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Purpose: To determine if an explainable artificial intelligence (XAI) model enhances the accuracy and transparency of predicting embryo ploidy status based on embryonic characteristics and clinical data., Methods: This retrospective study utilized a dataset of 1908 blastocyst embryos. The dataset includes ploidy status, morphokinetic features, morphology grades, and 11 clinical variables. Six machine learning (ML) models including Random Forest (RF), Linear Discriminant Analysis (LDA), Logistic Regression (LR), Support Vector Machine (SVM), AdaBoost (ADA), and Light Gradient-Boosting Machine (LGBM) were trained to predict ploidy status probabilities across three distinct datasets: high-grade embryos (HGE, n = 1107), low-grade embryos (LGE, n = 364), and all-grade embryos (AGE, n = 1471). The model's performance was interpreted using XAI, including SHapley Additive exPlanations (SHAP) and Local Interpretable Model-agnostic Explanations (LIME) techniques., Results: The mean maternal age was 38.5 ± 3.85 years. The Random Forest (RF) model exhibited superior performance compared to the other five ML models, achieving an accuracy of 0.749 and an AUC of 0.808 for AGE. In the external test set, the RF model achieved an accuracy of 0.714 and an AUC of 0.750 (95% CI, 0.702-0.796). SHAP's feature impact analysis highlighted that maternal age, paternal age, time to blastocyst (tB), and day 5 morphology grade significantly impacted the predictive model. In addition, LIME offered specific case-ploidy prediction probabilities, revealing the model's assigned values for each variable within a finite range., Conclusion: The model highlights the potential of using XAI algorithms to enhance ploidy prediction, optimize embryo selection as patient-centric consultation, and provides reliability and transparent insights into the decision-making process., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Epigenetics studies changes in gene activity without changes in the DNA sequence. Methylation is an epigenetic mechanism important in many pathways, such as biotic and abiotic stresses, cell division, and reproduction. Eragrostis curvula is a grass species reproducing by apomixis, a clonal reproduction by seeds. This work employed the MCSeEd technique to identify deferentially methylated positions, regions, and genes in the CG, CHG, and CHH contexts in E. curvula genotypes with similar genomic backgrounds but with different reproductive modes and ploidy levels. In this way, we focused the analysis on the cvs. Tanganyika INTA (4x, apomictic), Victoria (2x, sexual), and Bahiense (4x, apomictic). Victoria was obtained from the diploidization of Tanganyika INTA, while Bahiense was produced from the tetraploidization of Victoria. This study showed that polyploid/apomictic genotypes had more differentially methylated positions and regions than the diploid sexual ones. Interestingly, it was possible to observe fewer differentially methylated positions and regions in CG than in the other contexts, meaning CG methylation is conserved across the genotypes regardless of the ploidy level and reproductive mode. In the comparisons between sexual and apomictic genotypes, we identified differentially methylated genes involved in the reproductive pathways, specifically in meiosis, cell division, and fertilization. Another interesting observation was that several differentially methylated genes between the diploid and the original tetraploid genotype recovered their methylation status after tetraploidization, suggesting that methylation is an important mechanism involved in reproduction and ploidy changes., (© 2023. The Author(s).)

Purpose: Are human embryos arising from two plus one small pronucleated zygotes, called 2.1 pronuclei (PN), clinically useful?, Methods: In a retrospective embryo cohort study and prospective experimental study, a total of 287 cycles in which at least one 2.1PN was identified in the fertilization check were included. Embryonic development and clinical outcome were compared for the 1395 2PN zygotes and 304 2.1PN zygotes that were siblings. All embryos were individually cultured in time-lapse systems. Twenty-five 2.1PN-derived blastocysts, donated for research, were used in focused single-nucleotide variant ploidy analysis to identify the distribution pattern of heterozygosity., Results: The average diameter of PN was 24.9 ± 2.4 µm for large PN and 10.2 ± 2.4 µm for small PN; 79.9% of small PN was derived from female pronuclei. Blastocyst formation rate and good-quality blastocyst rate were significantly lower with 2.1PN embryos than with 2PN embryos (40.0% vs. 57.7%, 21.4% vs. 33.5%, respectively). A total of 13 embryos derived from 2.1PN were transferred, and three healthy babies were born. In ploidy constitutions of trophectoderm (TE), 2.1PN-derived blastocyst TE was shown to be mostly diploid (95.8%, 23/24), and only one blastocyst showed triploid., Conclusions: It was suggested that 2.1PN embryos have lower embryonic developmental potential than 2PN embryos, but most of the 2.1PN were diploid, indicating that they are likely to be clinically usable. It is recommended to perform embryo transfer following a combination of PGT-A and ploidy analysis., (© 2024. The Author(s).)

Apart from apomictic types, the Polygonum-type eight-nuclear embryo sac is considered to be dominant in grasses. A triploid endosperm is formed as a result of double fertilisation. This study showed, for the first time, the dominance of diploid nuclei in the syncytial stage of the central cell of embryo sac in oat species and amphiploids. The dominance of diploid nuclei, which were the basis for the formation of polyploid nuclei, was weaker in amphiploids due to aneuploid events. The genomic in situ hybridisation method applied in the study did not distinguish the maternal and paternal haploid nuclei of embryo sac. However, this method demonstrated the lack of a set of genomes of one haploid nucleus. Embryological analyses of the initial stages of oat endosperm development revealed a fertilised egg cell, and two polar nuclei differing in size. It can be assumed that the formation of diploid oat endosperm occurred after the fusion of one polar nucleus and the nucleus of a male gamete, while the second polar nucleus gave rise to 1n nuclei. The levels of ploidy of syncytial nuclei were not influenced by both aneuploid events and correlated with pollen developmental anomalies. The differences in the analysed cytogenetic events distinguished amphiploids and their parental species in the ordination space., (© 2023. The Author(s).)

Does the late follicular phase progesterone (P4) and the P4-to-follicle-ratio affect the ploidy state of the biopsied embryos? A retrospective observational study conducted at ART Fertility Clinics Abu Dhabi and Muscat, including all stimulation cycles performed between January 2015 and December 2019. In total, 975 cycles were considered for this study. Inclusion criteria were ovarian stimulation due to primary/secondary infertility, patient's age between 18 and 45 years, ICSI as fertilization method, and patients undergoing preimplantation genetic testing for aneuploidies (PGT-A). Patients with testicular sperm extraction (TESE) and warmed oocytes were excluded. Our results have shown that progesterone had no effect on the euploid rate (p = 0.371). However, when adding the ratio of P4 to the number of follicles that were bigger than 10 mm in the last scan, a negative effect on the euploid rate (p < 0.05) was observed. This study was able to show that the use of only P4 is unable to predict ploidy outcomes. However, by including the number of follicles > 10 mm, a clear association was observed between P4/Foll ratio and euploid rate per cycle. The use of both parameters could aid clinicians in their decision to trigger a patient or continue stimulation. Further prospective studies are warranted to confirm those results., (© 2023. The Author(s), under exclusive licence to Society for Reproductive Investigation.)

Understanding processes that govern and sustain biological diversity is a central goal of community ecology. Unisexual complexes, where reproduction depends on sperm from males of one or more bisexual host species, are rare and the processes driving their diversity and structure remain poorly understood. Unisexual Ambystoma salamanders produce distinct biotypes ('genomotypes') depending on which bisexual species they 'steal' sperm from. This reproductive mode should generate distinct assemblages depending on the locally available bisexual host species. Yet, how availability and relative abundance of multiple bisexual hosts influences composition and diversity of natural unisexual assemblages at local or regional scales remains unknown. We hypothesize that host identity most directly drives local assemblage composition, with host variation associated with increased beta and gamma diversity within unisexuals. We collected genetic samples from Ambystoma salamanders across Pelee Island, Ontario, Canada (2015-2022). Two host species were identified (A. texanum and A. laterale) with nine sites having a single host and one site having both. Unisexual assemblages were grouped into four clusters by similarity, with host identity being a key determinant. Gamma diversity increased as a result of distinct host-specific assemblages forming at different sites on the island (i.e., high beta diversity). Assemblage composition, but not diversity, was correlated with relative host abundance, which may reflect matching niche requirements between host and unisexual forms they produce. Our results demonstrate that diversity and structure of unisexual assemblages are clearly shaped by their host(s) and such systems may serve as models for studying how biotic interactions shape ecological communities., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Purpose: This retrospective observational study compares how different classes of blastocyst genotypes from egg donor cycles differentially blastulate and expand using a standard assay., Methods: Quantitative measurements of expansion utilized a customized neural network that segments all sequential time-lapse images during the first 10 h of expansion., Results: Analyses were performed using two developmental time perspectives using time-lapse imaging. The first was the time to blastocyst formation (tB), which broadly reflects variations in developmental rate. Euploidy peaked at 100-115 h from fertilization. In contrast, aneuploidy peaks flanked this interval bi-modally. These distributions limit ploidy discrimination based upon traditional standard grading features when assessed in real time. In contrast, from the second perspective of progressive blastocyst expansion that is normalized to each individual blastocyst's tB time, euploidy was significantly increased at expansion values > 20,000µ 2 across all tB intervals studied. A Cartesian coordinate plot graphically summarizes information useful to rank order blastocysts within cohorts for transfer. Defined aneuploidy subgroups, distinguished by the number and complexity of chromosomes involved, also showed distributive differences from both euploids and from each other. A small subset of clinically significant trisomies did not show discriminating features separating them from other euploids., Conclusion: A standard assay of blastocyst expansion normalized to each individual blastocyst's time of blastocyst formation more usefully discriminates euploidy from aneuploidy than real-time expansion comparisons using absolute developmental time from fertilization., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

This study examined blastomere exclusion which is seen during embryo development and could represent imperfect cell division or a mechanism of aneuploidy correction. This was a retrospective cohort study which included embryos cultured in a time-lapse incubator undergoing preimplantation genetic testing for aneuploidy (PGT-A) with trophectoderm biopsy. Embryos were evaluated for blastomere exclusion early in development, late in development, both, or neither. Blastomere exclusion was compared to embryo ploidy. Embryos with no blastomere exclusion had an aneuploidy rate of 52.9%, while embryos displaying blastomere exclusion at any stage had an aneuploidy rate of 68.5% (p < .001). Early blastomere exclusion was not significantly associated with an increased aneuploidy risk (59.2% vs. 52.9% in no blastomere exclusions; p = 0.22). However, embryos with late blastomere exclusion were significantly more likely to be aneuploid, compared to embryos with no blastomere exclusions (77.5% vs. 52.9%; p < 0.001) as were embryos with both early + late blastomere exclusions (71.2% vs. 52.9%; p < 0.001). Upon restricting the analysis to aneuploid embryos, the presence of any blastomere exclusion was not significantly associated with complex aneuploidy, defined as 2 more affected chromosomes (43.9% vs. 38.7%; p = 0.28). However, the proportion with adverse embryo genetics significantly increased with the timing of blastomere exclusion (38.7%, 37%, 45.5%, and 50% for none, early, late, and early + late; p = 0.043). Late blastomere exclusion or a combination of both early + late blastomere exclusion was associated with an increased risk of aneuploid embryo genetics. Embryo selection using time-lapse culture systems should incorporate these findings when untested embryos are transferred., (© 2022. The Author(s), under exclusive licence to Society for Reproductive Investigation.)

The majority of polyploids can withstand many stresses better than their monoploid counterparts; however, there is no proven mechanism that can fully explain the level of tolerance at the biochemical and molecular levels. Here, we make an effort to provide an explanation for this intriguing but perplexing issue using the antioxidant responses, genomic stability, DNA methylation pattern and yield in relation to ploidy level under the elevated level of ozone in Abelmoschus cytotypes. The outcome of this study inferred that the elevated ozone causes an increase in reactive oxygen species leading to enhanced lipid peroxidation, DNA damage and DNA de-methylation in all the Abelmoschus cytotypes. The monoploid cytotype of Abelmoschus, that is Abelmoschus moschatus L., experienced the highest oxidative stress under elevated O 3 , resulting in maximum DNA damage and DNA de-methylation leading to the maximum reduction in yield. While the diploid (Abelmoschus esculentus L.) and triploid (Abelmoschus caillei A. Chev.) cytotypes of Abelmoschus with lower oxidative stress result in lesser DNA damage and DNA de-methylation which ultimately leads to lower yield reduction. The result of this experiment explicitly revealed that polyploidy confers better adaptability in the case of Abelmoschus cytotypes under ozone stress. This study can further be used as a base to understand the mechanism behind the ploidy-induced stress tolerance in other plants mediated by gene dosage effect., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Introduction: Morphokinetic analysis using a closed time-lapse monitoring system (EmbryoScope + ™) provides quantitative metrics of meiotic progression and cumulus expansion. The goal of this study was to use a physiologic aging mouse model, in which egg aneuploidy levels increase, to determine whether there are age-dependent differences in morphokinetic parameters of oocyte maturation., Methods: Denuded oocytes and intact cumulus-oocyte complexes (COCs) were isolated from reproductively young and old mice and in vitro matured in the EmbryoScope + ™. Morphokinetic parameters of meiotic progression and cumulus expansion were evaluated, compared between reproductively young and old mice, and correlated with egg ploidy status., Results: Oocytes from reproductively old mice were smaller than young counterparts in terms of GV area (446.42 ± 4.15 vs. 416.79 ± 5.24 µm 2 , p < 0.0001) and oocyte area (4195.71 ± 33.10 vs. 4081.62 ± 41.04 µm 2 , p < 0.05). In addition, the aneuploidy incidence was higher in eggs with advanced reproductive age (24-27% vs. 8-9%, p < 0.05). There were no differences in the morphokinetic parameters of oocyte maturation between oocytes from reproductively young and old mice with respect to time to germinal vesicle breakdown (GVBD) (1.03 ± 0.03 vs. 1.01 ± 0.04 h), polar body extrusion (PBE) (8.56 ± 0.11 vs. 8.52 ± 0.15 h), duration of meiosis I (7.58 ± 0.10 vs. 7.48 ± 0.11 h), and kinetics of cumulus expansion (0.093 ± 0.002 vs. 0.089 ± 0.003 µm/min). All morphokinetic parameters of oocyte maturation were similar between euploid and aneuploid eggs irrespective of age., Conclusion: There is no association between age or ploidy and the morphokinetics of mouse oocyte in vitro maturation (IVM). Future studies are needed to evaluate whether there is an association between morphokinetic dynamics of mouse IVM and embryo developmental competence., (© 2023. The Author(s).)

Purpose: To determine if creating voting ensembles combining convolutional neural networks (CNN), support vector machine (SVM), and multi-layer neural networks (NN) alongside clinical parameters improves the accuracy of artificial intelligence (AI) as a non-invasive method for predicting aneuploidy., Methods: A cohort of 699 day 5 PGT-A tested blastocysts was used to train, validate, and test a CNN to classify embryos as euploid/aneuploid. All embryos were analyzed using a modified FAST-SeqS next-generation sequencing method. Patient characteristics such as maternal age, AMH level, paternal sperm quality, and total number of normally fertilized (2PN) embryos were processed using SVM and NN. To improve model performance, we created voting ensembles using CNN, SVM, and NN to combine our imaging data with clinical parameter variations. Statistical significance was evaluated with a one-sample t-test with 2 degrees of freedom., Results: When assessing blastocyst images alone, the CNN test accuracy was 61.2% (± 1.32% SEM, n = 3 models) in correctly classifying euploid/aneuploid embryos (n = 140 embryos). When the best CNN model was assessed as a voting ensemble, the test accuracy improved to 65.0% (AMH; p = 0.1), 66.4% (maternal age; p = 0.06), 65.7% (maternal age, AMH; p = 0.08), 66.4% (maternal age, AMH, number of 2PNs; p = 0.06), and 71.4% (maternal age, AMH, number of 2PNs, sperm quality; p = 0.02) (n = 140 embryos)., Conclusions: By combining CNNs with patient characteristics, voting ensembles can be created to improve the accuracy of classifying embryos as euploid/aneuploid from CNN alone, allowing for AI to serve as a potential non-invasive method to aid in karyotype screening and selection of embryos., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Caladium × hortulanum 'Fancy' is an important ornamental plant grown in pots and landscapes and known for its colorful leaves often used for interior decorations. In this work, we present a method of in vitro regeneration from three explants source through direct somatic embryogenesis (DSE) wherein the regenerated plants were screened for ploidy changes through flow cytometry analysis. Tuber, leaf and petiole explants were cultured on MS basal medium supplemented with 1-napthalene acetic acid (NAA), 6-benzyl amino purine (BAP) and N-phenyl-N'-1, 2,3-thiadiazol-5-ylurea (TDZ) concentrations. Tuber explants induced highest direct somatic embryos on NAA (1 mg L - 1 ) + BAP (0.5 mg L - 1 ) with 55.6 mean number of embryos per explant while as leaf and petiole explants amended with 1 mg L - 1 TDZ developed 18.7 and 12.27 mean number of embryos per explant respectively. The highest embryo conversion frequency was achieved on BAP (2 mg L - 1 ) + NAA (0.2 mg L - 1 ) with 44.2, 18.7 and 7.5 mean number of plantlets produced per tuber, leaf and petiole explant respectively after 4 weeks of culture. Plantlets were later rooted and maximum number of roots (6.33) per shoot was achieved on 2 mg L - 1 indolebutyric acid amended medium. Description of the process of DSE is presented through the histological and SEM evidences. The 2C DNA content of field grown plants and the DSE regenerants evaluated under flow cytometric analysis were 8.06 pg and 8.28 pg respectively showing no ploidy changes. Hence, a successful protocol of inducing direct somatic embryos from three explant types with efficient embryo conversion frequency was obtained with regenerants showing similar DNA ploidy as that of their parent plants., (© 2021. The Author(s), under exclusive licence to Institute of Plant Genetics Polish Academy of Sciences.)

Amyloidosis was modeled in young mice by administration of aqueous solution of soy cream substitute and then, amyloidogenesis was corrected by oral administration of dry red wine. In mice with amyloidosis, the area of megakaryocytes decreased by 1.69 times, the relative content of oxyphilic forms of megakaryocytes increased by 4.33 times, and ploidy did not change in comparison with the corresponding parameters in intact mice. Administration of red dry grape wine against the background of formation of systemic amyloidosis changed the reaction of megakaryocytes: the content of polychromatophilic forms was comparable to the level of intact control, the content of oxyphilic forms decreased by 3.11 times, the RNA and DNA content in megakaryocyte nuclei decreased proportionally, and the ploidy decreased. Thus, red grape wine reduced the response of the megakaryocyte genome to the external supply of amyloidogenic substance., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Key Message: A total of three QTLs, responsible for A. brassicae resistance were introgressed into S. alba - B. juncea introgression lines from S. alba and mapped through donor genome-specific SSR markers. Alternaria brassicae is a key pathogen of the Brassicaceae family causing severe blight disease to oilseed crops that leads to heavy yield losses due to lack of resistance source within cultivated Brassicas. However, the host resistance present in the Sinapis alba, an allied member of the Brassicaceae family is still unexplored precisely due to the unavailability of segregating population for Alternaria blight resistance and scarcity of donor genome-specific genetic markers. The present study was undertaken to identify quantitative trait loci governing resistance to Alternaria blight which was introgressed from S. alba to the backcross population of stable S. alba + B. juncea somatic hybrids (2n = 60; AABBSS). The second backcross population showed significant phenotypic variations for Alternaria blight ranging from immune to highly susceptible phenotype, thus suggesting quantitative nature of resistance for the disease. A subset of 154 BC 2 F 3-4 lines was used for disease screening and genotyping with 234 S. alba genome-specific microsatellite markers. As a result of the study, twelve linkage groups were developed corresponding to 12 chromosomes of S. alba (n = 12) covering a length of 1694.02 cM. The chromosomes 5 and 11 harbored a total of 1 (Abr-01), and 2 (Abr-02, and Abr-03) QTLs detected by ICIM-ADD mapping method at LOD score values 3.7, 5.12, and 6.74, respectively. The QTLs identified during the study have a range of 5.51-10.87 percent phenotypic variations for disease resistance. To the best of our knowledge, this is the first report of QTLs introgression for A. brassicae resistance in cultivated Brassica from an allied member of Brassicaceae., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Purpose: The goal is to determine if variations exist between male and female blastocysts in preimplantation measurements of quality and ploidy and in vitro fertilization elective single-embryo transfer (eSET) outcomes., Methods: A retrospective chart review was conducted from a private fertility center's database of blastocysts undergoing preimplantation genetic testing for aneuploidy, along with details of eSET from this screened cohort. Main outcomes included preimplantation embryo quality and sex-specific eSET outcomes., Results: A total of 3708 embryos from 578 women were evaluated, with 45.9% male and 54.1% female. The majority were High grade. No difference existed between embryo sex and overall morphological grade, inner cell mass or trophectoderm grade, or blastocyst transformation day. Female blastocysts had a higher aneuploidy rate than male blastocysts (P < 0.001). Five hundred thirty-nine eSETs from 392 women were evaluated, with High grade embryos more likely to have implantation (P < 0.001), clinical pregnancy (P < 0.001), and ongoing pregnancy (P = 0.018) than Mid or Low grade embryos. Day 5 blastocysts were more likely to have implantation (P = 0.018), clinical pregnancy (P = 0.005), and ongoing pregnancy (P = 0.018) than day 6 blastocysts. Male and female embryos had similar transfer outcomes, although female day 5 blastocysts were more likely to result in clinical pregnancy (P = 0.012), but not ongoing pregnancy, than female day 6 blastocysts. Male eSET outcomes did not differ by blastocyst transformation day., Conclusion: Male and female embryos have comparable grade and quality; however, female embryos were more likely to be aneuploid. Ongoing pregnancy rates did not differ by embryo sex. Day 5 embryos had more favorable transfer outcomes than day 6 embryos., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: To study the relationship between the migration speed of nucleolus precursor bodies (NPBs) in male and female pronuclei (mPN; fPN) and human embryo development during assisted reproduction., Methods: The migration speed of 263 NPBs from 47 zygotes was quantitated, and embryonic development was observed until the blastocyst stage. The central coordinates of mPN, fPN, and NPBs were noted at multiple timepoints. Then, the distance traveled by the NPBs between two sequential images was measured, and migration speed was calculated. Additionally, we investigated the relationship between NPB migration speed and ploidy status (N = 33) or live birth/ongoing pregnancy (LB/OP) (N = 60) after assisted reproduction., Results: The NPB migration speed in both mPN and fPN was significantly faster in the zygotes that developed into blastocysts (N = 25) than that in the zygotes that arrested (N = 22). The timing of blastulation was negatively correlated with NPB migration speed in the mPN. Faster NPB migration was significantly correlated with LB/OP. In multivariate logistic analysis, NPB migration speed in the mPN was the only morphokinetic parameter associated with LB/OP. In a receiver-operating characteristic curve analysis of LB/OP by the NPB migration speed in the mPN, the cut-off value was 4.56 μm/h. When this cut-off value was applied to blastocysts with preimplantation genetic testing for aneuploidy, 100% of the blastocysts faster than or equal to the cut-off value were euploid., Conclusion: The NPBs migrated faster in zygotes having the potential to develop into a blastocyst, and eventually into a baby. This predictor could be an attractive marker for non-invasive embryo selection., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objective: The primary objective of this study was to test the hypotheses that compared to IVF cycles undergoing preimplantation genetic testing for aneuploidy (PGT-A) with or without testing for monogenic disorders (PGT-M), IVF cycles undergoing PGT for structural rearrangements (PGT-SR) will have (1) a poorer blastocyst conversion rate and (2) fewer usable blastocysts available for transfer. Secondarily, the study aimed to compare pregnancy outcomes among PGT groups., Patients: Retrospective cohort study including cycles started from January 1, 2012, to March 30, 2020, with the intent of pursuing PGT-A, PGT-A with PGT-M, and PGT-SR, with trophectoderm biopsy on days 5 or 6., Results: A total of 658 women underwent 902 cycles, including 607 PGT-A, 216 PGT-A&M, and 79 PGT-SR cycles. When compared with the blastocyst conversion rate for the PGT-A group (59.4%), and after adjustment for patient age, total number of mature oocytes, BMI, and ICSI, there were no significant differences for either the PGT-A&M (69.7%, aRR 1.03, 95% CI 0.96-1.10) or PGT-SR (63.2%, aRR1.04, 95% CI 0.96-1.13) groups. Compared to the PGT-A group, the proportion of usable blastocysts was statistically significantly lower in the PGT-SR group: 35.1% versus 24.4% (aRR 0.57, 95% CI 0.46-0.71) and the PGT-A&M group: 35.1% versus 31.5% (aRR 0.68, 95% CI 0.58-0.81). Implantation, pregnancy, and miscarriage rates were equivalent for all groups., Conclusion: Patients with structural rearrangements have similar blastocyst development but significantly fewer usable blastocysts available for transfer compared to PGT-A testers. Nevertheless, with the transfer of a usable embryo, PGT-SR testers perform as well as those testing for PGT-A.

To determine whether embryo developmental stage or morphological grading can predict live birth rate (LBR) from a single blastocyst in nonbiopsied and biopsied frozen embryo transfer (FET) cycles. This retrospective study included 1336 nonbiopsied and 360 euploid FET cycles. Blastocysts were divided according to developmental stage (day 5 [D5] and day 6 [D6]) and morphology (good quality and low quality). Nonbiopsied cycles in which D5 blastocysts were transferred were associated with a significantly higher LBR than those in the D6 group (48.5 vs. 24.3%; p < 0.001), as well as in good-quality embryo transfer cycles than that in low-quality embryo cycles (52.6 vs. 25.3%; p < 0.001). Embryos reaching good-quality blastocysts on D5 yielded significantly higher LBR than those similar quality blastocysts on D6. The same trend was seen in low-quality embryos. Concerning only D5 or D6 blastocyst transfer, the LBRs of good-quality embryos were still superior to those of low-quality embryos. In the case of euploid embryo transfers, the LBR (48.9 vs. 44.9%, p = 0.444) of D5 blastocysts did not significantly differ from that of D6 blastocysts. Good-quality embryos showed a higher LBR than low-quality embryos (51.6 vs. 40.0%, p = 0.030); the adjusted odds ratio remained insignificant after controlling for confounders (aOR 1.56; 95% CI 0.99-2.45; p = 0.056). The LBRs in the same developmental stage or morphology subgroups were not statistically significant. Embryo developmental stage and morphological grade are useful predictors of LBR in nonbiopsied FET cycles. However, no association was found in euploid transfer cycles.

Purpose: To determine whether the blastocyst mitochondrial DNA (mtDNA) content is related to the miscarriage rate in patients undergoing single euploid frozen embryo transfer (SEFET)., Methods: A total of 355 single euploid frozen embryo transfer cycles were studied retrospectively between April 2017 and December 2018. A trophectoderm biopsy was performed on day 5/6 blastocysts. Post next-generation sequencing (NGS), the mtDNA content was calculated as the ratio of mitochondrial DNA over nuclear DNA, and the association between blastocyst mtDNA content and miscarriage rate was evaluated., Result(s): Three hundred fifty-five euploid blastocysts were selected for SEFET in 314 patients with an average age of 33.7 ± 5.6 years; 255 were biopsied on day 5 (71.8%) and 100 on day 6 (28.2%). Frozen embryo transfer (FET) was performed either in a hormone replacement therapy (HRT) cycle (71.8%; n = 255) or in a natural cycle (NC) (28.2%; n = 100). A pregnancy rate of 66.2% (235/355) was obtained with clinical pregnancy and miscarriage rates of 52.4% (n = 186) and 5.6% (n = 20), respectively. There was no significant difference neither between the blastocyst mtDNA content of pregnant and nonpregnant patients (27.7 ± 9.2 vs. 29.4 ± 8.6, P = 0.095) nor between patients with a clinical pregnancy and miscarriage (30.5 ± 9.3 vs. 27.3 ± 9.2, P = 0.136). Multivariate logistic regression analysis showed the same nonsignificant relationship, except for the miscarriage rate and BMI (OR 1.149, 95% CI 1.03-1.28; P = 0.012)., Conclusion(s): Mitochondrial DNA content is unable to predict the miscarriage of implanted human euploid blastocysts.

Purpose: To evaluate the factors that affect the incidence of euploid balanced embryos and interchromosomal effect (ICE) in carriers of different structural rearrangements., Methods: This retrospective study includes 95 couples with reciprocal translocations (RecT) and 36 couples with Robertsonian translocations (RobT) undergoing Preimplantation Genetic Testing for Structural Rearrangements (PGT-SR) between March 2016 and July 2019. Next-generation sequencing (NGS) was the technique used coupled with trophectoderm (TE) biopsy. Only cases with females under 38 years were included. A total of 532 blastocysts were evaluated., Results: The euploidy rate was similar in RobT when compared with RecT carriers [57/156 (36.5%) vs. 112/376 (29.8%), p = 0.127]. The pure ICE rate was significantly higher in RobT carriers [48/156 (30.8%) vs. 53/376 (14.1%), p < 0.001] than it was in RecT carriers. Female age was the independent factor for the probability of obtaining a euploid embryo in RecT and RobT carriers, and increasing female age decreases the probability of obtaining a euploid embryo. In RecT carriers, no significant differences were observed in euploidy rates, pure ICE, or combined ICE according to the length of the translocated fragment and the chromosome group. However, total ICE was significantly lower when there was a breakpoint in the short chromosome arm together with a breakpoint in the long arm [(44/158 (27.8%) for pq or qp, 51/155 (32.9%) for pp and 30/63 (47.6%) for qq; p = 0.02]., Conclusion: The incidence of euploid/balanced blastocysts was similar in both types of translocations. However, there was a significant increase in pure ICE in RobT compared to RecT carriers. In RecT carriers, the presence of the breakpoints in the long arm of the chromosomes involved in the rearrangement resulted in a higher total ICE.

Purpose: To investigate whether live birth rates from euploid blastocyst frozen-thawed embryo transfer (FET) cycles are associated with infertility diagnosis or oocyte source., Design: Retrospective analysis of FET cycles reported to SART CORS in 2014., Methods: Data from fresh IVF cycles with preimplantation genetic testing for aneuploidy (PGT-A), linked to the first FET cycles, were collected from the 2014 SART CORS database for autologous and donor oocyte cycles. Inclusion criteria were patients undergoing FET with euploid embryos (n = 4148). Demographic data including age, BMI, prior fertility, and etiology of infertility were collected from the retrieval cycle and analyzed. Patients with uterine anomalies, preimplantation genetic testing-mutation (PGT-M) for genetic diseases, gender selection, HLA determination, or systemic and immunologic disorders were excluded. The primary outcome measure was live birth (LB) rate. Potential confounders such as age, prior fertility, and maximum baseline FSH values were analyzed with regression models as indicated., Results: Though age, maximum baseline FSH, and infertility diagnosis were significantly different, LB was similar between patients undergoing autologous or donor oocyte FET cycles. Etiology of infertility was not significantly associated with LB in autologous cycles (p = 0.95). Potential confounders such as maternal age, prior fertility, and maximum baseline FSH were not associated with outcomes; however, maternal BMI was inversely related to LB in autologous cycles, with an odds ratio of 0.97 (95% CI: 0.96-0.98 (rho = - 0.08, p < 0.01))., Conclusions: After controlling for confounding variables, a euploid embryo derived from a donor or autologous oocyte results in similar LB in women with different infertility diagnoses.

Purpose: Embryo genotyping in IVF clinics aims to identify aneuploid embryos, and current methodologies rely on costly, invasive and time-consuming approaches such as PGT-A screening. MALDI-ToF-based mass spectral analysis of embryo culture has been demonstrated to be a non-invasive, affordable and accurate technique that is able to capture secretome profiles from embryo culture media extremely quick. Thus, aneuploid embryo genotypes can be distinguished from euploids from these profiles towards the development of novel embryo selection tools., Methods: A retrospective cohort study, including 292 spent media samples from embryo cultures collected from a single IVF clinic in USA. There were 149 euploid and 165 aneuploid embryos previously analysed by PGT-A next-generation sequencing techniques. Secretome mass spectra of embryos were generated using MALDI-ToF mass spectrometry in the UK. Data was systematically analysed using a fully automated and ultra-fast bioinformatic pipeline developed for the identification of mass spectral signatures., Results: Distinct spectral patterns were found for euploid and aneuploid genotypes in embryo culture media. We identified 12 characteristic peak signatures for euploid and 17 for aneuploid embryos. Data analysis also revealed a high degree of complementarity among regions showing that 22 regions are required to differentiate between genotypes with a sensitivity of 84% and a false positive rate of 18%., Conclusion: Ultra-fast and fully automated screening of an embryo genotype is possible based on multiple combinations of specific mass spectral peak signatures. This constitutes a breakthrough towards the implementation of non-invasive and ultra-fast tools for embryo selection immediately prior to transfer.

We studied the radioprotective effect of betaleukin administered to (CBA×C57Bl/6)F1 mice in single doses of 50 and 3 μg/kg 2 and 22 h, respectively, prior to long-term (21 h) whole-body low-intensity (10 mGy/min) γ-radiation ( 137 Сs; total dose 12.65 Gy). Hepatocyte ploidy, a biomarker of metabolic disorders of the liver, was evaluated, and nuclearity and ploidy indices were calculated. In 10 months after irradiation, a significant decrease in the ploidy index was revealed in the group of irradiated mice, while in animal receiving 3 or 50 μg/kg betaleukin, this parameter did not differ and even surpassed the control level, respectively. Thus, in vivo assessment of hepatocytic ploidy profile in mice revealed negative delayed effects of γ-irradiation in a dose of 12.65 Gy and a protective effect a single injection of immunomodulator betaleukin.

Key Message: One hundred and thirty four introgressions from Thinopyrum elongatum have been transferred into a wheat background and were characterised using 263 SNP markers. Species within the genus Thinopyrum have been shown to carry genetic variation for a very wide range of traits including biotic and abiotic stresses and quality. Research has shown that one of the species within this genus, Th. elongatum, has a close relationship with the genomes of wheat making it a highly suitable candidate to expand the gene pool of wheat. Homoeologous recombination, in the absence of the Ph1 gene, has been exploited to transfer an estimated 134 introgressions from Th. elongatum into a hexaploid wheat background. The introgressions were detected and characterised using 263 single nucleotide polymorphism markers from a 35 K Axiom ® Wheat-Relative Genotyping Array, spread across seven linkage groups and validated using genomic in situ hybridisation. The genetic map had a total length of 187.8 cM and the average chromosome length was 26.8 cM. Comparative analyses of the genetic map of Th. elongatum and the physical map of hexaploid wheat confirmed previous work that indicated good synteny at the macro-level, although Th. elongatum does not contain the 4A/5A/7B translocation found in wheat.

Key Message: A variety of Thinopyrum bessarabicum introgressions in both hexaploid and tetraploid wheats were generated and characterized by molecular cytogenetic analysis. Six wheat-J genome recombinants were identified with ND-FISH and GISH. Diploid wheatgrass, Thinopyrum bessarabicum (2n = 2x = 14, E b E b or J b J b or JJ), is a well-known alien source of salinity tolerance and disease resistance for wheat improvement. The true genetic potential and effect of such introgressions into wheat can be best studied in chromosomal addition or substitution lines. Here, we report the generation and characterization of various categories of Th. bessarabicum derivatives in both hexaploid and tetraploid cultivated wheats. Sequential non-denaturing fluorescence in situ hybridization (ND-FISH) and genomic in situ hybridization (GISH) are robust techniques to visualize the size of alien introgressions and breakpoints. We identified a complete set of monosomic addition lines into both bread wheat and durum wheat, except for 7J in durum wheat, by sequential ND-FISH and GISH. We also characterized alien derivatives belonging to various classes including mono-telosomic additions, disomic additions, monosomic substitutions, double monosomic substitutions, monosomic substitution-monosomic additions, double monosomic additions, and multiple monosomic additions into both bread and durum wheats. In addition, various wheat-Th. bessarabicum recombinant chromosomes were also detected in six alien derivatives. These wheat-Th. bessarabicum derivatives will provide useful cytogenetic resources for improvement of both hexaploid and tetraploid wheats.

Sequencing the entire RNA molecule leads to a better understanding of the transcriptome architecture. SMARTer (Switching Mechanism at 5'-End of RNA Template) is a technology aimed at generating full-length cDNA from low amounts of mRNA for sequencing by short-read sequencers such as those from Illumina. However, short read sequencing such as Illumina technology includes fragmentation that results in bias and information loss. Here, we built a pipeline, UNAGI or UNAnnotated Gene Identifier, to process long reads obtained with nanopore sequencing and compared this pipeline with the standard Illumina pipeline by studying the Saccharomyces cerevisiae transcriptome in full-length cDNA samples generated from two different biological samples: haploid and diploid cells. Additionally, we processed the long reads with another long read tool, FLAIR. Our strand-aware method revealed significant differential gene expression that was masked in Illumina data by antisense transcripts. Our pipeline, UNAGI, outperformed the Illumina pipeline and FLAIR in transcript reconstruction (sensitivity and specificity of 80% and 40% vs. 18% and 34% and 79% and 32%, respectively). Moreover, UNAGI discovered 3877 unannotated transcripts including 1282 intergenic transcripts while the Illumina pipeline discovered only 238 unannotated transcripts. For isoforms profiling, UNAGI also outperformed the Illumina pipeline and FLAIR in terms of sensitivity (91% vs. 82% and 63%, respectively). But the low accuracy of nanopore sequencing led to a closer gap in terms of specificity with Illumina pipeline (70% vs. 63%) and to a huge gap with FLAIR (70% vs 0.02%).

Purpose: To explore the whole-chromosome status, origins, and mechanisms of chromosomal abnormalities in good-quality cleavage embryos using multiple annealing and looping-based amplification cycle (MALBAC) sequencing., Methods: The embryos studied came from7 patients (maternal aged 26-35) who had healthy birth from the same IVF cycles. These 21 frozen day 3 good-quality embryos were thawed and disaggregated into individual blastomere. Each blastomere was collected and analyzed by MALBAC sequencing., Results: Conclusive results were obtained from a high percentage of blastomeres (95.3%). A total of 46.6% of blastomeres were diploid, 53.4% were abnormal, and 28.0% had complex aneuploidy. Out of 21 embryos, 3 (14.3%) were normal and 18 (85.7%) were mosaics, showing the occurrence of mitotic errors; aneuploidy was confirmed in all cells of 4 of the 18 embryos, which showed the coexistence of meiotic errors. Conclusive results were obtained from all blastomeres of 15 embryos (71.4%, 15/21), which enabled us to reconstruct the cell lineage on the basis of the chromosomal content of the blastomeres in each division. There were 9 mitotic errors (8.7%, 9/103): nondisjunction accounted for 88.9% (8/9), and endoreplication accounted for 11.1% (1/9)., Conclusions: In good-quality embryos, there was a high rate and diverse array of chromosomal abnormalities. Morphological evaluation does not appear to assist in the reduction in meiotic errors from parental origins. Mitotic errors were common, and nondisjunction was found to be the main mechanism causing malsegregation during the cleavage divisions.

Yeast Candida utilis is considered to be a potentially advantageous expression system for production of recombinant proteins utilizable for industrial and pharmaceutical purposes. As the scientific literature is not consistent in the ploidy of this yeast, in this work, we focused on resolving the problem via several methods such as the copy number determination of maltase gene by multiplex PCR, measuring α-glucosidase activity, the characterization of maltase gene copy number in deletion mutants using qPCR and flow cytometry. In context with the published data and results obtained in this study about the copy number of the maltase gene on C. utilis genome, we attempted to hypothesise and made conclusion about the ploidy of C. utilis. The results of this work, besides the biotechnological aspect, contribute to the elementary knowledge of C. utilis. The exact information about the ploidy or more specifically about the copy number of appropriate gene is essential for expression cassette dosage determination integrated into the chromosome of the host. In this study, we come to the conclusion that the maltase gene is present in C. utilis genome in four alleles, and in combination with flow cytometry, published information and the published genome sequences, the observations support the theory about tetraploidy of C. utilis.

Key Message: We introgressed wheatgrass germplasm from the octoploid amphiploid Triticum aestivum× Lophopyrum elongatum into wheat by manipulating the wheat Ph1 gene and discovered and characterized 130 introgression lines harboring single or, in various combinations, complete and recombined L. elongatum chromosomes. Diploid wheatgrass Lophopyrum elongatum (genomes EE) possesses valuable traits for wheat genetics and breeding. We evaluated several strategies for introgression of this germplasm into wheat. To detect it, we developed and validated multiplexed sets of Sequenom MassARRAY single nucleotide polymorphism (SNP) markers, which differentiated disomic and monosomic L. elongatum chromosomes from wheat chromosomes. We identified 130 introgression lines (ILs), which harbored 108 complete and 89 recombined L. elongatum chromosomes. Of the latter, 59 chromosomes were recombined by one or more crossovers and 30 were involved in centromeric (Robertsonian) translocations or were telocentric. To identify wheat chromosomes substituted for or recombined with L. elongatum chromosomes, we genotyped the ILs with the wheat 90-K Infinium SNP array. We found that most of the wheat 90-K probes correctly detected their targets in the L. elongatum genome and showed that some wheat SNPs are ancient and had originated prior to the divergence of the wheat and L. elongatum lineages. Of the 130 ILs, 52% were homozygous for Ph1 deletion and thus are staged to be recombined further. We failed to detect in the L. elongatum genome the 4/5 reciprocal translocation that has been reported in Thinopyrum bessarabicum and several other Triticeae genomes.

Purpose: The rate of embryonic aneuploidy increases with increasing female age and is the primary cause of lower pregnancy and live birth rates (LBR) in older reproductive age women. This retrospective cohort study evaluates single euploid embryo transfers to determine whether an age-related decline in reproductive efficiency persists., Methods: A total of 8175 non-donor single embryo transfers (SET) after pre-implantation testing for aneuploidy (PGT-A) and cryopreservation were included. These were divided into five groups by patient age: < 35 years old (n = 3789 embryos transferred), 35-37 (n = 2200), 38-40 (n = 1624), 41-42 (n = 319), and > 42 (n = 243). Implantation rate (IR), clinical pregnancy rate (CPR), and LBR were calculated for each group as a percentage of embryos transferred and compared. CPR was also analyzed as a percentage of implanted pregnancies, and LBR as a percentage of clinical pregnancies, to determine when age has the greatest impact. These results were then adjusted for confounding variables via a multivariate logistic regression model., Results: Implantation rates negatively correlated with age. After adjusting for confounders, women 38 years or older had a significantly lower IR than those under 35 (OR 0.85, 95%CI 0.73-0.99 for 38-40 years old; 0.69, 0.53-0.91 for 41-42, and 0.69, 0.51-0.94 for > 42). These differences are also apparent in CPR and LBR. The rates of progression to clinical pregnancy and live birth did not differ significantly by age group. Other factors observed to affect IR independently were anti-Müllerian hormone (AMH), day of embryo transfer, and embryo morphology., Conclusion: While selection of euploid embryos may be effective in overcoming a significant proportion of the age-related decline in reproductive efficiency, a decrease in IR, CPR, and LBR persists even when analyzing only euploid embryo transfers. The observed impact of aging is, therefore, independent of ploidy, as well as of other variables that affect reproductive efficiency. These results indicate that factors other than aneuploidy contribute to reproductive senescence.

Key Message: Novel major gene resistance against Potato virus Y in diploid populations of Solanum tuberosum Groups Phureja and Tuberosum was biologically and genetically characterised. Named Ry(o) phu , it mapped to chromosome 9. A new source of genetic resistance derived from Solanum tuberosum Group Phureja against Potato virus Y (PVY) was identified and genetically characterised in three diploid biparental potato populations. Segregation data for two populations (05H1 and 08H1) suggested the presence of a single dominant gene for resistance to PVY which, following DaRT analysis of the 08H1 cross, was mapped to chromosome 9. More detailed genetic analysis of resistance utilised a well-characterised SNP-linkage map for the 06H1 population, together with newly generated marker data. In these plants, which have both S. tuberosum Group Phureja and S. tuberosum Group Tuberosum in their pedigree, the resistance was shown to map to chromosome 9 at a locus not previously associated with PVY resistance, although there is evidence for at least one other genetic factor controlling PVY infection. The resistance factor location on chromosome 9 (named as Ry(o)phu) suggests a potential role of NB-LRR genes in this resistance. Phenotypic analysis using a GUS-tagged virus revealed that a small amount of PVY replication occurred in occasional groups of epidermal cells in inoculated leaves of resistant plants, without inducing any visible hypersensitive response. However, the virus did not enter the vascular system and systemic spread was completely prevented.

Purpose: To evaluate whether the mitoscore of cleavage stage embryos might correlate with developmental kinetics and the ploidy status., Materials: This retrospective single-center study involved all cycles between April 2016 and April 2018 in which preimplantation genetic testing for aneuploidy (PGT-A) on day 3 was performed. The mitochondrial DNA (mtDNA) content and embryo ploidy were determined on 375 single blastomere biopsies by next generation sequencing (NGS). After intracytoplasmic sperm injection, a time-lapse imaging system (embryoscope) was used to follow the development. The median mtDNA content of cleavage stage embryos (49.4) was used to stratify the embryos into two groups to compare embryo development and ploidy status: low mitoscore group (≤ 49.4) and high mitoscore group (> 49.4)., Results: The total number of euploid embryos was equal between both mitoscore groups (32.1% versus 33.5%; p = 0.854). However, embryos in the low mitoscore group had a significantly higher cell number on day 3 (8.13 ± 1.59 versus 7.62 ± 1.5; p = 0.0013) and showed a significantly faster development up until the 8-cell stage. Mitoscore was not different between euploid and aneuploid embryos, with the same blastomere number at the time of biopsy. Furthermore, absence of cavitation within 118 h after insemination was correlated with higher mitoscore values (60.22 ± 42.23 versus 50.97 ± 13.37; p = 0.006) and a lower chance of being euploid (17.1% versus 47.4%; p = 0.001)., Conclusion: mtDNA content of cleavage stage embryos correlates with time-lapse parameters. Early blastulation is correlated with a lower mtDNA content and a higher chance of euploidy.

Purpose: To investigate the effects of a carrier's sex and age on the segregation patterns of the trivalent of Robertsonian translocations., Methods: This retrospective study was designed to analyze the segregation patterns of the trivalent and euploidy rates of blastocysts. Data were collected from 154 couples with Robertsonian translocation (77 with a female carrier and 77 with a male carrier). Embryos were diagnosed via array comparative genomic hybridization between January 2013 and July 2017. The segregation patterns of the trivalent of 604 blastocysts were analyzed according to the carrier's sex and age., Results: The proportion of alternate segregation was significantly higher (82.9% vs. 55.2%) in the male carriers than in the female carriers of Robertsonian translocation, and the proportion of adjacent segregation was significantly lower (16.8% vs. 42.6%), with no difference in 3:0 segregation. The segregation patterns were similar in same-sex carriers when analyzed according to the type of translocation. The carrier's age had no influence on the segregation patterns of the trivalent., Conclusions: The proportions of the trivalent's meiotic segregation pattern differ significantly according to the carrier's sex in Robertsonian translocations and are independent of the carrier's age. A significantly higher proportion of alternate segregation for normal or balanced chromosome contents was observed in the blastocysts of the male carriers than in those of the female carriers.

Purpose: The aim was to evaluate mtDNA content and its dynamics in euploid and aneuploid embryos from cleavage to blastocyst stage following consecutive biopsies. The effect of female age on mtDNA content was evaluated by comparing reproductively younger (≤ 37 years) with older (> 37 years) women., Methods: A retrospective single-centre descriptive study was performed between August 2016 and January 2017. Forty patients, with 112 embryos, undergoing preimplantation genetic testing for aneuploidies (PGT-A) by next-generation sequencing (NGS) were included. Embryos that reached the blastocyst stage and were not selected for fresh embryo transfer were included following consecutive biopsies of a single blastomere on day 3 and trophectoderm biopsy of day 5 blastocysts., Results: Cleavage-stage mtDNA was significantly lower in fast cleaving embryos (p = 0.016). Based on the concordance between day 3 and day 5 biopsies, a difference was identified in blastocyst mtDNA content between groups (p = 0.019); true euploid blastocysts presented a lower mtDNA content. No association was identified between cleavage-stage mtDNA content and ploidy status (OR 1.008 [0.981-1.036], p = 0.565) nor between blastocyst mtDNA content and ploidy outcome (OR 0.954 [0.898-1.014], p = 0.129). No difference was found when comparing mtDNA content and ploidy outcome between the two reproductive age groups (p = 0.505 (cleavage stage) and p = 0.774 (blastocyst))., Conclusion: Mitochondrial DNA content of cleavage-stage embryos and blastocysts is unable to predict ploidy status. Subgroup analysis based on ploidy concordance between day 3 and day 5 revealed a significantly lower mtDNA content for true euploid blastocysts. Reproductive ageing does not affect mtDNA content.

Objective: To evaluate the feasibility of adjusted mitochondrial DNA quantification in human embryos as a biomarker for implantation potential., Design: Double-blind, observational, prospective analysis of an Asian population in a single university-affiliated in vitro fertilization center. A total of 1617 embryos derived from 380 infertile couples were collected. The DNA from blastomere biopsy (n = 99) or trophectoderm biopsy (n = 1518) were analyzed with next-generation sequencing., Results: The adjusted mtDNA quantification followed a non-normal distribution in both types of the embryos. When stratified by ploidy status, the adjusted mtDNA quantification was significantly higher in aneuploid trophectoderm than in euploid cells, but not in blastomeres. The adjusted mtDNA quantification of embryos showed significant but very weak positive correlation in total trophectoderm cells with maternal age (Spearman's correlation, r = 0.095, p = 0.0028) but neither in blastomeres nor stratified by ploidy status. The median adjusted mtDNA quantification was also significantly higher in aneuploid blastocysts than in euploid ones while corrected with embryo morphology. Viable embryos did not contain significantly different quantities of adjusted mtDNA compared with nonviable embryos (implanted n = 103, non-implanted n = 164; median 0.00097 vs. 0.00088, p = 0.21) in 267 transferred blastocysts., Conclusion: Quantification of adjusted mitochondria DNA in human embryos was significantly lower in euploid blastocysts than in aneuploid blastocysts. However, no statistically significant differences regarding implantation outcome were evident. To our best knowledge, this study provides the largest scale and the first correlation data between mitochondria copy number and human embryo implantation potential in Asians.

Key Message: Reproduction in triploid plants is important for understanding polyploid population dynamics. We show that genetically identical reciprocal F1 hybrid triploids can display transgenerational epigenetic effects on viable F2 seed development. The success or failure of reproductive outcomes from intra-species crosses between plants of different ploidy levels is an important factor in flowering plant evolution and crop breeding. However, the effects of inter-ploidy cross directions on F1 hybrid offspring fitness are poorly understood. In Arabidopsis thaliana, hybridization between diploid and tetraploid plants can produce viable F1 triploid plants. When selfed, such F1 triploid plants act as aneuploid gamete production "machines" where the vast majority of gametes generated are aneuploid which, following sexual reproduction, can generate aneuploid swarms of F2 progeny (Henry et al. 2009). There is potential for some aneuploids to cause gametophyte abortion and/or F2 seed abortion (Henry et al. 2009). In this study, we analyse the reproductive success of 178 self-fertilized inter-accession F1 hybrid triploids and demonstrate that the proportions of aborted or normally developed F2 seeds from the selfed F1 triploids depend upon a combination of natural variation and cross direction, with strong interaction between these factors. Single-seed ploidy analysis indicates that the embryonic DNA content of phenotypically normal F2 seeds is highly variable and that these DNA content distributions are also affected by genotype and cross direction. Notably, genetically identical reciprocal F1 hybrid triploids display grandparent-of-origin effects on F2 seed set, and hence on the ability to tolerate aneuploidy in F2 seed. There are differences between reciprocal F1 hybrid triploids regarding the proportions of normal and aborted F2 seeds generated, and also for the DNA content averages and distributions of the F2 seeds. To identify genetic variation for tolerance of aneuploidy in F2 seeds, we carried out a GWAS which identified two SNPs, termed MOT and POT, which represent candidate loci for genetic control of the proportion of normal F2 seeds obtained from selfed F1 triploids. Parental and grandparental effects on F2 seeds obtained from selfed F1 triploids can have transgenerational consequences for asymmetric gene flow, emergence of novel genotypes in polyploid populations, and for control of F2 seed set in triploid crops.

Purpose: Does blastocyst morphology following euploid elective single embryo transfer (eSET) after preimplantation genetic testing for aneuploidies (PGT-A) via next generation sequencing impact clinical outcome?, Methods: Two hundred ninety-six patients underwent PGT-A. Of 1549 blastocysts, 1410 blastocysts had a conclusive result after PGT-A and were included for analysis. An eSET policy was followed in a frozen embryo replacement cycle. A total of 179 euploid blastocysts were thawed and transferred. Clinical outcomes were categorized in four different embryo quality groups: excellent, good, average and poor., Results: Euploidy rate was 19/36 (52.7%, 95% CI 37-68), 199/470 (42.3%, 95% CI 38-47), 156/676 (23.0%, 95% CI 20-26) and 39/228 (17.1%, 95% CI 13-23) in the excellent, good, average and poor quality blastocyst groups, respectively. Fitted logistic regression analysis taking into account the following covariables: female, age, embryo chromosomal status and day of blastocyst development/biopsy showed that morphology was predictive of the comprehensive chromosome screening result (p < 0.05). A logistic regression analysis was also performed on clinical outcomes taking into account the effect of blastocyst morphology and day of blastocyst development/biopsy. None of the parameters were shown to be significant, suggesting morphology and day of blastocyst development/biopsy do not reduce the competence of euploid embryos (p > 0.05)., Conclusions: After eSET, implantation rate was 80-86%; live birth rate per embryo transfer was 60-73% and clinical miscarriage rate was found to be < 10% and were not significantly affected by the embryo morphology. Results are concordant with those reported when using aCGH and highlights the competence of poor-quality euploid embryos.

Purpose: To determine whether a selected set of mRNA biomarkers expressed in individual cumulus granulosa cell (CC) masses show association with oocyte developmental competence, embryo ploidy status, and embryo outcomes., Methods: This prospective observational cohort pilot study assessed levels of mRNA biomarkers in 163 individual CC samples from 15 women stimulated in antagonist cycles. Nineteen mRNA biomarker levels were measured by real-time PCR and related to the development of their corresponding individually cultured oocytes and subsequent embryos, embryo ploidy status, and live birth outcomes., Results: PAPPA mRNA levels were significantly higher in CC from oocytes that led to euploid embryos resulting in live births and aneuploid embryos compared to immature oocytes by ANOVA. LHCGR mRNA levels were significantly higher in CC of oocytes resulting in embryos associated with live birth compared to immature oocytes and oocytes resulting in arrested embryos by ANOVA. Using a general linearized mixed model to assess ploidy status, CC HSD3B mRNA levels in oocytes producing euploid embryos were significantly lower than other oocyte outcomes, collectively. When transferred euploid embryos outcomes were analyzed by ANOVA, AREG mRNA levels were significantly lower and PAPPA mRNA levels significantly higher in CC from oocytes that produced live births compared to transferred embryos that did not form a pregnancy., Conclusions: Collectively, PAPPA, LHCGR, and AREG mRNA levels in CC may be able to identify oocytes with the best odds of resulting in a live birth, and HSD3B1 mRNA levels may be able to identify oocytes capable of producing euploid embryos.

Relationship between the method of hypothermic exposure and ploidometric characteristics of hepatocyte nuclei of rats were studied immediately after hypothermic exposure and during the posthypothermic period. The following cooling modes were used: single submersion into 5°C water (until rectal temperature drop to 20-25°C; deep hypothermia) and single exposure at air temperature of -25°C (until rectal temperature drop to 30°C; moderate hypothermia). A manifest destructive effect of deep hypothermia on rat liver was detected: the exposure caused manifest alteration of hepatocytes (alteration index increased 81-fold immediately after the exposure and remained 16-fold higher than normally after 14 days) and an increase of DNA accumulation index with predominance of hepatocytes with 5c-8c nuclei (60% of the population) during the entire period of observation, which indicated intensification of the compensatory and adaptive processes in the liver with predominating high-ploid population of hepatocytes. Hepatocyte alteration processes in response to moderate hypothermia were less intense (hepatocyte alteration index increased 19-fold immediately after the exposure and remained 3-fold higher than normally after 14 days). The DNA accumulation index increased moderately immediately after the exposure and returned to normal after 14 days, with emergence of 2c hepatocytes and an increase in the level of 4c hepatocytes (51% of the population), which indicated realization of a different strategy of liver regeneration - recovery of the cell population intrinsic to intact animals.

Purpose: This paper aims to investigate the efficacy of IVF with preimplantation genetic testing for aneuploidy (PGT-A), using only best-scoring blastocysts from young (≤ 35 years) infertile patients undergoing single blastocyst frozen embryo transfers (FET)., Method: In this randomized controlled trial (RCT) registered 29 March 2017, 302 infertile patient-couples eligible to participate underwent autologous ICSI blastocyst freeze-all cycles. Two-hundred and twenty patient-couples satisfied the inclusion criteria (i.e., female age ≤ 35 years, two-day 5 ≥ 2BB blastocysts) and were randomized to either the PGT-A (PGT-A group, n = 109) selection arm or morphology score (morphology group, n = 111) selection arm. In both arms, the highest ranking (by morphological score) blastocysts were selected for FET., Results: Of the 109 best-scoring blastocysts that underwent PGT-A, 80 were predicted to be euploid (73.4%) and were transferred in FET (euploid subgroup). There was no statistical difference in LB rate between the euploid subgroup and morphology group (56.3% vs 58.6%, odds ratio 0.91 (95% CI 0.51-1.63), p = 0.750). In a multiple logistic regression, the transfer of euploid blastocysts was not found to be a significant predictor of LB when adjusting for female age, infertility duration, antral follicle count, and blastocyst quality, with the independent odds expressed as 0.91 (95% CI 0.50-1.66, p = 0.760)., Conclusion: In young (≤ 35 years) infertile patients with at least two ≥ 2BB blastocysts, PGT-A blastocyst selection does not result in an enhanced LB rate, with the evidence suggesting that the effectivity of PGT-A may be limited by the effectivity of TE biopsy., Trial Registration: ClinicalTrials.gov ID: NCT03095053.

Key Message: Application of a low-input chromatin profiling method, CUT&RUN, to FACS-purified Arabidopsis endosperm nuclei generates parental-specific genome-wide H3K27me3 landscapes with high sensitivity, specificity and reproducibility. Endosperm is an essential seed tissue with a unique epigenetic landscape. During endosperm development, differential epigenetic regulation of the maternal and paternal genomes plays important roles in regulating gene expression, especially at imprinted genes. In Arabidopsis, profiling the epigenetic landscape of endosperm on a genome-wide scale is challenging due to its small size, mode of development and close association with maternal tissue. Here, we applied a low-input chromatin profiling method, CUT&RUN (cleavage under targets and release using nuclease), to profile parental-specific chromatin modifications using limited numbers of Arabidopsis endosperm nuclei. We demonstrate that CUT&RUN generates genome-wide H3K27me3 landscapes with high sensitivity, specificity and reproducibility using around 20,000 endosperm nuclei purified by flow cytometry and fluorescence-activated cell sorting. H3K27me3 peaks identified by CUT&RUN and previous ChIP (chromatin immunoprecipitation) approaches were largely overlapping, with some distinctions in heterochromatin. The versatility and simplicity of CUT&RUN make it a viable alternative to ChIP, which requires greater amounts of starting material, and will enable further study of tissue- or cell-type-specific epigenomes in Arabidopsis and other plant species.

The karyotypes of Allium, a genus that comprises many crops and ornamental plants, are relatively poorly studied. To extend our knowledge on karyotype structure of the genus, the chromosomal organization of rRNA genes and CMA/DAPI bands was studied. Fluorescence in situ hybridization using 5S and 35S rDNA probes and banding methods (silver staining and CMA 3 /DAPI staining) were used to analyze the karyotypes of eight cultivated Allium L. species. Analyzed Allium taxa revealed three different basic chromosome numbers (x = 7, 8, 9) and three different ploidy levels (diploid, triploid, and tetraploid). The rDNA sites chromosomal organization is reported the first time for the six species (A. moly, A. oreophilum, A. karataviense, A. nigrum, A. sphaerocephalon, A. porrum). The Allium species that were analyzed showed a high level of interspecies polymorphism in the number and localization of the rDNA sites. The fluorescence in situ hybridization patterns of 35S rDNA sites were more polymorphic than those of the 5S rDNA in the diploid species. Several groups of similar chromosomes could be distinguished among the chromosomes that had rDNA sites in the polyploid species. Each of the groups had three chromosomes (triploid A. sphaerocephalon L.) or four chromosomes (tetraploid A. porrum L.) suggesting their autopolyploid origin. In the genomes of four of the analyzed species, only some of the 35S rDNA sites were transcriptionally active. Fluorochrome banding revealed that the CMA 3 + bands were associated with the 35S rDNA sites in all of the species that were analyzed, except A. fistulosum L. in which positive CMA 3 + bands were detected in the terminal position of all of the chromosome arms. The rDNA sequences, nucleolar organizer regions (NORs), and CMA/DAPI bands are very good chromosome markers that allowed to distinguished from two to five pairs of homologous chromosomes in analyzed Allium species. The karyotypes of the studied species could be clearly distinguished by the number and position of the rDNA sites, NORs, and CMA/DAPI bands, which revealed high interspecific differentiation among the taxa.

Purpose: The quantification of mtDNA in cumulus granulosa cells (CGCs) surrounding an oocyte has been positively linked with morphological embryonic quality. In the present study, we evaluated the link between the amount of mtDNA in CGCs surrounding an oocyte and the chances for the corresponding embryo of implanting and leading to an ongoing pregnancy., Methods: This is an observational study, performed on 84 oocyte-cumulus-complexes (OCCs) having led to the replacement of an embryo in the maternal uterus, retrieved from 71 patients undergoing IVF with intracytoplasmic sperm. The OCCs were classified in two groups, one including 26 OCCs having led to an implanted embryo and the other including 58 OCCs having led to a non-implanted embryo. The average mtDNA content of CGCs was assessed by using a quantitative real-time PCR technique., Results: Significantly higher mtDNA copy numbers in CGCs were associated with implanted embryos than with non-implanted embryos (mean 215 [sd 375] and 59 [sd 72], respectively; p < 10 4 ). Multivariate analysis, taking into account the women's age, the embryo quality, and the AMH level, suggests an independent relationship between the mtDNA content of CGCs and the potential of embryo implantation., Conclusion: During in vitro fertilization (IVF) procedures, the probability of the implantation of the embryo appears to be closely correlated to the mtDNA copy numbers in the CGCs. Our results highlight the interest of mtDNA quantification in GCGs as a biomarker of the potential of embryo implantation.

Objective: To study if the number of trophectoderm (TE) biopsied cells has an impact on implantation rates., Design: A retrospective cohort study in a single-center study., Setting: In vitro fertilization center., Patients: Patients who underwent PGT-A from January 2013 to March 2016. In total, 482 vitrified/warmed single embryo transfers were included., Interventions: None., Main Outcome Measures: Clinical pregnancies rate, implantation rate., Results: Overall, clinical pregnancies per embryo transfer were higher when a regular TE were biopsied compared to larger size biopsy cells (66% (175/267) vs 53% (115/215) (p < 0.005) respectively). Pregnancy rates were also analyzed according to embryo morphology at the moment of embryo biopsy, when a good-quality embryo was transferred the clinical outcome was 75% (81/108) in group 1 and 61% (60/99) in group 2 (p < 0.05). Data was also stratified by age in patients ≤ 35 years and > 35 years. The clinical pregnancy was 67% (51/76) in women ≤ 35 years and 65% (124/191) in women > 35 years when a regular size biopsy was performed. These results significantly reduced when a larger size biopsy was performed 54% (49/91) and 53% (66/124), respectively (p < 0.05). Further investigation indicated that miscarriage rate was similar between these groups (4% (7/182) in group 1 and 5% (6/121) in group 2)., Conclusions: These findings underscore that when a large amount of TE cells are biopsied, it may negatively affect implantation rates, but once implanted, the embryos have the same chance to miscarry or reach term.

Purpose: We investigated if automated TLI selection may be a valuable strategy to identify those euploid embryos with the best chances of success., Methods: This is a unicentric and retrospective study involving 244 patients undergoing preimplantational genetic screening (PGS) cycles with autologous oocytes or oocyte donation (OD) with single euploid embryo transferred. We examined euploid embryos selected for transfer based on morphology evaluation alone (PGS-only; control group) or by assessment using an automated TLI system (Eeva™; PGS-TLI group)., Results: In both, autologous oocytes and OD patients, significantly better implantation and clinical and ongoing pregnancy rates were obtained in the PGS-TLI group when euploid embryos with high implantation potential as predicted by the automated TLI System (Eeva™) were transferred compared with the PGS-only group. This improvement was also observed when only transfers of good morphological quality embryos were compared. TLI categories showed significant differences on blastocyst formation and euploidy rate., Conclusions: Automated TLI combined with PGS is a useful prognostic tool to identify euploid embryos with the highest potential for implantation and pregnancy. Further, these results provide evidence that a healthy pregnancy does not only depend upon normal chromosomal status.