Search Results (9)

Preimplantation genetic testing for monogenic disorders (PGT-M) is a powerful tool for identifying genetic disorders prior to gestation. For hemoglobinopathies like thalassemias and sickle cell disease, PGT-M offers a preventative strategy to ensure that only embryos deemed genetically healthy are transferred. A comprehensive review of 22 original articles explores and summarizes the existing evidence on PGT-M techniques in hemoglobinopathies. The review focuses on key aspects such as accuracy, benefits, and drawbacks related to various hemoglobinopathies. Given the limited quantity of DNA obtained from an embryo biopsy, whole genome amplification (WGA) is a critical step for amplifying the sample. One of the available methods of WGA, multiple displacement amplification (MDA) is one of the most widely adopted method with acceptable allele drop-out (ADO) rates for hemoglobinopathies compared with traditional methods. Dealing with ADO constitutes a primary technical obstacle in PGT-M. The failure to amplify one allele in single-cell analysis is a major factor limiting the overall diagnostic accuracy of the procedure. To mitigate this issue, PCR-based and next-generation sequencing (NGS)-based approaches are employed. These methods incorporate linkage analysis with genetic markers such as short tandem repeats (STRs) or single-nucleotide polymorphisms (SNPs) to reduce the risk of incorrect interpretations from ADO and enhance the proportion of conclusive results. A future direction for PGT-M that involves the development of non-invasive methods (niPGT) will be included and discussed. Full article

Background: Recurrent pregnancy loss (RPL) and recurrent implantation failure (RIF) are significant challenges in reproductive medicine. For both, embryonic aneuploidy is the leading etiological factor. Preimplantation genetic testing for aneuploidy (PGT-A) via trophectoderm biopsy is the current standard for embryo selection. However, it is limited by its invasiveness, potential for embryo damage, and diagnostic errors due to mosaicism. Rationale/Objectives: This review critically evaluates the emerging role of noninvasive PGT (niPGT). NiPGT analyzes cell-free DNA from spent blastocyst culture media, thus, it is a potential alternative for managing RPL and RIF. Hence, the primary objective is to determine whether current evidence supports niPGT as a reliable replacement for conventional biopsy-based PGT-A in these high-risk populations. Outcomes: The analysis reveals that niPGT offers significant theoretical advantages. These include complete non-invasiveness, enhanced embryo preservation, and high patient acceptability. However, its clinical application is hampered by substantial limitations. Key amongst them is the inconsistent and often suboptimal diagnostic accuracy (sensitivity 70–85%, specificity 88–92%) compared to biopsy. Other significant factors include the high rates of amplification failure (10–50%), vulnerability to maternal DNA contamination, as well as low DNA yield. Crucially, there is a definitive lack of robust, prospective randomized controlled trial (RCT) data demonstrating improved live birth rates or reduced miscarriage rates specifically in RPL and RIF cohorts. As such, niPGT is not yet ready to be a standalone clinical adoption in RPL and RIF cases. However, it may serve as a valuable adjunct for rescue scenarios following biopsy failure or for ethical reasons. Wider Implications: The integration of niPGT with artificial intelligence, time-lapse imaging, and multi-omics profiling underlies a promising future. However, its transition from a predominantly research tool to a clinical standard necessitates various critical undertakings. These include rigorous multicenter RCTs, standardizing international protocol, and tailoring validation for the RPL and RIF subgroups. This review highlights the need for cautious optimism, positing that evidence-based integration, rather than premature adoption, is essential to realizing niPGT’s full potential without compromising patient care in these complex fertility scenarios. Full article

Background: Preimplantation genetic testing for aneuploidy (PGT-A) is a popular approach in assisted reproductive technology that improves embryo selection and implantation rates. Traditional approaches rely on trophectoderm (TE) biopsy, which is an invasive procedure that might jeopardize embryo integrity and create technical constraints such as mosaicism-related misclassification. Non-invasive preimplantation genetic testing (niPGT) has emerged as a possible alternative, using embryonic cell-free DNA (cfDNA) extracted from wasted culture media or blastocoel fluid to assess chromosomal status without requiring direct embryo manipulation. Methods: This systematic study investigates the molecular mechanisms behind cfDNA release, its biological properties, and the technological concerns that influence its utilization in niPGT. We look at recent advances in next-generation sequencing (NGS), whole-genome amplification (WGA), and bioinformatic techniques that improve cfDNA-based aneuploidy detection. In addition, we compare the sensitivity, specificity, and concordance rates of niPGT to conventional TE biopsy, highlighting the major aspects impacting its diagnostic performance. Results: The release of cfDNA from embryos is influenced by apoptotic and necrotic processes, active DNA shedding, and extracellular vesicle secretion, which results in fragmented chromosomal material of different qualities and quantities. While niPGT has shown promise as a noninvasive screening approach, significant variability in cfDNA yield, maternal DNA contamination, and sequencing biases all have an impact on test accuracy. Studies show that niPGT and TE biopsies have moderate-to-high concordance, although there are still issues in detecting mosaicism, segmental aneuploidies, and DNA degradation artifacts. Conclusions: NiPGT is a safer and less intrusive alternative to TE biopsy, with potential clinical benefits. However, technical advancements are required to improve cfDNA collecting procedures, reduce contamination, and improve sequencing accuracy. Additional large-scale validation studies are needed to create standardized methodologies and ensure that niPGT achieves the diagnostic reliability requirements required for widespread clinical deployment in IVF programs. Full article

Background/Objectives: To assess the ploidy status of embryos via preimplantation genetic testing for aneuploidy (PGT-A), a biopsy of trophectoderm (TE) cells can be performed. However, this approach is considered invasive, and therefore the aim of this study was to identify the optimal sample type and sampling day for non-invasive or minimally invasive PGT-A (ni/miPGT-A) in terms of data quality and concordance rates with TE biopsies derived from the same embryos. Methods: This study was performed using 239 embryo cultures. After optimization using 96 embryos, non-invasive spent culture media (SCM) and a minimally invasive combination of blastocoel fluid and SCM (BF+SCM), along with the corresponding TE samples, were collected from 143 embryos cultured for 5 days (n = 70) or 6 days (n = 73), and all were subjected to ni/miPGT-A with whole-genome amplification followed by next-generation sequencing. Results: The amplification failure rate was lower for SCM samples than for BF+SCM (SCM: 0.7%, 1/143 vs. BF+SCM: 7.7%, 11/143; p = 0.005). The rate of ploidy concordance with TE was significantly higher for SCM samples than for BF+SCM samples (SCM: 83.7%, 118/141 vs. BF+SCM: 58%, 76/131; p < 0.001). Among SCM samples, concordance rates were higher for samples derived from embryos cultured for 6 days (87.5%, 63/72) than for 5 days (79.7%, 55/69). In the embryos cultured for 6 days, discordant cases included five (6.9%) SCM samples with falsely negative (euploid) results that were deemed to be mosaic according to TE and four (5.6%) samples falsely found to be aneuploid. Conclusions: SCM samples derived from embryos cultured for 6 days can be applied in niPGT-A with subsequent verification of aneuploid samples using TE biopsy. Full article

Preimplantation genetic testing for aneuploidy (PGT-A) has become a useful approach for embryo selection following IVF and ICSI. However, the biopsy process associated with PGT-A is expensive, prone to errors in embryo ploidy determination, and potentially damaging, impacting competence and implantation potential. Therefore, a less invasive method of PGT-A would be desirable and more cost-effective. Noninvasive methods for PGT-A (ni-PGT-A) have been well-studied but present limitations in terms of cf-DNA origin and diagnostic accuracy. Minimally invasive pre-implantation genetic testing (mi-PGT-A) for frozen-thawed embryo transfer is a promising, less studied approach that utilizes a combination of spent culture media (SCM) and blastocoelic fluid (BF)-derived cell-free (CF)-DNA for genetic testing. This study aimed to optimize the effectiveness of mi-PGT-A for aneuploidy diagnosis by investigating the optimal temporal sequence for this protocol. SCM+BF was collected at either 48 or 72 h of culture after thawing day 3 preimplantation embryos. cf-DNA in the SCM+BF was amplified, analyzed by next-generation sequencing (NGS) and compared with results from the corresponding whole embryos (WEs) obtained from human embryos donated for research. Fifty-three (42 expanded blastocysts, 9 early blastocysts, and 2 morula) WE and SCM+BF samples were analyzed and compared. The overall concordance rate between SCM+BF and WE was 60%. Gender and ploidy concordance improved with extended culture time from 48 h (73% and 45%) to 72 h (100% and 64%), respectively. These results demonstrate that SCM+BF-derived cf-DNA can be successfully used for mi-PGT-A. Our findings indicate that longer embryo culture time prior to SCM+BF-derived cf-DNA analysis improves DNA detection rate and concordance with WEs and decreases the proportion of false positive results. Full article

Embryo selection is needed to optimize the chances of pregnancy in assisted reproduction technology. This study aimed to validate non-invasive preimplantation genetic testing for aneuploidy (niPGT-A) using a routine IVF laboratory workflow. Can niPGT-A combined with time-lapse morphokinetics provide a better embryo-selection strategy? A total of 118 spent culture mediums (SCMs) from 32 couples were collected. A total of 40 SCMs and 40 corresponding trophectoderm (TE) biopsy samples (n = 29) or arrested embryos (n = 11) were assessed for concordance. All embryos were cultured to the blastocyst stage (day 5 or 6) in a single-embryo culture time-lapse incubator. The modified multiple annealing and looping-based amplification cycle (MALBAC) single-cell whole genome amplification method was used to amplify cell-free DNA (cfDNA) from the SCM, which was then sequenced on the Illumina MiSeq system. The majority of insemination methods were conventional IVF. Low cfDNA concentrations were noted in this study. The amplification niPGT-A and conventional PGT-A was 67.7%. Based on this study, performing niPGT-A without altering the daily laboratory procedures cannot provide a precise diagnosis. However, niPGT-A can be applied in clinical IVF, enabling the addition of blastocysts with a better prediction of euploidy for transfer. Full article

This review focuses on recent findings in the preimplantation genetic testing (PGT) of embryos. Different preimplantation genetic tests are presented along with different genetic materials and their analysis. Original material concerning preimplantation genetic testing for aneuploidy (PGT-A) was sourced by searching the PubMed and ScienceDirect databases in October and November 2021. The searches comprised keywords such as ‘preimplantation’, ‘cfDNA’; ‘miRNA’, ‘PGT-A’, ‘niPGT-A’, ‘aneuploidy’, ‘mosaicism’, ‘blastocyst biopsy’, ‘blastocentesis’, ‘blastocoel fluid’, ‘NGS’, ‘FISH’, and ‘aCGH’. Non-invasive PGT-A (niPGT-A) is a novel approach to the genetic analysis of embryos. The premise is that the genetic material in the spent embryo culture media (SECM) corresponds to the genetic material in the embryo cells. The limitations of niPGT-A are a lower quantity and lesser quality of the cell-free genetic material, and its unknown origin. The concordance rate varies when compared to invasive PGT-A. Some authors have also hypothesized that mosaicism and aneuploid cells are preferentially excluded from the embryo during early development. Cell-free genetic material is readily available in the spent embryo culture media, which provides an easier, more economic, and safer extraction of genetic material for analysis. The sampling of the SECM and DNA extraction and amplification must be optimized. The origin of the cell-free media, the percentage of apoptotic events, and the levels of DNA contamination are currently unknown; these topics need to be further investigated. Full article

Although non-invasive pre-implantation genetic testing for aneuploidy (NIPGT-A) is potentially appropriate to assess chromosomal ploidy of the embryo, practical application of it in a routine IVF centre have not been started in the absence of a recommendation. Our objective in this study was to provide a comprehensive workflow for a clinically applicable strategy for NIPGT-A based on next-generation sequencing (NGS) technology with the corresponding bioinformatic pipeline. In a retrospective study, we performed NGS on spent blastocyst culture media of Day 3 embryos fertilised with intracytoplasmic sperm injection (ICSI) with quality score on morphology assessment using the blank culture media as background control. Chromosomal abnormalities were identified by an optimised bioinformatics pipeline applying copy number variation (CNV) detecting algorithm. In this study, we demonstrate a comprehensive workflow covering both wet- and dry-lab procedures supporting a clinically applicable strategy for NIPGT-A that can be carried out within 48 h, which is critical for the same-cycle blastocyst transfer. The described integrated approach of non-invasive evaluation of embryonic DNA content of the culture media can potentially supplement existing pre-implantation genetic screening methods. Full article

Since the birth of the first IVF baby, Louise Brown, in 1978, researchers and clinicians have sought ways to improve pregnancy outcomes through embryo selection. In the 1990s, blastomere biopsy and fluorescence in situ hybridization (FISH) were developed in human embryos for the assessment of aneuploidy and translocations. Limitations in the number of chromosomes that could be assayed with FISH lead to the development of comparative genomic hybridization (CGH); however, pregnancy rates overall were not improved. The later development of trophectoderm biopsy with comprehensive chromosome screening (CCS) technologies, as well as the subsequent development of next-generation sequencing (NGS), have shown much greater promise in improving pregnancy and live birth rates. Recently, many studies are focusing on the utilization of non-invasive preimplantation genetic testing (niPGT) in an effort to assess embryo ploidy without exposing embryos to additional interventions. Full article