OBJECTIVE:To study the maternal contribution to early human embryogenesis by describing the transcriptional dynamics and regulatory roles of maternal effect genes (MEGs) and transcription factors (TFs) throughout the first four cell cycles. This will be achieved using parthenogenotes, such as the human uniparental bioconstruct model. DESIGN:Descriptive observational study based on single-cell transcriptomic analysis. SUBJECTS:A total of 19 single human parthenocytes were derived from six parthenogenotes at the first (n = 2), third (n = 2), and fourth (n = 2) cell cycles. EXPOSURE:Transcriptomic changes occurring during early embryonic development in the absence of paternal genomic input. MAIN OUTCOME MEASURES:Transcript abundance of MEGs, expression levels of key TFs, number and identity of differentially expressed genes (DEGs), pathway enrichment associated with DEGs, transcriptional complexity, temporal patterns of MEG transcript decay and persistence and timing of embryonic genome activation (EGA). RESULTS:Transcriptomic analysis revealed progressive increases in transcriptional complexity, with major shifts between the third and fourth cell cycles coinciding with EGA. A total of 212 and 1,515 DEGs were identified in the third and fourth cycles, respectively (fold change ≥|2| vs. first cycle), predominantly involved in ribonucleic acid biosynthesis and cell proliferation pathways. Principal component and hierarchical clustering analyses showed distinct transcriptomic profiles by cell cycle and oocyte origin. Key TFs (DUXA, DUX4, Elk-1, E2F-1, Sp1) were implicated in cell cycle regulation. The MEG analysis revealed decay of transcripts associated with messenger ribonucleic acid clearance, alongside sustained expression of MEGs linked to cell cycle progression and spindle assembly, suggesting a nonrandom, structured maternal regulatory program. CONCLUSION:This study provides the first comprehensive single-cell transcriptomic characterization of early human parthenogenotes, suggesting a structured, genome-driven maternal program that governs early embryonic development in the absence of paternal input. The identification of key TFs and MEG signature highlights the pivotal regulatory role of the maternal genome before EGA and may inform strategies to improve outcomes in assisted reproductive technologies.

The implementation of non-invasive PGT-A offers a new strategy to genetically assess the preimplantation embryo and to enhance IVF results. The extraction of DNA from the embryo culture medium has been sufficiently demonstrated, and the ability to obtain chromosomal information as a result is particularly interesting. As morphological criteria have proven to have a weak correlation with embryo ploidy status, this technique emerges as a promising alternative for embryo selection. It also appears reasonable that avoiding biopsy may enhance further embryo development. However, there are growing concerns regarding several aspects of this technique, such as the origin of this cell free DNA, the degree of representativeness of the whole embryo, the need for extended culture or the absence of standardized protocols. Despite the published data on good prognosis couples are promising, niPGT-A is yet to be considered a substitute for trophectoderm biopsy. The current SWOT analysis aims to summarize both resolved and unresolved issues, as well as limiting aspects of niPGT-A.

Autosomal recessive or X-linked disorders are passed from parents to offspring through Mendelian inheritance patterns and may lead to severe clinical manifestations in early childhood development. Together, the Spanish Association of Human Genetics (AEGH), Association for the Study of Reproductive Biology (ASEBIR), Spanish Association of Genetic Counselling (SEAGEN), Spanish Fertility Society (SEF), Spanish Society of Clinical Genetics and Dysmorphology (SEGCD), and the Spanish Association of Prenatal Diagnostics (AEDP) developed a consensus statement for population-based genetic carrier screening (GCS). The presented opinion statement recommends that preconception GCS services be included in the public healthcare system to support couples’ reproductive autonomy and timely medical decision-making. Program design and implementation strategies, as well as key technical, ethical, and legal considerations are discussed.

STUDY QUESTION:How do transcriptomics vary in haploid human androgenote embryos at single cell level in the first four cell cycles of embryo development? SUMMARY ANSWER:Gene expression peaks at the fourth cell cycle, however some androcytes exhibit unique transcriptional behaviors. WHAT IS KNOWN ALREADY:The developmental potential of an embryo is determined by the competence of the oocyte and the sperm. However, studies of the contribution of the paternal genome using pure haploid androgenotes are very scarce. STUDY DESIGN, SIZE, DURATION:This study was performed analyzing the single-cell transcriptomic sequencing of 38 androcytes obtained from 10 androgenote bioconstructs previously produced in vitro (de Castro et al., 2023). These results were analyzed through different bioinformatics software such as g: Profiler, GSEA, Cytoscape, and Reactome. PARTICIPANTS/MATERIALS, SETTING, METHODS:Single cell sequencing was used to obtain the transcriptomic profiles of the different androcytes. The results obtained were compared between the different cycles studied using the DESeq2 program and functional enrichment pathways using g: Profiler, Cytoscape, and Reactome. MAIN RESULTS AND THE ROLE OF CHANCE:A wave of paternally driven transcriptomic activation was found during the third-cell cycle, with 1128 upregulated and 225 downregulated genes and the fourth-cell cycle, with 1373 upregulated and 286 downregulated genes, compared to first-cell cycle androcytes. Differentially expressed routes related to cell differentiation, DNA-binding transcription, RNA biosynthesis and RNA polymerase II transcription regulatory complex, and cell death were found in the third and fourth with respect to the first-cell cycle. Conversely, in the fourth cell cycle, 153 downregulated and 332 upregulated genes were found compared with third cell cycle, associated with differentially expressed processes related to E-box binding and zinc finger protein 652 (ZNF652) transcription factor. Further, significant overexpression of LEUTX, PRAMEF1, DUXA, RFPL4A, TRIM43, and ZNF675 found in androgenotes, compared to biparental embryos, highlights the paternal contributions to zygote genome activation. LARGE SCALE DATA:All raw sequencing data are available through the Gene Expression Omnibus (GEO) under accessions number: GSE216501. LIMITATIONS, REASONS FOR CAUTION:Extrapolation of biological events from uniparental constructs to biparental embryos should be done with caution. Maternal and paternal genomes do not act independently of each other in a natural condition. The absence of one genome may affect gene transcription of the other. In this sense, the haploid condition of the bioconstructs could mask the transcriptomic patterns of the single cells. WIDER IMPLICATIONS OF THE FINDINGS:The results obtained demonstrated the level of involvement of the human paternal haploid genome in the early stages of embryo development as well as its evolution at the transcriptomic level, laying the groundwork for the use of these bioconstructs as reliable models to dispel doubts about the genetic role played by the paternal genome in the early cycles of embryo development. STUDY FUNDING/COMPETING INTEREST(S):This study was funded by Instituto de Salud Carlos III (ISCIII) through the project 'PI22/00924', co-funded by European Regional Development Fund (ERDF); 'A way to make Europe'. F.D. was supported by the Spanish Ministry of Economy and Competitiveness through the Miguel Servet program (CPII018/00002). M.J.E. was supported by Instituto de Salud Carlos III (PI19/00577 [M.J.E.]) and FI20/00086. P.dC. was supported by a predoctoral grant for training in research into health (PFIS PI19/00577) from the Instituto de Salud Carlos III. All authors declare having no conflict of interest with regard to this trial.

Objective: To unravel the differential transcriptomic behavior of human androgenotes (AGs) and parthenogenotes (PGs) throughout the first cell cycles, analyze the differential expression of genes related to key biologic processes, and determine the time frame for em-bryonic genome activation (EGA) in AGs and PGs.Design: Laboratory study.Setting: Private fertility clinic.Patient(s): Mature oocytes were retrieved from healthy donors and subjected to artificial oocyte activation using calcium ionophore and puromycin to generate PGs (n = 6) or enucleated and subjected to intracytoplasmic sperm injection to generate AGs (n = 10). Intervention(s): Uniparental constructs at different early stages of development were disaggregated into constituent single cells (we suggest the terms parthenocytes and androcytes) to characterize the single-cell transcriptional landscape using next-generation sequencing.Main Outcomes Measure(s): Transcriptomic profiles comparison between different stages of early development in AGs and PGs. Result(s): The uniparental transcriptomic profiles at the first cell cycle showed 68 down-regulated and 26 up-regulated differentially expressed genes (DEGs) in PGs compared with AGs. During the third cell cycle, we found 60 up-regulated and 504 down-regulated DEGs in PGs compared with AGs. In the fourth cell cycle, 1,771 up-regulated and 1,171 down-regulated DEGs were found in PGs compared with AGs. The AGs and PGs had reduced EGA profiles during the first 3 cell cycles, and a spike of EGA at the fourth cell cycle was observed in PGs.Conclusion(s): Transcriptomic analysis of AGs and PGs revealed their complementary behavior until the fourth cell cycle. Androge-notes undergo a low wave of transcription during the first cell cycle, which reflects the paternal contribution to cell cycle coordination, mechanics of cell division, and novel transcription regulation. Maternal transcripts are most prominent in the third and fourth cell cy-cles, with amplification of transcription related to morphogenic progression and embryonic developmental competence acquisition. Regarding EGA, in PGs, a primitive EGA begins at the 1-cell stage and gradually progresses until the 4-cell stage, when crucial epigenetic reprogramming (through methylation) is up-regulated. In addition, our longitudinal single-cell transcriptomic analysis challenges that the zygote and early cleavage stages are the only totipotent entities, by revealing potential totipotency in cleavage -stage AGs and implications of paternal transcripts. (Fertil Sterile 2023;119:675-87. (c) 2022 by American Society for Reproductive Medicine.)El resumen esta disponible en Espanol al final del articulo.

To find out, at the transcriptomic level, the role played by the maternal haploid genotype in early embryo development using haploid Parthenogenotes (PGs) and to compare it with biparental embryos. Thirteen oocytes were subjected to AOA, by incubation in 6 μM calcium ionophore for 5 minutes, followed by 10 mg/mL for 5 hours. PGs were cultured in a time-lapse system, and only those that had extruded the second polar body and had a single pronucleus after 16-20 hours, were cultured for three additional days. Developmental stages analysed were: pronuclear stage (1st cell cycle (cc), n=2), 4-cell stages (3rd cc, n=2) and 7/10-celll stages (4th cc, n=2). At different developmental stages, every uniparental embryo was disaggregated into constituent blastomeres (parthenocytes) for RNAseq. Functional enrichment of biological pathways were performed using g:Profiler, Reactome and Cytoscape. As embryo development progressed, statistically significant differences in transcriptome profiles were observed in PGs. When pronuclear stage was compared with the 3rd and 4th cell cycle, PGs showed a higher number of upregulated genes. Functional enrichment analysis highlighted pathways related to "cellular death processes", "RNA and developmental processes", "cell and systems differentiation", among others. These differences were maintained between the 3rd and 4th cell cycle. Comparisons with biparental blastomeres showed significant differences in several transcendental genes. Also, there was a variable number of cells within each PGs that, transcriptomically, tend to behave differently from their sister cells, maintained in a state of higher totipotency or lower indifferentiation. The transcription level of PGs remains relatively constant at 1st and 3rd cc showing a large increase at 4th. In turn, numerous metabolic pathways involved in cell development were found to be affected throughout the first four cc. Finally, PGs showed significant differences in the expression levels of certain key genes for embryonic development compared to biparentals.

Preimplantation genetic testing is implemented worldwide as an effective tool to avoid transmission of single gene disorders and/or chromosome abnormalities. This approach requires the obtention of representative samples from embryos in order to infer its genetic status. Nowadays, the embryo biopsy is the first-choice method for embryo sampling. Biopsy procedures are safe and widely performed in the clinical routine. However, this intervention is invasive and requires trained personnel and investment in specific equipment. Recently, new sampling methods have been suggested under the term of “non-invasive”. These approaches are based on the existence of cell-free DNA into the embryo or its environment. An increasing number of studies suggest the collection of fluid from embryos or spent culture medium to obtain cell-free DNA to assess the genetic condition of preimplantation embryos avoiding embryo biopsy. The reliability of this attractive idea needs to be confirmed and validated. In this sense, this work offers a deep review of data published to date. Several methods of DNA detection, quantification and amplification have been tested and different protocols and culture systems, with or without additional embryo manipulations, are being investigated. In general terms, an enormous variability among published results is noteworthy. Central aspects as DNA detection rates, contamination with extraembryonic DNA and concordance between results (cell-free DNA versus embryo samples) are centring proofs-of-concept and validation experiments. However, basic questions as the biological origin and representativeness of cell-free DNA are pending to be answered. Solved, unsolved and serious limitations of the new approaches are discussed. A final reflection respecting the state of the technique is offered.

Objective: To quantify the percentage of monopronuclear-derived blastocysts (MNBs) that are potentially useful for reproductive purposes using classic and state-of-the-art chromosome analysis approaches, and to study chromosomal distribution in the inner cell mass (ICM) and trophectoderm (TE) for intertissue/intratissue concordance comparison. Design: Prospective experimental study. Setting: Single-center in vitro fertilization clinic and reproductive genetics laboratory. Patient(s): A total of 1,128 monopronuclear zygotes were obtained between June 2016 and December 2018. Intervention(s): MNBs were whole -fixed or biopsied to obtain a portion of ICM and 2 TE portions (TE1 and TE2) and were subsequently analyzed by fluorescence in situ hybridization, new whole-genome sequencing, and fingerprinting by single-nucleotide polymorphism array-based techniques (a-SNP). Main Outcome Measure(s): We assessed MNB rate, ploidy rate, and chromosomal constitution by new whole-genome sequencing, and parental composition by comparative a-SNP, performed in a "trio"-format (embryo/parents). The 24-chromosome distribution was compared between the TE and the ICM and within the TE. Result(s): A total of 18.4% of monopronuclear zygotes progressed to blastocysts; 77.6% of MNBs were diploid; 20% of MNBs were male and euploid, which might be reproductively useful. Seventy-five percent of MNBs were biparental and half of them were euploid, indicating that 40% might be reproductively useful. Intratissue concordance (TE1/TE2) was established for 93.3% and 73.3% for chromosome matching. Intertissue concordance (TE/ICM) was established for 78.8%, but 57.6% for chromosome matching. When segmental aneuploidy was not considered, intratissue concordance and chromosome matching increased to 100% and 80%, respectively, and intertissue concordance and chromosome matching increased to 84.8% and 75.8%, respectively. Conclusion(s): The a-SNP-trio strategy provides information about ploidy, euploidy, and parental origin in a single biopsy. This approach enabled us to identify 40% of MNBs with reproductive potential, which can have a significant effect in the clinical setting. Additionally, segmental aneuploidy is relevant for mismatched preimplantation genetic testing of aneuploidies, both within and between MNB tissues. Repeat biopsy might clarify whether segmental aneuploidy is a prone genetic character. (C) 2021 by American Society for Reproductive Medicine.

Rescate de blastocistos humanos derivados de monopronucleados: un modelo para estudiar la topografía cromosómica y la huella genética.

Recommendations are made for the genetic screening of gamete donors. These recommendations are the result of the consensus reached by a work group consisting of representatives from the Spanish Fertility Society, the Spanish Association of Andrology, the Spanish Association of Medical Biopathology and Laboratory Medicine, the Association for the Study of Reproductive Biology and the Spanish Association of Human Genetics and were subsequently reviewed and approved by the executive boards of each of these associations. This document describes 2 types of genetic screening: a basic mandatory screening of all donors and an extended genetic screening for donor-recipient genetic matching. The importance of pre- and post-screening genetic counselling, the management of the occurrence of adverse reactions of a genetic nature and the use of informed consent from donors who accept the management of their DNA samples stored in the centre's bank are emphasized. The role of informed consent to find out patients’ opinions on what type of screening they want to be carried out, their desire to know the results of the screening and aptitude for future genetic data is also highlighted.

To describe the ploidy and euploidy, chromosomal concordance between different regions of the trophectoderm (TE) and also between TE and inner cell mass (ICM). Besides, we aimed to identify chromosomal inheritance (paternal/maternal) of haploid, diploid and poliploid blastocysts derived from monopronuclear (MPN) zygotes. Additionally, it will be discussed the eventual "rescue" of these blastocysts for reproductive purposes. Prospective experimental study that includes 910 ICSI cycles from 892 couples registered from April 2016 to December 2018. 1081 MPN zygotes (1.2%) were obtained. A total of 199 zygotes reached the blastocyst stage (18.5%, blastocyst rate). Seventy-six blastocysts were assigned to three experimental series, according to the genetic analysis performed (ploidy, topography and parental inheritance). The study was carried out in 3 series. Series 1: 26 blastocysts were fixed by FISH (chromosomes X, Y, and 18) to assess ploidy. Series 2: 35 blastocysts were biopsied in three samples, two from TE (TE1 and TE2) and ICM. TE1 let us to determinate ploidy by FISH (chromosomes X, Y, 18); TE2 and ICM were used for 24-chromosomes study by NGS. Series 3: 15 blastocysts were biopsied as described in Series 2. TE1: study of 24 chromosomes by NGS. TE2: study of 24 chromosomes and SNPs (single nucleotide polymorphisms) by SNP-array of 750K in a "trio" format (simultaneous study of paternal/maternal/TE2 DNAs) and bioinformatic analysis. R-package for statistical analysis. The rest of the embryo was used for ploidy determinations by FISH (chromosomes X, Y, 18). 80.5% of MPN-derived blastocysts were diploid, 8% mosaic and 11.5% haploid (P<0.01). Diploid blastocysts showed a normal sex ratio (1:1); 50% diploid blastocysts were aneuploid. In relation to chromosomal topography, results showed different patterns, according to the chromosomal instability grade. Correlation between compartments (TE and ICM) was perfectly matched when both compartments were euploid or whole-chromosome aneuploid (trisomies and monosomies). Incomplete matching between compartments was observed in complex (>3 chromosomes involved), segmental or mosaic samples, which were more frequently observed in those from TE. 70% MPN-derived blastocysts showed two copies of both parental genomes. In relation to parental inheritance, 40% of blastocysts were diploid heteroparental. The MPN experimental model confirms the chromosomal correlation between ICM and different regions of TE, in cases of euploidy or pure aneuploidy. The chromosomal instability associated to segmental aneuploidy seems to be confined equally to both TE and ICM compartments. A high percentage of MPN-derived blastocysts showed two copies of both parental and euploid genomes. These data re-open debate on their convenience for clinical reproductive use.

PURPOSE:Fluorescence in situ hybridization (FISH) in spermatozoa provides an estimate of the frequency of chromosomal abnormalities, but there is not a clinical consensus on how to statistically analyze sperm FISH results. We therefore propose a statistical approach to establish sperm aneuploidy thresholds in a fertile population.METHODS:We have determined the distribution and variation of the frequency of nullisomy, disomy, and diploidy for a set of 13 chromosomes (1, 2, 9, 13, 15, 16, 17, 18, 19, 21, 22, X, and Y) in sperm nuclei from 14 fertile men by means of automatized FISH. The dispersion of data has been analyzed by the non-parametric Wilcoxon Rank Sum test. We have established the threshold values for each chromosome and aneuploidy type on the basis of the confidence interval values (99.9%).RESULTS:Nullisomy thresholds ranged from 0.49% for chromosome 19 to 3.09% for chromosome 22; disomy thresholds ranged from 0.30% for chromosome 21 to 1.47% for chromosome 15; diploidy thresholds ranged from 0.24% for the 9/19 chromosome set to 1.21% for the 13/21 chromosome set.CONCLUSIONS:Applying this approach with clinical purposes will enable us to categorize the patient as altered or normal regarding his sperm aneuploidy. Any result surpassing the cited threshold values indicates a 99.9% probability of being significantly different from fertile controls.

To determine the optimal concentration of 6-DMAP to synchronize human zygotes at the pronuclear stage (presumably at the G2-phase of the cell-cycle) without compromising subsequent development to blastocyst, as possible pre-treatment to enhance the natural DSB repair pathways in CRISPR-Cas9 technology. This study used mono- (MPN; n=580) and tripronuclear (TPN; n=261) human embryos. They were incubated for 6hrs in different 6-DMAP concentrations, in order to assess the arresting rate. After 6-DMAP treatment, zygotes were cultured to the blastocyst stage, in order to assess the effect of 6-DMAP on subsequent developmental competence. MPN and TPN zygotes were incubated in 0mM (control), 0.24mM, 0.48mM or 0.60mM 6-DMAP, in GEMS medium (Genea Biomedx) for 6h at 37ºC, 6%CO2 and 5%O2. Arresting rate was calculated as percentage of zygotes, blocked at PN stage when 6-DMAP treatment had finished. Then, MPN/TPN were cultured in a time-lapse incubator in 20μL GEMS for 5 days. Blastocyst rate was calculated as a percentage of blastocysts per number of pronuclear-arrested zygotes. Morphokinetic variables included the precise occurrence time of pronuclear fading and cleavage (6h after 6-DMAP treatment, t0). Concerning MPN zygotes, higher arresting rates were observed in 0.48mM and 0.60mM 6-DMAP groups (averaged: 86.1%) than in 0.24mM (44.4%; p= 0.004). In 0.24mM and 0.48mM 6-DMAP groups, some zygotes exhibited an anomalous pronuclear fragmentation at the end of 6-DMAP treatment (27.8% and 7.1%, respectively). This event was never observed in 0.60mM or control groups. Morphokinetic analysis showed that regardless 6-DMAP concentration, PNF and cleavage occurred at comparable timings (averaged: 3.9h and 8.3h, respectively). Regardless of 6-DMAP concentration, arrested MPN cleaved (78.3%) and progressed to the blastocyst stages (18.2%) at comparable rates to controls (77.8%; p=0.3 and 18.6%; p=0.96, respectively). As regards TPN zygotes, they were arrested at the pronuclear stage efficiently (averaged, 92.3%), regardless 6-DMAP concentration. No PN fragmentation was observed at any 6-DMAP concentration or controls. However, at 0.24mM and 0.48mM concentrations pronuclei faded significantly latter than 0.60mM group did (2.8-8.0h vs. 1.6-4.0h; p=0.03). Concerning developmental competence, TPN zygotes cleaved (83.0%) and progressed to the blastocyst stage (33.6%) at comparable rates to control (81.7%; p=0.34 and 33.6%; p=0.6, respectively), regardless 6-DMAP concentration. MPN and TPN zygotes, incubated in 0.6mM 6-DMAP for 6h did efficiently arrest the first cell-cycle at the G2-phase without compromising subsequent development. This finding could have a potential applicability in CRISPR-Cas9 technology due to DSB repair pathways are dependent of the cell-cycle stage.

Abstract Background Microarray-based and next generation sequencing (NGS) technologies have revealed that segmental aneuploidy is frequently present in human oocytes, cleavage-stage embryos and blastocysts. However, very little research has analyzed the type, size, chromosomal distribution and topography of the chromosomal segments at the different stages of development. Methods This is a retrospective study of 822 PGT-A (preimplantation genetic test for aneuploidies) performed on trophectoderm samples from 3565 blastocysts biopsied between January 2016 and April 2017. The cycles in question had been initiated for varying clinical indications. Samples were analyzed by next generation sequencing-based technology. Segmental aneuploidies were evaluated when fragment size was > 5 Mb. Blastocysts presenting a single segmental aneuploidy (SSA), without any additional whole-chromosome gain/loss, were statistically analyzed for incidence, type, size and chromosomal emplacement. Segment sizes relative to the whole chromosome or arm (chromosome- and arm-ratios) were also studied. Results 8.4% (299/3565) of blastocysts exhibited segmental aneuploidy for one or more chromosomes, some of which were associated with whole-chromosome aneuploidy while others were not. Nearly half of them (4.5%: 159/3565 of blastocysts) exhibited pure-SSA, meaning that a single chromosome was affected by a SSA. Segments were more frequent in medium-sized metacentric or submetacentric chromosomes and particularly in q-chrmosome arms, variables that were related to trophectoderm quality. SSA size was related to a greater extent to chromosome number and the arm affected than it was to SSA type. In absolute values (Mb), SSA size was larger in large chromosomes. However, the SSA:chromosome ratio was constant across all chromosomes and never exceeded 50% of the chromosome. Conclusions SSA frequency is chromosome- and topographically dependent, and its incidence is not related to clinical or embryological factors, but rather to trophectoderm quality. SSA might be originated by chromosome instability in response to chromothripsis, bias introduced by the biopsy and/or iatrogenic effects. Trial registration Retrospectively registered.

Our objective is to describe quantitatively and qualitatively segmental aneuploidies (SA) in trophoectoderm samples, defined as a loss or gain of a chromosomal fragment and its relationship with clinical and embryological parameters. Clinical retrospective historical study 3628 blastocysts were studied of 844 cycles of PGT-A.Trophoectoderms were analyzed by NGS (next generation sequencing).SA was considered, if the lost / gained fragment measured was higher than 5 Mb.The diagnosed aneuploidies were classified as: complete chromosome, single segmentals (only a segmented chromosome, with or without complete chromosome aneuploidy) and pure segmentals (PSA, segmented chromosome unique without additional aneuploidy).We defined: prevalence, type, size, distribution and chromosomal topology (arm p or q) and its relation to: clinical indication, blastocyst stage and quality of the MCI and of the trophoectoderm. 8.6% (314/3628) of blastocysts showed SA associated or not with complete chromosome aneuploidy;7.9% (288/3628) exhibited unique SA, and 4.4% (161/3628) PSA. The incidence of PSA was not related to clinical or embryological parameters, except for the quality of the trophoectoderm.Chromosomes 19, 22, and Y did not exhibit PSA. PSAs were more frequent in the q arm of the metacentric and submetacentric chromosomes. Its size was greater in q than in p. The PSA/ chromosome ratio was constant. The PSA in q was greater than in p.The ratio PSA / arm was lower in arm q. The description of the PSAs only relates to intrachromosome topographic parameters PSA is chromosome-dependent with clear topographic effect. In addition, it does not vary with maternal age, but it does vary with the morphology of the blastocyst, as a possible indicator of chromosomal inestability in the trophoectoderm.

To describe the 1PN embryos population from a morphometric and morphokinetic point of view, according to their ploidy. Basic research study including 249 1PN zygotes recruited during 12 months. Pronucleus (PN) and zygote (ZY) diameters were sized in 217 zygotes and the corresponding area and volume were calculated. After sizing, 1PN-zygotes were fixed for ploidy determinations by FISH, using 5-chromosome probes. The remaining 1PN-zygotes (n=217) were cultured in a time-lapse incubator for 5 or 6 days until the blastocyst stage in order to assess the direct variables: timing for PN appearance (tPNA) and fading (tPNF), cleavage to the 2-, 3-, 4-, 5-, 6-, 7-, 8-cell stages, morula and blastocyst (t2, t3, t4, t5, t6, t7, t8, tM, tB, respectively) which led us to calculate the indirect variables: duration of the first cycle S-phase (PNF-PNA), duration of the second and third cell cycles (t4-t2 and t8-t5, respectively). Ploidy was determined by FISH on 17 blastocysts. Once the ploidy of 1PN-zygotes and blastocysts were known, morphometrics and morphokinetics were compared by test-t. FISH results on zygotes showed that 56.5% were haploid, 34.8% diploid and 8.7% mosaic. Pronuclear diameter, area and volume were significantly larger (p<0.05) in diploid zygotes (28.5±1.5 μm; 6.3±0.9x100 μm2; 17.1±4.8 x10⁴ μm3) than in haploid ones (25.2±1.9 μm, 5±0.8 x100 μm2, 9.9±4 x10⁴ μm3). After culture, 26.3% (57/217) of 1PN-zygotes reached the blastocyst stage. FISH analysis showed that 82.3% blastocysts were diploid (7XX and 7XY) and three were female mosaic (XX/XXXX). None haploid blastocyst was observed. In 1PN-zygotes that had progressed to blastocysts, pronuclear fading (21.6±3.5hrs) and t2 (26.4±5.8hrs) occurred earlier (p<0.05) than in those arrested at cells (24.9±7.5hrs; 28.2±5.2hrs, respectively). In such blastocysts, the first S-phase duration was significantly shorter (p<0.05) than in arrested 1PN-embryos (14.3±3.2hrs vs. 17.1±7.5hrs, respectively). The ploidy condition (haploid vs. diploid) of 1PN zygotes could be inferred by morphometrics. The haploid condition impairs embryo developmental competence to progress to the blastocyst stage which was observable by delayed morphokinetics. Nevertheless, more data to confirm and assess the predictable value of these preliminary observations are required. Furthermore, complementary genetic analysis on euploidy and heteroparentality of 1PN-derived blastocysts are also required in order to use such 1PN embryos for reproductive purposes.

Currently, we are witnessing revolutionary advances in the analytical power of genetic tools. An enormous quantity of data can now be obtained from samples; however, the translation of genetic findings to the general status of individuals, or their offspring, should be done with caution. This is especially relevant in the reproductive context, where the concepts of “transmission” and “inheritability” of a trait are crucial. Against this background, we offer new insight based on a systemic view of genetic constitution in the compartmentalized organism, that is, the human body. This model considers the coexistence of “different” genomes in the same individual and the repercussion of this on reproductive efficacy and offspring. Herein, we review the major differences between somatic, germinal, embryonic, and fetal/placental genomes and their contribution to the next generation and its reproductive efficacy. The major novelty of our approach is the holistic interaction between microsystems within a macrosystem (i.e., the reproductive system). This panoramic model allows us to sketch the future implications of genetic results in function of the origin (compartment) of the sample: peripheral blood or other somatic tissues, gametes, zygotes, preimplantation embryos, fetus, or placenta. We believe this perspective can be of great use in the context of reproductive genetic counseling.

STUDY QUESTION:Has PGD-HLA been successful relative to diagnostic and clinical efficacy?SUMMARY ANSWER:The diagnostic efficacy of PGD-HLA protocols was found lower in this study in comparison to published PGD-HLA protocols and to that reported for general PGD by ESHRE (78.5 vs 94.1% and vs 92.6%, respectively), while the clinical efficacy has proven very difficult to assess due to inadequate follow-up of both the ART/PGD and HSCT procedure outcomes.WHAT IS KNOWN ALREADY:The first clinical cases for PGD-HLA were reported in 2001. It is now a well-established procedure, with an increasing number of cycles performed every year. However, PGD-HLA is still offered by relatively few PGD centres, the currently available data is fragmented and most reports on PGD-HLA applications are limited in number and scope. Published systematic details on methodology, diagnostic results, overall ART success and haematopoietic stem cell transplantation (HSCT) outcomes are limited, precluding an evaluation of the true clinical utility of PGD-HLA cycles.STUDY DESIGN, SIZE, DURATION:This retrospective multi-centre cohort study aimed to investigate the diagnostic and clinical efficacy of the PGD-HLA procedure and the aspects of PGD-HLA cycles influencing positive outcomes: birth of genetically suitable donor-baby (or babies) and HSCT. In April 2014, 32 PGD centres (Consortium members and non-members) with published/known PGD-HLA activity were invited to participate. Between February and September 2015, 14 centres submitted their data, through a custom-designed secure database, with unique login access for each centre. Data parameters covered all aspects of PGD-HLA cycles (ART, embryology and genetic diagnosis), donor-babies born and HSCT.PARTICIPANTS/MATERIALS, SETTING, METHODS:From 716 cycles submitted by 14 centres (performed between August 2001 and September 2015), the quality evaluation excluded 12 cycles, leaving 704, from 364 couples. The online database, based on REDCap, a free, secure, web-based data-capture application, was customized by Centre for Clinical Epidemiology and Outcomes Research (CLEO), Athens. Continuous variables are presented using mean, standard deviation, median and interquartile range, and categorical variables are presented as absolute and relative frequencies.MAIN RESULTS AND THE ROLE OF CHANCE:The data included 704 HLA-PGD cycles. Mean maternal age was 33.5 years. Most couples (81.3%) requested HLA-typing with concurrent exclusion of a single monogenic disease (58.6% for beta-thalassaemia). In 92.5% couples, both partners were fertile, with an average 1.93 HLA-PGD cycles/couple. Overall, 9751 oocytes were retrieved (13.9/cycle) and 5532 embryos were analysed (7.9/cycle). Most cycles involved fresh oocytes (94.9%) and Day 3 embryo biopsy (85.3%). In 97.5% of cycles, the genotyping method involved PCR only. Of 4343 embryos diagnosed (78.5% of analysed embryos), 677 were genetically suitable (15.4% of those analysed for HLA alone, 11.6% of those analysed for HLA with exclusion of monogenic disease). Of the 364 couples, 56.6% achieved an embryo transfer (ET) and 598 embryos were transferred in 382 cycles, leading to 164 HCG-positive pregnancies (pregnancy rate/ET 41.3%, pregnancy rate/initiated cycle 23.3%) and 136 babies born (live birth rate/ET 34.3%, live birth rate/initiated cycle 19.3%) to 113 couples. Data analysis identified the following limitations to the overall success of the HLA-PGD procedure: the age of the mother undergoing the treatment cycle, the number of oocytes collected per cycle and genetic chance. HSCT was reported for 57 cases, of which 64.9% involved combined umbilical cord-blood and bone marrow transplantation from the HLA-identical sibling donor; 77.3% of transplants reported no complications.LIMITATIONS REASONS FOR CAUTION:The findings of the study may be limited as not all PGD centres with PGD-HLA experience participated. Reporting bias on completion of the online database may be another potential limitation. Furthermore, the study is based on retrospective data collection from centres with variable practices and strategies for ART, embryology and genetic diagnosis.WIDER IMPLICATIONS OF THE FINDINGS:This is the first multi-centre study evaluating the clinical utility of PGD-HLA, indicating variations in practice and outcomes throughout 15 years and between centres. The study highlights parameters important for positive outcomes and provides important information for both scientists and couples interested in initiating a cycle. Above all, the study underlines the need for better collaboration between all specialists involved in the ART-PGD/HLA procedure, as well as the need for comprehensive and prospective long-term data collection, and encourages all specialists to aim to properly evaluate and follow-up all procedures, with the ultimate aim to promote best practice and encourage patient informed decision making.STUDY FUNDING/COMPETING INTEREST(S):The study wishes to acknowledge ESHRE for funding the customization of the REDCap database. There are no competing interests.TRIAL REGISTRATION NUMBER:N/A.