Search Results (24)

Preimplantation genetic testing (PGT) reports play a decisive role in determining the fate of IVF-generated embryos. The identification of a chromosomal or genetic abnormality that could impact the health of the resulting newborn often leads to embryo disposal or indefinite storage in cryogenic containers. As a growing proportion of IVF cycles include PGT assessment, greater scrutiny is being placed on its clinical validity. Initially developed to detect monogenic disorders (PGT-M) and later expanded to identify full chromosomal aneuploidies, PGT is primarily used to identify embryos unlikely to implant (aneuploid), those that would lead to miscarriage, or those causing chromosomal syndromes or monogenic conditions. Advancements in genetic analysis now allow for the assessment of more complex traits and chromosomal features from a trophectoderm biopsy, including segmental aneuploidies, chromosomal mosaicism, and polygenic conditions. However, as technology pushes the limits of biological resolution, questions arise regarding the accuracy, clinical utility, and representativeness of these findings for the entire embryo. This article reviews the gold standards for validating clinical findings and reporting strategies, aiming to maximize diagnostic utility while minimizing false positives towards appropriately defined reproductive outcomes and phenotypes. 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

Preimplantation genetic testing for aneuploidy (PGT-A) is widely used to select euploid embryos for in vitro fertilization (IVF), but its accuracy in predicting the implantation potential for full segmental aneuploid (Seg-A) embryos remains unclear. In this study, we investigated chromosomal concordance between clinically biopsied trophectoderm (TE) and inner cell mass (ICM) in 175 donated blastocysts, which comprised those clinically diagnosed as euploid (13), Seg-A (36), segmental mosaicism (Seg-M) (60), whole-chromosome aneuploid (Who-A) (52), and whole-chromosome mosaicism (14). Using next-generation sequencing (NGS), we found that TE–ICM concordance rates were higher for euploid (85%) and Who-A (94%) embryos but significantly lower for Seg-A (25%) and Seg-M embryos (33%). For Seg-A, the euploidy rate in the ICM was 19% and the euploidy rate in the ICM was 63% for Seg-M. These low concordance rates may be due to technical and biological artifacts of PGT-A for Seg-A. Despite the significant discordance between TE and ICM, a subset of Seg-A embryos demonstrated euploidy. While clinically diagnosed euploid embryos remain the preferred choice, Seg-A embryos should be considered as having implantation potential. In particular, Seg-A results should be clearly distinguished from Who-A results and not routinely categorically discarded. Further research is required to refine the selection criteria, aided by parental karyotyping or re-biopsy, and to develop more reliable embryo assessment methods to ensure the accurate evaluation of reproductive potential and support shared decision making between doctors and patients. Full article

To minimise the influence of chromosomal abnormalities during IVF treatment, embryos can be screened before transfer using preimplantation genetic testing. This typically involves an invasive trophectoderm biopsy at the blastocyst stage, where 4–8 cells are collected and analysed. However, emerging evidence indicates that, as embryos develop in vitro in culture media, they release cell-free DNA into the media, providing an alternative source of genetic material that can be accessed non-invasively. Spent blastocyst media samples that contain embryo cell-free DNA demonstrate high informativity rates and ploidy concordance when compared with the corresponding trophectoderm, inner cell mass, or whole blastocyst results. However, optimising this non-invasive approach requires several changes to embryo culture protocols, including additional embryo washes to tackle contamination and extending embryo culture time to maximise the amount of cell-free DNA released into the culture media. In this review, we discuss this novel non-invasive approach for aneuploidy detection and embryo prioritisation, as well as the current data and future prospects for utilising cell-free DNA analysis to identify structural rearrangements and single gene disorders. Full article

Alpha-fetoprotein (AFP) is measured during pregnancy in maternal serum to screen for, and in amniotic fluid to test for, neural tube defects. This study aimed to determine whether or not AFP is expressed in blastocoel fluid-conditioned media (BFCM) at the blastocyst stage of embryonic development. For this in vitro study, BFCM was obtained from blastocyst stage embryos following standard embryology laboratory processes. Good quality blastocysts (n = 40) had trophectoderm biopsy for preimplantation genetic testing for aneuploidy (PGT-A) with subsequent blastocyst vitrification and BFCM collection. BFCM samples (n = 40) were analyzed for human AFP protein via an AFP Human ELISA Kit. Statistical analysis was performed with Fisher’s exact test. AFP was expressed in 12.5% (5/40) of BFCM samples (range = 1.69–20.5 pg/mL). Of blastocysts with AFP in BFCM, 80% (4/5) had aneuploid PGT-A results; of blastocysts with no AFP in BFCM, 57% (20/35) had aneuploid PGT-A results, with no difference between groups (p = 0.63). Our study demonstrates AFP expression in BFCM. To our knowledge, this is the first study to report the detection of AFP at the embryonic blastocyst stage in vitro. Future studies are needed and underway to determine whether assessment of AFP at the embryonic stage can improve embryo transfer outcomes. Full article

Trophectoderm (TE) biopsy is at present the most widely used procedure for preimplantation genetic testing (PGT). At the blastocyst stage, more TE cells (five to seven) can be obtained for genetic analysis. While removing TE cells and not touching the inner cell mass (ICM), the procedure is less invasive. Due to a natural selection happening between day 3 and day 5, 6 or 7 of human embryo development, fewer embryos will have to be biopsied and tested. An additional benefit, especially in view of aneuploidy testing (PGT-A), is the lower level of mosaicism present at the blastocyst stage. The biopsy procedure involves two steps: laser-assisted zona pellucida (ZP) opening and the excision of five to eight TE cells from the blastocyst with or without additional laser energy. Different protocols have emerged over time with variations regarding the technique, the exact moment of ZP opening, and the method of cell removal. The ‘pulling’ method involves laser excision, whereas the ‘flicking’ method represents a mechanical approach with or without laser assistance. Embryo developmental speed reaching the full/expanded or hatching/hatched blastocyst stage dictates the timing of the procedure, mostly on day 5 post-insemination, and to a lesser extent on day 6 or even on day 7. The inclusion of lesser quality or delayed blastocysts may impact the quality of the TE sample as well as the clinical outcome. Intracytoplasmic sperm injection (ICSI) is still the preferred method of fertilization for PGT-M (monogenic disorders) and PGT-SR (structural rearrangements). However, conventional in vitro fertilization (IVF) seems feasible for PGT-A (aneuploidy testing). In the absence of a (conclusive) genetic result, the re-biopsy of cryopreserved blastocysts is possible, however, with reduced clinical outcomes. So far, neonatal outcome post-TE biopsy has so far been reassuringly documented. Full article

Objective: Abnormally fertilized embryos are often discarded during in vitro fertilization due to the fact that known chromosomal ploidy abnormalities lead to implantation failure or pregnancy loss. The objective of this study was to determine if pronuclear numeration (PN) observed at fertilization check is representative of the true ploidy status of the subsequent developing blastocyst in order to maximize the number of viable embryos available for infertility patients and increase their chances of conception. Methods: Upon successful fertilization, pronuclear numeration was noted, and zygotes were cultured to the blastocyst stage. Biopsied trophectoderm cells were then lysed, and the isolated DNA was whole-genome amplified followed by library preparation. Next-generation sequencing was performed for PGT-A, and excess whole-genome amplified DNA was utilized for single nucleotide polymorphism beadchip array analysis. Results: At the time of fertilization check on day 1 of embryo development, when there were no visible pronuclei (n = 291), 56% of these 0PN blastocysts were confirmed to be diploid and normally fertilized. The remaining 41.9% were aneuploid, and 2.1% of the 0PN blastocysts contained only 23 haploid chromosomes. Upon analysis of the 1PN blastocysts (n = 217), just over a third (36.4%) only contained 23 haploid chromosomes (23XO), with another third (31.8%) identified as aneuploid, and surprisingly, the remaining third (31.8%) confirmed to be diploid and normally fertilized. In contrast, 50% of the 3PN blastocysts (n = 172) showed the presence of a third set of 23 parental chromosomes and were confirmed to be triploid (69XXY = 59.3% and 69XXX = 40.7%), with 41.9% identified as aneuploid and, interestingly, a small percentage (8.1%) confirmed to be diploid with normal fertilization. A very small proportion of biopsied blastocysts (0.63%) displaying the correct number of pronuclei for normal fertilization (2PN) were also identified as triploid with a third set of 23 parental chromosomes. To date, there have been 74 euploid embryo transfers from zygotes originally identified with an alternate pronuclear numeration, resulting in 16 ongoing pregnancies and 32 healthy live births, rates that match those typically observed with normally fertilized 2PN zygotes (>60%). Conclusions: A surprising number of blastocysts that were identified to have alternate pronuclear numeration at fertilization check on day 1 of embryo development were actually determined to be diploid with normal fertilization after molecular analysis. Accurate identification of haploid and tripoid zygotes is critical to prevent implantation failure and pregnancy loss and allows for the identification of all euploid embryos in a cohort, which has the potential to increase cumulative live birth rates for infertility patients. 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

The evolution of preimplantation genetic testing for aneuploidy (PGT-A) techniques has been crucial in assisted reproductive technologies (ARTs), improving embryo selection and increasing success rates in in vitro fertilization (IVF) treatments. Techniques ranging from fluorescence in situ hybridization (FISH) to next-generation sequencing (NGS) have relied on cellular material extraction through biopsies of blastomeres at the cleavage stage on day three or from trophectoderm (TE) cells of the blastocyst. However, this has raised concerns about its potential impact on embryo development. As a result, there has been growing interest in developing non-invasive techniques for detecting aneuploidies, such as the analysis of blastocoel fluid (BF), spent culture medium (SCM), and artificial intelligence (AI) models. Non-invasive methods represent a promising advancement in PGT-A, offering the ability to detect aneuploidies without compromising embryo viability. This article reviews the evolution and principles of PGT-A, analyzing both traditional techniques and emerging non-invasive approaches, while highlighting the advantages and challenges associated with these methodologies. Furthermore, it explores the transformative potential of these innovations, which could optimize genetic screening and significantly improve clinical outcomes in the field of assisted reproduction. Full article

Background: To investigate prenatal ultrasound findings and the chromosomal outcomes of mosaic embryo transfer. Methods: This retrospective study was conducted on pregnant women who underwent mosaic embryo transfer following blastocyst-stage preimplantation genetic testing for aneuploidy (PGT-A) at CHA Gangnam Medical Center from January 2021 to July 2024. Trophectoderm biopsy specimens were collected using standard protocols, and next-generation sequencing profiles were defined as mosaics when displaying copy number counts in the 20–80% range. The results of the PGT-A, the amniocentesis results, the findings of prenatal ultrasounds, and the pregnancy outcomes were analyzed. Results: A total of 88 mosaic embryos were transferred, of which 77 embryos were successfully implanted. Sixty-seven embryo-maintained pregnancies went beyond 11 weeks (87.0%), all among 58 patients with singleton pregnancies. The chaotic subtype showed the lowest ongoing pregnancy rate, and high-level mosaicism was less frequent in the ongoing group, compared to the total study group and the successful implantation group. Amniocentesis was performed on 33 mothers (56.9%), revealing two cases with abnormal findings that did not correlate with the PGT-A results. Two cases showed abnormalities in the second trimester detailed ultrasound, and both subsequently demonstrated normal findings in the third trimester and after birth. The average gestational age at birth was 38.4 weeks, and the average birth weight was 3313 g. No congenital anomalies were detected in 16 postnatal cases. Conclusions: Our study indicated that mosaic embryos can develop into euploid healthy infants with various levels or types of mosaicism, although the postnatal follow-up data are limited. This study is invaluable for counseling clinical results after mosaic embryo transfer, reassuring that, if patients do not have euploid embryos available, mosaic embryos can also be a viable option for transfer. Full article

Background: In recent years, preimplantation genetic testing for aneuploidies (PGT-A) has become widespread in assisted reproduction. However, contrary to expectations, PGT-A does not significantly improve the clinical outcomes of assisted reproductive technologies. One of the underlying reasons is the discordance between the PGT-A results and the true chromosomal constitution of the blastocyst. In this case series, we re-examined the PGT-A results in trophectoderm (TE) re-biopsies and in the two isolated blastocyst compartments—the TE and the inner cell mass (ICM). Methods: This study enrolled 23 human blastocysts from 17 couples who were referred for assisted reproduction. The blastocysts were unsuitable for uterine transfer due to the chromosomal imbalance revealed by PGT-A using array comparative genomic hybridization (aCGH) (n = 11) or next-generation sequencing (NGS) (n = 12). The re-examination of the PGT results involved two steps: (1) a TE re-biopsy with subsequent aCGH and (2) blastocyst separation into the TE and the ICM with a subsequent cell-by-cell analysis of each isolated compartment by fluorescence in situ hybridization (FISH) with the DNA probes to chromosomes 13, 16, 18, 21, and 22 as well as to the PGT-A detected imbalanced chromosomes. Results: In 8 out of 23 cases, the PGT-A results were concordant with both the re-biopsy and the isolated TE and ICM analyses. The latter included the diagnoses of full non-mosaic aneuploidies (five cases of trisomies and two cases of monosomies). In one case, the results of PGT-A, aCGH on the TE re-biopsy, and FISH on the isolated TE showed Xp tetrasomy, which contrasted with the FISH results on the isolated ICM, where this chromosomal pathology was not detected. This case was classified as a confined mosaicism. In 4 out of 23 cases, the results were partially discordant. The latter included one case of trisomy 12, which was detected as non-mosaic by PGT-A and the re-biopsy and as mosaic by FISH on the isolated TE and ICM. This case was classified as a true mosaicism with a false negative PGT-A result. In 11 out of 23 cases, the re-examination results were not concordant with the PGT-A results. In one of these discordant cases, non-mosaic tetraploidy was detected by FISH in the isolated TE and ICM, whereas the PGT-A and the TE re-biopsy failed to detect any abnormality, which advocated for their false negative result. In two cases, the re-examination did not confirm full aneuploidies. In eight cases, full or partial mosaic aneuploidies as well as chaotic mosacism were not confirmed in the isolated TE nor the isolated ICM. Thus, in 47.8% of cases, the PGT-A results did not reflect the true chromosomal constitution of a blastocyst. Conclusions: The PGT results may have different prognostic value in the characterization of the chromosomal constitution of a blastocyst. The detected non-mosaic aneuploidies have the highest prognostic value. In stark contrast, most PGT-identified mosaic aneuploidies fail to characterize the true chromosomal constitution of a blastocyst. Once detected, a differential diagnosis is needed. Full article

Huntington’s disease (HD) arises from expanded CAG repeats in exon 1 of the Huntingtin (HTT) gene. The resultant misfolded HTT protein accumulates within neuronal cells, negatively impacting their function and survival. Ultimately, HTT accumulation results in cell death, causing the development of HD. A nonhuman primate (NHP) HD model would provide important insight into disease development and the generation of novel therapies due to their genetic and physiological similarity to humans. For this purpose, we tested CRISPR/Cas9 and a single-stranded DNA (ssDNA) containing expanded CAG repeats in introducing an expanded CAG repeat into the HTT gene in rhesus macaque embryos. Analyses were conducted on arrested embryos and trophectoderm (TE) cells biopsied from blastocysts to assess the insertion of the ssDNA into the HTT gene. Genotyping results demonstrated that 15% of the embryos carried an expanded CAG repeat. The integration of an expanded CAG repeat region was successfully identified in five blastocysts, which were cryopreserved for NHP HD animal production. Some off-target events were observed in biopsies from the cryopreserved blastocysts. NHP embryos were successfully produced, which will help to establish an NHP HD model and, ultimately, may serve as a vital tool for better understanding HD’s pathology and developing novel treatments. Full article

The implementation of next generation sequencing (NGS) in preimplantation genetic testing for aneuploidy (PGT-A) has led to a higher prevalence of mosaic diagnosis within the trophectoderm (TE) sample. Regardless, mosaicism could potentially increase the rate of live-born children with chromosomic syndromes, though available data from the transfer of embryos with putative PGT-A mosaicism are scarce but reassuring. Even with lower implantation and higher miscarriage rates, mosaic embryos can develop into healthy live births. Therefore, this urges an explanation for the disappearance of aneuploid cells throughout development, to provide guidance in the management of mosaicism in clinical practice. Technical overestimation of mosaicism, together with some sort of “self-correction” mechanisms during the early post-implantation stages, emerged as potential explanations. Unlike the animal model, in which the elimination of genetically abnormal cells from the future fetal lineage has been demonstrated, in human embryos this capability remains unverified even though the germ layer displays an aneuploidy-induced cell death lineage preference with higher rates of apoptosis in the inner cell mass (ICM) than in the TE cells. Moreover, the reported differential dynamics of cell proliferation and apoptosis between euploid, mosaic, and aneuploid embryos, together with pro-apoptosis gene products (cfDNA and mRNA) and extracellular vesicles identified in the blastocoel fluid, may support the hypothesis of apoptosis as a mechanism to purge the preimplantation embryo of aneuploid cells. Alternative hypotheses, like correction of aneuploidy by extrusion of a trisomy chromosome or by monosomic chromosome duplication, are even, though they represent an extremely rare phenomenon. On the other hand, the technical limitations of PGT-A analysis may lead to inaccuracy in embryo diagnoses, identifying as “mosaic” those embryos that are uniformly euploid or aneuploid. NGS assumption of “intermediate copy number profiles” as evidence of a mixture of euploid and aneuploid cells in a single biopsy has been reported to be poorly predictive in cases of mosaicism diagnosis. Additionally, the concordance found between the TE and the ICM in cases of TE biopsies displaying mosaicism is lower than expected, and it correlates differently depending on the type (whole chromosome versus segmental) and the level of mosaicism reported. Thus, in cases of low-/medium-level mosaicism (<50%), aneuploid cells would rarely involve the ICM and other regions. However, in high-level mosaics (≥50%), abnormal cells in the ICM should display higher prevalence, revealing more uniform aneuploidy in most embryos, representing a technical variation in the uniform aneuploidy range, and therefore might impair the live birth rate. Full article

Preimplantation genetic testing for structural rearrangements (PGT-SR) was one of the first applications of PGT, with initial cases being worked up in the Delhanty lab. It is the least well-known of the various forms of PGT but nonetheless provides effective treatment for many carrier couples. Structural chromosomal rearrangements (SRs) lead to infertility, repeated implantation failure, pregnancy loss, and congenitally affected children, despite the balanced parent carrier having no obvious phenotype. A high risk of generating chromosomally unbalanced gametes and embryos is the rationale for PGT-SR, aiming to select for those that are chromosomally normal, or at least balanced like the carrier parent. PGT-SR largely uses the same technology as PGT-A, i.e., initially FISH, superseded by array CGH, SNP arrays, Karyomapping, and, most recently, next-generation sequencing (NGS). Trophectoderm biopsy is now the most widely used sampling approach of all PGT variants, though there are prospects for non-invasive methods. In PGT-SR, the most significant limiting factor is the availability of normal or balanced embryo(s) for transfer. Factors directly affecting this are rearrangement type, chromosomes involved, and sex of the carrier parent. De novo aneuploidy, especially for older mothers, is a common limiting factor. PGT-SR studies provide a wealth of information, much of which can be useful to genetic counselors and the patients they treat. It is applicable in the fundamental study of basic chromosomal biology, in particular the purported existence of an interchromosomal effect (ICE). An ICE means essentially that the existence of one chromosomal defect (e.g., brought about by malsegregation of translocation chromosomes) can perpetuate the existence of others (e.g., de novo aneuploidy). Recent large cohort studies of PGT-SR patients seem, however, to have laid this notion to rest, at least for human embryonic development. Unless new evidence comes to light, this comprehensive review should serve as a requiem. 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