Search Results (204)

Puccinia graminis f. sp. tritici (Pgt) is responsible for stem rust in wheat, a disease with worldwide occurrence. Ethylene response factors (ERFs), a group of transcription factors (TFs) responsive to ethylene, are essential for managing stress signaling under biotic and abiotic challenges. However, our understanding of ERF TFs’ function in wheat (Triticum aestivum L.) resistance against the obligate biotrophic Puccinia graminis f. sp. tritici remains limited. In this work, we report our findings of the TaERF109 gene, which is transcriptionally up-regulated by ethylene or Pgt infection. TaERF109 is localized in the nucleus of rice protoplasts. Results obtained using the yeast one-hybrid (Y1H) assay support the conclusion that TaERF109 interacts with the AGCCGCC sequence (GCC-box). Transient knockdown of TaERF109 via virus-induced gene silencing (VIGS) increased wheat susceptibility to Pgt, accompanied by the down-regulation of three pathogenesis-related (PR) genes, TaPR1, TaPR2, and TaPR10, as confirmed via real-time quantitative PCR. In contrast, the Agrobacterium-mediated overexpression of TaERF109 potentiated resistance of transgenic wheat against Pgt. Overall, these results expand the current understanding of the TaERF109 gene’s function in wheat resistance to Pgt. Full article

Background/Objectives: In the last decade, non-invasive methods for aneuploidy detection have been explored. The most successful approach involves analyzing the cell-free DNA (cfDNA) released by the embryo into the culture medium. The main objective of this study is to examine the technical feasibility of this new approach called non-invasive PGT-A or niPGT-A. In addition, as an exploratory objective, the impact of the niPGT-A results on clinic outcomes will be assessed. Methods: This was a multicenter, international study that included 716 patients and 2539 blastocysts (ClinicalTrials.gov: NCT03520933). Each embryo was cultured following a specific protocol for niPGT-A. Individual spent blastocyst medium (SBM) and trophectoderm (TE) biopsy were obtained, analyzed, and compared to assess concordance. In a subset of embryos, the comparison also included an inner cell mass (ICM) biopsy. Clinical outcomes from the embryo transfers performed (all based on the TE result) were registered, and results were analyzed blindly regarding the impact of aneuploidies in the culture medium. Results: The concordance rate between SBM and TE was 79.1% (range: 74.1–82.1; cycles with autologous oocytes). This value increased to 87.0% when comparing SBM and ICM. Applying an adapted embryo culture protocol to collect the SBM for niPGT-A did not affect blastocyst quality. Analysis of the embryo transfers performed (n = 265) revealed a trend towards lower miscarriage rate in blastocysts where both TE and SBM were concordant and euploid (13.0%), compared to blastocysts with a euploid TE and an aneuploid SBM (22.2%). Conclusions: The results obtained show a high concordance between the SBM and TE biopsies. Although additional refinement of the technique would further increase niPGT-A’s performance, the results obtained support the potential use of this non-invasive approach for aneuploidy detection. The high concordance of the cfDNA present in the SBM with the corresponding ICM biopsy and the miscarriage rate observed in cases with an aneuploid SBM, despite the euploid TE results, also support niPGT-A’s capacity to assess embryo aneuploidies and its potential as a prioritization system for selecting blastocysts to transfer. This approach could hold special interest in patients with no PGT-A indications, couples that prefer not to biopsy their embryos or those who do not have access to invasive PGT-A. Full article

Objective: To investigate the relationship between the number of previous implantation failures (IFs) and embryo ploidy status, as well as subsequent clinical outcomes, in women with recurrent implantation failure (RIF) undergoing preimplantation genetic testing for aneuploidy (PGT-A). Methods: This retrospective cohort study included 422 women with RIF who underwent their first PGT-A cycle between 2017 and 2022. Participants were stratified by maternal age (<38 years, n = 292; ≥38 years, n = 130) and by the number of previous IFs, categorized as 3, 4, or ≥5. The primary outcomes were embryo ploidy rates (euploidy, aneuploidy, and mosaicism). Secondary outcomes included reproductive outcomes after single euploid blastocyst transfer (biochemical pregnancy, clinical pregnancy, ongoing pregnancy, live birth, and pregnancy loss) and neonatal birth weight. Results: Women aged ≥38 years had a significantly lower euploidy rate than those <38 years (24.8% vs. 47.3%, p < 0.001). Ploidy distribution did not differ significantly across IF categories. Among women aged <38 years with ≥5 IFs, a greater number of previous embryo transfer attempts was independently associated with higher odds of live birth after euploid embryo transfer (adjusted OR = 1.258, 95% CI: 1.051–1.505; p = 0.012). Neonatal weight did not differ significantly across IF categories. Conclusions: The number of previous IFs was not independently associated with embryo ploidy or clinical outcomes after euploid transfer, whereas advanced maternal age was strongly associated with a lower likelihood of obtaining euploid embryos. In younger women with ≥5 IFs, a greater number of previous embryo transfer attempts was associated with live birth after euploid transfer; however, this exploratory subgroup finding should be interpreted cautiously and requires prospective validation. Because this study did not directly evaluate therapeutic strategies, any potential role for individualized endometrial evaluation or optimization should be considered as hypothesis-generating rather than supported by the present data. Full article

Background/Objectives: Laser-assisted zona pellucida (ZP) drilling on day 4 embryos is routinely performed in IVF laboratories to facilitate trophectoderm (TE) herniation for blastocyst biopsy. Nevertheless, inner cell mass (ICM) herniation through the initial ZP opening occasionally occurs and may interfere with standard TE biopsy. Methods: This retrospective study assessed the clinical and obstetric safety of a double ZP drilling strategy for TE biopsy in preimplantation genetic testing for aneuploidy (PGT-A) cycles. A total of 560 single euploid embryo transfer cycles were analyzed. Blastocysts were categorized (Groups 1–6) based on ICM/TE herniation patterns and the corresponding biopsy approach. Clinical outcomes were compared between cycles undergoing TE biopsy through a single ZP opening (TE hatching with ICM remaining within the ZP) and cycles requiring a second opening to relocate the biopsy site when the ICM herniated through the original opening or was positioned externally. Results: The single-opening approach of Group 1 accounted for 295 cycles (52.7%), with implantation, miscarriage, and live birth rates of 65.4%, 14.0%, and 56.3%, respectively. The double-opening approach of Group 3 was applied in 21 cycles (3.8%), yielding implantation, miscarriage, and live birth rates of 66.7%, 0%, and 66.7%, respectively. No significant differences were observed between the two strategies in implantation, miscarriage, or live birth rates. Obstetric and neonatal outcomes, including gestational age, birth weight, and monozygotic twinning incidence, were comparable. Fifteen healthy infants were delivered following TE biopsy using the double-opening strategy. Conclusions: These data support incorporating ICM position into TE biopsy decision-making and suggest that creating a second ZP opening to reposition the biopsy site is clinically feasible and does not compromise reproductive or obstetric outcomes in PGT-A cycles. Full article

Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant neurodegenerative disorder caused by a pathogenic CAG trinucleotide repeat expansion in the ATXN2 gene. At-risk couples can embark on unaffected pregnancies through preimplantation genetic testing of monogenic disorders (PGT-M) of SCA2, which should involve accurate repeat expansion detection together with risk haplotype tracking using informative linked markers. Two couples underwent SCA2 PGT-M involving analysis of whole genome amplified embryonic trophectoderm cells by ATXN2 (CAG)n triplet-primed PCR (TP-PCR) and linkage-based risk allele genotyping using customized markers. To simplify and expedite the identification of informative markers for future PGT-M cases, putative microsatellite markers closely linked to ATXN2 were initially screened for polymorphism using a small set of anonymous DNA samples obtained from Coriell Cell Repository. Shortlisted markers with high polymorphism likelihood were then multiplexed in a single-tube reaction and genotyped on 190 anonymous DNA samples to determine their polymorphic information content. Across both SCA2 PGT-M clinical cases, the linked marker genotypes corroborated the TP-PCR results, allowing clear differentiation between unaffected and affected embryos. In both cases, transfer of an unaffected embryo led to a successful pregnancy and live birth of a healthy baby. In silico mining, filtering, and curation identified 287 microsatellites located within 1.65 Mb of either side of the ATXN2 CAG repeat. Of these, eight upstream and nine downstream polymorphic markers were successfully co-amplified in a single-tube assay and demonstrated high overall heterozygosity in both Chinese and Caucasian populations. Conclusion: To ensure high diagnostic accuracy for PGT-M of SCA2, we developed a heptadecaplex microsatellite marker panel for haplotype-based linkage analysis to complement TP-PCR-based direct detection of the ATXN2 CAG repeat. The panel can rapidly identify informative markers from virtually any couple, and it works equally well on MDA-amplified DNAs for embryonic haplotype analysis. Full article

Background and Objectives: Aneuploidy is the leading cause of implantation failure and miscarriage, with prevalence increasing with maternal age. Embryos classified as chaotic, characterized by the presence of five or more chromosomal abnormalities, and those with complex aneuploidies, defined by two to four abnormalities, represent a controversial category in preimplantation genetic testing for aneuploidy (PGT-A), as the potential for misclassification remains a significant concern. Materials and Methods: We performed a retrospective study at the Calla IVF Center, Oradea, analyzing 230 blastocysts grouped by maternal age (25–30, 31–35, 36–40, and 41–50 years). A trophoblast biopsy was performed on days 5–7, and the samples were analyzed by next-generation sequencing (NGS). Embryos were classified as euploid, aneuploid, mosaic, or chaotic. The 19 embryos initially diagnosed as chaotic were thawed and subjected to re-biopsy. Statistical analysis included descriptive statistics (chi-square tests and ANOVA) and multivariable regression models, with p < 0.05 as the criterion for statistical significance. Results: Aneuploidy increased with maternal age, from 29.6% in women aged 25–30 years to 68.7% in those aged 41–50 (p = 0.002). Poor-quality blastocysts exhibited higher aneuploidy rates (72.4%) than good-quality embryos (34.6%; p = 0.004). Chaotic embryos comprised 8.3% of the cohort. Upon re-biopsy, none were confirmed as euploid; all remained abnormal and were reassigned to aneuploid, mosaic, or persistently chaotic categories. This finding suggests that apparent euploid results reported elsewhere may reflect technical variability and sampling limitations in PGT-A rather than accurate chromosomal normalization. Conclusions: The prevalence of aneuploid embryos showed a progressive increase with advancing maternal age. Chaotic embryos are heterogeneous, and re-biopsy may help refine the interpretation of complex PGT-A profiles, supporting its role as a diagnostic and quality control tool rather than a strategy to identify euploid embryos. Our study offers novel insights through age-stratified analysis, the integration of morphology with genetics in a Romanian IVF cohort, and a detailed evaluation of chaotic embryos, providing clinical recommendations for patient counseling and embryo selection. Full article

Background: Major Depressive Disorder (MDD) is a leading global health concern, yet its pharmacological management is hampered by a «trial-and-error» approach, with a significant proportion of patients failing to achieve remission with initial therapy. This challenge stems from the disorder’s marked biological heterogeneity, which is poorly captured by current broad diagnostic categories. This literature review synthesizes the latest evidence across three complementary fields poised to revolutionize MDD treatment: pharmacogenetics testing (PGT), therapeutic drug monitoring (TDM), and artificial intelligence (AI). We hypothesize that integrating all three facilitates the transition from empirical prescribing to model-informed precision dosing (MIPD), enabling prediction of optimal antidepressant selection and dosage before the first dose is administered. The convergence of these technologies, supported by an interdisciplinary framework, has the potential to enhance current treatment strategies and contribute to more individualized psychiatric care. Conclusions: Antidepressant therapy for MDD may be further optimized through the combined use of TDM, PGT, and digital tools. However, the development of this field requires ongoing research and interdisciplinary work. Full article

This paper focuses on analyzing and implementing a numerical technique using the Petrov–Galerkin technique (PGT) to solve the time fractional cable problem (TFCP). The trial functions are a modified set of shifted Legendre polynomials ($\mathsf{LPs}$). An appropriate numerical approach can be used to solve the linear algebraic equations resulting from the application of the PGT. With error bounds, we discuss the truncation estimation and stability in the $L_2$ norm. We apply some inequalities on the modified set of shifted $\mathsf{LPs}$ to this research. Numerical experiments include benchmark issues for which exact solutions are presented to show how efficient and accurate the method is. Comparisons with different techniques in the literature are used to support our examples. Full article

Alpine tundra’s harsh conditions challenge plants, but Rhododendron’s adaptive mechanisms remain unclear. This study explored phenotypic/transcriptomic adaptations of three Rhododendron species (R. aureum, R. lapponicum, R. redowskianum) in Changbai Mountains’ tundra vs. timberline. Mature leaves were sampled for leaf length and leaf width measurement and RNA-seq. Results showed leaf width (not leaf length uniformly) reduced in tundra across all species. RNA-seq identified 2399–5716 DEGs per species; plant dwarfism DEGs (e.g., DELLA, EDS1) were up-regulated. Shared DEGs were enriched in carbon/nitrogen metabolism and stress response; IPUT1 (DUH022406.1) and PGT1 (DUH001929.1) were consistently down-regulated (linked to dwarfism). Species-specific responses included R. aureum’s light adaptation, R. lapponicum’s freezing/hypoxia response, and R. redowskianum’s sugar/UV/microbial regulation. Rhododendron adapts to tundra via leaf width adjustment, metabolic optimization, and IPUT1/PGT1-mediated dwarfism, with conserved core mechanisms and species specialization, supporting climate change response predictions and conservation. Full article

Fusobacterium nucleatum (Fn) is increasingly recognized as a cancer-associated bacterium, yet reliable quantification in human specimens is challenging due to low bacterial burden and abundant host DNA. We analyzed 145 Fusobacterium genomes to design primers targeting conserved regions of the fadA adhesin gene and developed a duplex quantitative real-time PCR (qPCR) assay for simultaneous detection of fadA and a human PGT as an internal control. Analytical sensitivity, specificity, precision, and reproducibility were evaluated using serially diluted Fn DNA, spike-in experiments with human DNA, and cross-platform/operator validation. Clinical performance was assessed in colorectal cancer patient tissues, including fresh tissue (n = 24) and formalin-fixed paraffin-embedded (FFPE) samples (n = 22), using 16S rRNA-based methods as references. The assay successfully detected all four major Fn subspecies (nucleatum, animalis, polymorphum, and vincentii). The limit of detection was ≤0.1 pg, with no interference between duplex targets. Spike-in experiments demonstrated consistent target detection in human-DNA-rich samples, with strong linearity (R² = 0.998) across dilutions. High precision (coefficient of variations < 5%) was observed across intra-day, inter-day, inter-instrument, and inter-operator evaluations. In fresh tissues, the assay yielded 86% sensitivity, 94% specificity, and 92% accuracy. Using the FFPE samples, the assay achieved 91% sensitivity and 100% specificity, confirming robust classification in both clinical samples. This duplex qPCR assay enables broad detection of Fn with high analytical performance in both fresh and FFPE tissues. Its simplicity, reproducibility, and compatibility with pathology workflows support deployment in multi-center studies and downstream applications in diagnostic studies and prognostic modeling. Full article

Objectives: This study aimed to determine whether chromosomal mosaicism in blastocysts is associated with a distinct morphokinetic signature. Methods: Preimplantation genetic testing for aneuploidy (PGT-A) was performed on 182 human embryos via trophectoderm biopsy on day 5 and analyzed by next-generation sequencing. Embryos were classified as euploid (n = 55), mosaic (n = 39: 21 low-grade, 18 high-grade), or aneuploid (n = 88), of which 18 with concurrent mosaicism. Prior to biopsy, embryos were cultured in a time-lapse system (EmbryoScope), and 12 morphokinetic parameters were assessed, including pronuclei fading (tPNf), cleavage times (t2–t9), morula formation (tM), blastulation start (tSB), and full blastocyst formation (tB). These parameters were compared according to ploidy status. Results: Patients with euploid and mosaic embryos were comparable in terms of maternal age, ART indication and embryo quality (p > 0.05). In contrast, aneuploid embryos were obtained from older patients and had lower morphological grades. Mosaic embryos showed delayed tPNf (24.8 ± 6.5 vs. 22.8 ± 2.3 h, p = 0.03) and t2 (27.6 ± 6.6 vs. 25.4 ± 2.5 h, p = 0.02) compared to euploid embryos, mainly attributable to low-grade mosaic embryos. Whole-chromosome mosaicism, but not segmental mosaicism, was associated with delayed embryo development at several intermediate cleavage time points (t3, t4, t6, t7 and t9). Aneuploid embryos showed significant delays at later stages versus euploid embryos, particularly aneuploid embryos with mosaicism at t7 (56.6 ± 8.3 vs. 52 ± 5.6 h, p = 0.02), t8 (59.1 ± 9.6 vs. 54.8 ± 6.7 h, p = 0.04), tM (90.3 ± 7.7 vs. 83.6 ± 8.2 h, p = 0.006) and tB (113.0 ± 11.6 vs. 106.6 ± 8.9 h, p = 0.03). Conclusions: Mosaic embryos exhibit delays in early development (tPNf, t2) but reach later morphokinetic milestones at rates similar to euploid embryos. In contrast, aneuploid embryos, especially those with mosaicism, exhibit marked developmental delays at later stages (t7, t8, tM, tB). Full article

Within the field of assisted reproductive technologies (ARTs), embryologists regularly face the critical task of identifying embryos with the highest likelihood of implantation and survival. To help aid and standardize this practice, many embryo selection strategies have been developed to give the best chance of pregnancy success. Over the years, there has been a large increase in experimental studies conducted within this area of research. This increase has allowed for the formation of significant and plausible theories of embryo development, especially in cases where the most prominent factors seem identical. These advancements have both expanded the typical process of traditional treatments and have even paved the way for new techniques. The exact combination of all these relevant factors has not been fully elucidated into a single all-encompassing scheme for embryo decision. Morphological, genetic, and developmental indicators are well-studied individually, but the exact methods that should be prioritized in each scenario may change with respect to an individual patient. Deciding whether factors like age, egg quality, lifestyle choices, or previous medical history should alter methods of embryo ranking can result in conflict, especially in the case where a choice is being made between two similar embryos. This article reviews the conventional methods along with emerging technologies that provide the tools for embryologists to evaluate and rank embryos with high implantation potential (HIP). By showcasing these methods, including their respective benefits and drawbacks, this article provides information to allow clinicians to make effective decisions by integrating multiple approaches to embryo selection. Full article

Background/Objectives: The timing of blastocyst formation is an important factor in in vitro fertilization (IVF) outcomes. While many studies have shown similar pregnancy rates for euploid blastocyst transfers occurring on day 5 or 6, controversy remains, especially regarding patients with recurrent implantation failure (RIF). This study aimed to evaluate whether day 5 (D5) and day 6 (D6) euploid blastocysts differ in terms of the clinical outcomes achieved after frozen–thawed euploid embryo transfer, with subgroup analysis by maternal age in RIF patients. Methods: This retrospective analysis included a cohort of 514 patients who experienced RIF, categorized into two distinct groups: the initial group consisting of individuals who underwent embryo biopsy on day 5 (n = 456 euploid transfers) and the subsequent group comprising patients who underwent biopsy on day 6 (n = 58 euploid transfers) following ovum retrieval. These groups were utilized based on the completion of blastocyst development and the eventual pregnancy outcomes after the first post-frozen–thawed euploid embryo transfer confirmed by PGT-A. Results: Out of the total cohort of 514 RIF patients, 472 individuals were younger than 40 years of age, representing 91.8% of the sample population. The IVF process yielded successful positive pregnancy outcomes in 85.4% of the cases (439 out of 514)., and there was no statistically significant difference in maternal ages between the day 5 and day 6 biopsy cohorts (p = 0.286). The proportion of successful clinical pregnancies per transfer was markedly higher among those who underwent biopsy on day 5 in comparison to those subjected to biopsy on day 6 (79.2% vs. 58.6%, p = 0.002). In patients aged 35 years and younger who underwent biopsy on day 5, the rates of successful clinical pregnancy outcomes were superior to those who underwent biopsy on day 6 (79.8% vs. 56.5% p = 0.016). In individuals older than 35 years of age, no significant discrepancies in clinical pregnancy rates were observed between the two groups. Conclusions: Day 6 euploid embryo transfer was associated with lower clinical pregnancy rates per transfer compared with those on day 5, particularly in patients younger than 35 years of age. In women ≥ 35 years of age, outcomes did not differ significantly. These outcomes suggest that developmental timing interacts with maternal age in determining clinical outcomes. Further prospective studies with larger and more balanced cohorts are needed to confirm these findings. Full article

Lombardy is Italy’s leading region in primary agricultural production, yet it faces a significant decline in agricultural soil, primarily due to urban expansion. This land consumption largely affects arable areas, as land is repurposed for low-density residential developments, roads, logistics, and commercial or industrial hubs. The reduction in agricultural land threatens regional food security and increases dependency on external markets. This study determines the long-term sustainability of this trend by estimating the actual quantity of agricultural land required to satisfy the food demand of the region’s citizens. The research employed a two-part georeferenced analysis. First, a cross-tabulation matrix quantified the land consumption over two decades. Second, the Planning Forecasts Map was analyzed, coupled with new road projects, to estimate future potential land consumption embedded in Land Use Plans (PGT). Finally, food consumption was converted into the required hectares of agricultural land per capita and compared to the current stock of agricultural land to quantify the deficit by municipality. The dramatic spatial deficit confirms that the current trajectory of land consumption is unsustainable, leaving Lombardy’s food security highly dependent on imports. While regional laws have reduced planned urbanization, the limitation of land take remains far from the goals. The results highlight the urgent need for effective compensatory measures and mitigation strategies that account for the true magnitude and spatial distribution of the agricultural land deficit, particularly in the most critical urban and peri-urban areas. Full article

Assisted reproduction is a rapidly expanding pillar of medical tourism. Greece combines a liberal legal framework, internationally accredited clinics, and comparatively competitive costs, attracting cross-border patients seeking ART services. Following the 2022 amendment (Law 4958/2022) which amends the original law n.3305/2005, treatment is permitted up to age 54 under specific authorization, while court-approved surrogacy, anonymous gamete donation, and the adoption of decision-support technologies (e.g., AI-assisted embryo assessment, PGT-A) underpin the sector’s growth. This review synthesizes legal, medical, economic, and social dimensions, drawing on Q1 literature and official datasets (WHO, OECD, ESHRE/ICMART), and compares Greece with Spain, the USA, the Czech Republic, and Ukraine. Quantitative indicators include age-stratified success rates and indicative treatment costs. We discuss benefits and risks for patients and the health system, highlighting policy options for sustainable, ethically robust reproductive tourism in Greece. Full article