Healthy newborn delivered after very high segmental mosaic embryo transfer- a case report.

Reflections on preimplantation genetic testing for aneuploidy and mosaicism: how did we get here, and what does it mean clinically?

text Recent advances in preimplantation genetic testing for aneuploidy (PGT-A) and time-lapse imaging have improved our understanding of the early human embryo confirming the variable patterns of development and chromosomal status. Aneuploidy is common and increased sensitivity in PGT-A allows the non-binary reporting of euploid-aneuploid mosaicism. The PGT-A result is the inference of the biopsied embryo’s ploidy status at a point in time, by assessment of a small percentage of cells, and, whilst concordance with the rest of the embryo is high; it is not absolute. Many reports have demonstrated that, with the transfer of embryos with increasing severity and complexity of mosaicism, comes compromised implantation, reduced ongoing pregnancy rates and increased miscarriage rates. Segmental mosaic embryos have been reported to have slightly reduced implantation potential compared with euploid counterparts. However, complex mosaic embryos are widely reported to result in severely reduced implantation success, if transferred. Outside of PGT-A treatment cycles, undoubtedly fertility clinics are unwittingly transferring mosaic and aneuploid embryos daily, with variable success. The transfer of embryos in which mosaicism has been detected, although associated with lower implantation and higher miscarriage rates than euploid embryos, can lead to normal pregnancies and healthy births. We know that the placenta can harbour chromosomal aberrations which are absent from the fetus, and there are few reports of births with demonstrably high levels of mosaicism through fetal development. This raises the question as to whether correction mechanisms exist. In other words, do conceptuses become chromosomally more normal as development progresses, and what are the mechanisms, if so? PGT-A, time lapse, novel live cell imaging and in vitro model techniques have enabled a more detailed study of early embryo development and consideration of the phenomenon of self-correction. This has provided insights and hypotheses surrounding the mechanisms of development and of self-correction. The relatively lower levels of chromosome abnormality in the blastocyst, compared with cleavage stage, are well documented and indicative of some form of correction. A recent investigation reported that a large proportion of embryos initially diagnosed as mosaic were later diagnosed as euploid when assessed at day 12 of development; providing evidence of the depletion of abnormal cells throughout the early post-implantation stages. There are many time-lapse reports of anomalous ‘direct’ or multichotomous blastomere divisions being associated with aneuploidy, and leading to developmental arrest or reduced implantation potential and of temporal delays in aneuploid embryos compared with their euploid counterparts. It is possible, therefore, that errors and even attempts to repair them, in individual cells in the rapidly developing embryo; which involve complex biochemical systems, could delay karyo- and cytokinesis, resulting in these detectable delays. The embryonic mortality model suggests that there is selection against embryos based on their degree of aneuploidy, such that aneuploid cell lines are lost during implantation. We know that irregularities in blastomere cleavage can generate chromosome segregation errors but these may sporadically be confined to cells excluded, or extruded, from the morula or from the blastocyst; a possible exhibition of the clonal depletion or embryo mortality model. The trisomic/monosomic rescue model suggests that aneuploid cells can give rise to diploid cells (and possibly uniparental disomy) through mitotic chromosome losses or gains. We know that an abnormal number of pronuclei does not always produce an aneuploid blastocyst and that early embryos exhibiting multinucleation can result in healthy live births. Finally, the preferential allocation of aneuploid cells to the trophectoderm model is based on the hypothesis that euploid cells are preferentially retained in the ICM in order to achieve viability. This presentation aims to consider what we know, and discuss the theories and available evidence for self-correction.

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

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

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

Study question Is the iDAScore v1.0 score correlated with pregnancy outcomes in single embryo transfer (SET) cycles following preimplantation genetic testing for aneuploidy (PGT-A)? Summary answer We demonstrated that elevated iDAScore v1.0 scores are positively correlated with the probabilities of pregnancy and live birth (LB) in SETs following PGT-A. What is known already To select the blastocysts with high development potential in advance, several groups have proposed the implementation of artificial intelligence (AI)-based software is capable of predicting implantation in women undergoing in vitro fertilization (IVF). It has also been known that using high resolution next generation sequencing preimplantation genetic screening (hr-NGS) for PGT-A can enable the exclusion of aneuploid embryos before embryo transfers and thus improve pregnancy outcomes of SETs. However, the capacity of AI-based assessments has remained unclear in IVF cycles with PGT-A. Study design, size, duration This retrospective study, approved by the Institutional Review Board of Chung Sun Medical University, was performed to assess the dataset of 317 SET cycles after PGT-A from January 2017 to September 2019. A single euploid or mosaic blastocyst with an AI score calculated using the iDAScore software (v1.0) was selected for frozen embryo transfer according to the given priority of blastocyst morphology. The iDAScore groups were categorized by quartiles of AI scores. Participants/materials, setting, methods Embryos were cultured in a time-lapse incubator and qualified blastocysts underwent next generation sequencing (NGS)-based PGT-A. The confounding factors associated with LB were evaluated using logistic regression analysis in generalized estimating equations (GEEs). The differences between iDAScore groups were assessed using the Mann–Whitney U test or Fisher’s exact test, as applicable. The receiver operating characteristic (ROC) curve analysis was used to estimate the predictive powers. A P value &amp;lt; 0.05 was considered statistically significant. Main results and the role of chance The results revealed the patient age, anti-Müllerian hormone, body mass index, oocyte sources (autologous or donor), PGT-A results (euploidy, low-level mosaicism, or high-level mosaicism), aberrant chromosome types (none, whole chromosome, segmental chromosome, or whole with segmental chromosome), and aberrant chromosome sites (0, 1, 2, or &amp;gt; 2) were not correlated with LB. However, embryo biopsy days (day 5 vs. day 6, OR = 2.271, 95% CI = 1.534–14.824), blastocyst morphology scores (OR = 1.185, 95% CI = 1.034–1.359), and AI scores (OR = 1.640, 95% CI = 1.231–2.183) were significantly correlated with LB. The AI scores were then divided into quartiles (group 1: 5.0–7.9; group 2: 8.0–8.5; group 3: 8.6–8.9; and group 4: 9.0–9.5). The ongoing pregnancy (29.1% vs. 54.2%–57.5%) and live birth (27.8% vs. 52.8%–56.3%) rates of group 1 were significantly lower than other groups. The blastocyst morphology score (5.3 ± 1.2), and KIDScore D5 scores (v1 = 3.0 ± 1.7; v3 = 4.6 ± 1.5) were also lowest in group 1. The ROC curve analysis confirmed a significant but limited LB prediction capability for iDAScore v1.0 (AUC = 0.6), which was similar to the KIDScore D5 (AUC = 0.61–0.62). Limitations, reasons for caution Because of the retrospective nature, the major limitation was the lack of randomization, which may present a risk of selection bias. According to the criteria of blastocyst selection for SETs, the embryos with low (&amp;lt; 5.0) or high (&amp;gt; 9.5) AI scores were not present in this dataset. Wider implications of the findings Although prediction capability of iDAScore remains perfectible, the AI score is still significantly associated with LB probability. Euploid or mosaic blastocysts with low AI scores (&amp;lt;8.0) processed a decreased LB rate, indicating the potential of annotation-free iDAScore system as a decision support tool for deselecting embryos with poor post-implantation development. Trial registration number not applicable

Study question To investigate the effect of embryo mosaicism on clinical IVF outcomes in single and double frozen embryo transfer (FET). Summary answer There is no statistical difference between the low/high mosaic and euploid embryos in regards to the pregnancy and miscarriage rate, in single and double FETs. What is known already During mitosis and meiosis, mis-segregation of chromosomes causes aneuploidy. Recent studies have shown that next-generation sequencing (NGS) based pre-implantation genetic test for aneuploidy (PGT-A) improve clinical IVF outcomes by detecting chromosomal abnormalities in human embryos. Euploid embryos have been confirmed to achieve a higher pregnancy, implantation and live birth rate than aneuploid embryos. However, mosaic embryos, which contain two or more cell lines with different chromosomal set, are yet controversial in the assisted reproductive medical field. Hence, comprehensive studies on the clinical IVF outcomes of mosaic embryo transfers (METs) are necessary to resolve the concerns of embryo mosaicism. Study design, size, duration This single centre retrospective study included 93 low mosaic embryos, which were transferred in 64 FET cycles (35 single and 29 double FET), and 27 high mosaic embryos from 18 FET cycles (9 single and 9 double FET), from January 2018 to October 2021. The pregnancy and miscarriage rate of the METs were compared to an euploid control group, which comprised of 184 euploid embryos from 124 FET cycles (64 single and 60 double FET). Participants/materials, setting, methods All embryos were cultured to blastocyst stage and trophectoderm (TE) biopsy was performed on day 5 or 6. NGS based PGT-A was conducted to detect chromosomal abnormalities. According to the Preimplantation Genetic Diagnosis International Society guideline, mosaic embryos were categorized into 2 groups: low (20-39% mosaicism) and high mosaic embryos (40-80% mosaicism). Pearson chi-square test was performed to compare the pregnancy and miscarriage rate between low/high METs and euploid group, in single and double FETs. Main results and the role of chance In single FET cycles, patients with low mosaic embryos transferred achieved a higher pregnancy rate than the patients with euploid (60% versus 42%) and high mosaic (60% versus 33%) embryos transferred. In addition, the miscarriage rate for low mosaic embryos (9%) was the lowest, compared to the the euploid (11%) and high mosaic (22%) embryos. On the other hand, the highest pregnancy and lowest miscarriage rate in double FET cycles were achieved by patients with euploid embryos transferred. Results showed that patients with euploid embryos transferred possessed a pregnancy rate of 68%, whereas patients with low and high mosaic embryos transferred showed a pregnancy rate of 48% and 56% respectively. As mentioned above, the euploid embryos resulted in a lower miscarriage rate than the low (3% versus 10%) and high mosaic (3% versus 11%) embryos in double FET cycles. However, it is important to highlight that the above mentioned differences between the low/high METs and euploid control group in respect of the pregnancy and miscarriage rate were not statistically significant, in both single and double FET cycles (p &amp;lt; 0.05). Limitations, reasons for caution The factors of patient’s age and blastocyst grading were not taken into account in this study. Hence, a larger-scale randomized trial is a need to further identify and resolve the concerns of embryo mosaicism in the assisted reproductive medical field . Wider implications of the findings In the PGT-A analysis, only a fraction of the TE within an embryo was tested. Thus, misclassification of embryos might occur. It is suggested that clinical management of mosaic embryos should be carefully reviewed in the case of patients with no euploid embryos to be transferred. Trial registration number NA

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

Natural fecundity of women decreases gradually and more rapidly after age 37 years. This decrease is accompanied by rising aneuploidy rates of pregnancies and can also be observed in products of conception of spontaneous abortions [1]. These observations lead to the hypothesis that transferring only euploid embryos in association with in vitro fertilization (IVF) might decrease miscarriages and increase live birth rates (LBRs), attesting-procedure now called preimplantation genetic testing (of embryos) for aneuploidy (PGT-A), until recently generally referred to as preimplantation genetic screening (PGS). Verlinsky and Kuliev further proposed that the removal of all aneuploid embryos prior to transfer would improve implantation rates and live birth rates and suggested that the diagnosis be made via biopsy of both polar bodies [2]. Polar body biopsy, however, proved technically too difficult for general IVF practice and would have revealed only meiotic aneuploidies. The procedure was, therefore, initially performed biopsying 1–2 blastomeres of day-3 cleavage-stage embryos, often given the acronym PGS 1.0. This form of embryo testing has, since, been replaced by PGS 2.0, with the embryo biopsy being moved from day-3 cleavage stage to trophectoderm biopsy of blastocyst-stage embryos on days 5–6 after fertilization. In July 2016, another major change in PGT-A was announced, for the first time introducing the concept pf “mosaic” embryos (also called PGS 3.) (Preimplantation Genetic Diagnosis Society (PGDIS) position statement on chromosome mosaicism and preimplantation aneuploidy testing at the blastocyst stage, Chicago, IL; July 19, 2016 http://pgdis.org/docs/newsletter_071816.html). After almost two decades of PGS 1.0 through PGS 3.0, the procedure has, however, still been unable to demonstrate the promised improvements in live births and anticipated declines in miscarriage rates [3–5]. Several studies, even summarized in a meta-analysis [6], have claimed improved clinical IVF outcomes following PGT-A. They, however, reported IVF outcomes with reference point embryo transfer rather than cycle start (intent-to-treat) and, therefore, by excluding poorer prognosis patients, were severely biased [7]. The STAR study This is why the recently published STAR study [8] attracted special attention: It avoided at least some patient selection biases of earlier fresh-cycle studies by being prospectively randomized and reporting on IVF outcomes from transfers of only single frozen-thawed embryos at blastocyst stage. That qualifying patients required having at least two frozen embryos from a prior fresh cycle, however, still demonstrates a favorable patient selection bias. Importantly, however, the study at least analyzed outcomes for study and control groups with reference point initial first cycle start [7]. In doing so, the study convincingly revealed no improvements in live birth rates and no reduction in miscarriage rates when cycle outcomes were compared in singe-embryo transfers at blastocyst stage between women, randomized to either PGT-A or only morphological assessments of a single embryo prior to transfers [8]. For no declared reason, the authors then, however, performed a post hoc sub-group analysis based on age and reported, between ages 35 and 40 years, that PGT-A, still, offered significant increases in ongoing pregnancy rate (OPR). In the discussion of their manuscript, they emphasized this finding as “continuous evidence” for the clinical utility of PGT-A in at least that age group. Again, in contrast to the overall study that had been performed with reference point cycle start (intent-to-treat), their post hoc analysis was performed with reference embryo transfer and, therefore, statistically suspect. Because results of the STAR study are already impacting IVF practice worldwide, we here offer a statistically corrected analysis of the STAR study, reaffirming the study’s overall findings by refuting the results of the post hoc analysis and its interpretation by the authors, claimed benefits for PGT-A utilization for all age groups.

DOES THE MOSAIC EMBRYO RATIO OF PGT-A CYCLES IMPACT THE LIVE BIRTH CHANCE EVEN WHEN AN EUPLOID EMBRYO IS TRANSFERRED?

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

Study question Is the size of TE biopsy specimens related to the chromosomal status of the blastocysts in PGT-A? Summary answer Smaller biopsy specimens are associated with higher proportion and altered composition of mosaic blastocysts in PGT-A. What is known already Concordance between TE biopsy and inner cell mass or whole embryos is influenced by mosaicism type, degree, biopsy location, number of cells biopsied, and cell loading. Particularly, mosaic embryos and those with structural rearrangements have a higher level of inconsistency. Recent studies have demonstrated the potential reclassification of chaotic embryos as euploid, leading to successful live births. This study aimed to investigate the potential influence of TE biopsy specimen size on the chromosomal status of blastocysts undergoing PGT-A. Study design, size, duration This retrospective study, conducted from October, 2023, to January, 2024, included 246 blastocysts from patients undergoing TE biopsy and PGT-A at the blastocyst stage on days 5, 6, or 7. Next-generation sequencing (NGS) was used to obtain PGT-A results, categorizing blastocysts into euploid, aneuploid, and mosaic based on their genetic composition. Participants/materials, setting, methods The biopsy specimen area was measured using the Image J program. Mosaic blastocysts were categorized into either a chaotic mosaic group or another group, which included blastocysts with fewer than three chromosomal aneuploidies and segmental mosaicism. Further analysis was conducted by classifying blastocysts into high-level mosaic (&amp;gt;30% aneuploidy) and low-level mosaic (≤30% aneuploidy) groups. Group differences were analyzed using t-tests and chi-square, with a significance threshold set at p&amp;lt;0.05. Main results and the role of chance In the analysis of expanded mosaic blastocysts, the chaotic mosaic group had smaller biopsy specimens than those in the comparison group (p&amp;lt;0.05). The high-level mosaic group also had significantly smaller biopsy specimens than the low-level mosaic group (p&amp;lt;0.05). When categorizing the biopsied sample size into two groups—below 1350 µM2 (n = 98) and above 1500 µM2 (n = 97)—the rates of mosaic embryos were 37% (36/98) and 30% (29/97), respectively, showing a numerical difference without statistical significance. These findings suggest a correlation between the size of biopsy specimens and the chromosomal status of mosaic blastocysts, particularly indicating that a smaller sample size is associated with an elevated chance of mosaic embryos showing chaotic or high-level mosaic characteristics. Limitations, reasons for caution The biopsy specimens were photographed, and their areas were measured using the Image J program, lacking a comprehensive representation of the three-dimensional sample morphology. This constraint could potentially affect the accuracy and in-depth understanding of the spatial characteristics of the biopsy specimens. Wider implications of the findings The study emphasizes the crucial role of optimizing biopsy specimen size in PGT-A. The identified correlation with the chromosomal status in mosaic embryos, especially the association with chaotic or high-level mosaic embryo outcomes in smaller samples, highlights the need for careful size considerations. Trial registration number not applicable

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

Study question Do donor oocyte recipients benefit from preimplantation genetic testing for aneuploidy (PGT-A)? Summary answer The PGT-A does not improve the likelihood of live birth and time to pregnancy for recipients of vitrified donor oocytes. What is known already Oocyte vitrification has led to increased live birth from cryopreserved oocytes and has led to widespread use of this technology in donor egg IVF programs. However, oocyte cryopreservation has the potential to disrupt the meiotic spindle leading to abnormal segregation of chromosomes during meiosis II and may increase aneuploidy in the blastocyst. Therefore, PGT-A might have benefits in vitrified donor egg cycles. However, blastocysts derived from young donor oocytes are expected to be predominantly euploid, and trophectoderm biopsy may harm blastocysts compared to embryo transfer without PGT-A. Study design, size, duration Retrospective single-center study encompassing 2233 vitrified-warmed donor oocyte cycles conducted between March 2021 and August 2024 at a private Italian IVF clinic. The study included 299 donor cycles with and 1934 without PGT-A. Vitrified donor oocyte cycles were analyzed for live birth as the main outcome measure. Secondary outcomes were time to achieve pregnancy defined as the days from the egg thawing until a live birth achieved, clinical pregnancy, ongoing pregnancy miscarriage rates. Participants/materials, setting, methods The study included women aged 30-49 who underwent blastocyst single embryo transfer (SET). Trophectoderm biopsy was performed on day 5 or 6 based on embryo development. Both natural and artificial cycle SETs were considered. Exclusions were women with fibroids &amp;gt;3 cm, severe adenomyosis, or male partners with sperm concentration &amp;lt;1 million/ml. Statistical analyses included chi-square and Student’s t-tests for group comparisons. Logistic regression adjusted for confounders was used to analyze live birth rates (LBR). Main results and the role of chance The fertilization and blastulation rates were similar in both groups with PGT and no-PGT-A, respectively p = 0.24 and p = 0.49.The mean euploidy rate per recipient was 75.3% in the PGT-A group.No statistical differences were reported for age of the donor type of endometrial preparation (natural/artificial), endometrial thickness, and days of endometrial preparation.Regarding the sperm parameter in the PGT-A, the sperm concentration (mil/mL) and sperm motility was lower than no-PGT-A (p &amp;lt; 0.001).The live birth rate was not different in the PGT-A group 39.9% (CI95%35.31-44.74) vs no-PGT-A 42.9% (40.95-44.87), p = 0.27.The days to reach a live birth was higher in the group with PGT-A 65.5 (CI 95% 44.31-86.77) than no PGT-A 49.7 (CI95%42.52-56.95), p = 0.48.The pregnancy rate was lower in the PGT-A group 53.3% than in no-PGT-A 62.3% (p &amp;lt; 0.01), while no statistical differences were reported for the clinical pregnancy rate 52.33% (CI95% 47.72-56.92) vs 56.6% (CI95%54.79-58.50), p = 0.08.The miscarriage rate calculated on the pregnancy rate was 21.37%(CI95%16.44-27.00) in the PGT-A group vs 22.44%(CI95% 20.46-24.53) in the no-PGT-A group, p = 0.76.The multivariate analysis adjusted for several confounders (patients and oocyte age, BMI, male age, sperm characteristics, day of embryo transfer, endometrial thickness, PGT-A) confirmed that these factors do not influence the live birth rate. Limitations, reasons for caution The nature of the retrospective study and two different laboratories used for the PGT-A represent the principal limitation of the study. Wider implications of the findings PGT-A testing in donor oocyte-recipient cycles does not improve the chance for live birth nor decrease the risk of miscarriage. The use of PGT-A in the donor cycle does not reduce the time to reach a live birth. Further large studies are required to confirm these results. Trial registration number No

Study question How does female age affect ratio of patients with at least one euploid embryo (≥1EE) and at least one mosaic embryo without any euploid ones(≥1ME)? Summary answer Percentage of patients receiving mosaic embryo transfers increases with female age. What is known already Introduction of Next Generation Sequencing (NGS) to the area of Preimplantation Genetic Testing for Aneuploidy (PGT-A) has enabled detection of mosaicism in human embryos. Studies reporting the birth of healthy babies after the transfer of embryos with a chromosomal mosaic result on PGT-A have been published (Greco et al., 2015; Kahraman et al., 2020; Viotti et al., 2021 and 2023). Data suggested lower implantation rates and higher miscarriage rates when mosaics were compared with euploid embryo transfers. The percentage of mosaic embryos in overall tested embryos decreases with increased maternal age, similar to euploid embryos’ decline (Sanders et al., 2023). Study design, size, duration This retrospective study includes 7287 cycles with PGT-A performed between January 2017 and December 2023. Number of biopsied blastocysts was 21,277 and all were tested with NGS. All PGT-A testing was conducted on Ion Torrent S5 (Thermo Fisher Scientific) according to the manufacturer guidelines. The mosaic embryo reporting thresholds were determined as 20 to 80%. Participants/materials, setting, methods Euploidy ratio (ER) was defined as (euploid embryos (n)/ all tested embryos(n)). Mosaicism ratio (MR) was defined as (mosaic embryos (n)/all tested embryos(n)). Alternative mosaicism ratio (AMR) was defined as the number of mosaic embryos divided by the sum of euploid embryos and mosaic embryos. In other words, AMR can be defined as the number of mosaic embryos divided by non-aneuploid embryos. Female ages were divided into five categories: FAG1:&amp;lt;35, FAG2:35-37, FAG3:38-40, FAG4:41-42, FAG5:&amp;gt;42. Main results and the role of chance The number of cycles, total number and average biopsied embryos with standard deviations were highest in FA1 and declined with AFA: FAG1:2537, 9298, 3.7±2.2; FAG2:1348, 4140, 3.1±1.7; FAG3:1701, 4298, 2.5±1.6; FAG4: 958,2116, 2.2±1.5; FAG5:738, 2407, 1.9±1.2. ER and MR were observed to decrease with AFA: FAG1:%48.4, %15.2; FAG2:%39.0, %12.7; FAG3:%26.5, %10.5; FAG4:%14.9, %6.0; FAG5:%5.0, %2.6. This decrease was statistically significant (p &amp;lt; 0.0001, Chi-Square Test). However, when AMR was calculated among age groups, AMR was increasing with AFA: FAG1:%23.9; FAG2:%24.6; FAG3:%28.5;FAG4:%28.7; FAG5:%34.5 and this was also statistically significant (p = 0.0002, Chi-Square Test). Correlation of MR and AMR with female age groups were tested with Pearson correlation coefficient and MR was found to be negatively correlated with AFA whereas AMR was positively correlated (r=-0.19, p &amp;lt; 0.0001; r = 0.07, p &amp;lt; 0.0001, respectively). The ratio of patients with ≥1EE and ≥1ME was found to be decreasing with AFA when compared to overall tested cycles: FAG1: %83%, %7; FAG2: %69, %9; FAG3: %47, %11; FAG4: %28, %9; FAG5: %9, %4. However, the ratio of ≥ 1ME in cycles with transferrable embryos significantly increased with AFA consisting of more than one fourth of embryo transfer cycles above 38 years of age: FAG1: %7; FAG2: %12; FAG3: %19; FAG4: %24; FAG5: 31%. Limitations, reasons for caution These are the results of a single ART and Reproductive Genetics Center with its strictly defined standard operating procedures for PGT-A testing and ovarian stimulation protocols. Although not expected, different PGT-A testing methodologies or stimulation protocols may influence the above-mentioned outcomes. Wider implications of the findings The alternative mosaicism ratio defined here is generally overlooked but impacts the treatment strategies of ART patients, specifically for the advanced maternal age groups. Trial registration number not applicable