Relationship between a novel assessment scoring system in pre-implantation genetic testing for aneuploidy and clinical outcomes after embryo transfer: a single-center retrospective cohort study

Relationship between a novel assessment scoring system in pre-implantation genetic testing for aneuploidy and clinical outcomes after embryo transfer: a single-center retrospective cohort study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Relationship between a novel assessment scoring system in pre-implantation genetic testing for aneuploidy and clinical outcomes after embryo transfer: a single-center retrospective cohort study Yoshihisa Harada, Emi Fukunaga, Tomoyo Maeda, Hiyori Sasagawa, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4653808/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background The evaluation and interpretation of mosaicism in pre-implantation genetic testing for aneuploidy (PGT-A) can be complex, and no consensus or standardized criteria are available for its assessment. We investigated whether mosaicism as assessed by the proprietary Knowledge-based Aneuploidy Theoretical Score (KAT-Score) in PGT-A correlates with clinical pregnancy and live birth rates. Methods This retrospective cohort study was conducted in a single in vitro fertilization center between August 2020 and March 2023. A total of 124 single vitrified-warmed blastocyst transfer cycles were analyzed for clinical outcomes stratified by KAT-Score. We also analyzed the correlations between KAT-Score and clinical pregnancy or live birth rates. Results We found no significant difference in the KAT-Score between the pregnancy and non-pregnancy groups. However, the KAT-Score was significantly lower in the live birth group than in the non-live birth group. Moreover, the KAT-Score was a predictive indicator of live birth (area under the curve = 0.64, P < 0.01) but not of clinical pregnancy (area under the curve = 0.56, P = 0.1597). Multivariate logistic regression analysis, which included maternal age, previous embryo transfer cycles, endometrial thickness, and morphological grade as confounding factors, showed that lower KAT-Scores significantly correlated with the live birth rate (adjusted odds ratio: 0.83, 95% confidence interval: 0.70–0.99, P = 0.0403). No eventual live births were recorded in the group with a KAT-Score indicative of high chromosomal mosaic gain (KAT-Score ≥ 7). Conclusions This study suggests that the KAT-Score correlates with live birth but not with clinical pregnancy. The live birth rate differed according to type of mosaicism. PGT-A mosaicism embryo biopsy a novel scoring system blastocyst quality embryo selection Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION In assisted reproductive technology (ART), multiples have historically been a serious problem resulting from the transfer of two or more embryos into the uterus [ 1 , 2 ]. In modern ART clinics, the aim is to achieve a healthy live birth using single embryo transfer. In recent years, there have been dramatic advances in the culture media and environment, meaning that embryos cultured for 5–6 days in vitro will frequently provide multiple blastocysts for embryo transfer [ 3 ]. In this context, the issue arises of how best to rank embryos in terms of their quality to determine which have the highest likelihood of leading to a successful pregnancy. Pre-implantation genetic testing for aneuploidy (PGT-A) is a technique for selecting high-quality embryos by evaluating the number of chromosomes in multiple blastocysts [ 4 ]. From the perspective of embryo selection, a normal chromosomal constitution defined by PGT-A is the strongest predictive factor for a successful pregnancy. However, PGT-A also detects chromosomal mosaicism, in addition to euploidy and aneuploidy [ 5 , 6 ]. Excluding mosaic blastocysts may compromise treatment outcomes by reducing the number of embryos available for transfer. Chromosomal mosaicism involves a mixture of euploid and aneuploid cells and results in mitotic errors [ 7 ]. The evaluation and interpretation of mosaicism in embryos can be complex, and there is no consensus or standardized criteria to assess it. The incidence of mosaicism varies depending on the embryonic stage, with a reported incidence of 4–22% among embryos at the blastocyst stage depending on the clinic [ 8 , 9 ]. Earlier technologies for PGT-A had limitations in detecting mosaicism, but next-generation sequencing (NGS) has become widely used as a more accurate method for this purpose [ 10 ]. Viotti et al. retrospectively analyzed 1000 mosaic embryo transfers and found that the pregnancy and live birth rates differed in a stepwise manner depending on the type and extent of mosaicism [ 11 ]. This suggests that the outcome of mosaic embryo transfer may vary depending on the specific characteristics of the mosaicism. The Knowledge-based Aneuploidy Theoretical Score (KAT-Score) model was developed as a novel approach to assess mosaicism. In this model, the type and extent of mosaicism are represented by a numerical scoring system [ 11 ]. Viotti et al. focused on the implantation, ongoing pregnancy, and live birth rates when mosaic embryos were transferred and showed the influence of mosaicism status (i.e., number of chromosomes showing chromosomal or segmental mosaicism). Several reports have also addressed mosaic trisomy and monosomy, showing that mosaic trisomy has a higher miscarriage risk than mosaic monosomy [ 9 , 12 – 17 ]. Furthermore, mosaicism in multiple chromosomes has a lower ongoing implantation rate than mosaicism in one or two chromosomes [ 18 – 20 ]. In case of multiple embryos with the same mosaicism status, the mosaic rate is also useful and is added as a decimal point to the risk score to support judging the order of transfer. Numerical PGT-A of embryos may also be useful for comparative evaluation of embryos. Maximizing the clinical outcome of PGT-A is crucial to increasing the chances of implantation. The current study assessed the correlation between KAT-Score and pregnancy outcomes by scoring PGT-A cycle embryos. The purpose of this study was to investigate whether the KAT-Score, as an indicator of the likelihood of mosaicism, can be used as a predictor of pregnancy outcomes. MATERIALS AND METHODS Patients and study design A total of 124 cycles of single vitrified-warmed blastocyst transfer performed between October 2020 and March 2023 in a single in vitro fertilization (IVF) center (Kinutani Women’s Clinic, Hiroshima, Japan) were included in the analysis. All transferred blastocysts from the PGT-A cycles were evaluated using the KAT-Score (v2, Varinos Inc, Tokyo, Japan). However, cases with pre-implantation genetic testing for structural chromosome rearrangements or multiple embryo transfer cycles including two-step embryo transfer were excluded from the analysis (Fig. 1 ). The main outcome measures were clinical pregnancy (presence or absence of gestational sac) and live birth (presence or absence of live birth). This study was approved by the Ethics Committee of Kinutani Women’s Clinic, Hiroshima, Japan. Informed consent was obtained from all patients included in this study. Ovarian stimulation and oocyte retrieval Patients were treated with gonadotropin hormone-releasing hormone (GnRH) agonist using a short or long protocol, following a progestin-primed ovarian stimulation protocol, in accordance with each patient’s ovarian response and medical history of IVF treatment. Serum estradiol levels were monitored and oocyte growth evaluated by transvaginal ultrasound. In some cycles, the minimal stimulation protocol and natural cycle were used. When a follicle reached a mean diameter ≥ 18 mm, human chorionic gonadotropin (Aska Pharmaceutical, Tokyo, Japan), 250 µg of choriogonadotropin alpha (Ovidrel; Merck Serono, Darmstadt, Germany), or GnRH agonist was administered. Transvaginal oocyte retrieval was performed 35–37 h after administration. Fertilization procedure, embryo culture, time-lapse monitoring Prior to conventional IVF, collected cumulus–oocyte complexes were cultured for approximately 4 h in G-IVF medium (Vitrolife, Gothenburg, Sweden) at 37°C under an atmosphere of 6% CO 2 , 5% O 2 , and 89% N 2 . Cumulus–oocyte complexes were denuded by pipetting using 80 IU/ml hyaluronidase (Irvine Scientific, Santa Ana, CA, USA). The maturity of denuded oocytes was assessed by visualization of the first polar body. Only metaphase II oocytes were used for intracytoplasmic sperm injection (ICSI). Embryos fertilized by ICSI or conventional IVF were cultured in DNP dishes (Dai Nippon Printing Co., Ltd., Tokyo, Japan) with SAGE 1 Step (Origio, Målöv, Denmark) at 37°C under an atmosphere 6% CO 2 , 5% O 2 , and 89% N 2 for 5–7 days and evaluated for development to the blastocyst stage. The oocytes were cultured individually in a time-lapse system (CCM-iBIS; Astec, Fukuoka, Japan). A total of 1–25 embryos were placed in a 100 µl drop of medium, and images of the embryos were recorded automatically at 15-min intervals. Blastocyst biopsy protocols and NGS analysis Trophectoderm (TE) biopsy was performed on expanded blastocysts on days 5–7 regardless of morphological grade. First, artificial shrinkage was performed using a laser system (Zilos-TK™; Hamilton Thorn Bioscience Inc., Beverly, MA, USA) to create a space between the zona pellucida and TE cells. While holding the blastocyst in an inner cell mass (ICM) at a position of approximately 8 to 9 o’clock, serial laser pulses were used to create a small hole (~ 10 µm) in the zona pellucida at a position of approximately 3 to 4 o’clock. The biopsy pipette was pushed into the hole created by the laser to aspirate the TE cells. Five to 10 TE cells were aspirated into the biopsy pipette and collected by flicking the holding pipette and biopsy pipette with the aid of several laser pulses. Biopsied samples were processed for whole-genome amplification and NGS at Varinos Laboratory. The VeriSeq PGS kit (Illumina K.K., San Diego, CA, USA) on the MiSeq system (Illumina K.K., San Diego, CA, USA) was used in 24-sample runs following the manufacturer’s protocol. The copy number of each sample was analyzed using BluFuse Multi Software (Illumina K.K., San Diego, CA, USA). A molecular karyotype profile consistent with mosaicism was detected when a whole chromosome or chromosomal segment resulted in intermediate copy number levels of 20–80% between whole numbers according to the guidelines of the Preimplantation Genetic Diagnosis International Society [ 21 ]. Vitrification and warming of blastocysts and embryo transfer Blastocyst vitrification was performed using the method reported by Hiraoka et al. [ 22 ] within 1 h after embryo biopsy. The blastocysts were placed in equilibration solution containing 7.5% (v/v) ethylene glycol (Sigma-Aldrich, St. Louis, MO, USA) and 7.5% (v/v) dimethyl sulfoxide (Nacalai Tesque Inc., Kyoto, Japan) in mHTF (Irvine Scientific, Santa Ana, CA, USA) supplemented with 20% (v/v) serum protein substitute (Origio, Målöv, Denmark) at 37°C. Blastocysts were then transferred into vitrification solution containing 15% (v/v) ethylene glycol, 15% (v/v) dimethyl sulfoxide, and 0.5 M sucrose (Sigma-Aldrich, St. Louis, MO, USA) in mHTF supplemented with 20% (v/v) serum protein substitute for 1 min at 37°C. The blastocysts were loaded onto Cryotops (Kitazato Corporation, Fujinomiya, Japan) at a minimum volume and immediately immersed in liquid nitrogen at − 196°C. For the warming protocol, the tip of the Cryotop was immersed directly in 1.0 M sucrose solution for 1 min at 37°C. The blastocyst was transferred to 0.5 M sucrose solution for 3 min and washed twice in mHTF supplemented with 20% serum protein substitute for 5 min at 37°C. Prior to embryo transfer, the warmed blastocyst was cultured for approximately 3–4 h in EmbryoGlue (Vitrolife, Gothenburg, Sweden). Luteal phase support was started using intravaginal micronized progesterone or natural cycles. Embryo transfers were performed under ultrasound guidance using a soft catheter (Origio, Målöv, Denmark). KAT-Score-based scoring of embryos Embryos were scored 0 (complete euploidy) to 10 (complete aneuploidy) using the KAT-Score v2 (Table 1 ). First, a score from 1 to 8 was assigned for each type of mosaicism and a score of 9 for segmental aneuploidy. Next, a score of 0.2–0.8 was added to the total depending on the level of mosaicism in the range of 20–80%. When mosaicism was present on multiple chromosomes, the score of the chromosome with the highest level of mosaicism was assigned as the mosaicism score. Figure 2 shows an example of the scoring. Table 1 Knowledge-based Aneuploidy Theoretical Score (KAT-Score) for pre-implantation genetic testing. Mosaic type Score Aneuploid 10 Segmental aneuploid 9 High chromosomal mosaic complex 8 High chromosomal mosaic gain 7 High chromosomal mosaic loss 6 Low chromosomal mosaic complex 5 Low chromosomal mosaic gain 4 Low chromosomal mosaic loss 3 High segmental mosaic gain/loss 2 Low segmental mosaic gain/loss 1 Euploid 0 Mosaic level Score 80% 0.8 70% 0.7 60% 0.6 50% 0.5 40% 0.4 30% 0.3 20% 0.2 Note : Low=low-level mosaicism >20% to <50% , High=high-level mosaicism ≥50% to <80% , Complex=mosaicism of monosomies and trisomies found on multiple chromosomes, Gain=trisomy , Loss=monosomy. Statistical analysis Statistical analysis was performed using JMP 14.0 software (SAS Institute, Inc., Cary, NC, USA) and GraphPad PRISM 6.03 software (GraphPad Inc., San Diego, CA, USA). Patient characteristics were presented as means and standard deviation (SD). The KAT-Score data were not normally distributed, so the Wilcoxon rank-sum test was used to compare the scores as non-parametric continuous variables. We calculated the area under the receiver operating characteristic curve (AUC) of the KAT-Score for predicting clinical pregnancy and ongoing pregnancy. Adjusted odds ratios (aORs) were calculated using multivariate logistic regression analysis. Differences were considered significant at P < 0.05. RESULTS Participant characteristics and KAT-Score distribution Table 2 shows the participant characteristics for embryo transfer. A total of 124 cycles of single vitrified-warmed blastocyst transfer were performed using euploid and mosaic embryos, mostly under hormone replacement cycles. Of these 124 cycles, 60 clinical pregnancies were achieved, 50 of which resulted in live births. PGT-A for the 124 cycles was classified by the KAT-Score before embryo transfer. The KAT-Scores for the 124 blastocysts ranged from 0 to 9 and were distributed as follows: 0, n = 51; 1–2, n = 22; 2, n = 1; 3–4, n = 6; 4–5, n = 19; 5–6, n = 8; 6–7, n = 8; 7–8, n = 7; and 9, n = 2. Figure 3 shows the clinical outcomes after single vitrified-warmed blastocyst transfer stratified by KAT-Score. Clinical pregnancy was confirmed for KAT-Scores of 1–7. There were no transfers with a KAT-Score of 8. One case with a KAT-Score of 2 (high segmental mosaic gain/loss) led to pregnancy but did not result in live birth. No cases with a KAT-Score of 9 (segmental aneuploidy) led to pregnancy. Clinical pregnancies were confirmed even in mosaic embryos with a KAT-Score of 7 (high chromosomal mosaic gain). However, there were no live births among these cases. Table 2 Characteristics of participants for embryo transfer cycles. Characteristic of embryo transfer cycles No. of patients 84 No. of transfer cycles 124 Maternal age at ET (years) 38.8 ± 4.00 Maternal age at OR (years) 38.0 ± 3.87 Previous embryo transfer (cycles) 5.15 ± 3.04 Endometrial thickness (mm) 10.5 ± 1.88 Hormone replacement cycles 108 (87.1) Natural cycles 15 (12.1) Aromatase inhibitor cycles 1 (0.8) Clinical pregnancies 60 (48.4) Miscarriages 10 (8.1) Live births 50 (40.3) Day of blastocyst Day 5 74 (59.7) Day 6 45 (36.3) Day 7 5 (4.0) Morphology Good (AA/AB/BA) 72 (58.1) Fair (BB) 36 (29.0) Poor (BC/CB/AC/CC) 16 (12.9) KAT-Score distribution Euploid (0) 51 (41.2) Segmental mosaic (1–2) 23 (18.5) Low level mosaic (3–5) 33 (26.6) High level mosaic (6–8) 15 (12.1) Segmental aneuploid (9) 2 (1.6) Note : Values are given as n (%) or mean ± SD unless otherwise noted. ET=embryo transfer, OR=oocyte retrieval. Correlations between KAT-Score and clinical pregnancy/live birth Continuous variables were compared between two groups using the Wilcoxon signed-rank test (Fig. 4 ). No significant difference in the mean KAT-Score was observed between the clinical pregnancy and non-clinical pregnancy groups (1.99 ± 2.59 vs. 2.67 ± 2.76; P = 0.2008; Fig. 4 a). The KAT-Score was significantly lower in the live birth group than in the non-live birth group (1.37 ± 2.07 vs. 3.00 ± 2.87; P < 0.01; Fig. 4 b). Figure 5 shows the results of univariate logistic regression analyses for the probabilities of clinical pregnancy and live birth. The utility of the KAT-Score as a predictive measure of clinical pregnancy or live birth was evaluated using the AUC. The KAT-Score of embryos was a predictive indicator of live birth (AUC = 0.64, P < 0.01) but not of clinical pregnancy (AUC = 0.56, P = 0.067; Fig. 5 a, b). Table 3 shows the results of multivariate logistic regression analyses, which included maternal age, previous ET cycles, endometrial thickness, and morphological grade as confounding factors, for the probabilities of clinical pregnancy and live birth. The number of previous ET cycles significantly correlated with a negative probability of clinical pregnancy (aOR 0.82, 95% CI 0.69–0.96, P < 0.01). The morphological grade of the blastocyst significantly correlated with a positive probability of clinical pregnancy (aOR 4.26, 95% CI 1.31–16.8, P < 0.05). On the other hand, the probability of live birth negatively correlated with the number of previous ET cycles (aOR 0.79, 95% CI 0.66–0.94, P < 0.01) and KAT-Score (aOR 0.83, 95% CI 0.70–0.99, P < 0.05). Table 3 Multivariate logistic regression analysis of several factors associated with clinical pregnancy and live birth in patients who underwent PGT-A. Clinical pregnancy Live birth Factor aOR 95% CI P value aOR 95% CI P value Maternal age 0.94 0.85–1.05 0.2964 0.95 0.85–1.06 0.3563 No. of previous ET cycles 0.82 0.69–0.96 0.0077 0.79 0.66–0.94 0.0048 Endometrial thickness 1.15 0.94–1.42 0.1736 1.19 0.96–1.48 0.1012 Morphological grade (≥ 3BB) 4.26 1.31–16.8 0.0246 2.84 0.70–11.4 0.0676 KAT-Score 0.98 0.83–1.14 0.7576 0.83 0.70–0.99 0.0403 underwent PGT-A. Note : aOR=adjusted odds ratio, CI=confidence interval, ET=embryo transfer. Neonatal outcomes stratified by KAT-Score Table 4 shows a comparison of neonatal outcomes among the KAT-Score groups after single vitrified-warmed blastocyst transfer. The neonatal outcomes were divided into three groups according to the KAT-Score: euploid, 0; low KAT-Score, 1–5; high KAT-Score, 6–9. We found no significant difference in the clinical pregnancy rates between the three groups. Miscarriage rates were significantly increased in the low KAT-Score and high KAT-Score groups [0% (0/27) vs. 33.3% (5/25) vs. 62.5% (5/8); P < 0.05]. The live birth rate among embryos with a high KAT-Score was 17.6% (3/17), which was significantly lower than that of embryos with complete euploidy [52.9% (27/51)]. There were no significant differences in β-hCG and maternal age in the three groups. Moreover, we did not find significant differences in the mean birth weight among the three groups (3117 ± 373.5 vs. 3073 ± 502.0 vs. 2621 ± 730.9 g, respectively). Table 4 Neonatal outcome stratified by Knowledge-based Aneuploidy Theoretical Score (KAT-Score). KAT-Score group 0 1–5 6–9 P value Transfers 51 (41.1) 56 (45.2) 17 (13.7) - Previous ET cycles 4.53 ± 2.74 5.36 ± 2.73 6.35 ± 4.39 N.S.** Maternal age at ET (years) 37.3 ± 4.11 38.4 ± 3.90 42.0 ± 2.00 N.S.** Endometrial thickness (mm) 10.4 ± 1.84 10.5 ± 1.94 10.3 ± 1.88 N.S.** Good morphology blastocyst rate (≥ 3BB) 90.2 (46/51) 85.7 (48/56) 82.4 (14/17) N.S* Clinical pregnancy rate 52.9 (27/51) 44.6 (25/56) 47.1 (8/17) N.S.* Miscarriage rate 0 (0/27) a 33.3 (5/25) b 62.5 (5/8) c < 0.05* Live birth rate 52.9 (27/51) a 35.7 (20/56) ab 17.6 (3/17) b < 0.01* Singleton 27 20 3 - β-hCG (mIU/ml) 539.3 ± 399.9 492.1 ± 353.2 349.5 ± 197.9 N.S.** Birth weight (g) 3117 ± 373.5 3073 ± 502.0 2621 ± 730.9 N.S.** Male infant 13 (48.1) 10 (50.0) 0 (0.0) - Female infant 14 (51.9) 10 (50.0) 3 (100.0) - Note : Values are given as n (%) or mean ± SD unless otherwise noted. ET=embryo transfer, SD=standard deviation, N.S.=not significant. *Fisher’s exact test, **one-way ANOVA and Tukey–Kramer test. a, b, c Different superscripts indicate a significant difference ( P < 0.05) DISCUSSION This study examined whether the KAT-Score correlates with pregnancy prognosis. PGT-A is used to assess embryonic aneuploidy to increase pregnancy rates, decrease abortion rates, and prioritize the highest quality embryos for transfer. In this study, the embryos from 124 cycles of PGT-A, including mosaic embryos, resulted in the birth of 50 healthy children. The KAT-Score is a scoring method originally developed by Varinos Inc. based on a report by Viotti et al. that allows scoring according to the type and extent of mosaicism [ 11 ]. Statistical analyses clarified that this scoring is associated with type of mosaicism and the rate of live birth, but not with clinical pregnancy. Multivariate logistic regression analysis, which included various confounding factors, showed that the KAT-Score and previous ET cycles correlated with the live birth rate. The possibility of viable embryos being discarded because of concerns about mosaicism is one of the challenges currently facing PGT-A. The transfer of embryos classified as mosaic by PGT-A was reported for the first time in 2015 and is now routinely performed in clinical practice [ 23 ]. The fertility potential of mosaic embryos remains controversial [ 13 , 24 ]. When analyzing the mosaicism status using techniques such as amplification or NGS, noise or artifacts may be indistinguishable from true mosaicism [ 25 ]. However, several studies have suggested that embryos classified as mosaic have reduced reproductive potential compared with embryos classified as euploid [ 9 , 26 , 27 ]. In these previous studies, mosaicism was mostly diagnosed on the basis of its presence or absence. The general definition of a mosaic embryo does not involve stringent thresholds (mosaicism level: 20–80%), but it is unclear which type or level of mosaicism is suitable for implantation [ 21 ]. Girardi et al. found that, to maximize the accuracy of PGT-A, single TE biopsy results should be reported according to the euploid/aneuploid classification based on a single cut-off of 50% [ 28 ]. The current study divided the neonatal outcomes into three groups based on a mosaicism level of 50% and compared them. Although there was a slight decrease in the live birth rate from the euploid group (KAT-Score 0) to the group with low-level mosaicism (KAT-Score 1–5), no significant difference was observed. A lower level of mosaicism is traced in subsequent TE biopsy, and many embryos are uniformly euploid [ 29 ]. The incidence of chromosomal mosaicism has also raised concerns about the possibility that a considerable number of PGT-A results are false-positive errors [ 30 ]. If the level of mosaicism is < 50%, the embryo may have the same developmental potential as a euploid embryo. The live birth rate was significantly decreased in the group with a KAT-Score of 6–9, 17.6% compared with 52.9% in the group with a KAT-Score of 0. Interestingly, no live births were recorded in the group with a KAT-Score ≥ 7 (high chromosomal mosaic gain). These results may differ depending on whether the case involves monosomy or trisomy. The results of TE biopsies are not always concordant with the chromosomal constitution of the ICM in mosaic embryos [ 31 ]. Confirming mosaicism ≥ 50% and trisomy mosaicism in TE biopsy suggests that the chromosomal composition of the majority of these embryos shares a uniform aneuploid configuration [ 32 ]. A previous study showed that, when trisomy abnormalities are present in the ICM, implantation is possible, but miscarriage occurs in most cases [ 33 ]. Although it is clear that mosaic embryos have significant developmental potential, those with high-level trisomy are not suitable for transfer. Currently, TE biopsies are performed at the blastocyst stage because the incidence of mosaicism decreases between day 3 (cleavage stage) and day 5 (blastocyst stage) [ 34 ] due to a self-corrective mechanism during the division process [ 35 ]. In human embryos, aneuploid cells are sequestered from the ICM during the progression from day 3 to day 5 and are partially sequestered in the TE surrounding the blastocyst [ 36 ]. Although neonatal outcome data from transferred mosaic embryos need to be comprehensively analyzed, 23 newborns from the mosaic group in this study were born healthy. To use the KAT-Score as a tool that allows mosaic embryos to be ranked for their suitability for implantation and increase the likelihood of a positive clinical outcome, it is necessary to develop an algorithm that includes the morphological and kinetic characteristics of the embryo. Most of the blastocysts used for transfer in this study were day 5 blastocysts with good morphology (≥ BB). Blastocysts that reach this stage later or have poor morphology are more likely to be aneuploid and, therefore, were not selected for transfer. Currently, clear chromosomal abnormalities, such as mosaicism and chromosomal imbalances, can only be detected by PGT-A, but the live birth rate of embryos transferred after PGT-A was limited approximately 40% in this study. In ART, it is vital to develop a large number of embryos with high developmental competence. Further research is required to gain a more complete understanding of whether the KAT-Score is associated with clinical pregnancy and live birth. CONCLUSIONS In conclusion, this study suggests that the KAT-Score can be assessed during PGT-A as an indicator to predict live birth. Embryos with high segmental mosaic gain/loss (KAT-Score 2), segmental aneuploidy (KAT-Score 9), and high chromosomal mosaic gain (KAT-Score 7) are not suitable for transfer. The scoring of 124 embryo transfers by the KAT-Score provides statistically valid evidence for the use of this tool to rank mosaic embryos in infertility clinics. Abbreviations KAT-Score Knowledge-based Aneuploidy Theoretical Score ART assisted reproductive technology PGT-A pre-implantation genetic testing for aneuploidy NGS next-generation sequencing IVF in vitro fertilization GnRH gonadotropin hormone-releasing hormone C-IVF conventional IVF ICSI intracytoplasmic sperm injection TE trophectoderm ICM inner cell mass aOR adjusted odds ratio AUC area under the curve SD standard deviation. Declarations ACKNOWLEDGEMENTS The authors thank the Kinutani Women’s Clinic embryologist team for acquiring data and the participating couples and gynecologists at the infertility clinic. We also thank Varinos Inc. ( https://varinos.com ) CEO Yoshiyuki Sakuraba for help with this paper and the development of the KAT-Score. AUTHORS’ CONTRIBUTIONS KA invented the KAT-Score model. YH interpreted the data collection and analysis of data and participated in designing the study. EF, TM, HS, MI, RS, SO, and MK assisted in acquiring and interpreting data. YS, ST, and MS revised the manuscript. All authors have read and approved the final manuscript. FUNDING No funding was received related to this study. AVAILABILITY OF DATA AND MATERIALS The dataset used and/or analyzed in this manuscript will be available from the corresponding author upon reasonable request. DISCLARATIONS Conflict of interest: The authors declare no conflict of interest. Human rights statements and informed consent: All procedures were performed in accordance with the ethical standards of the institutional ethical committee and with the Helsinki Declaration of 1964 and its later amendments. Informed consent was obtained from all patients in this study, and the study design was approved by the ethics committee of Kinutani Women’s Clinic, Hiroshima, Japan. Animal studies: This article does not describe any study involving animals performed by any of the authors. Declarations of interest: none. 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Culture and transfer of human blastocysts increases implantation rates and reduces the need for multiple embryo transfers. Fertil Steril. 1998;69(1):84–8. https://doi.org/10.1016/s0015-0282(97)00438-x . Scott RT, Upham KM, Forman EJ, Hong KH, Scott KL, Taylor D, Tao X, Treff R. Blastocyst biopsy with comprehensive chromosome screening and fresh embryo transfer siginificantly increases in vitro fertilization implantation and delivery rates: a randomized controlled trial. Fertil Steril. 2013;100:697–703. https://doi.org/10.1016/j.fertnstert.2013.04.035 . Rodrigo L, Clemente-Ciscar M, Campas-Galindo I, Peinado V, Simon C, Rubio C. Characteristics of the IVF cycle that contribute to the incidence of mosaicism. Gene (Basel). 2020;11(10):1151. https://doi.org/10.3390/genes11101151 . Vera-Rodriguez M, Rubio C. Assessing the true incidence of mosaicism in preimplantation embryos. Fertil Steril. 2017;107(5):1107–12. https://doi.org/10.1016/j.fertnstert.2017.03.019 . 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Fertil Steril. 2018;109(4):627–32. https://doi.org/10.1016/j.fertnstert.2017.12.017 . Viotti M, Victor AR, Barnes FL, Zouves CG, Besser AG, Grifo JA, Cheng EH, Lee MS, Horcajadas JA, Corti L, Fiorentino F, Spinella F, Minasi MG, Greco E, Munne S. Using outcome data from one thousand mosaic embryo tranfers to formulate an embryo ranking system for clinical use. Fertil Steril. 2021;115(5):1212–24. https://doi.org/10.1016/j.fertnstert.2020.11.041 . PGDIS Newsletter. (2016) PGDIS Position Statement on Chromosome Mosacism and Preimplantation Aneuploidy Testing at the Blastocyst Stage. Spinella F, Fiorentino F, Biricik A, Bono S, Ruberti A, Cotroneo E, Baldi M, Cursio E, Minasi MG, Greco E. Extent of chromosomal mosaicism influences the clinical outcome of in vitro fertilization treatments. Fertil Steril. 2018;109:77–83. https://doi.org/10.1016/j.fertnstert.2017.09.025 . Zore T, Kroener LL, Wang C, Liu L, Buyalos R, Hubert G, Shamonki M. Transfer of embryos with segmental mosaicism is associated with a significant reduction in live birth rate. Fertil Steril. 2019;111(1):69–76. https://doi.org/10.1016/j.fertnstert.2018.08.057 . Gavin SE, Chatzicharalampous C, Puscheck E. Reflections on preimplantation genetic testing for aneuploidy and mosaicism: how did we get here, and what does it mean clinically? Fertil Steril. 2019;111(1):45–7. https://doi.org/10.1016/j.fertinstert.2018.11.006 . Willian HK, Ralph SP, Melissa KM, Elias MD. The role of genetic analysis of products conception and preimplantation genetic testing in the management of early pregnancy loss. Reprod Biomed Online. 2023;103738. https://doi.org/10.1016/j.rbmo.2023.103738 . Lai HH, Chuang TH, Wong LK, Lee MJ, Hsieh CL, Wang HL, Chen SU. Identification of mosaic and segmental aneuploidies by next-generation sequencing in preimplantation genetic screening can improve clinical outcomes compared to array-comparative genomic hybridization. Mol Cytogen. 2017;10:14. https://doi.org/10.1186/s13039-017-0315-7 . Fragouli E, Alfarawati S, Spath K, Babariya D, Tarozzi N, Borini A, Wells D. Analysis of implantation and ongoing pregnancy rates following the transfer of mosaic diploid-aneuploid blastocysts. Hum Gen. 2017;136(7):805–19. https://doi.org/10.1007/s00439-017-1797-4 . Munne S, Blazek J, Large M, Martinez-Ortiz PA, Nisson H, Liu E, Tarozzi N, Borini A, Becker A, Zhang J, Maxwell S, Grifo J, Babariya D, Wells D, Fragouki E. Detailed investigation into the cytogentic constitution and pregnancy outcome of replacing mosaic blastocysts detected with the use of high-resolution next-generation sequencing. Fertil Steril. 2017;108(1):62–71. https://doi.org/10.1016/j.fertnstert.2017.05.002 . Nakhuda G, Jing C, Butler R, Guimond C, Hitkari J, Taylor E, Tallon N, Yuzpe A. Frequencies of chromosome-specific mosaicism in trophectoderm biopsied detected by next-generation sequencing. Fertil Steril. 2018;109(5):857–65. https://doi.org/10.1016/j.fertnstert.2018.01.001 . Leigh D, Cram DS, Rechitsky S, Handyside A, Wells D, Munne S, Kahraman S, Grifo J, Katz-Jaffe M, Rubio C, Viotti M, Forman E, Xu E, Gordon T, Madjunkova S, Qiao J, Chen ZJ, Harton G, Gianoroli L, Simon C, Scott R, Simpson JL, Luliev A. PDGIS position statement on the transfer of mosaic embryos 2021. Reprod Biomed Online. 2022;45:19–25. https://doi.org/10.1016/j.rbmo.2022.03.013 . Hiraoka K, Hiraoka K, Kinutani M, Kinutani K. Blastocoele collapse by micropipetting prior to vitrification gives excellent survival and pregnancy outcomes for human day 5 and 6 expanded blastoctsts. Hum Reprod. 2004;19(12):2884–8. https://doi.org/10.1093/humrep/deh504 . Greco E, Minasi MG, Fiorentino F. Healthy babies after intrauterine transfer of mosaic aneuploid blastocysts. N Engl J Med. 2015;373:2089–90. https://doi.org/10.1056/NEJMc1500421 . Kushnir VA, Darmon SK, Barad DH, Gleicher N. Degree of mosaicism in trophectoderm does not predict pregnancy potential: a corrected analysis of pregnancy outcomes following tranfer of mosaic embryos. Reprod Biol Endocrinol. 2018;16:6. https://doi.org/10.1186/s12958-018-0322-5 . Thornhill AR, deDie-Smulders CE, Geraedts JP, Harper JC, Harton GL, Lavery SA, Moutou C, Robinson MD, Schmutzler AG, Scriven PN, Sermon KD, Wilton L. ESHRE PGD Consortium Best practice guidelines for clinical preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS). Hum Reprod. 2005;20:35–48. https://doi.org/10.1093/humrep/deh579 . Iwasa T, Kawahara A, Takeshita T, Taniguchi Y, Mikami M, Irahara M. Preimplantation genetic testing aneuploidy and chrmosomal structual rearrangement: A summary of a nationwide study by the Japan Society of Obsteristics and Gynecology. Reprod Med Biol. 2023;22(1):e12518. https://doi.org/10.1002/rmb2.12518 . Munne S, Blazek J, Large M, Martinez-Ortiz PA, Nisson H, Liu E, Tarizzi N, Borini A, Becker A, Zhang J, Maxwell S, Grifo J, Babariya D, Wells D, Fragouli E. Detailed investigation into cytogenetic constitution and pregnancy outcome of replacing mosaic blastocysts detected with th use of high-resolution next-generation sequencing. Fertil Steril. 2017;108(1):62–e718. https://doi.org/10.1016/j.fertnstert.2017.05.002 . Girardi L, Figliuzzi M, Poli M, Serdarogullari M, Patassini C, Caroselli S, Pergher I, Cogo F, Coban O, Boynukalin FK, Bahceci M, Navarro R, Rubio C, Findikli N, Simon C, Capalbo A. The use of copy number loads to designate mosaicism in blastocyst stage PGT-A cycles: fewer is better. Hum Reprod. 2023;38(5):982–91. https://doi.org/10.1093/humrep/dead049 . Martin D, Xu J, Treff NR. Preimplantation genetic testing for aneuploidy: A review of published blastocyst analysis concordance data. Prenat Diagn. 2020;41(5):545–53. https://doi.org/10.1002/pd.5828 . Wu L, Jin L, Chen W, Liu JM, Hu J, Yu Q, Ren KL, Huang B, He H. The true incidence of chromosomal mosaicism after preimplantation genetic testing much lower than that indicated by trophectoderm biopsy. Hum Reprod. 2021;36(6):1691–701. https://doi.org/10.1093/humrep/deab064 . Popovic M, Dhaenens L, Boel A, Menten B, Heindryckk B. Chromosomal mosaicism in human blastocysts: The ultimate diagnostic dilemma. Hum Reprod Updat. 2020;26:313–34. https://doi.org/10.1093/humupd/dmz050 . Capalbo A, Poli M, Rienzi L, Girardi L, Patassini C, Fabiani M, Cimadomo D, Benini F, Farcomeni A, Cuzzi J, et al. Mosaic human preimplantation embryos and their developmental potential in a prospective, non-selection clinical trial. Am J Hum Genet. 2021;108:2238–47. https://doi.org/10.1016/j.ajhg.2021.11.002 . Grati FR, Gallazzi G, Branca L, Maggi F, Simoni G, Yaron Y. An evidence-based scoring system for prioritizing mosaic aneuploid embryos following preimplatation genetic screening. Reprod Biomed Online. 2018;36(4):442–9. https://doi.org/10.1016/j.rbmo.2018.01.005 . Harton GL, Munne S, Surrey M, Grifo J, Kaplan B, MuCulloh DH, Griffin DK, Wells D, Group PP, PGD Practitioner Group. Diminished effect of maternal age on implantation after preimplantation genetic diagnosis with array comparative genomic hybridization. Fertil Steril. 2013;100:1695–703. https://doi.org/10.1016/j.fertnstert.2013.07.2002 . Coticchio G, Barrie A, Lagalla C, Borini A, Fishel S, Griffin D, Campbell A. Plasticity of the human preimplantation embryo: developmental dogmas, variations on themes and self-correction. Hum Reprod Update. 2021;27(5):848–65. https://doi.org/10.1093/humupd/dmab016 . Griffin DK, Brezina PR, Tobler K, Zhao Y, Silvestri G, Mccoy RC, Anchan R, Benner A, Cutting GR, Kearns WG. The human embryonic genome is karyotypically complex, with chromosomally abnormal cells preferentially located away from the developing fetus. Hum Reprod. 2023;38(1):180–8. https://doi.org/10.1093/humrep/deac238 . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4653808","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":322164326,"identity":"f9598b3d-acd4-4461-9e00-ee5468cec516","order_by":0,"name":"Yoshihisa Harada","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIiWNgGAWjYBACxgYYi5mx8QGQ4uEjWgsPO3OzAYhmI9o6Hn72NgkQg6AW5vbeg495ahjs9jMztlV+zbGTYWNgfvjoBj6H9ZxLNuY5xpDcA9RyW3ZbMtBhbMbGOfi0zMgxk5zBxpDMA9IiuY0ZqIWHTRqvlvlvgFr+QbQUS26rJ0LLDB4ziY9tDHYgLYwftx0mQktPjrHBxz6JBJ7DjM3SjNuO87AxE/CLYfsZwwcJ32zs2fuPP/z4c1u1PT9788PHeLU0gCmJRBDNzANiM+NRDgLyUNoe7MofBFSPglEwCkbByAQAK98+b6pWxt8AAAAASUVORK5CYII=","orcid":"","institution":"Kinutani Women’s Clinic","correspondingAuthor":true,"prefix":"","firstName":"Yoshihisa","middleName":"","lastName":"Harada","suffix":""},{"id":322164329,"identity":"4db3c8d5-c9e4-4880-9911-c92be02442c4","order_by":1,"name":"Emi Fukunaga","email":"","orcid":"","institution":"Kinutani Women’s Clinic","correspondingAuthor":false,"prefix":"","firstName":"Emi","middleName":"","lastName":"Fukunaga","suffix":""},{"id":322164330,"identity":"2f36d24f-15ee-4318-a214-266966b4f393","order_by":2,"name":"Tomoyo Maeda","email":"","orcid":"","institution":"Kinutani Women’s Clinic","correspondingAuthor":false,"prefix":"","firstName":"Tomoyo","middleName":"","lastName":"Maeda","suffix":""},{"id":322164332,"identity":"03d3ef13-1bc5-47fd-92d9-eeb9fe7a1cd3","order_by":3,"name":"Hiyori Sasagawa","email":"","orcid":"","institution":"Kinutani Women’s Clinic","correspondingAuthor":false,"prefix":"","firstName":"Hiyori","middleName":"","lastName":"Sasagawa","suffix":""},{"id":322164334,"identity":"fbd95d59-e37e-4a13-ad7f-f2155c894992","order_by":4,"name":"Maki Ikeda","email":"","orcid":"","institution":"Kinutani Women’s Clinic","correspondingAuthor":false,"prefix":"","firstName":"Maki","middleName":"","lastName":"Ikeda","suffix":""},{"id":322164335,"identity":"4c0b7d1c-fd55-48ca-9d8a-02f4dec64706","order_by":5,"name":"Reiko Shiba","email":"","orcid":"","institution":"Kinutani Women’s Clinic","correspondingAuthor":false,"prefix":"","firstName":"Reiko","middleName":"","lastName":"Shiba","suffix":""},{"id":322164336,"identity":"60cf693f-55d9-4c5a-8b56-1a7f1b737109","order_by":6,"name":"Shinichiro Okano","email":"","orcid":"","institution":"Kinutani Women’s Clinic","correspondingAuthor":false,"prefix":"","firstName":"Shinichiro","middleName":"","lastName":"Okano","suffix":""},{"id":322164337,"identity":"34c302a9-b336-4a5a-bda6-b29087e16873","order_by":7,"name":"Masayuki Kinutani","email":"","orcid":"","institution":"Kinutani Women’s Clinic","correspondingAuthor":false,"prefix":"","firstName":"Masayuki","middleName":"","lastName":"Kinutani","suffix":""},{"id":322164338,"identity":"1596e168-7927-4baf-8c09-7f3cc1eb2779","order_by":8,"name":"Suguru E. Tanaka","email":"","orcid":"","institution":"Varinos Inc","correspondingAuthor":false,"prefix":"","firstName":"Suguru","middleName":"E.","lastName":"Tanaka","suffix":""},{"id":322164339,"identity":"7e23ef91-6ce1-4423-85ea-f1df7d98405a","order_by":9,"name":"Miho Shimada","email":"","orcid":"","institution":"Varinos Inc","correspondingAuthor":false,"prefix":"","firstName":"Miho","middleName":"","lastName":"Shimada","suffix":""},{"id":322164340,"identity":"245b905e-6f7f-4443-9e13-af8a7270cced","order_by":10,"name":"Kyota Ashikawa","email":"","orcid":"","institution":"Varinos Inc","correspondingAuthor":false,"prefix":"","firstName":"Kyota","middleName":"","lastName":"Ashikawa","suffix":""},{"id":322164341,"identity":"faa35dc3-94d0-4bfe-b4ee-07689480a4cc","order_by":11,"name":"Yoshiyuki Sakuraba","email":"","orcid":"","institution":"Varinos Inc","correspondingAuthor":false,"prefix":"","firstName":"Yoshiyuki","middleName":"","lastName":"Sakuraba","suffix":""}],"badges":[],"createdAt":"2024-06-28 09:51:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4653808/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4653808/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60816514,"identity":"5d430dd8-d00a-4876-aeda-29553fb95236","added_by":"auto","created_at":"2024-07-22 12:01:33","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":291521,"visible":true,"origin":"","legend":"\u003cp\u003eFlowchart of study inclusion. ET = embryo transfer, PGT-SR = preimplantation genetic testing for structural rearrangements, KAT-Score = Knowledge-based Aneuploidy Theoretical Score\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4653808/v1/2d111b5d44a49f8922c2d49a.jpg"},{"id":60816519,"identity":"94723c8b-e606-4e25-9471-293885697f3f","added_by":"auto","created_at":"2024-07-22 12:01:33","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1173737,"visible":true,"origin":"","legend":"\u003cp\u003eExample of scoring the\u003cstrong\u003e \u003c/strong\u003eKnowledge-based Aneuploidy Theoretical Score (KAT-Score). (a) Complete euploidy. (b) Low chromosomal mosaic gain. Chromosome 1 and chromosome 5, 20% gain. (c) High chromosomal mosaic loss. Chromosome 4, 60% loss. Arrows refer to chromosome mosaic derived use of intermediate copy number thresholds to determine as mosaic.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4653808/v1/de00fcaceafa5c376fb1c09b.jpg"},{"id":60816515,"identity":"84348588-813d-4bbf-b282-1343f484612f","added_by":"auto","created_at":"2024-07-22 12:01:33","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":394601,"visible":true,"origin":"","legend":"\u003cp\u003ePregnancy rate and live birth rate for each Knowledge-based Aneuploidy Theoretical Score (KAT-Score). (a)\u003cstrong\u003e \u003c/strong\u003eClinical pregnancy rate and (b) live birth rate for each KAT-Score.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4653808/v1/7ec3c78be48c0375685def60.jpg"},{"id":60816516,"identity":"dd4593a1-6cbb-43d3-bfcb-3edb94d4363c","added_by":"auto","created_at":"2024-07-22 12:01:33","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":302125,"visible":true,"origin":"","legend":"\u003cp\u003eBox plot of the KAT-Score distribution. (a) Clinical pregnancy. + indicates presence. (b) Live birth. + indicates detection.\u003cstrong\u003e \u003c/strong\u003eN.S. = not significant, Wilcoxon rank-sum test.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4653808/v1/1d3d2bdd4821987b75d63809.jpg"},{"id":60816517,"identity":"785746f1-e12c-496c-a9a9-4e832efb1396","added_by":"auto","created_at":"2024-07-22 12:01:33","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":485951,"visible":true,"origin":"","legend":"\u003cp\u003eReceiver operating characteristic curve analysis. (a) KAT-Score and clinical pregnancy. (b) KAT-Score and live birth. AUC = area under the curve, N.S. = not significant.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4653808/v1/161f30ed4dc3b15675ee6294.jpg"},{"id":77283267,"identity":"8ee2dd2d-7cfa-4ab3-8c3a-fee327c3c31d","added_by":"auto","created_at":"2025-02-27 04:46:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3606189,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4653808/v1/9851d987-e8f2-4d25-a4af-45ea396d71e8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Relationship between a novel assessment scoring system in pre-implantation genetic testing for aneuploidy and clinical outcomes after embryo transfer: a single-center retrospective cohort study","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eIn assisted reproductive technology (ART), multiples have historically been a serious problem resulting from the transfer of two or more embryos into the uterus [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In modern ART clinics, the aim is to achieve a healthy live birth using single embryo transfer. In recent years, there have been dramatic advances in the culture media and environment, meaning that embryos cultured for 5\u0026ndash;6 days in vitro will frequently provide multiple blastocysts for embryo transfer [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In this context, the issue arises of how best to rank embryos in terms of their quality to determine which have the highest likelihood of leading to a successful pregnancy. Pre-implantation genetic testing for aneuploidy (PGT-A) is a technique for selecting high-quality embryos by evaluating the number of chromosomes in multiple blastocysts [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. From the perspective of embryo selection, a normal chromosomal constitution defined by PGT-A is the strongest predictive factor for a successful pregnancy. However, PGT-A also detects chromosomal mosaicism, in addition to euploidy and aneuploidy [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Excluding mosaic blastocysts may compromise treatment outcomes by reducing the number of embryos available for transfer. Chromosomal mosaicism involves a mixture of euploid and aneuploid cells and results in mitotic errors [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The evaluation and interpretation of mosaicism in embryos can be complex, and there is no consensus or standardized criteria to assess it. The incidence of mosaicism varies depending on the embryonic stage, with a reported incidence of 4\u0026ndash;22% among embryos at the blastocyst stage depending on the clinic [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Earlier technologies for PGT-A had limitations in detecting mosaicism, but next-generation sequencing (NGS) has become widely used as a more accurate method for this purpose [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Viotti et al. retrospectively analyzed 1000 mosaic embryo transfers and found that the pregnancy and live birth rates differed in a stepwise manner depending on the type and extent of mosaicism [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. This suggests that the outcome of mosaic embryo transfer may vary depending on the specific characteristics of the mosaicism. The Knowledge-based Aneuploidy Theoretical Score (KAT-Score) model was developed as a novel approach to assess mosaicism. In this model, the type and extent of mosaicism are represented by a numerical scoring system [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Viotti et al. focused on the implantation, ongoing pregnancy, and live birth rates when mosaic embryos were transferred and showed the influence of mosaicism status (i.e., number of chromosomes showing chromosomal or segmental mosaicism). Several reports have also addressed mosaic trisomy and monosomy, showing that mosaic trisomy has a higher miscarriage risk than mosaic monosomy [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan additionalcitationids=\"CR13 CR14 CR15 CR16\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Furthermore, mosaicism in multiple chromosomes has a lower ongoing implantation rate than mosaicism in one or two chromosomes [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In case of multiple embryos with the same mosaicism status, the mosaic rate is also useful and is added as a decimal point to the risk score to support judging the order of transfer. Numerical PGT-A of embryos may also be useful for comparative evaluation of embryos.\u003c/p\u003e \u003cp\u003eMaximizing the clinical outcome of PGT-A is crucial to increasing the chances of implantation. The current study assessed the correlation between KAT-Score and pregnancy outcomes by scoring PGT-A cycle embryos. The purpose of this study was to investigate whether the KAT-Score, as an indicator of the likelihood of mosaicism, can be used as a predictor of pregnancy outcomes.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatients and study design\u003c/h2\u003e \u003cp\u003eA total of 124 cycles of single vitrified-warmed blastocyst transfer performed between October 2020 and March 2023 in a single in vitro fertilization (IVF) center (Kinutani Women\u0026rsquo;s Clinic, Hiroshima, Japan) were included in the analysis. All transferred blastocysts from the PGT-A cycles were evaluated using the KAT-Score (v2, Varinos Inc, Tokyo, Japan). However, cases with pre-implantation genetic testing for structural chromosome rearrangements or multiple embryo transfer cycles including two-step embryo transfer were excluded from the analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The main outcome measures were clinical pregnancy (presence or absence of gestational sac) and live birth (presence or absence of live birth).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e This study was approved by the Ethics Committee of Kinutani Women\u0026rsquo;s Clinic, Hiroshima, Japan. Informed consent was obtained from all patients included in this study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eOvarian stimulation and oocyte retrieval\u003c/h2\u003e \u003cp\u003ePatients were treated with gonadotropin hormone-releasing hormone (GnRH) agonist using a short or long protocol, following a progestin-primed ovarian stimulation protocol, in accordance with each patient\u0026rsquo;s ovarian response and medical history of IVF treatment. Serum estradiol levels were monitored and oocyte growth evaluated by transvaginal ultrasound. In some cycles, the minimal stimulation protocol and natural cycle were used. When a follicle reached a mean diameter\u0026thinsp;\u0026ge;\u0026thinsp;18 mm, human chorionic gonadotropin (Aska Pharmaceutical, Tokyo, Japan), 250 \u0026micro;g of choriogonadotropin alpha (Ovidrel; Merck Serono, Darmstadt, Germany), or GnRH agonist was administered. Transvaginal oocyte retrieval was performed 35\u0026ndash;37 h after administration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eFertilization procedure, embryo culture, time-lapse monitoring\u003c/h2\u003e \u003cp\u003ePrior to conventional IVF, collected cumulus\u0026ndash;oocyte complexes were cultured for approximately 4 h in G-IVF medium (Vitrolife, Gothenburg, Sweden) at 37\u0026deg;C under an atmosphere of 6% CO\u003csub\u003e2\u003c/sub\u003e, 5% O\u003csub\u003e2\u003c/sub\u003e, and 89% N\u003csub\u003e2\u003c/sub\u003e. Cumulus\u0026ndash;oocyte complexes were denuded by pipetting using 80 IU/ml hyaluronidase (Irvine Scientific, Santa Ana, CA, USA). The maturity of denuded oocytes was assessed by visualization of the first polar body. Only metaphase II oocytes were used for intracytoplasmic sperm injection (ICSI). Embryos fertilized by ICSI or conventional IVF were cultured in DNP dishes (Dai Nippon Printing Co., Ltd., Tokyo, Japan) with SAGE 1 Step (Origio, M\u0026aring;l\u0026ouml;v, Denmark) at 37\u0026deg;C under an atmosphere 6% CO\u003csub\u003e2\u003c/sub\u003e, 5% O\u003csub\u003e2\u003c/sub\u003e, and 89% N\u003csub\u003e2\u003c/sub\u003e for 5\u0026ndash;7 days and evaluated for development to the blastocyst stage. The oocytes were cultured individually in a time-lapse system (CCM-iBIS; Astec, Fukuoka, Japan). A total of 1\u0026ndash;25 embryos were placed in a 100 \u0026micro;l drop of medium, and images of the embryos were recorded automatically at 15-min intervals.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eBlastocyst biopsy protocols and NGS analysis\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eTrophectoderm (TE) biopsy was performed on expanded blastocysts on days 5\u0026ndash;7 regardless of morphological grade. First, artificial shrinkage was performed using a laser system (Zilos-TK\u0026trade;; Hamilton Thorn Bioscience Inc., Beverly, MA, USA) to create a space between the zona pellucida and TE cells. While holding the blastocyst in an inner cell mass (ICM) at a position of approximately 8 to 9 o\u0026rsquo;clock, serial laser pulses were used to create a small hole (~\u0026thinsp;10 \u0026micro;m) in the zona pellucida at a position of approximately 3 to 4 o\u0026rsquo;clock. The biopsy pipette was pushed into the hole created by the laser to aspirate the TE cells. Five to 10 TE cells were aspirated into the biopsy pipette and collected by flicking the holding pipette and biopsy pipette with the aid of several laser pulses. Biopsied samples were processed for whole-genome amplification and NGS at Varinos Laboratory. The VeriSeq PGS kit (Illumina K.K., San Diego, CA, USA) on the MiSeq system (Illumina K.K., San Diego, CA, USA) was used in 24-sample runs following the manufacturer\u0026rsquo;s protocol. The copy number of each sample was analyzed using BluFuse Multi Software (Illumina K.K., San Diego, CA, USA). A molecular karyotype profile consistent with mosaicism was detected when a whole chromosome or chromosomal segment resulted in intermediate copy number levels of 20\u0026ndash;80% between whole numbers according to the guidelines of the Preimplantation Genetic Diagnosis International Society [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eVitrification and warming of blastocysts and embryo transfer\u003c/h2\u003e \u003cp\u003eBlastocyst vitrification was performed using the method reported by Hiraoka et al. [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] within 1 h after embryo biopsy. The blastocysts were placed in equilibration solution containing 7.5% (v/v) ethylene glycol (Sigma-Aldrich, St. Louis, MO, USA) and 7.5% (v/v) dimethyl sulfoxide (Nacalai Tesque Inc., Kyoto, Japan) in mHTF (Irvine Scientific, Santa Ana, CA, USA) supplemented with 20% (v/v) serum protein substitute (Origio, M\u0026aring;l\u0026ouml;v, Denmark) at 37\u0026deg;C. Blastocysts were then transferred into vitrification solution containing 15% (v/v) ethylene glycol, 15% (v/v) dimethyl sulfoxide, and 0.5 M sucrose (Sigma-Aldrich, St. Louis, MO, USA) in mHTF supplemented with 20% (v/v) serum protein substitute for 1 min at 37\u0026deg;C. The blastocysts were loaded onto Cryotops (Kitazato Corporation, Fujinomiya, Japan) at a minimum volume and immediately immersed in liquid nitrogen at \u0026minus;\u0026thinsp;196\u0026deg;C. For the warming protocol, the tip of the Cryotop was immersed directly in 1.0 M sucrose solution for 1 min at 37\u0026deg;C. The blastocyst was transferred to 0.5 M sucrose solution for 3 min and washed twice in mHTF supplemented with 20% serum protein substitute for 5 min at 37\u0026deg;C. Prior to embryo transfer, the warmed blastocyst was cultured for approximately 3\u0026ndash;4 h in EmbryoGlue (Vitrolife, Gothenburg, Sweden). Luteal phase support was started using intravaginal micronized progesterone or natural cycles. Embryo transfers were performed under ultrasound guidance using a soft catheter (Origio, M\u0026aring;l\u0026ouml;v, Denmark).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eKAT-Score-based scoring of embryos\u003c/h2\u003e \u003cp\u003eEmbryos were scored 0 (complete euploidy) to 10 (complete aneuploidy) using the KAT-Score v2 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). First, a score from 1 to 8 was assigned for each type of mosaicism and a score of 9 for segmental aneuploidy. Next, a score of 0.2\u0026ndash;0.8 was added to the total depending on the level of mosaicism in the range of 20\u0026ndash;80%. When mosaicism was present on multiple chromosomes, the score of the chromosome with the highest level of mosaicism was assigned as the mosaicism score. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows an example of the scoring.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eKnowledge-based Aneuploidy Theoretical Score (KAT-Score) for pre-implantation genetic testing.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMosaic type\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eScore\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAneuploid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSegmental aneuploid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHigh chromosomal mosaic complex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHigh chromosomal mosaic gain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHigh chromosomal mosaic loss\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLow chromosomal mosaic complex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLow chromosomal mosaic gain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLow chromosomal mosaic loss\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHigh segmental mosaic gain/loss\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLow segmental mosaic gain/loss\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEuploid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMosaic level\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eScore\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e80%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e70%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e60%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e50%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e40%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e\u003cem\u003eNote\u003c/em\u003e: Low=low-level mosaicism \u0026gt;20% to \u0026lt;50%\u003cem\u003e,\u0026nbsp;\u003c/em\u003eHigh=high-level mosaicism \u0026ge;50% to \u0026lt;80%\u003cem\u003e,\u0026nbsp;\u003c/em\u003eComplex=mosaicism of monosomies and trisomies found on multiple chromosomes, Gain=trisomy\u003cem\u003e,\u0026nbsp;\u003c/em\u003eLoss=monosomy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using JMP 14.0 software (SAS Institute, Inc., Cary, NC, USA) and GraphPad PRISM 6.03 software (GraphPad Inc., San Diego, CA, USA). Patient characteristics were presented as means and standard deviation (SD). The KAT-Score data were not normally distributed, so the Wilcoxon rank-sum test was used to compare the scores as non-parametric continuous variables. We calculated the area under the receiver operating characteristic curve (AUC) of the KAT-Score for predicting clinical pregnancy and ongoing pregnancy. Adjusted odds ratios (aORs) were calculated using multivariate logistic regression analysis. Differences were considered significant at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eParticipant characteristics and KAT-Score distribution\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the participant characteristics for embryo transfer. A total of 124 cycles of single vitrified-warmed blastocyst transfer were performed using euploid and mosaic embryos, mostly under hormone replacement cycles. Of these 124 cycles, 60 clinical pregnancies were achieved, 50 of which resulted in live births. PGT-A for the 124 cycles was classified by the KAT-Score before embryo transfer. The KAT-Scores for the 124 blastocysts ranged from 0 to 9 and were distributed as follows: 0, n\u0026thinsp;=\u0026thinsp;51; 1\u0026ndash;2, n\u0026thinsp;=\u0026thinsp;22; 2, n\u0026thinsp;=\u0026thinsp;1; 3\u0026ndash;4, n\u0026thinsp;=\u0026thinsp;6; 4\u0026ndash;5, n\u0026thinsp;=\u0026thinsp;19; 5\u0026ndash;6, n\u0026thinsp;=\u0026thinsp;8; 6\u0026ndash;7, n\u0026thinsp;=\u0026thinsp;8; 7\u0026ndash;8, n\u0026thinsp;=\u0026thinsp;7; and 9, n\u0026thinsp;=\u0026thinsp;2. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows the clinical outcomes after single vitrified-warmed blastocyst transfer stratified by KAT-Score. Clinical pregnancy was confirmed for KAT-Scores of 1\u0026ndash;7. There were no transfers with a KAT-Score of 8. One case with a KAT-Score of 2 (high segmental mosaic gain/loss) led to pregnancy but did not result in live birth. No cases with a KAT-Score of 9 (segmental aneuploidy) led to pregnancy. Clinical pregnancies were confirmed even in mosaic embryos with a KAT-Score of 7 (high chromosomal mosaic gain). However, there were no live births among these cases.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCharacteristics of participants for embryo transfer cycles.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCharacteristic of embryo transfer cycles\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo. of patients\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e84\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo. of transfer cycles\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e124\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaternal age at ET (years)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e38.8\u0026thinsp;\u0026plusmn;\u0026thinsp;4.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaternal age at OR (years)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e38.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.87\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrevious embryo transfer (cycles)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.15\u0026thinsp;\u0026plusmn;\u0026thinsp;3.04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEndometrial thickness (mm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.88\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHormone replacement cycles\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e108 (87.1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNatural cycles\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15 (12.1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAromatase inhibitor cycles\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (0.8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClinical pregnancies\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e60 (48.4)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMiscarriages\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10 (8.1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLive births\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50 (40.3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDay of blastocyst\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDay 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e74 (59.7)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDay 6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e45 (36.3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDay 7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5 (4.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMorphology\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGood (AA/AB/BA)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e72 (58.1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFair (BB)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36 (29.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePoor (BC/CB/AC/CC)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16 (12.9)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eKAT-Score distribution\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEuploid (0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e51 (41.2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSegmental mosaic (1\u0026ndash;2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23 (18.5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLow level mosaic (3\u0026ndash;5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33 (26.6)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHigh level mosaic (6\u0026ndash;8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15 (12.1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSegmental aneuploid (9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 (1.6)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e\u003cem\u003eNote\u003c/em\u003e: Values are given as n (%) or mean \u0026plusmn; SD unless otherwise noted. ET=embryo transfer, OR=oocyte retrieval.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCorrelations between KAT-Score and clinical pregnancy/live birth\u003c/h2\u003e \u003cp\u003eContinuous variables were compared between two groups using the Wilcoxon signed-rank test (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). No significant difference in the mean KAT-Score was observed between the clinical pregnancy and non-clinical pregnancy groups (1.99\u0026thinsp;\u0026plusmn;\u0026thinsp;2.59 vs. 2.67\u0026thinsp;\u0026plusmn;\u0026thinsp;2.76; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.2008; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). The KAT-Score was significantly lower in the live birth group than in the non-live birth group (1.37\u0026thinsp;\u0026plusmn;\u0026thinsp;2.07 vs. 3.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.87; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e shows the results of univariate logistic regression analyses for the probabilities of clinical pregnancy and live birth. The utility of the KAT-Score as a predictive measure of clinical pregnancy or live birth was evaluated using the AUC. The KAT-Score of embryos was a predictive indicator of live birth (AUC\u0026thinsp;=\u0026thinsp;0.64, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) but not of clinical pregnancy (AUC\u0026thinsp;=\u0026thinsp;0.56, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.067; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b). Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows the results of multivariate logistic regression analyses, which included maternal age, previous ET cycles, endometrial thickness, and morphological grade as confounding factors, for the probabilities of clinical pregnancy and live birth. The number of previous ET cycles significantly correlated with a negative probability of clinical pregnancy (aOR 0.82, 95% CI 0.69\u0026ndash;0.96, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The morphological grade of the blastocyst significantly correlated with a positive probability of clinical pregnancy (aOR 4.26, 95% CI 1.31\u0026ndash;16.8, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). On the other hand, the probability of live birth negatively correlated with the number of previous ET cycles (aOR 0.79, 95% CI 0.66\u0026ndash;0.94, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and KAT-Score (aOR 0.83, 95% CI 0.70\u0026ndash;0.99, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMultivariate logistic regression analysis of several factors associated with clinical pregnancy and live birth in patients who underwent PGT-A.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eClinical pregnancy\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e \u003cp\u003eLive birth\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFactor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eaOR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e95% CI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eaOR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e95% CI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaternal age\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.85\u0026ndash;1.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.2964\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.85\u0026ndash;1.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.3563\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo. of previous ET cycles\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.69\u0026ndash;0.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0077\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.66\u0026ndash;0.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.0048\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEndometrial thickness\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.94\u0026ndash;1.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1736\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.96\u0026ndash;1.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.1012\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMorphological grade (\u0026ge;\u0026thinsp;3BB)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.31\u0026ndash;16.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0246\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.70\u0026ndash;11.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.0676\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKAT-Score\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.83\u0026ndash;1.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.7576\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.70\u0026ndash;0.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.0403\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003cp\u003eunderwent PGT-A.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNote\u003c/em\u003e: aOR=adjusted odds ratio, CI=confidence interval, ET=embryo transfer.\u003c/p\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eNeonatal outcomes stratified by KAT-Score\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows a comparison of neonatal outcomes among the KAT-Score groups after single vitrified-warmed blastocyst transfer. The neonatal outcomes were divided into three groups according to the KAT-Score: euploid, 0; low KAT-Score, 1\u0026ndash;5; high KAT-Score, 6\u0026ndash;9. We found no significant difference in the clinical pregnancy rates between the three groups. Miscarriage rates were significantly increased in the low KAT-Score and high KAT-Score groups [0% (0/27) vs. 33.3% (5/25) vs. 62.5% (5/8); \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05]. The live birth rate among embryos with a high KAT-Score was 17.6% (3/17), which was significantly lower than that of embryos with complete euploidy [52.9% (27/51)]. There were no significant differences in β-hCG and maternal age in the three groups. Moreover, we did not find significant differences in the mean birth weight among the three groups (3117\u0026thinsp;\u0026plusmn;\u0026thinsp;373.5 vs. 3073\u0026thinsp;\u0026plusmn;\u0026thinsp;502.0 vs. 2621\u0026thinsp;\u0026plusmn;\u0026thinsp;730.9 g, respectively).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eNeonatal outcome stratified by Knowledge-based Aneuploidy Theoretical Score (KAT-Score).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKAT-Score group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u0026ndash;5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6\u0026ndash;9\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTransfers\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e51 (41.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e56 (45.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17 (13.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrevious ET cycles\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.53\u0026thinsp;\u0026plusmn;\u0026thinsp;2.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.36\u0026thinsp;\u0026plusmn;\u0026thinsp;2.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.35\u0026thinsp;\u0026plusmn;\u0026thinsp;4.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN.S.**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaternal age at ET\u003c/p\u003e \u003cp\u003e(years)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e37.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e38.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e42.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN.S.**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEndometrial thickness\u003c/p\u003e \u003cp\u003e(mm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN.S.**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGood morphology blastocyst\u003c/p\u003e \u003cp\u003erate (\u0026ge;\u0026thinsp;3BB)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e90.2 (46/51)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e85.7 (48/56)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e82.4 (14/17)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN.S*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClinical pregnancy rate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e52.9 (27/51)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e44.6 (25/56)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e47.1 (8/17)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN.S.*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMiscarriage rate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 (0/27) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33.3 (5/25) \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e62.5 (5/8) \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.05*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLive birth rate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e52.9 (27/51) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35.7 (20/56) \u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17.6 (3/17) \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSingleton\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eβ-hCG (mIU/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e539.3\u0026thinsp;\u0026plusmn;\u0026thinsp;399.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e492.1\u0026thinsp;\u0026plusmn;\u0026thinsp;353.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e349.5\u0026thinsp;\u0026plusmn;\u0026thinsp;197.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN.S.**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBirth weight (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3117\u0026thinsp;\u0026plusmn;\u0026thinsp;373.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3073\u0026thinsp;\u0026plusmn;\u0026thinsp;502.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2621\u0026thinsp;\u0026plusmn;\u0026thinsp;730.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN.S.**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMale infant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13 (48.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10 (50.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0 (0.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFemale infant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14 (51.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10 (50.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3 (100.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\u003cp\u003e\u003cem\u003eNote\u003c/em\u003e: Values are given as n (%) or mean \u0026plusmn; SD unless otherwise noted. ET=embryo transfer, SD=standard deviation, N.S.=not significant.\u003c/p\u003e\n\u003cp\u003e*Fisher\u0026rsquo;s exact test,\u0026nbsp;**one-way ANOVA and Tukey\u0026ndash;Kramer test.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ea, b, c\u003c/sup\u003e Different superscripts indicate a significant difference (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05)\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis study examined whether the KAT-Score correlates with pregnancy prognosis. PGT-A is used to assess embryonic aneuploidy to increase pregnancy rates, decrease abortion rates, and prioritize the highest quality embryos for transfer. In this study, the embryos from 124 cycles of PGT-A, including mosaic embryos, resulted in the birth of 50 healthy children. The KAT-Score is a scoring method originally developed by Varinos Inc. based on a report by Viotti et al. that allows scoring according to the type and extent of mosaicism [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Statistical analyses clarified that this scoring is associated with type of mosaicism and the rate of live birth, but not with clinical pregnancy. Multivariate logistic regression analysis, which included various confounding factors, showed that the KAT-Score and previous ET cycles correlated with the live birth rate.\u003c/p\u003e \u003cp\u003eThe possibility of viable embryos being discarded because of concerns about mosaicism is one of the challenges currently facing PGT-A. The transfer of embryos classified as mosaic by PGT-A was reported for the first time in 2015 and is now routinely performed in clinical practice [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The fertility potential of mosaic embryos remains controversial [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. When analyzing the mosaicism status using techniques such as amplification or NGS, noise or artifacts may be indistinguishable from true mosaicism [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. However, several studies have suggested that embryos classified as mosaic have reduced reproductive potential compared with embryos classified as euploid [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In these previous studies, mosaicism was mostly diagnosed on the basis of its presence or absence. The general definition of a mosaic embryo does not involve stringent thresholds (mosaicism level: 20\u0026ndash;80%), but it is unclear which type or level of mosaicism is suitable for implantation [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGirardi et al. found that, to maximize the accuracy of PGT-A, single TE biopsy results should be reported according to the euploid/aneuploid classification based on a single cut-off of 50% [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The current study divided the neonatal outcomes into three groups based on a mosaicism level of 50% and compared them. Although there was a slight decrease in the live birth rate from the euploid group (KAT-Score 0) to the group with low-level mosaicism (KAT-Score 1\u0026ndash;5), no significant difference was observed. A lower level of mosaicism is traced in subsequent TE biopsy, and many embryos are uniformly euploid [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The incidence of chromosomal mosaicism has also raised concerns about the possibility that a considerable number of PGT-A results are false-positive errors [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. If the level of mosaicism is \u0026lt;\u0026thinsp;50%, the embryo may have the same developmental potential as a euploid embryo. The live birth rate was significantly decreased in the group with a KAT-Score of 6\u0026ndash;9, 17.6% compared with 52.9% in the group with a KAT-Score of 0. Interestingly, no live births were recorded in the group with a KAT-Score\u0026thinsp;\u0026ge;\u0026thinsp;7 (high chromosomal mosaic gain). These results may differ depending on whether the case involves monosomy or trisomy. The results of TE biopsies are not always concordant with the chromosomal constitution of the ICM in mosaic embryos [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Confirming mosaicism\u0026thinsp;\u0026ge;\u0026thinsp;50% and trisomy mosaicism in TE biopsy suggests that the chromosomal composition of the majority of these embryos shares a uniform aneuploid configuration [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. A previous study showed that, when trisomy abnormalities are present in the ICM, implantation is possible, but miscarriage occurs in most cases [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Although it is clear that mosaic embryos have significant developmental potential, those with high-level trisomy are not suitable for transfer.\u003c/p\u003e \u003cp\u003eCurrently, TE biopsies are performed at the blastocyst stage because the incidence of mosaicism decreases between day 3 (cleavage stage) and day 5 (blastocyst stage) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] due to a self-corrective mechanism during the division process [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In human embryos, aneuploid cells are sequestered from the ICM during the progression from day 3 to day 5 and are partially sequestered in the TE surrounding the blastocyst [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Although neonatal outcome data from transferred mosaic embryos need to be comprehensively analyzed, 23 newborns from the mosaic group in this study were born healthy.\u003c/p\u003e \u003cp\u003eTo use the KAT-Score as a tool that allows mosaic embryos to be ranked for their suitability for implantation and increase the likelihood of a positive clinical outcome, it is necessary to develop an algorithm that includes the morphological and kinetic characteristics of the embryo. Most of the blastocysts used for transfer in this study were day 5 blastocysts with good morphology (\u0026ge;\u0026thinsp;BB). Blastocysts that reach this stage later or have poor morphology are more likely to be aneuploid and, therefore, were not selected for transfer. Currently, clear chromosomal abnormalities, such as mosaicism and chromosomal imbalances, can only be detected by PGT-A, but the live birth rate of embryos transferred after PGT-A was limited approximately 40% in this study. In ART, it is vital to develop a large number of embryos with high developmental competence. Further research is required to gain a more complete understanding of whether the KAT-Score is associated with clinical pregnancy and live birth.\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eIn conclusion, this study suggests that the KAT-Score can be assessed during PGT-A as an indicator to predict live birth. Embryos with high segmental mosaic gain/loss (KAT-Score 2), segmental aneuploidy (KAT-Score 9), and high chromosomal mosaic gain (KAT-Score 7) are not suitable for transfer. The scoring of 124 embryo transfers by the KAT-Score provides statistically valid evidence for the use of this tool to rank mosaic embryos in infertility clinics.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKAT-Score\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eKnowledge-based Aneuploidy Theoretical Score\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eART\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eassisted reproductive technology\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePGT-A\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epre-implantation genetic testing for aneuploidy\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNGS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enext-generation sequencing\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIVF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ein vitro fertilization\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGnRH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003egonadotropin hormone-releasing hormone\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eC-IVF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003econventional IVF\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eICSI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eintracytoplasmic sperm injection\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etrophectoderm\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eICM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003einner cell mass\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eaOR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eadjusted odds ratio\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAUC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003earea under the curve\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003estandard deviation.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank the Kinutani Women\u0026rsquo;s Clinic embryologist team for acquiring data and the participating couples and gynecologists at the infertility clinic. We also thank Varinos Inc. (\u003cu\u003ehttps://varinos.com\u003c/u\u003e) CEO Yoshiyuki Sakuraba for help with this paper and the development of the KAT-Score.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHORS\u0026rsquo; CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKA invented the KAT-Score model. YH interpreted the data collection and analysis of data and participated in designing the study. EF, TM, HS, MI, RS, SO, and MK assisted in acquiring and interpreting data. YS, ST, and MS revised the manuscript. All authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funding was received related to this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAVAILABILITY OF DATA AND MATERIALS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe dataset used and/or analyzed in this manuscript will be available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDISCLARATIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConflict of interest: The authors declare no conflict of interest. Human rights statements and informed consent: All procedures were performed in accordance with the ethical standards of the institutional ethical committee and with the Helsinki Declaration of 1964 and its later amendments. Informed consent was obtained from all patients in this study, and the study design was approved by the ethics committee of Kinutani Women\u0026rsquo;s Clinic, Hiroshima, Japan. Animal studies: This article does not describe any study involving animals performed by any of the authors. Declarations of interest: none. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR DETAILS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eKinutani Women\u0026rsquo;s Clinic, 8-23-4F Hondori, Naka-ku, Hiroshima 730-0035, Japan.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u003c/sup\u003e Varinos Inc., 21\u003csup\u003est\u003c/sup\u003e Floor, Diver City Tokyo Office Tower, 1-1-20 Aomi, Koto-ku, Tokyo 135-0064, Japan.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePinborg A, Loft A, Schmidt L, Andersen AN. Morbidity in a Danish national cohort of 472 IVF/ICSI twins, 1132 non-IVF/ICSI twins and 634 IVF/ICSI singletons: health-related and social implications for the children and their families. 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Hum Reprod. 2023;38(1):180\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/humrep/deac238\u003c/span\u003e\u003cspan address=\"10.1093/humrep/deac238\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"PGT-A, mosaicism, embryo biopsy, a novel scoring system, blastocyst quality, embryo selection","lastPublishedDoi":"10.21203/rs.3.rs-4653808/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4653808/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe evaluation and interpretation of mosaicism in pre-implantation genetic testing for aneuploidy (PGT-A) can be complex, and no consensus or standardized criteria are available for its assessment. We investigated whether mosaicism as assessed by the proprietary Knowledge-based Aneuploidy Theoretical Score (KAT-Score) in PGT-A correlates with clinical pregnancy and live birth rates.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThis retrospective cohort study was conducted in a single in vitro fertilization center between August 2020 and March 2023. A total of 124 single vitrified-warmed blastocyst transfer cycles were analyzed for clinical outcomes stratified by KAT-Score. We also analyzed the correlations between KAT-Score and clinical pregnancy or live birth rates.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe found no significant difference in the KAT-Score between the pregnancy and non-pregnancy groups. However, the KAT-Score was significantly lower in the live birth group than in the non-live birth group. Moreover, the KAT-Score was a predictive indicator of live birth (area under the curve\u0026thinsp;=\u0026thinsp;0.64, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) but not of clinical pregnancy (area under the curve\u0026thinsp;=\u0026thinsp;0.56, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.1597). Multivariate logistic regression analysis, which included maternal age, previous embryo transfer cycles, endometrial thickness, and morphological grade as confounding factors, showed that lower KAT-Scores significantly correlated with the live birth rate (adjusted odds ratio: 0.83, 95% confidence interval: 0.70\u0026ndash;0.99, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0403). No eventual live births were recorded in the group with a KAT-Score indicative of high chromosomal mosaic gain (KAT-Score\u0026thinsp;\u0026ge;\u0026thinsp;7).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThis study suggests that the KAT-Score correlates with live birth but not with clinical pregnancy. The live birth rate differed according to type of mosaicism.\u003c/p\u003e","manuscriptTitle":"Relationship between a novel assessment scoring system in pre-implantation genetic testing for aneuploidy and clinical outcomes after embryo transfer: a single-center retrospective cohort study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-22 12:01:28","doi":"10.21203/rs.3.rs-4653808/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"35f0c7c2-f7c8-4cfd-8d59-3b8afa99695f","owner":[],"postedDate":"July 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-02-27T04:38:37+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-22 12:01:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4653808","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4653808","identity":"rs-4653808","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}