Influence of the number of washings for embryos on non-invasive preimplantation chromosome screening results.

Objective: To explore the effect of varying numbers of embryo washings prior to blastocyst formation in non-invasive preimplantation chromosome screening (NICS) on the accuracy of NICS results. Methods: In this study, 68 blastocysts from preimplantation genetic testing (PGT)-assisted pregnancy were collected at our institution. On the fourth day of embryo culture, the embryos were transferred to a new medium for blastocyst culture and were washed either three times (NICS1 group) or ten times (NICS2 group). A trophectoderm (TE) biopsy was performed on the blastocysts, and the corresponding embryo culture media were collected for whole genome amplification (WGA) and high-throughput sequencing. Results: The success rate of WGA was 100% (TE biopsy), 76.7% (NICS1 group), and 89.5% (NICS2 group). The success rate of WGA in embryo medium on days 5 and 6 of culture was 75.0% (33/44) and 100% (24/24), respectively. Using TE as the gold standard, the karyotype concordance rate between the results of the NICS1 and NICS2 groups' embryo culture medium samples and TE results was 43.5% (10/23) and 73.5% (25/34), respectively. The sensitivity and specificity of detecting chromosomal abnormalities were higher in the NICS2 group than in the NICS1 group when TE was used (83.3% vs 60.0%; 62.5% vs 30.8%, respectively). The false-positive rate and false-negative rate (i.e., misdiagnosis rate and missed diagnosis rate, respectively) were lower in the NICS2 group than in the NICS1 group (37.5% vs 69.2%; 16.7% vs 40.0%, respectively). Conclusion: The NICS yielded favorable results after ten washings of the embryos. These findings provide a novel method for lowering the amount of cell-free DNA contamination from non-embryonic sources in the medium used for embryo development, optimizing the sampling procedure and improving the accuracy of the NICS test.

[Analysis of related factors influencing the detection rate of mosaic embryo and the pregnancy outcomes with mosaic embryo transfers].

Objective: To explore the related factors influencing the detection rate of mosaic embryo and the pregnancy outcomes of mosaic embryo transfer in preimplantation genetic testing for aneuploidy (PGT-A) based on next generation sequencing (NGS) technology. Methods: A retrospective study was performed to analyze the clinical data of patients in 745 PGT-A cycles from January 2019 to May 2023 at Chongqing Health Center for Women and Children, including 2 850 blastocysts. The biopsy cells were tested using NGS technology, and the embryos were divided into three groups based on the test results, namely euploid embryos, aneuploid embryos and mosaic embryos. The influence of population characteristics and laboratory-related parameters on the detection rate of mosaic embryo were analyzed, and the pregnancy outcomes of 98 mosaic embryo transfer cycles and 486 euploid embryo transfer cycles were compared during the same period, including clinical pregnancy rate and live birth rate. Results: Among the embryos tested (n=2 850), the number and proportion of euploid embryos, aneuploid embryos and mosaic embryos were 1 489 (52.2%, 1 489/2 850), 917 (32.2%, 917/2 850) and 444 (15.6%, 444/2 850), respectively. Among mosaic embryos, 245 (55.2%, 245/444) were segmental mosaic embryos, 118 (26.6%, 118/444) were whole-chromosome mosaic embryos, and 81 (18.2%, 81/444) were complex mosaic embryos. NGS technology was performed in 4 genetic testing institutions and the detection rate of mosaic embryo fluctuated from 13.5% to 27.0%. The distributions of female age, level of anti-Müllerian hormone, PGT-A indications, ovulation-inducing treatments, gonadotropin (Gn) dosage, Gn days, inner cell mass grade, trophectoderm cell grade, genetic testing institutions and developmental stage of blastocyst were significantly different among the three groups (all P<0.05). Multi-factor analysis showed that the trophectoderm cell grade and genetic testing institutions were significantly related to the detection rate of mosaic embryo; compared with the trophectoderm cell graded as A, the detection rate of mosaic embryo was significantly increased in the trophectoderm cell graded as B-(OR=1.59, 95%CI: 1.04-2.44, P=0.033); compared with genetic testing institution a, the detection rate of mosaic embryo was significantly higher (OR=2.89, 95%CI: 2.10-3.98, P<0.001) in the testing institution c. The clinical pregnancy rate and live birth rate with mosaic embryos transfer were significantly lower than those of euploid embryos transfer (clinical pregnancy rate: 51.0% vs 65.2%, P=0.008; live birth rate: 39.4% vs 53.2%, P=0.017). After adjustment for age, PGT-A indications, trophectoderm cell grade and days of embryo culture in vitro, the clinical pregnancy rate and live birth rate with mosaic embryos transfer were significantly lower than those of euploid embryos transfer (clinical pregnancy rate: OR=0.52, 95%CI: 0.32-0.83, P=0.007; live birth rate: OR=0.50, 95%CI: 0.31-0.83, P=0.007). Conclusions: The trophectoderm cell grade and genetic testing institutions are related to the detection rate of mosaic embryo. Compared with euploid embryos transfer, the clinical pregnancy rate and live birth rate with mosaic embryos transfer are significantly reduced. For infertile couple without euploid embryos, transplantable mosaic embryos could be recommended according to the mosaic ratio and mosaic type in genetic counseling to obtain the optimal pregnancy outcome.

Clinical Guideline for Preimplantation Genetic Testing in Inherited Cardiac Diseases.

BACKGROUND: Preimplantation genetic testing (PGT) is a reproductive technology that selects embryos without (familial) genetic variants. PGT has been applied in inherited cardiac disease and is included in the latest American Heart Association/American College of Cardiology guidelines. However, guidelines selecting eligible couples who will have the strongest risk reduction most from PGT are lacking. We developed an objective decision model to select eligibility for PGT and compared its results with those from a multidisciplinary team. METHODS: All couples with an inherited cardiac disease referred to the national PGT center were included. A multidisciplinary team approved or rejected the indication based on clinical and genetic information. We developed a decision model based on published risk prediction models and literature, to evaluate the severity of the cardiac phenotype and the penetrance of the familial variant in referred patients. The outcomes of the model and the multidisciplinary team were compared in a blinded fashion. RESULTS: Eighty-three couples were referred for PGT (1997-2022), comprising 19 different genes for 8 different inherited cardiac diseases (cardiomyopathies and arrhythmias). Using our model and proposed cutoff values, a definitive decision was reached for 76 (92%) couples, aligning with 95% of the multidisciplinary team decisions. In a prospective cohort of 11 couples, we showed the clinical applicability of the model to select couples most eligible for PGT. CONCLUSIONS: The number of PGT requests for inherited cardiac diseases increases rapidly, without the availability of specific guidelines. We propose a 2-step decision model that helps select couples with the highest risk reduction for cardiac disease in their offspring after PGT.

Effects of first and second division modes on euploidy acquisition in human embryo.

The aim of this study was to non-invasively investigate euploid embryos using methods other than pre-implantation genetic testing for aneuploidy. The study focused on direct cleavage (DC) observed during early embryo development. We also investigated the relationship between the mode of early embryo division and embryo ploidy. Embryos were divided into the normal cleavage (NC) and DC groups, and the DC group was further subdivided into the DC-First (DC-F) and DC-Second (DC-S) groups, depending on whether DC was observed at the first or second cleavage, respectively. The acquisition rates of euploid embryos and embryos appropriate for transfer were compared between the groups. Our results revealed that the timing of the first division did not differ between blastocyst grades or in embryos with varying degrees of ploidy. Further, the timing of the first cleavage did not affect the acquisition rate of embryos appropriate for transfer and euploid embryo formation rate did not significantly differ between the DC and NC groups. We also noted that for embryos appropriate for transfer, euploidy acquisition rate did not differ significantly between the DC and NC groups. Further, the euploidy acquisition rate of embryos did not differ between the DC-F and DC-S groups. However, the acquisition rate of embryos appropriate for transfer, including those with low mosaicism, was significantly higher in the DC-S group than in the DC-F group. These findings indicated that the number of good-quality blastocysts formed was significantly higher in the NC group than in the DC group and the acquisition rate of embryos appropriate for transfer, including those with low mosaicism, was significantly higher in the DC-S group than in the DC-F group.

Agarose amplification based sequencing characterization cell-free RNA in preimplantation spent embryo medium.

BACKGROUND: The cell-free RNA (cf-RNA) of spent embryo medium (SEM) has aroused a concern of academic and clinical researchers for its potential use in non-invasive embryo screening. However, comprehensive characterization of cf-RNA from SEM still presents significant technical challenges, primarily due to the limited volume of SEM. Hence, there is urgently need to a small input liquid volume and ultralow amount of cf-RNA library preparation method to unbiased cf-RNA sequencing from SEM. (75) RESULT: Here, we report a high sensitivity agarose amplification-based cf-RNA sequencing method (SEM-Acf) for human preimplantation SEM cf-RNA analysis. It is a cf-RNA sequencing library preparation method by adding agarose amplification. The agarose amplification sensitivity (0.005 pg) and efficiency (105.35 %) were increased than that of without agarose addition (0.45 pg and 96.06 %) by âˆ¼ 90 fold and 9.29 %, respectively. Compared with SMART sequencing (SMART-seq), the correlation of gene expression was stronger in different SEM samples by using SEM-Acf. The cf-RNA number of detected and coverage uniformity of 3' end were significantly increased. The proportion of 5' end adenine, alternative splicing events and short fragments (<400 bp) were increased. It is also found that 4-mer end motifs of cf-RNA fragments was significantly differences between different embryonic stage by day3 spent cleavage medium and day5/6 spent blastocyst medium. (141) SIGNIFICANCE: This study established an efficient SEM amplification and library preparation method. Additionally, we successfully described the characterizations of SEM cf-RNA in preimplantation embryo using SEM-Acf, including expression features and fragment lengths. SEM-Acf facilitates the exploration of cf-RNA as a noninvasive embryo screening biomarker, and opens up potential clinical utilities of small input liquid volume and ultralow amount cf-RNA sequencing. (59).

Blastocysts from partial compaction morulae are not defined by their early mistakes.

RESEARCH QUESTION: Is partial compaction during morula formation associated with an embryo's developmental ability and implantation potential? DESIGN: Retrospective analysis of data from 196 preimplantation genetic testing for aneuploidy (PGT-A) cycles. Embryos starting compaction were grouped according to the inclusion or not of all the blastomeres in the forming morula (full compaction or partial compaction). The possible effect of maternal age and ovarian response on compaction was analysed. Morphokinetic characteristics, blastocyst formation rate, morphology and cytogenetic constitution of the obtained blastocysts were compared. Comparisons of reproductive outcomes after the transfer of euploid blastocysts from both groups were established. Finally, in a subset of embryos, the chromosomal constitution concordance of the abandoned cells and the corresponding blastocyst through trophectoderm biopsies was assessed. RESULTS: A total of 430 embryos failed to include at least one cell during compaction (partial compaction group [49.3%]), whereas the 442 remaining embryos formed a fully compacted morula (full compaction group [50.7%]). Neither female age nor the number of oocytes collected affected the prevalence of partial compaction morulae. Morphokinetic parameters were altered in embryos from partial compaction morulae compared with full compaction. Although an impairment in blastocyst formation rate was observed in partial compaction morulae (57.2% versus 70.8%, P < 0.001), both chromosomal constitution (euploidy rate: partial compaction [38.4%] versus full compaction [34.2%]) and reproductive outcomes (live birth rate: partial compaction [51.9%] versus full compaction [46.2%]) of the obtained blastocysts were equivalent between groups. A high ploidy correlation of excluded cells-trophectoderm duos was observed. CONCLUSIONS: Partial compaction morulae show a reduced developmental ability compared with full compaction morulae. Resulting blastocysts from both groups, however, have similar euploidy rates and reproductive outcomes. Cell exclusion might be a consequence of a compromised embryo development regardless of the chromosomal constitution of the excluded cells.

Trophectoderm cells of human mosaic embryos display increased apoptotic levels and impaired differentiation capacity: a molecular clue regarding their reproductive fate?

STUDY QUESTION: Are there cell lineage-related differences in the apoptotic rates and differentiation capacity of human blastocysts diagnosed as euploid, mosaic, and aneuploid after preimplantation genetic testing for aneuploidy (PGT-A) based on concurrent copy number and genotyping analysis? SUMMARY ANSWER: Trophectoderm (TE) cells of mosaic and aneuploid blastocysts exhibit significantly higher levels of apoptosis and significantly reduced differentiation capacity compared to those of euploid blastocysts. WHAT IS KNOWN ALREADY: Embryos diagnosed as mosaic after PGT-A can develop into healthy infants, yet understanding the reasons behind their reproductive potential requires further research. One hypothesis suggests that mosaicism can be normalized through selective apoptosis and reduced proliferation of aneuploid cells, but direct evidence of these mechanisms in human embryos is lacking. Additionally, data interpretation from studies involving mosaic embryos has been hampered by retrospective analysis methods and the high incidence of false-positive mosaic diagnoses stemming from the use of poorly specific PGT-A platforms. STUDY DESIGN, SIZE, DURATION: Prospective cohort study performing colocalization of cell-lineage and apoptotic markers by immunofluorescence (IF). We included a total of 64 human blastocysts donated to research on Day 5 or 6 post-fertilization (dpf) by 43 couples who underwent in vitro fertilization treatment with PGT-A at IVI-RMA Valencia between September 2019 and October 2022. A total of 27 mosaic blastocysts were analyzed. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study consisted of two phases: Phase I (caspase-3, n = 53 blastocysts): n = 13 euploid, n = 22 mosaic, n = 18 aneuploid. Phase II (terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL), n = 11 blastocysts): n = 2 euploid, n = 5 mosaic, n = 4 aneuploid. Following donation for research, vitrified blastocysts were warmed, cultured until re-expansion, fixed, processed for IF, and imaged using confocal microscopy. For each blastocyst, the following cell counts were conducted: total cells (DAPI+), TE cells (GATA3+), inner cell mass (ICM) cells (GATA3-/NANOG+), and apoptotic cells (caspase-3+ or TUNEL+). The incidence of apoptosis was calculated for each blastocyst by dividing the number of caspase-3+ cells (Phase I) or TUNEL+ cells (Phase II) by the number of TE or ICM cells. Statistical analysis was performed according to data type and distribution (P < 0.05 was considered statistically significant). MAIN RESULTS AND THE ROLE OF CHANCE: Phase I: Mosaic blastocysts displayed a similar number of total cells (49.6 ± 15 cells at 5 dpf; 58.8 ± 16.9 cells at 6 dpf), TE cells (38.8 ± 13.7 cells at 5 dpf; 49.2 ± 16.2 cells at 6 dpf), and ICM cells (10.9 ± 4.2 cells at 5 dpf; 9.7 ± 7.1 cells at 6 dpf) compared to euploid and aneuploid blastocysts (P > 0.05). The proportion of TE cells retaining NANOG expression increased gradually from euploid blastocysts (9.7% = 63/651 cells at 5 dpf; 0% = 0/157 cells at 6 dpf) to mosaic blastocysts (13.1% = 104/794 cells at 5 dpf; 3.4% = 12/353 cells at 6 dpf) and aneuploid blastocysts (27.9% = 149/534 cells at 5 dpf; 4.6% = 19/417 cells at 6 dpf) (P < 0.05). At the TE level, caspase-3+ cells were frequently observed (39% = 901/2310 cells). The proportion of caspase-3+ TE cells was significantly higher in mosaic blastocysts (44.1% ± 19.6 at 5 dpf; 43% ± 16.8 at 6 dpf) and aneuploid blastocysts (45.9% ± 16.1 at 5 dpf; 49% ± 15.1 at 6 dpf) compared to euploid blastocysts (26.6% ± 16.6 at 5 dpf; 17.5% ± 14.8 at 6 dpf) (P < 0.05). In contrast, at the ICM level, caspase-3+ cells were rarely observed (1.9% = 11/596 cells), and only detected in mosaic blastocysts (2.6% = 6/232 cells) and aneuploid blastocysts (2.5% = 5/197 cells) (P > 0.05). Phase II: Consistently, TUNEL+ cells were only observed in TE cells (32.4% = 124/383 cells). An increasing trend was identified toward a higher proportion of TUNEL+ cells in the TE of mosaic blastocysts (37.2% ± 21.9) and aneuploid blastocysts (39% ± 41.7), compared to euploid blastocysts (23% ± 32.5), although these differences did not reach statistical significance (P > 0.05). LIMITATIONS, REASONS FOR CAUTION: The observed effects on apoptosis and differentiation may not be exclusive to aneuploid cells. Additionally, variations in aneuploidies and unexplored factors related to blastocyst development and karyotype concordance may introduce potential biases and uncertainties in the results. WIDER IMPLICATIONS OF THE FINDINGS: Our findings demonstrate a cell lineage-specific effect of aneuploidy on the apoptotic levels and differentiation capacity of human blastocysts. This contributes to unravelling the biological characteristics of mosaic blastocysts and supports the concept of clonal depletion of aneuploid cells in explaining their reproductive potential. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by grants from Centro para el Desarrollo Tecnológico Industrial (CDTI) (20190022) and Generalitat Valenciana (APOTIP/2019/009). None of the authors has any conflict of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Preimplantation genetic testing for X-linked chronic granulomatous disease induced by a CYBB gene variant: A case report.

INTRODUCTION: X-linked recessive chronic granulomatous disease (XR-CGD) is a severe primary immunodeficiency principally caused by a CYBB (OMIM: 300481) gene variant. Recurrent fatal bacterial or fungal infections are the main clinical manifestations of XR-CGD. PATIENT CONCERNS: In the current case, in vitro fertilization (IVF) associated with preimplantation genetic testing for monogenic disorder (PGT-M) was applied for a Chinese couple who had given birth to a boy with XR-CGD. DIAGNOSIS: Next-generation sequencing-based SNP haplotyping and Sanger-sequencing were used to detect the CYBB gene variant (c.804 + 2T>C, splicing) in this family. INTERVENTIONS: The patient was treated with IVF and PGT-M successively. OUTCOMES: In this IVF cycle, 7 embryos were obtained, and 2 of them were euploid and lacked the CYBB gene variant (c.804 + 2T>C). The PGT results were verified by prenatal diagnosis after successful pregnancy, and a healthy girl was eventually born. CONCLUSION: PGT-M is an effective method for helping families with these fatal and rare inherited diseases to have healthy offspring. It can availably block the transmission of disease-causing loci to descendant.

Improved prediction of clinical pregnancy using artificial intelligence with enhanced inner cell mass and trophectoderm images.

This study aimed to assess the performance of an artificial intelligence (AI) model for predicting clinical pregnancy using enhanced inner cell mass (ICM) and trophectoderm (TE) images. In this retrospective study, we included static images of 2555 day-5-blastocysts from seven in vitro fertilization centers in South Korea. The main outcome of the study was the predictive capability of the model to detect clinical pregnancies (gestational sac). Compared with the original embryo images, the use of enhanced ICM and TE images improved the average area under the receiver operating characteristic curve for the AI model from 0.716 to 0.741. Additionally, a gradient-weighted class activation mapping analysis demonstrated that the enhanced image-trained AI model was able to extract features from crucial areas of the embryo in 99% (506/512) of the cases. Particularly, it could extract the ICM and TE. In contrast, the AI model trained on the original images focused on the main areas in only 86% (438/512) of the cases. Our results highlight the potential efficacy of using ICM- and TE-enhanced embryo images when training AI models to predict clinical pregnancy.

Preimplantation genetic testing in the current era, a review.

BACKGROUND: Preimplantation genetic testing (PGT), also referred to as preimplantation genetic diagnosis (PGD), is an advanced reproductive technology used during in vitro fertilization (IVF) cycles to identify genetic abnormalities in embryos prior to their implantation. PGT is used to screen embryos for chromosomal abnormalities, monogenic disorders, and structural rearrangements. DEVELOPMENT OF PGT: Over the past few decades, PGT has undergone tremendous development, resulting in three primary forms: PGT-A, PGT-M, and PGT-SR. PGT-A is utilized for screening embryos for aneuploidies, PGT-M is used to detect disorders caused by a single gene, and PGT-SR is used to detect chromosomal abnormalities caused by structural rearrangements in the genome. PURPOSE OF REVIEW: In this review, we thoroughly summarized and reviewed PGT and discussed its pros and cons down to the minutest aspects. Additionally, recent studies that highlight the advancements of PGT in the current era, including their future perspectives, were reviewed. CONCLUSIONS: This comprehensive review aims to provide new insights into the understanding of techniques used in PGT, thereby contributing to the field of reproductive genetics.

To transfer or not to transfer: the dilemma of mosaic embryos - a narrative review.

A frequent finding after preimplantation genetic diagnostic testing for aneuploidies using next-generation sequencing is an embryo that is putatively mosaic. The prevalence of this outcome remains unclear and varies with technical and external factors. Mosaic embryos can be classified by the percentage of cells affected, type of chromosome involvement (whole or segmental), number of affected chromosomes or affected cell type (inner mass cell, trophectoderm or both). The origin of mosaicism seems to be intrinsic as a post-zygotic mitotic error, but some external factors can play a role. As experience has increased with the transfer of mosaic embryos, clinical practice has gradually become more flexible in recent years. Nevertheless, clinical results show lower implantation, pregnancy and clinical pregnancy rates and higher miscarriage rates with mosaic embryo transfer when compared with the transfer of euploid embryos. Prenatal diagnosis is highly recommended after the transfer of mosaic embryos. This narrative review is intended to serve as reference material for practitioners in reproductive medicine who must manage a mosaic embryo result after preimplantation genetic testing for aneuploidies.

The reproductive potential of vitrified-warmed euploid embryos declines following repeated uterine transfers.

BACKGROUND: Recurrent implantation failure (RIF) represents a vague clinical condition with an unclear diagnostic challenge that lacks solid scientific underpinning. Although euploid embryos have demonstrated consistent implantation capabilities across various age groups, a unanimous agreement regarding the advantages of preimplantation genetic testing for aneuploidy (PGT-A) in managing RIF is absent. The ongoing discussion about whether chromosomal aneuploidy in embryos significantly contributes to recurrent implantation failure remains unsettled. Despite active discussions in recent times, a universally accepted characterization of recurrent implantation failure remains elusive. We aimed in this study to measure the reproductive performance of vitrified-warmed euploid embryos transferred to the uterus in successive cycles. METHODS: This observational cohort study included women (n = 387) with an anatomically normal uterus who underwent oocyte retrieval for PGT-A treatment with at least one biopsied blastocyst, between January 2017 and December 2021 at a university-affiliated public fertility center. The procedures involved in this study included ICSI, blastocyst culture, trophectoderm biopsy and comprehensive 24-chromosome analysis of preimplantation embryos using Next Generation Sequencing (NGS). Women, who failed a vitrified-warmed euploid embryo transfer, had successive blastocyst transfer cycles (FET) for a total of three using remaining cryopreserved euploid blastocysts from the same oocyte retrieval cycle. The primary endpoints were sustained implantation rate (SIR) and live birth rate (LBR) per vitrified-warmed single euploid embryo. The secondary endpoints were mean euploidy rate (m-ER) per cohort of biopsied blastocysts from each patient, as well as pregnancy and miscarriage rates. RESULTS: The mean age of the patient population was 33.4 years (95% CI 32.8-33.9). A total of 1,641 embryos derived from the first oocyte retrieval cycle were biopsied and screened. We found no associations between the m-ER and the number of previous failed IVF cycles among different ranges of maternal age at oocyte retrieval (P = 0.45). Pairwise comparisons showed a significant decrease in the sustained implantation rate (44.7% vs. 30%; P = 0.01) and the livebirth rate per single euploid blastocyst (37.1% vs. 25%; P = 0.02) between the 1st and 3rd FET. The cumulative SIR and LBR after up to three successive single embryo transfers were 77.1% and 68.8%, respectively. We found that the live birth rate of the first vitrified-warmed euploid blastocyst transferred decreased significantly with the increasing number of previously failed IVF attempts by categories (45.3% vs. 35.8% vs. 27.6%; P = 0.04). A comparable decrease in sustained implantation rate was also observed but did not reach statistical significance (50% vs. 44.2 vs. 37.9%; P = NS). Using a logistic regression model, we confirmed the presence of a negative association between the number of previous IVF failed attempts and the live birth rate per embryo transfer cycle (OR = 0.76; 95% CI 0.62-0.94; P = 0.01). CONCLUSIONS: These findings are vital for enhancing patient counseling and refining management strategies for individuals facing recurrent implantation failure. By tailoring interventions based on age and ovarian reserve, healthcare professionals can offer more personalized guidance, potentially improving the overall success rates and patient experiences in fertility treatments. TRIAL REGISTRATION NUMBER: N/A.

Should non-invasive prenatal testing be recommended for patients who achieve pregnancy with PGT?

OBJECTIVE: To determine whether non-invasive prenatal testing is an alternative testing option to preimplantation genetic testing (PGT) in pregnant patients. METHODS: This was a retrospective study of the clinical outcomes of patients who underwent PGT and invasive or non-invasive pregnancy testing after euploid blastocyst transfer at our IVF centre between January 2017 and December 2022. RESULTS: In total, 321 patients were enrolled in this study, 138 (43.0%) received invasive pregnancy testing, and 183 (57.0%) patients underwent non-invasive testing. The mean age of the patients in Group 2 was higher than that of the patients in Group 1 (35.64 ± 4.74 vs. 31.04 ± 4.15 years, P < 0.001). The basal LH and AMH levels were higher in Group 1 than in Group 2 (4.30 ± 2.68 vs. 3.40 ± 1.88, P = 0.003; 5.55 ± 11.22 vs. 4.09 ± 3.55, P = 0.012), but the clinical outcomes were not significantly different. Furthermore, the clinical outcomes of patients undergoing invasive testing were similar to those of patients undergoing non-invasive testing with the same PGT indication. CONCLUSION: Our results suggest that non-invasive pregnancy testing is a suitable alternative option for detecting the foetal chromosomal status in a PGT cycle. However, the usefulness of non-invasive testing in PGT-M patients is still limited.

Mosaic embryo transfer versus additional IVF with PGT-A Cycle: a decision model comparing live birth rate and cost.

PURPOSE: To evaluate the relative live birth rate and net cost difference between mosaic embryo transfer and an additional cycle of IVF with PGT-A for patients whose only remaining embryos are non-euploid. METHODS: A decision analytic model was designed with model parameters varying based on discrete age cutoffs (<35, 35-37, 38-39, 40-42, 43-44, >44). Model inputs included probabilities of successful IVF, clinical pregnancy, and live birth as well as costs of IVF with PGT-A, embryo transfer, live birth, amniocentesis, and dilation and curettage. All costs were modeled from the healthcare system perspective and adjusted for inflation to 2023 $USD. Model outcomes were sub-stratified by degree and type of mosaicism. RESULTS: For patients younger than 43, an additional cycle of IVF with PGT-A resulted in a higher relative live birth rate (<35, +20%; 35-37, +15%; 38-39, +17%; 40-42, +6%; average, +14.5%) compared to mosaic embryo transfer with an average additional cost of $16,633. For patients older than 42, mosaic embryo transfer resulted in a higher live birth rate (43-44, +5%; >44, +3%; average, +4%) while on average costing $9572 less than an additional cycle of IVF with PGT-A. CONCLUSION: Mosaic embryo transfers are a superior alternative to an additional cycle of IVF with PGT-A for patients older than 42 whose only remaining embryos are non-euploid. Mosaic embryo transfers also should be considered for patients younger than 42 who are unable to pursue additional autologous IVF cycles. Counseling and care should be personalized to individual patients and embryos.

Extended application of PGT-M strategies for small pathogenic CNVs.

PURPOSE: The preimplantation genetic testing for aneuploidy (PGT-A) platform is not currently available for small copy-number variants (CNVs), especially those < 1 Mb. Through strategies used in PGT for monogenic disease (PGT-M), this study intended to perform PGT for families with small pathogenic CNVs. METHODS: Couples who carried small pathogenic CNVs and underwent PGT at the Reproductive and Genetic Hospital of CITIC-Xiangya (Hunan, China) between November 2019 and April 2023 were included in this study. Haplotype analysis was performed through two platforms (targeted sequencing and whole-genome arrays) to identify the unaffected embryos, which were subjected to transplantation. Prenatal diagnosis using amniotic fluid was performed during 18-20 weeks of pregnancy. RESULTS: PGT was successfully performed for 20 small CNVs (15 microdeletions and 5 microduplications) in 20 families. These CNVs distributed on chromosomes 1, 2, 6, 7, 13, 15, 16, and X with sizes ranging from 57 to 2120 kb. Three haplotyping-based PGT-M strategies were applied. A total of 89 embryos were identified in 25 PGT cycles for the 20 families. The diagnostic yield was 98.9% (88/89). Nineteen transfers were performed for 17 women, resulting in a 78.9% (15/19) clinical pregnancy rate after each transplantation. Of the nine women who had healthy babies, eight accepted prenatal diagnosis and the results showed no related pathogenic CNVs. CONCLUSION: Our results show that the extended haplotyping-based PGT-M strategy application for small pathogenic CNVs compensated for the insufficient resolution of PGT-A. These three PGT-M strategies could be applied to couples with small pathogenic CNVs.

Single-cell DNA sequencing reveals a high incidence of chromosomal abnormalities in human blastocysts.

Aneuploidy, a deviation from the normal chromosome copy number, is common in human embryos and is considered a primary cause of implantation failure and early pregnancy loss. Meiotic errors lead to uniformly abnormal karyotypes, while mitotic errors lead to chromosomal mosaicism: the presence of cells with at least 2 different karyotypes within an embryo. Knowledge about mosaicism in blastocysts mainly derives from bulk DNA sequencing (DNA-Seq) of multicellular trophectoderm (TE) and/or inner cell mass (ICM) samples. However, this can only detect an average net gain or loss of DNA above a detection threshold of 20%-30%. To accurately assess mosaicism, we separated the TE and ICM of 55 good-quality surplus blastocysts and successfully applied single-cell whole-genome sequencing (scKaryo-Seq) on 1,057 cells. Mosaicism involving numerical and structural chromosome abnormalities was detected in 82% of the embryos, in which most abnormalities affected less than 20% of the cells. Structural abnormalities, potentially caused by replication stress and DNA damage, were observed in 69% of the embryos. In conclusion, our findings indicated that mosaicism was prevalent in good-quality blastocysts, whereas these blastocysts would likely be identified as normal with current bulk DNA-Seq techniques used for preimplantation genetic testing for aneuploidy.

Associations between the artificial intelligence scoring system and live birth outcomes in preimplantation genetic testing for aneuploidy cycles.

BACKGROUND: Several studies have demonstrated that iDAScore is more accurate in predicting pregnancy outcomes in cycles without preimplantation genetic testing for aneuploidy (PGT-A) compared to KIDScore and the Gardner criteria. However, the effectiveness of iDAScore in cycles with PGT-A has not been thoroughly investigated. Therefore, this study aims to assess the association between artificial intelligence (AI)-based iDAScore (version 1.0) and pregnancy outcomes in single-embryo transfer (SET) cycles with PGT-A. METHODS: This retrospective study was approved by the Institutional Review Board of Chung Sun Medical University, Taichung, Taiwan. Patients undergoing SET cycles (n = 482) following PGT-A at a single reproductive center between January 2017 and June 2021. The blastocyst morphology and morphokinetics of all embryos were evaluated using a time-lapse system. The blastocysts were ranked based on the scores generated by iDAScore, which were defined as AI scores, or by KIDScore D5 (version 3.2) following the manufacturer's protocols. A single blastocyst without aneuploidy was transferred after examining the embryonic ploidy status using a next-generation sequencing-based PGT-A platform. Logistic regression analysis with generalized estimating equations was conducted to assess whether AI scores are associated with the probability of live birth (LB) while considering confounding factors. RESULTS: Logistic regression analysis revealed that AI score was significantly associated with LB probability (adjusted odds ratio [OR] = 2.037, 95% confidence interval [CI]: 1.632-2.542) when pulsatility index (PI) level and types of chromosomal abnormalities were controlled. Blastocysts were divided into quartiles in accordance with their AI score (group 1: 3.0-7.8; group 2: 7.9-8.6; group 3: 8.7-8.9; and group 4: 9.0-9.5). Group 1 had a lower LB rate (34.6% vs. 59.8-72.3%) and a higher rate of pregnancy loss (26% vs. 4.7-8.9%) compared with the other groups (p < 0.05). The receiver operating characteristic curve analysis verified that the iDAScore had a significant but limited ability to predict LB (area under the curve [AUC] = 0.64); this ability was significantly weaker than that of the combination of iDAScore, type of chromosomal abnormalities, and PI level (AUC = 0.67). In the comparison of the LB groups with the non-LB groups, the AI scores were significantly lower in the non-LB groups, both for euploid (median: 8.6 vs. 8.8) and mosaic (median: 8.0 vs. 8.6) SETs. CONCLUSIONS: Although its predictive ability can be further enhanced, the AI score was significantly associated with LB probability in SET cycles. Euploid or mosaic blastocysts with low AI scores (≤ 7.8) were associated with a lower LB rate, indicating the potential of this annotation-free AI system as a decision-support tool for deselecting embryos with poor pregnancy outcomes following PGT-A.

Outcomes of BRCA pre-implantation genetic testing according to the parental mutation origin: a cohort study.

BACKGROUND: The process of gamete formation and early embryonic development involves rapid DNA replication, chromosome segregation and cell division. These processes may be affected by mutations in the BRCA1/2 genes. The aim of this study was to evaluate BRCA mutation inheritance and its effect on early embryonic development according to the parental origin of the mutation. The study question was approached by analyzing in vitro fertilization cycles (IVF) that included pre-implantation testing (PGT-M) for a BRCA gene mutation. METHODS: This retrospective cohort study compared cycles of pre-implantation genetic testing for mutations (PGT-M) between male and female patients diagnosed with BRCA 1/2 mutations (cases), to a control group of two other mutations with dominant inheritance (myotonic dystrophy (MD) and polycystic kidney disease (PKD)). Results were compared according to mutation type and through a generalized linear model analysis. RESULTS: The cohort included 88 PGT-M cycles (47 BRCA and 41 non-BRCA) among 50 patients. Maternal and paternal ages at oocyte retrieval were comparable between groups. When tested per cycle, FSH dose, maximum estradiol level, oocytes retrieved, number of zygotes, and number of embryos available for biopsy and affected embryos, were not significantly different among mutation types. All together 444 embryos were biopsied: the rate of affected embryos was comparable between groups. Among BRCA patients, the proportion of affected embryos was similar between maternal and paternal mutation origin (p = 0.24). In a generalized linear model analysis, the relative oocyte yield in maternal BRCA patients was significantly lower (0.7, as related to the non BRCA group)(p < 0.001). Zygote formation and blastulation were not affected by the BRCA gene among paternal cases (P = 0.176 and P = 0.293 respectively), nor by paternal versus maternal BRCA carriage (P = 0.904 and P = 0.149, respectively). CONCLUSIONS: BRCA PGT-M cycles performed similarly compared to non-BRCA cycles. Inheritance rate and cycle parameters were not affected by the parental origin of the mutation.

Optimizing non-invasive preimplantation genetic testing: investigating culture conditions, sample collection, and IVF treatment for improved non-invasive PGT-A results.

PURPOSE: This study aimed to optimize the non-invasive preimplantation genetic testing for aneuploidy (niPGT-A) in the laboratory by comparing two collection timing of the spent culture medium (SCM), two embryo rinsing protocols, and the use of conventional insemination instead of intracytoplasmic sperm injection (ICSI). METHODS: Results of two embryo rinsing methods (one-step vs sequential) and SCM collected on day 5 vs day 6 after retrieval were compared against trophectoderm (TE) biopsies as reference. Results from day 6 SCM in cycles fertilized by conventional insemination were compared with PGT-A using ICSI. RESULTS: The rate of concordance was higher in day 6 samples than in day 5 samples when the sequential method was used, in terms of total concordance (TC; day 6 vs day 5: 85.0% vs 60.0%, p = 0.0228), total concordance with same sex (TCS, 82.5% vs 28,0%, p < 0.0001), and full concordance with same sex (FCS, 62.5% vs 24.0%, p = 0.0025). The sequential method significantly out-performed the one-step method when SCM were collected on day 6 (sequential vs one-step, TC: 85.0% vs 64.5%, p = 0.0449; TCS: 82.5% vs 54.8%, p = 0.0113; FCS: 62.5% vs 25.8%, p = 0.0021). There was no significant difference in niPGT-A results between cycles fertilized by the conventional insemination and ICSI. CONCLUSION: We have shown a higher concordance rate when SCM was collected on day 6 and the embryos were rinsed in a sequential manner. Comparable results of niPGT-A when oocytes were fertilized by conventional insemination or ICSI. These optimization steps are important prior to commencement of a randomized trial in niPGT-A.

A clinical predictive model for live birth in women of advanced age undergoing PGT cycles.

PURPOSE: The trend of delaying childbirth has resulted in a growing number of advanced-aged women who are opting for preimplantation genetic testing (PGT) to screen for monogenic diseases or structural chromosomal rearrangements (PGT-M and PGT-SR). This increase in demand necessitates the development of a clinical predictive model for live birth outcomes in these women. Therefore, the objective of this study is to construct a comprehensive predictive model that assesses the likelihood of achieving a successful live birth in advanced-aged women undergoing PGT-M and PGT-SR treatments. METHODS: A retrospective cohort study of 37-45-year-old women undergoing preimplantation genetic testing for monogenic disease or structural chromosomal rearrangement cycles from 2010 to 2021 was conducted at a university hospital reproductive centre. The purpose was to develop a clinical predictive model for live birth in these women. The main outcome studied was the cumulative live birth rate in the first or subsequent cycles. Developing a decision tree enabled a comprehensive study of clinical parameters and expected outcomes. RESULTS: The analysis included 158 women undergoing 753 preimplantation genetic testing cycles. The cumulative live birth rate was 37.342% (59/158). Decision tree analysis revealed that women aged ≤ 40.1 or women > 40.1 with one or more top-quality transferable embryos in their first cycle had the best chance for a live baby (56% and 41%, respectively). Those older than 40.1 without top-quality embryos and seven or fewer dominant follicles had no live births. A Kaplan-Meier curve showed that for autosomal dominant diseases, there was a negligible increase in live birth rate after three cycles, compared to six cycles in autosomal recessive inheritance. CONCLUSION: In older women, the chance of delivering after repeated cycles is higher in those with at least one top-quality unaffected embryo in their first preimplantation genetic testing cycle. Additional preimplantation genetic testing cycles after three in carriers of an autosomal dominant disorder and six in those with an autosomal recessive disorder should be considered prudently.