosome morphologies with no crossovers. Furthermore, the spatial positions of all 46 chromosomes revealed a pattern showing a gene density-based correlation and a neighborhood map of individual chromosomes based on their relative spatial positioning. A comprehensive picture of 3D chromosomal organization at the nanometer level in a single human lymphocyte cell is presented.

Patterns of microchromosome organization remain highly conserved throughout avian evolution.

The structure and organization of a species genome at a karyotypic level, and in interphase nuclei, have broad functional significance. Although regular sized chromosomes are studied extensively in this regard, microchromosomes, which are present in many terrestrial vertebrates, remain poorly explored. Birds have more cytologically indistinguishable microchromosomes (~ 30 pairs) than other vertebrates; however, the degree to which genome organization patterns at a karyotypic and interphase level differ between species is unknown. In species where microchromosomes have fused to other chromosomes, they retain genomic features such as gene density and GC content; however, the extent to which they retain a central nuclear position has not been investigated. In studying 22 avian species from 10 orders, we established that, other than in species where microchromosomal fusion is obvious (Falconiformes and Psittaciformes), there was no evidence of microchromosomal rearrangement, suggesting an evolutionarily stable avian genome (karyotypic) organization. Moreover, in species where microchromosomal fusion has occurred, they retain a central nuclear location, suggesting that the nuclear position of microchromosomes is a function of their genomic features rather than their physical size.

Upgrading short-read animal genome assemblies to chromosome level using comparative genomics and a universal probe set.

Most recent initiatives to sequence and assemble new species' genomes de novo fail to achieve the ultimate endpoint to produce contigs, each representing one whole chromosome. Even the best-assembled genomes (using contemporary technologies) consist of subchromosomal-sized scaffolds. To circumvent this problem, we developed a novel approach that combines computational algorithms to merge scaffolds into chromosomal fragments, PCR-based scaffold verification, and physical mapping to chromosomes. Multigenome-alignment-guided probe selection led to the development of a set of universal avian BAC clones that permit rapid anchoring of multiple scaffolds to chromosomes on all avian genomes. As proof of principle, we assembled genomes of the pigeon (Columbia livia) and peregrine falcon (Falco peregrinus) to chromosome levels comparable, in continuity, to avian reference genomes. Both species are of interest for breeding, cultural, food, and/or environmental reasons. Pigeon has a typical avian karyotype (2n = 80), while falcon (2n = 50) is highly rearranged compared to the avian ancestor. By using chromosome breakpoint data, we established that avian interchromosomal breakpoints appear in the regions of low density of conserved noncoding elements (CNEs) and that the chromosomal fission sites are further limited to long CNE "deserts." This corresponds with fission being the rarest type of rearrangement in avian genome evolution. High-throughput multiple hybridization and rapid capture strategies using the current BAC set provide the basis for assembling numerous avian (and possibly other reptilian) species, while the overall strategy for scaffold assembly and mapping provides the basis for an approach that (provided metaphases can be generated) could be applied to any animal genome.

Preliminary assessment of aneuploidy rates between the polar, mid and mural trophectoderm.

The objective of this study is to compare aneuploidy rates between three distinct areas of the human trophectoderm: mural, polar and a region in between these two locations termed the 'mid' trophectoderm. This is a cohort study on in vitro fertilization (IVF) patients undergoing comprehensive chromosome screening at the blastocyst stage at a private IVF clinic. All embryos underwent assisted hatching on day 3 with blastocyst biopsy and comprehensive chromosome screening. Biopsied blastocysts were divided into three groups depending on which area (polar, mid, or mural) of the trophectoderm was protruding from the zona pellucida and biopsied. Aneuploidy rates were significantly higher with cells from the polar region of the trophectoderm (56.2%) compared with cells removed from the mural region of the trophectoderm (30.0%; P = 0.0243). A comparison of all three areas combined also showed a decreasing trend, but this did not reach clinical significance, polar (56.2%), mid (47.4%) and mural trophectoderm (30.0%; P = 0.1859). The non-concordance demonstrated between polar and mural trophectoderm can be attributed to biological occurrences including chromosomal mosaicism or procedural differences between embryologists.

Genome-wide association studies targeting the yield of extraembryonic fluid and production traits in Russian White chickens.

BACKGROUND: The Russian White is a gene pool breed, registered in 1953 after crossing White Leghorns with local populations and, for 50 years, selected for cold tolerance and high egg production (EL). The breed has great potential in meeting demands of local food producers, commercial farmers and biotechnology sector of specific pathogen-free (SPF) eggs, the former valuing the breed for its egg weight (EW), EL, age at first egg (AFE), body weight (BW), and the latter for its yield of extraembryonic fluid (YEF) in 12.5-day embryos, ratio of extraembryonic fluid to egg weight, and embryo mass. Moreover, its cold tolerance has been presumably associated with day-old chick down colour (DOCDC) - white rather than yellow, the genetic basis of these traits being however poorly understood. RESULTS: We undertook genome-wide association studies (GWASs) for eight performance traits using single nucleotide polymorphism (SNP) genotyping of 146 birds and an Illumina 60KBeadChip. Several suggestive associations (p < 5.16*10- 5) were found for YEF, AFE, BW and EW. Moreover, on chromosome 2, an association with the white DOCDC was found where there is an linkage disequilibrium block of SNPs including genes that are responsible not for colour, but for immune resistance. CONCLUSIONS: The obtained GWAS data can be used to explore the genetics of immunity and carry out selection for increasing YEF for SPF eggs production.

The role of chromosome segregation and nuclear organisation in human subfertility.

Spermatogenesis is central to successful sexual reproduction, producing large numbers of haploid motile male gametes. Throughout this process, a series of equational and reductional chromosome segregation precedes radical repackaging of the haploid genome. Faithful chromosome segregation is thus crucial, as is an ordered spatio-temporal 'dance' of packing a large amount of chromatin into a very small space. Ergo, when the process goes wrong, this is associated with an improper chromosome number, nuclear position and/or chromatin damage in the sperm head. Generally, screening for overall DNA damage is relatively commonplace in clinics, but aneuploidy assessment is less so and nuclear organisation studies form the basis of academic research. Several studies have focussed on the role of chromosome segregation, nuclear organisation and analysis of sperm morphometry in human subfertility observing significant alterations in some cases, especially of the sex chromosomes. Importantly, sperm DNA damage has been associated with infertility and both extrinsic (e.g. lifestyle) and intrinsic (e.g. reactive oxygen species levels) factors, and while some DNA-strand breaks are repaired, unexpected breaks can cause differential chromatin packaging and further breakage. A 'healthy' sperm nucleus (with the right number of chromosomes, nuclear organisation and minimal DNA damage) is thus an essential part of reproduction. The purpose of this review is to summarise state of the art in the fields of sperm aneuploidy assessment, nuclear organisation and DNA damage studies.

Assessment of aneuploidy concordance between clinical trophectoderm biopsy and blastocyst.

STUDY QUESTION: Is a clinical trophectoderm (TE) biopsy a suitable predictor of chromosomal aneuploidy in blastocysts? SUMMARY ANSWER: In the analyzed group of blastocysts, a clinical TE biopsy was an excellent representative of blastocyst karyotype in cases of whole chromosome aneuploidy, but in cases of only segmental (sub-chromosomal) aneuploidy, a TE biopsy was a poor representative of blastocyst karyotype. WHAT IS KNOWN ALREADY: Due to the phenomenon of chromosomal mosaicism, concern has been expressed about the possibility of discarding blastocysts classified as aneuploid by preimplantation genetic testing for aneuploidy (PGT-A) that in fact contain a euploid inner cell mass (ICM). Previously published studies investigating karyotype concordance between TE and ICM have examined small sample sizes and/or have utilized chromosomal analysis technologies superseded by Next Generation Sequencing (NGS). It is also known that blastocysts classified as mosaic by PGT-A can result in healthy births. TE re-biopsy of embryos classified as aneuploid can potentially uncover new instances of mosaicism, but the frequency of such blastocysts is currently unknown. STUDY DESIGN, SIZE, DURATION: For this study, 45 patients donated 100 blastocysts classified as uniform aneuploids (non-mosaic) using PGT-A by NGS (n = 93 whole chromosome aneuploids, n = 7 segmental aneuploids). In addition to the original clinical TE biopsy used for PGT-A, each blastocyst was subjected to an ICM biopsy as well as a second TE biopsy. All biopsies were processed for chromosomal analysis by NGS, and karyotypes were compared to the original TE biopsy. PARTICIPANTS/MATERIALS, SETTING, METHODS: The setting for this study was a single IVF center with an in-house PGT-A program and associated research laboratory. MAIN RESULTS AND THE ROLE OF CHANCE: When one or more whole chromosomes were aneuploid in the clinical TE biopsy, the corresponding ICM was aneuploid in 90 out of 93 blastocysts (96.8%). When the clinical TE biopsy contained only segmental (sub-chromosomal) aneuploidies, the ICM was aneuploid in three out of seven cases (42.9%). Blastocysts showing aneuploidy concordance between clinical TE biopsy and ICM were also aneuploid in a second TE biopsy in 86 out of 88 cases (97.7%). In blastocysts displaying clinical TE-ICM discordance, a second TE biopsy was aneuploid in only two out of six cases (33.3%). LIMITATIONS, REASONS FOR CAUTION: All embryos in this study had an initial classification of 'aneuploid' and not 'euploid' or 'mosaic'. Therefore, the findings of this study refer specifically to a TE biopsy predicting aneuploidy in the remaining blastocyst, and cannot be extrapolated to deduce the ability of a TE biopsy to predict euploidy in the blastocyst. No conclusions should be drawn from this study about the ability of a mosaic TE biopsy to predict the karyotype of the corresponding blastocyst. Caution should be exercised in generalizing the findings of the sample group of this study to the general IVF blastocyst population. The segmental aneuploidy group only contained seven samples. WIDER IMPLICATIONS OF THE FINDINGS: The high rate of intra-blastocyst concordance observed in this study concerning whole chromosome aneuploidy contributes experimental evidence to the validation of PGT-A at the blastocyst stage. Concomitantly, the results suggest potential clinical value in reassessing blastocysts deemed aneuploid by TE re-biopsy in select cases, particularly in instances of segmental aneuploidies. This could impact infertility treatment for patients who only have blastocysts classified as aneuploid by PGT-A available. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Zouves Foundation for Reproductive Medicine and Zouves Fertility Center. The authors have no competing interest to disclose. TRIAL REGISTRATION NUMBER: Not applicable.