Embryo Mosaicism Rate in National Referral Hospital of Indonesia Detected Using Next-Generation Sequencing: A Retrospective Study.

The primary objective underlying the practice of preimplantation genetic testing for aneuploidy (PGT-A) is to enhance the overall success rate of in vitro fertilization (IVF) procedures. This is accomplished by strategically sidestepping the transfer of embryos afflicted by aneuploidy, an aberration characterized by an abnormal number of chromosomes. By meticulously identifying and excluding such embryos, PGT-A aims to elevate the likelihood of achieving a successful pregnancy outcome following IVF. Next-generation sequencing (NGS) technology is currently available, which provides better resolution of chromosome analysis and may detect mosaicism in chromosomes as low as 20% ( 1 ). Mosaicism is defined as the occurrence of two or more karyotypically distinct cell lines in a single embryo ( 2 ). Unlike uniform aneuploidy which is caused by failure of meiosis affecting the entire embryo, mosaicism usually occurs due to failure of mitosis of 2 or more cell populations in one embryo ( 3 ). The mechanism of mosaicism formation comprises anaphase lagging, endoreplication, non-disjunction, chromosome demolition, cytokinesis errors, or cell fusion ( 4 ). Generally, mosaicism can be classified into several groups. The first group is aneuploid mosaicism, which refers to the combination of different aneuploid complements. The second group, diploid-aneuploid mosaics, comprises both normal and aneuploid cells. It typically occurs due to mitotic errors in a cell derived from a diploid zygote. Next are ploidy mosaics, alternatively referred to as mixoploid embryos, which consist of cells with varying haploid chromosome numbers. Such mosaics may encompass a combination of haploidy, diploidy, and polyploidy. Lastly, chaotic mosaics, exhibit a significant irregularity pattern, with many chromosomes being impacted and each cell carrying a random chromosome set ( 5 ). With the use of NGS technology, more and more mosaic embryos are found nowadays ( 6 ). Several recent studies have shown that mosaic embryos could still result in live births but with lower rates of pregnancy and higher rates of pregnancy loss than euploidy embryos ( 6 , 7 ). However, it is still unclear what type of mosaicism is related to it, the levels of mosaicism, the type, and the number of chromosomes involved ( 8 ). Studies showed that mosaicism degrees affect the success rate of IVF, but several other studies did not support the conclusion ( 9 - 11 ). Several factors are thought to play a role in the emergence of embryo mosaicism. It is still debatable whether maternal age affected embryo mosaicism. Some publications showed that maternal age was not correlated with a higher prevalence of mosaicism ( 12 , 13 ). However, other publications found that embryo mosaicism was higher in advanced maternal age compared to the younger population (less than 30 years old) ( 14 , 15 ). This finding was also confirmed by another study that showed complex mosaicism increased with maternal age ( 16 ). Other studies have also shown that embryo mosaicism was related to the male sperm factor especially segmental mosaicism ( 17 , 18 ) and the sex of the embryo ( 19 ). The results of each center on embryo mosaicism may vary from center to center. The incidence of mosaicism varies from 2-40% ( 20 , 21 ). Although the majority of clinics reported an incidence of mosaicism of 5-10% ( 14 , 19 , 22 , 23 ), the existing ambiguity and widespread occurrence necessitate a comprehensive investigation to ascertain the existence of a correlation between clinical conditions and embryo mosaicism. As far as the researcher’s knowledge, this study is the first exploration within Indonesia’s research population, delving into the prevalence of embryo mosaicism. The focus encompasses the involved chromosomes and their characteristics, filling a significant gap in current knowledge.

Of the 196 embryos assessed from 61 patients, 106 (54.1%) had chromosomal abnormalities ranging from low mosaicism to whole chromosome aneuploidy. Low (12.8%) and high (4.1%) mosaicism rates were found in 25 and 8 embryos, respectively, with the overall incidence of mosaicism being 16.3%. We found 75 (38.3%) whole chromosome aneuploidy and no segmental aneuploidy case in all 196 embryos assessed. Details of the chromosomal abnormalities found can be seen in Table 1. Chromosomal abnormalities of embryos assessed Descriptive analysis using the IBM SPSS Statistics Program version 25. We conduct the classification of the affected chromosomes by mosaicism and aneuploidy. The most common chromosomes affected by mosaicism were chromosome 9, 8 and 6, while the most abnormalities (n=20, 10.2%) were found in chromosome 21. Table 2 shows the complete profile of chromosomes affected by the abnormality. Chromosomal abnormalities based on chromosome number Data are presented as n (%). Descriptive analysis using the IBM SPSS Statistics Program version 25. We also compared maternal and paternal age, embryo sex, and indication of PGT-A toward chromosomal abnormalities. There was no significant difference between mean maternal age in low mosaicism, high mosaicism rate, and normal chromosome (33.88 vs. 35 vs. 33.26 years old, respectively). Still, there was a statistically significant difference in mean maternal age (35.84 vs. 33.26 years) between aneuploidy (monosomy or trisomy) and normal chromosomes. The paternal age is statistically higher in the group with chromosomal abnormalities than in the normal group. We detected a significant difference in high mosaicism rates for patients with unexplained infertility (P<0.05). Table 3 shows all the detailed information regarding factors that may affect chromosomal abnormalities. Chromosomal abnormalities and their characteristics *; P<0.05, Mann-Whitney U test and **; P<0.05, Fisher-Exact test. Data are presented as n (%).

In the current investigation, upon including all samples, the observed incidence proportions of embryo mosaicism were found to be 16.3%. This value was notably higher (6.99%) compared to the findings reported by Heiser et al. ( 19 ) and the study conducted by Rodrigo et al. ( 14 ). The prevalence of mosaicism in IVF procedures for assisted reproduction is generally low, with rates estimated to be approximately 4 to 5% of trophectoderm biopsy samples. These levels of mosaicism have been found to have minimal impact on the accuracy of PGT-A diagnosis ( 24 ). The range of mosaicism may vary across laboratories due to the NGS approach use. In this study, we choose 30% as the threshold for determining mosaicism. The determination of the mosaicism threshold may affect the rates of mosaicism diagnosed besides clinical factors. A slight difference in determining the threshold of mosaicism (20 vs. 30%) might result in a significant difference in mosaicism rate and decision in selecting the embryo for transfer. The previous study found that with the use of a 30% threshold of mosaicism, it was found that 8.4% of embryos had mosaicism. This number was significantly different when the threshold was modified to 20% (20.2% embryos affected) ( 25 ). This threshold (30%) was also chosen due to the consideration that potential technical reasons might affect the result including contamination, amplification bias, or biopsy technique ( 26 ). Despite the high threshold of mosaicism copy numbers, we found a mosaicism rate of 16.3%. Compared to the international result, the incidence of mosaicism varies from 2-40% ( 20 , 21 ) with the majority of clinics reporting an incidence of mosaicism of 5-10% ( 14 , 19 , 22 , 23 ). Hence, our number might be considered relatively high with the use of a 30% threshold of mosaicism copy number. Our study’s elevated prevalence of mosaicism may be attributed to unexplained infertility causes, including genetic abnormalities. Additionally, it explained the increased prevalence of high mosaicism in the group with unexplained infertility indications for PGT-A. Nevertheless, the chromosomal abnormalities observed in our investigation, specifically whole chromosome aneuploid embryos (monosomy or trisomy), exhibited a similar pattern to a prior study conducted in Brazil ( 19 ). We found no segmental aneuploidy in 196 embryos examined. This result differs from the study by Heiser et al. ( 19 ) which analyzed biopsy samples from 166 IVF clinics across Brazil. This may be due to the low number of samples in comparison to those who used 106,777 trophectoderm biopsies and found 5.30% segmental aneuploidy. Our study revealed a positive correlation between decreasing chromosome size and the occurrence of complete aneuploidy (whole chromosome aneuploid). Notably, chromosomes 13-22 exhibited the highest frequency of complete aneuploidy. Previous studies have reported higher rates of aneuploidy in chromosomes with sizes less than 90 Mbp, suggesting that smaller-sized chromosomes are more prone to errors during cell division ( 27 ). Inter-chromosomal heterogeneity arises from structural variability, including differences in arm lengths and centromere sizes ( 28 ). Mosaicism in our study was found to occur with the highest frequency on chromosomes 9, 8, and 6. In contrast, the preceding study identified chromosomes 20, 22, and 19 exhibiting the highest mosaicism prevalence ( 19 ). According to the findings of Osman et al. ( 29 ), chromosomes 22, 4, and 19 are the ones that exhibit mosaicism the most frequently. Nakhuda et al. ( 13 ) also noted a greater occurrence of mosaicism on chromosomes 21, 22, and 2. Furthermore, Chuang et al. ( 27 ) found a higher incidence of mosaicism on chromosomes 14, 1, and 9. The absence of a widespread agreement on the chromosomes most commonly impacted by mosaicism may be attributed to variations in the methodology employed for PGT-A and the specific criteria utilized by different study centers to identify mosaicism. The probability of embryonic aneuploidy is influenced by increasing maternal age. For complete aneuploidy (monosomy and trisomy), we observed a slight but significant increase in mean maternal age. We also found that embryos with chromosomal abnormalities had a slight increase in mean paternal age. However, we discovered no significant difference in the mean maternal age between mosaic and normal embryos. Rodrigo et al. discovered a higher prevalence of high-degree mosaicism and complete aneuploidies in older women and men ( 14 ). Furthermore, compared to younger patients, women over 37 had only slightly lower mosaicism ( 30 ). Mitotic errors after fertilization cause chromosomal mosaicism during the first embryonic cleavages ( 31 ), while chromosomal segregation defects cause complete aneuploidies during meiotic division for gamete formation ( 32 ). The previous study demonstrated that maternal age can independently affect mosaicism. For each one-year rise in maternal age, the risk of low and high mosaic concerning whole aneuploidy increases ( 19 ). Before undergoing PGT-A, patients must be informed of its advantages, disadvantages, and limitations ( 33 ). Couples should be informed that PGT-A only evaluates trophectoderm cells and cannot diagnose an embryo’s chromosomal condition. The laboratory’s reported mosaicism frequency, challenges in interpreting results from clinical and technical perspectives, and limited data on transfer procedure outcomes like congenital anomalies and other adverse perinatal outcomes should be discussed during pre-test counseling. In certain situations, such as when there is a lack of euploid embryos following an IVF/PGT-A cycle, with or without PGT for monogenic disorders (PGT-M) or PGT for structural rearrangements (PGT-SR), couples may consider transferring mosaic embryos during post-test counseling ( 33 ). The analysis of mosaic embryo transfer data is problematic for post-test counseling. In a multicenter prospective study, Capalbo et al. ( 34 ) transferred 484 euploid embryos, 282 low mosaic embryos (20-30% aneuploid cells), and 131 moderate mosaic embryos (30-50% aneuploid cells). The researchers discovered that poor/low-grade mosaics had an 11% miscarriage rate and moderate mosaics had a 12.7%. Viotti et al. ( 35 ) conducted a retrospective multicenter analysis from 2015 to 2020, comparing the transfer outcomes of over 5,500 euploid and 1,000 mosaic embryos. The mosaics were categorized as less than 50% or greater than 50%. The researchers found that mosaics have different miscarriage rates depending on the defect. The low mosaic embryos could be considered to be transferred if there were no other embryos left after informed consent. Additionally, mosaics at higher levels may have a higher risk of unfavorable outcomes than those at lower levels. It is important to note that this claim lacks data. The level of mosaicism seems to be a better indicator of success than the chromosome involved ( 34 ). Research by Viotti et al. ( 35 ) and Capalbo et al. ( 34 ) discovered that low-level mosaicism is capable of enhancing clinical outcomes. Thus, embryos with the lowest mosaics have promising embryo transfer potential and should be carefully examined. However, prenatal testing should follow PGT-A or IVF for every pregnancy. The couple must also receive genetic counseling before deciding on prenatal testing ( 36 ). In summary, the prevalence of mosaicism seen at our center exhibits a comparatively elevated level in relation to findings reported in other investigations. The chromosomes most often involved in mosaicism were 9, 8, and 6. A notable disparity was seen in the prevalence of high mosaicism rates among cases of unexplained infertility indications for PGT-A. Further study is required to expand the study, including a comprehensive nationwide investigation to better assess Indonesia’s mosaicism rate.

This study involved a retrospective investigation of trophectoderm embryo biopsies obtained during PGTA procedures carried out between 2020 and 2022. We analyzed 196 embryos for this study. This study was conducted in the Human Reproduction, Infertility, and Family Planning cluster of the Indonesian Medical Education and Research Institute, Faculty of Medicine, University of Indonesia. The Ethics Committee of the Faculty of Medicine, Universitas Indonesia, has approved this study under document number KET-1591/UN2.F1/ ETIK/PPM.00.02/2023. NGS was performed on trophectoderm biopsies obtained from blastocysts at day 5 or 6. All the embryos that underwent the biopsy were from fresh embryos. After the embryos had been biopsied, the embryos were frozen on day 5 or 6. The embryos were stored in the nitrogen tank for up to two or three months before embryo transfer. VeriSeq library preparation was used to detect each trophectoderm’s chromosomal status according to the manufacturer’s instructions (Illumina, Inc., California, USA). Negative controls from the genetics and embryology labs were also created to ensure no contamination throughout the preparation procedure. All samples underwent library preparation processing, including Whole Genome Amplification (WGA), unpurified Sureplex product quantification, tagmentation, polymerase chain reaction (PCR) amplification, PCR clean-up, normalization and pooling. After that, the samples were sequenced using Illumina’s Miseq NGS. All 24 chromosomes, including the sex chromosomes, might be screened using this technique. BlueFuse Software (Illumina Inc.) was used to evaluate the generated bioinformatics data. The analysis was performed using the human genome reference known as hg19. An embryo is classified as aneuploid when the trophectoderm biopsy reveals a deviation from the anticipated two copies, manifesting as one (monosomy) or three (trisomy). Mosaicism levels can vary between biopsies and may not accurately represent the overall mosaicism in the embryo. Mosaicism detection in a trophectoderm biopsy suggests a heightened likelihood that the embryo exhibits true mosaic characteristics. Samples exhibiting a "low degree of mosaicism," sometimes called low mosaicism, are characterized by aneuploid cells ranging from more than 30% to less than 50% in the biopsy. In cases where the biopsy reveals the existence of aneuploid cells ranging from more than 50% to less than 70%, it is referred to as a "high degree of mosaicism" or simply "high mosaicism." Biopsies exhibiting fewer than 30% of aneuploid cells were classified as euploid, while those demonstrating more than 70% of aneuploid cells were categorized as whole chromosome aneuploid. To classify as mosaic segmental aneuploidy, the partial deletion or duplication size must exceed 10 Mb and exhibit segmental aneuploidy ranging from 50 to 70%. The focal point of the analysis revolved around several key clinical factors, including maternal and paternal age, embryo sex, implicated chromosomes, and the underlying indication linked to the distinct aneuploidy categories. The clinical applications of PGT-A encompass various indications, including aneuploidy screening, male factor infertility, recurrent pregnancy loss (RPL), uterus/ ovarian factor infertility, endometriosis, and unexplained infertility. The analysis involved the examination of biopsies taken from the trophectoderm of blastocysts on day 5 or day 6. The statistical analyses were conducted using the SPSS Statistics program version 25 (IBM, USA). The clinical characteristics of samples from different types of outcomes (low mosaic, high mosaic, euploid, entire aneuploidy, and segmental) were summarized and compared using mean, standard deviation, and proportions. Pairwise Chi-squared comparisons were conducted to analyze the relationship between categorical variables, specifically embryo sex and indication, by comparing proportions. Pairwise t tests were conducted to examine continuous variables, specifically maternal and paternal age, across different group levels.