PGT-A reveals triploid embryos among normally fertilized oocytes: frequency and blastocyst features.

Study question Is there any embryo parameter that could differentiate 2 pronucleus (PN) triploid from 2 PN euploid embryos? Summary answer Accuracy in the fertilization check is still necessary as 2 PN but triploid embryos are due mostly to retention of the second polar body (PB). What is known already Since blastocyst culture has evolved and the development of genetic technologies enables to know the karyotipe of the embryos, the concept of normal fertilization is being revised. Commonly, correct fertilization is denoted by the appearance of 2 PN 16-18h after insemination. Deviation from 2PN is considered evidence of abnormal fertilization and ploidy anomalies. However, not all oocytes exhibiting 2 PN are euploids. After performing NGS in trophectoderm biopsies for preimplantation genetic testing for aneuploidy (PGT-A), a percentage of embryos are categorized as triploids. Study design, size, duration Retrospective study including 1862 cycles from 1605 patients aged 18-45 years old who underwent PGT-A with NGS of trophectoderm biopsies in our IVF center from 2019 to 2023. From 6526 analyzed blastocysts in an independent Genetics laboratory, 59 were categorized as triploids. These 59 blastocysts were obtained from 57 PGT-A cycles and belonged to 54 women. In total, these patients had 266 biopsied blastocysts. From the remaining 207 embryos, 42 were called as euploids. Participants/materials, setting, methods In embryos cultured in time-lapse, fertilization was re-evaluated to check the appearance of 2PN and the second PB extrusion. Genetic triploid embryos were analysed in relation to the day of biopsy (day 5 vs day 6), the trophectoderm quality and the timing of the first cell divisions (time of cleavage from 2 to 6 cells: t2, t3, t4, t5, t6). Categorical variables are shown as rate. T-student tests were performed. P-value<0.05 was considered statistically significant. Main results and the role of chance The re-evaluation of the fertilization of the 59 triploids categorized as 2 PN revealed 50 oocytes 2 PN, 8 embryos 3 PN and 1 embryo 2 PN + 1 micronuclei configuration. Therefore, the prevalence of real triploidy in 2PN embryos was 0,8% (n = 50/6526; CI95%:0,6-1,0%). These data showed that 2PN zygotes may give rise to embryos with altered ploidy. From 27 triploid 2PN embryos was possible to access to the whole development time-lapse images. Just 33,3% of triploid embryos exhibited the extrusion of the second PB, while 100% of euploid sibling embryos showed this extrusion (N = 9/27 vs N = 24/24,;p<0,005). Comparing 2 PN triploid and the rest of the embryos from the same cohorts, there were no differences regarding the day of biopsy (N = 31/176 day 5 biopsy, 17.6% vs N = 19/81 day 6 biopsy, 23.5%; p > 0.05). On the contrary, a worse blastocyst quality showed a significant relation with triploidy (N = 23/161,14,2% of good quality embryos are triploid vs N = 27/96, 38,6% of fair quality embryos are triploids; p:0.006).Embryos cleavage time points up to 6-cell stage did not show any significative difference between triploid and euploid embryos (t2: 27.36h vs 25.8h; t3: 38.5h vs 35.6h; t4 39.4h vs 37.2h; t5 52.8h vs 47.0h; t6 56.2h vs 52.1h; all comparisons p > 0.05). Limitations, reasons for caution The incidence of triplody from 2PN zygotes may vary among IVF laboratory settings. The small sample size may be responsible of no difference in morphokinetics. Wider implications of the findings Fertilization check should persist in IVF procedures. Special consideration should be given to the second PB extrusion as triploidy is the most common form of aneuploidy observed in the first trimester miscarriages. The role of diploid spermatozoa, the size of the pronucleus and location should be explored in further studies. Trial registration number NOT APPLICABLE

Noninvasive preimplantation genetic testing for aneuploidy in spent culture medium as a substitute for trophectoderm biopsy

Is a clinical trophectoderm (TE) biopsy a suitable predictor of chromosomal aneuploidy in blastocysts? In the analyzed group of blastocysts, a clinical TE biopsy was an excellent representative of blastocyst karyotype in cases of whole chromosome aneuploidy, but in cases of only segmental (sub-chromosomal) aneuploidy, a TE biopsy was a poor representative of blastocyst karyotype. Due to the phenomenon of chromosomal mosaicism, concern has been expressed about the possibility of discarding blastocysts classified as aneuploid by preimplantation genetic testing for aneuploidy (PGT-A) that in fact contain a euploid inner cell mass (ICM). Previously published studies investigating karyotype concordance between TE and ICM have examined small sample sizes and/or have utilized chromosomal analysis technologies superseded by Next Generation Sequencing (NGS). It is also known that blastocysts classified as mosaic by PGT-A can result in healthy births. TE re-biopsy of embryos classified as aneuploid can potentially uncover new instances of mosaicism, but the frequency of such blastocysts is currently unknown. For this study, 45 patients donated 100 blastocysts classified as uniform aneuploids (non-mosaic) using PGT-A by NGS (n = 93 whole chromosome aneuploids, n = 7 segmental aneuploids). In addition to the original clinical TE biopsy used for PGT-A, each blastocyst was subjected to an ICM biopsy as well as a second TE biopsy. All biopsies were processed for chromosomal analysis by NGS, and karyotypes were compared to the original TE biopsy. The setting for this study was a single IVF center with an in-house PGT-A program and associated research laboratory. When one or more whole chromosomes were aneuploid in the clinical TE biopsy, the corresponding ICM was aneuploid in 90 out of 93 blastocysts (96.8%). When the clinical TE biopsy contained only segmental (sub-chromosomal) aneuploidies, the ICM was aneuploid in three out of seven cases (42.9%). Blastocysts showing aneuploidy concordance between clinical TE biopsy and ICM were also aneuploid in a second TE biopsy in 86 out of 88 cases (97.7%). In blastocysts displaying clinical TE-ICM discordance, a second TE biopsy was aneuploid in only two out of six cases (33.3%). All embryos in this study had an initial classification of 'aneuploid' and not 'euploid' or 'mosaic'. Therefore, the findings of this study refer specifically to a TE biopsy predicting aneuploidy in the remaining blastocyst, and cannot be extrapolated to deduce the ability of a TE biopsy to predict euploidy in the blastocyst. No conclusions should be drawn from this study about the ability of a mosaic TE biopsy to predict the karyotype of the corresponding blastocyst. Caution should be exercised in generalizing the findings of the sample group of this study to the general IVF blastocyst population. The segmental aneuploidy group only contained seven samples. The high rate of intra-blastocyst concordance observed in this study concerning whole chromosome aneuploidy contributes experimental evidence to the validation of PGT-A at the blastocyst stage. Concomitantly, the results suggest potential clinical value in reassessing blastocysts deemed aneuploid by TE re-biopsy in select cases, particularly in instances of segmental aneuploidies. This could impact infertility treatment for patients who only have blastocysts classified as aneuploid by PGT-A available. This study was supported by the Zouves Foundation for Reproductive Medicine and Zouves Fertility Center. The authors have no competing interest to disclose. Not applicable.

STUDY QUESTIONCan third-generation sequencing (TGS)-based polar body (PB) analysis serve as a viable alternative to conventional trophectoderm (TE) biopsy for preimplantation genetic testing for aneuploidy (PGT-A)?SUMMARY ANSWERThis study demonstrates the feasibility of using TGS and PB biopsy for clinical PGT-A, particularly in advanced maternal age (AMA) patients.WHAT IS KNOWN ALREADYTE biopsy, the current standard approach for PGT-A, is limited by embryonic mosaicism. Mosaicism potentially leads to false-positive aneuploidy diagnoses, resulting in the discard of genetically normal embryos and compromising the cumulative live birth rates (CLBRs).STUDY DESIGN, SIZE, DURATIONA total of 125 oocytes were collected from 30 couples. First (PB1) and second (PB2) polar bodies from 89 oocytes were individually amplified and sequenced, while those from the remaining 36 oocytes were processed jointly (PB1 + PB2). Then, 74 oocytes were successfully fertilized and developed into blastocysts (59.2% blastulation rate), and from these, corresponding TE biopsies were obtained.PARTICIPANTS/MATERIALS, SETTING, METHODSBoth PB and TE samples underwent whole-genome amplification (WGA) using multiple annealing and looping-based amplification cycles (MALBAC), followed by next-generation sequencing (NGS; all samples) and TGS (PB samples only). Copy number variation (CNV) analysis was performed for aneuploidy screening. Single-nucleotide polymorphisms (SNPs) from TE samples and parental peripheral blood were analyzed to determine the origin of CNVs and investigate discrepancies between TE- and PB-based PGT-A results. Clinical information from patients was collected for inter-group statistical comparisons.MAIN RESULTS AND THE ROLE OF CHANCEThe amplification success rate was 97.75% (87/89) for PB1 and 92.13% (82/89) for PB2, while the amplification success rate for PB1 + PB2 was 97.22% (35/36), comparable to that of TE-biopsied cells. The concordance rate of CNV results between NGS and TGS was 96.81%, with observed discrepancies primarily attributed to differences in the sizes of segmental imbalances and varying levels of intermediate copy numbers. However, when inferring oocyte ploidy status from PB analysis, the concordance rate with TE-biopsied CNV results (blastocyst formation rate 59.2%) was 75.68% (56/74). Among the 56 embryos with consistent results, the CNV profiles of 40 embryos were identical, while the remaining 16 were embryos with paternal meiotic or mitotic abnormalities. Our results demonstrated a higher euploidy rate with PB-based PGT-A (55.4%) compared to blastocyst-stage PGT-A (43.1%). Additionally, the euploidy rate in oocytes from AMA patients (>38 years) was 40%, which was lower than that in younger patients (≤38 years; 64%).LIMITATIONS, REASONS FOR CAUTIONPGT-A via PB biopsy is subject to specific technical limitations and clinical risks that warrant attention. PB biopsy for PGT-A can only detect maternal meiotic abnormalities; it cannot detect mitotic errors or paternal meiotic abnormalities. Moreover, PBs are single cells with limited DNA quantity and are prone to degradation over time. The timing and technical execution of the biopsy are critical for the success of WGA. Amplification failure can occur due to sample loss, experimental error, or the absence of genetic material in PBs resulting from meiotic errors.WIDER IMPLICATIONS OF THE FINDINGSPB analysis represents a minimally invasive strategy that reduces the number of cryopreserved aneuploid embryos, decreases the number of embryo transfers required per live birth, and lowers miscarriage rates. Furthermore, TGS improves the cost-effectiveness and shortens the turnaround time of PB-based PGT-A, making it particularly suitable for fresh cleavage-stage (D3) transfers. This TGS-enhanced PB approach offers a clinically viable alternative to conventional TE biopsy by effectively combining the historical benefits of PB analysis with recent technological advances. The method shows significant promise for optimizing both clinical outcomes and laboratory efficiency in ART practice. Beyond PGT-A, the future may see the development of comprehensive, integrated PGT platforms (including PGT-M for monogenic disorders and PGT-SR for structural rearrangements) based on PB biopsy.STUDY FUNDING/COMPETING INTEREST(S)This work was supported by the National Natural Science Foundation of China (Grant No. 82273465) and the Pioneer and Leading Goose R&D Program of Zhejiang (Grant No. 2023C03034). All authors declare no competing interests.TRIAL REGISTRATION NUMBERN/A.

To assess the accuracy and reliability of comprehensive chromosome screening by next-generation sequencing (NGS) of human trophectoderm (TE) biopsy specimens. The reliability and accuracy of diagnoses made by preimplantation genetic testing for aneuploidy (PGT-A) from TE biopsy were tested. Repeat biopsies of TE and inner cell mass (ICM) samples were obtained from thawed blastocysts previously tested by NGS. To test for the reliability of the NGS assay, biopsy samples were compared with the original PGT-A results. Prior NGS testing classified the TE samples as euploid, aneuploid, or aneuploid-mosaic. The resulting re-biopsied samples underwent SurePlex whole genome amplification followed by NGS via the MiSeq platform, with copy number value (CNV) determined using BlueFuse Multi Software. The primary outcome measure was reliability, defined as concordance between initial TE result and the repeat biopsies. Accuracy was determined by concordance between the TE and ICM samples, and compared between three chromosome types (disomic, aneuploid, and mosaic). Re-biopsies were performed on 32 embryos with prior PGT-A showing euploidy (10 embryos), aneuploidy of one or two chromosomes (4 embryos), or aneuploid-mosaic with one aneuploid chromosome and one mosaic chromosome (18 embryos). One hundred twenty-nine biopsy samples completed NGS (90 TE and 39 ICM biopsies) and 105 biopsy results were included in the analysis. TE biopsies provide a highly accurate test of the future fetus, with the ICM disomic concordance rate of 97.6%. Clinical concordance rates indicate that TE biopsies provide a reliable test when the result is euploid (99.5%) or aneuploid (97.3%), but less reliable when the result is mosaic (35.2%). TE biopsies predict euploidy or aneuploidy in the ICM with a high degree of accuracy. PGT-A with NGS of TE biopsies is shown to be highly reliable, with clinically relevant concordance rates for aneuploidy and euploidy over 95%. TE biopsies indicating mosaicism were less reliable (35.2%), presumably because mitotic non-disjunction events are not uniformly distributed throughout the blastocyst. However, classification of TE biopsy of PGT-A with NGS results as either aneuploid or euploid provides a highly reliable test.

Study question How can SNPs analysis assist in indicating or contraindicating embryo transfer with euploid or intermediate copy number PGT-A results based on Next Generation Sequencing (NGS)? Summary answer The analysis of polymorphic SNPs applied as a complementary PGT-A technique provides information on ploidy alterations and the presence of contamination in trophectoderm (TE) biopsies. What is known already The PGT-A is commonly applied to detect numerical chromosomal abnormalities in TE to minimize the risks of transferring genetically abnormal embryos. However, PGT-A has limitations that do not allow the detection of numerical alterations involving genome-wide ploidy and the detection of contaminating DNA. Triploidy is an important cause of miscarriage and SNPs analysis in TE biopsies would allow us to optimize clinical outcomes. Study design, size, duration With the aim of detecting ploidy alterations and dubious results due to exogeneous DNA contamination presence, a set of SNPs were analyzed combined with PGT-A test in 1,174 TE biopsies received between January and December 2024. Subsequently, the frequencies of ploidy status (haploidy, diploidy and triploidy) and signs of exogeneous DNA contamination in TE biopsies, derived from normal and atypical fertilization, with euploid and intermediate copy number results in PGT-A test by NGS were described. Participants/materials, setting, methods TE biopsies received for PGT-A test plus SNPs analysis were evaluated. NGS-based PGT-A was performed by Ion Reproseq PGS kit and Ion Torrent S5 platform. Then, to infer contamination and ploidy level, a SNPs panel was used. A descriptive analysis of ploidy frequencies found in each PGT-A result was performed. The interference of triploidy in PGT-A intermediate copy number results, and the association between abnormal fertilization and ploidy were evaluated. Main results and the role of chance Of the total biopsies analyzed, 1,174 cases, we obtained 99% of informative results and in all of them we had access to fertilization information obtained by morphological analysis of pronuclei number (PN). PGT-A followed by SNPs analysis showed: A) Aneuploid results: 559 (48%) with mean maternal age (MMA) 39.0±3.5 years: Diploid (n = 388, MMA=39±3.5): (3.1% 0PN; 4.4% 1PN; 84% 2PN; 8.2% 3PN; 0.3% 4PN). Triploid (n = 61 MMA=39.9±4.0): (3.3% 1PN; 24.6% 2PN; 70.5% 3PN; 1.6%4PN). Haploid (n = 12, MMA= 39.5±3.3): (50% 1PN; 50% 2PN). Contaminated (n = 1): (100% 2PN). Non-informative ploidy (n = 97). B) Intermediate copy number results: 142 (12%) MMA=38.2±4.0 years: Diploid (n = 72, MMA=37.3±4.1): (4.2% 0PN; 4.2% 1PN; 87.5% 2PN; 4.2% 3PN). Triploid (n = 50, MMA=39.7±3.6): (2% 1PN; 14% 2PN; 82% 3PN; 2% 4PN). Haploid (n = 4, MMA=35.7±3.8): (50% 1PN; 50% 2PN). Contaminated (n = 2): (100% 2PN). Non-informative ploidy (n = 14). C) Euploid results: 460 (39%) euploid, MMA=37.4±4.2 years: Diploid (n = 381, MMA=37.3±4.3): (2.4% 0PN; 3.7% 1PN; 91.6% 2PN; 2.4% 3PN). Triploid (n = 36, MMA=38.5±3.6): (2.8% 0PN; 2.8% 1PN; 11.1% 2PN; 83.3%3PN). Haploid (n = 25, MMA=37.0±3.8): (92% 1PN; 8% 2PN). Contaminated (n = 4): (75% 2PN; 8% 3PN). Non-informative ploidy (n = 14). The intermediate copy number result was significantly associated (p < 0.05) to triploidy, either from 2PN or 3PN derived blastocyst, when compared with aneuploid or euploid results. A significant association was detected between the ploidy level and the PN described (p < 0.05). Limitations, reasons for caution Haploidy and contamination results in TE biopsies by SNPs analysis could not be included in statistical analysis due to small sample size. Lack information about embryo quality, culture conditions and the possibility of checking fertilization by time-lapse. Wider implications of the findings The SNPs analysis reveals that few embryos considered euploid may not be viable for transfer due to ploidy abnormalities, not detected in NGS-based PGT-A. More remarkably, uniform aneuploidy could be masked by triploidy or DNA contamination, and being considered as intermediate copy number results, if SNPs analysis is not included. Trial registration number No

There is insufficient good-quality evidence of a difference in cumulative live birth rate, live birth rate after the first embryo transfer, or miscarriage rate between IVF with and IVF without PGT-A as currently performed. No data were available on ongoing pregnancy rates. The effect of PGT-A on clinical pregnancy rate is uncertain. Women need to be aware that it is uncertain whether PGT-A with the use of genome-wide analyses is an effective addition to IVF, especially in view of the invasiveness and costs involved in PGT-A. PGT-A using FISH for the genetic analysis is probably harmful. The currently available evidence is insufficient to support PGT-A in routine clinical practice.

How PGS/PGT-A laboratories succeeded in losing all credibility

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.

Prognostic data in fertility preservation: the role of preimplantation genetic testing for aneuploidy (PGT-A) among cancer patients undergoing embryo banking (EB)

Are pregnancies after preimplantation genetic testing for aneuploidy (PGT-A) with trophectoderm (TE) biopsy and preferable elective freeze-all procedure associated with an increased risk of adverse obstetric and neonatal outcomes compared to pregnancies after IVF/ICSI without PGT-A? In this systematic review and meta-analysis specifically focusing on comparing a homogenous infertile population treated with IVF/ICSI, PGT-A was not associated with any adverse obstetric or neonatal outcomes. An increased risk of hypertensive disorders of pregnancy (HDP) following IVF/ICSI with PGT in general as compared to IVF/ICSI without PGT has been reported. However, people undergoing PGT for monogenic diseases or structural rearrangements are often fertile in contrast to infertile people undergoing general IVF/ICSI treatment. A systematic literature search was performed in PubMed, Embase, and Cochrane Library on 15 November 2024 and the search was updated on 11 June 2025. Inclusion criteria were: (i) randomized clinical trials or cohort studies comparing IVF/ICSI with or without PGT-A, (ii) embryo cultivation until the blastocyst stage, (iii) TE biopsy, and (iv) vitrification as cryopreservation method. Exclusion criteria were: (i) case series and case reports, (ii) polar body- or cleavage-stage biopsy, (iii) slow freeze, (iv) use of donor oocytes, or (v) natural conception controls. The main outcomes were HDP, preterm delivery, abnormal placentation, low birth weight, very low birth weight, and small for gestational age. Meta-analyses were performed for outcomes reported in ≥3 of the included studies. Frozen embryo transfer (FET)-cycles only subgroup-analyses were performed if the data were available in ≥3 of the included studies. Newcastle-Ottawa quality assessment score and Risk-of-Bias 2 were used to assess potential bias in the individual studies, and the GRADE approach was used to assess the certainty of evidence. A total of 2260 records were screened, and 12 studies comprising 56113 live births were included, of which 17254 resulted from PGT-A and 38859 resulted from IVF/ICSI without PGT-A. No outcomes differed significantly between the PGT-A and non-PGT-A group in the main analyses or the subgroup-analyses on FET-cycles. The included studies were mainly retrospective, and the number of cases was low for some outcomes. Current evidence does not suggest adverse obstetric or neonatal effects of PGT-A, though further research is needed, particularly for rare outcomes. This study was not funded. A.P. has received independent research grants and lecture fees from Abbott, IBSA, Gedeon Richter, Ferring, and Merck A/S. A.P. is part of research advisory boards for Gedeon Richter and Ferring Pharmaceuticals A/S. K.L. has received an independent research grant from Gedeon Richter and lecture fees from Ferring Pharmaceuticals. K.L. participated in a research advisory board for Ferring Pharmaceuticals A/S. N.F.W. has received a speaker's fee from Ferring Pharmaceuticals and travel support paid to institution from Gedeon Richter. The other authors had no conflict of interest to declare in relation to this work. PROSPERO No: CRD42024599519.

A review of factors influencing the implantation of euploid blastocysts after in vitro fertilization

Evaluation of chromosomal constitution in choronic villi samples of aborted fetuses after embryo transfers of euploid blastocysts

Preimplantation genetic testing for aneuploidy (PGT-A) with trophectoderm (TE) biopsy is widely applied in in vitro fertilization (IVF) to identify aneuploid embryos. However, potential safety concerns regarding biopsy and restrictions to only those embryos suitable for biopsy pose limitations. In addition, embryo mosaicism gives rise to false positives and false negatives in PGT-A because the inner cell mass (ICM) cells, which give rise to the fetus, are not tested. Here, we report a critical examination of the efficacy of noninvasive preimplantation genetic testing for aneuploidy (niPGT-A) in the spent culture media of human blastocysts by analyzing the cell-free DNA, which reflects ploidy of both the TE and ICM. Fifty-two frozen donated blastocysts with TE biopsy results were thawed; each of their spent culture medium was collected after 24-h culture and analyzed by next-generation sequencing (NGS). niPGT-A and TE-biopsy PGT-A results were compared with the sequencing results of the corresponding embryos, which were taken as true results for aneuploidy reporting. With removal of all corona-cumulus cells, the false-negative rate (FNR) for niPGT-A was found to be zero. By applying an appropriate threshold for mosaicism, both the positive predictive value (PPV) and specificity for niPGT-A were much higher than TE-biopsy PGT-A. Furthermore, the concordance rates for both embryo ploidy and chromosome copy numbers were higher for niPGT-A than TE-biopsy PGT-A. These results suggest that niPGT-A is less prone to errors associated with embryo mosaicism and is more reliable than TE-biopsy PGT-A.

Background: Preimplantation genetic testing for aneuploidy (PGT-A) is increasingly applied in assisted reproductive technologies, particularly among individuals with advanced maternal age or a history of recurrent pregnancy loss. By enabling the selection of chromosomally normal embryos, PGT-A may improve implantation rates and pregnancy outcomes following the initial embryo transfer. This study aims to compare ICSI outcomes, blastocyst quality, and vitrification timing between PGT-A and control groups to assess the reproductive outcomes Methods: A retrospective cohort study was conducted at a MHRT Hospital and Research Center, Hyderabad, involving patients undergoing Intracytoplasmic Sperm Injection (ICSI) cycles between March 2022 and March 2025. The study included 52 PGT-A and 52 control patients, with Baseline characteristics, embryological parameters, blastocyst quality, vitrification timing, and reproductive outcomes were assessed. The PGT-A group included embryos that underwent aneuploidy testing via trophectoderm biopsy. Blastocyst expansion, inner cell mass (ICM) quality, trophectoderm (TE) grading, and vitrification day were compared between the two groups. Results: Patients in the PGT-A group were older and had higher rates of primary and female-factor infertility. Hormonal profiles and oocyte retrieval outcomes were comparable between groups. The PGT-A group was older (mean age 36.2 vs. 33.7 years) and had higher proportions of primary infertility (71.15% vs. 59.61%) and female infertility (61.53% vs. 42.3%). Both groups had more or less similar oocyte retrieval numbers, but the Control group had more prior ICSI cycles. Regarding blastocyst quality, the PGT-A group exhibited higher proportions of grade 3 and grade 6 blastocysts, with a better ICM grade (B) and TE grade (A). The Control group had a higher proportion of grade 4 blastocysts. Additionally, more embryos in the Control group were vitrified on Day 5 (76.92% vs. 61.53%), while the PGT-A group showed a higher proportion vitrified on Day 6 (38.46% vs. 23.07%), these differences did not translate into superior clinical outcomes. The cumulative live-birth rate was slightly lower in the PGT-A group (75%) compared to controls (83.69%). Secondary outcomes, including biochemical, clinical, and ongoing pregnancy rates, were also lower in the PGT-A group. Singleton and twin birth weights were marginally reduced in the PGT-A cohort. Conclusion: PGT-A is associated with higher-quality blastocysts, particularly in terms of ICM and TE grading. It also influences vitrification strategies, with PGT-A embryos more likely to be vitrified on Day 6. These findings suggest that PGT-A may improve embryo quality, potentially enhancing ICSI outcomes, especially in older women or those with recurrent implantation failure. However, further studies are necessary to refine patient selection criteria and evaluate cost-effectiveness and ethical concerns associated with PGT-A.