Aneuploidy Rates Inversely Correlate with Implantation during In Vitro Fertilization Procedures: In Favor of PGT

Aneuploidy, the hold of an abnormal number of chromosomes that differs from the normal karyotype, is a recognized leading cause of miscarriage and congenital disabilities. In human gametes and embryos, aneuploidy rates are prevalent, and these rates increase with advanced maternal age; additionally, it has been suggested that hormonal stimulation for achieving in vitro fertilization (IVF) protocols further increases aneuploidy rates. Although about 65% of chromosomally abnormal embryos culminate in spontaneous miscarriages, there is still evidence of live births harboring crucial aneuploidies. Furthermore, although some frequent aneuploidies are consistent, others differ between countries, making it harder to focus on a specific set of anomalies but vital to focus regionally on those more prevalent. Preimplantation genetic testing (PGT) is a highly endorsed technique in assisted reproductive treatments to evaluate possible embryo aneuploidies, genetic defects, and congenital disorders. On this subject, this study shows that IVF aneuploidy rates in embryo cohorts of high morphological quality are inversely associated with implantation rates. In its entirety, this study reinforces the utility of PGT for embryo evaluation.


Introduction
Aneuploidy is defined as a chromosome number that is not an exact multiple of the usually haploid number [1]. The terms haploid and diploid that describe single (n) and double (2n) chromosome sets in cells originate from the Greek terms haplóos meaning single and diplóos meaning double. The term ploidy was subsequently derived to describe the total chromosome content of cells. Consequently, the term euploid refers to a chromosome with an exact multiple of the haploid number [2]. Human body cells (somatic cells) are diploid, carrying two complete sets of chromosomes: one set of 23 chromosomes from their father and one set of 23 chromosomes from their mother; the two sets combined provide a full complement of 46 chromosomes. Human gametes (or sex cells), sperm and oocytes, are haploid and contain only one set of 23 chromosomes.
Aneuploidies can occur either by chromosome gains (trisomies) and losses (monosomies) due to chromosome segregation errors, the so-called "whole chromosomal" aneuploidy or due to rearrangements of chromosomal parts, often accompanied by deletions, amplifications, or translocations of large regions of the genome that is referred to as a "structural" or "segmental" aneuploidy [3]. Whole chromosomal aneuploidies might arise due to random and sporadic chromosome missegregation events that occur with low frequency during any cell division. The missegregation levels range from 1/1000 to 1/10,000 in human cells [4].
Meiosis generates haploid gametes through a specialized cell division process that consists of one round of DNA replication followed by two cell divisions. The first division, or meiosis I (MI), involves the segregation of homologous chromosomes from each other, whereas meiosis II (MII) involves the segregation of the sister chromatids. Missegregation can also occur in germline cells, and the errors that arise in meiosis result in aneuploid embryos [5]. This chapter aims to provide evidence that supports the use of PGT for embryo evaluation and euploid embryo selection due to a positive correlation with fertilization rates.

Incidence of aneuploidy
Errors in meiotic chromosome segregation frequently occur during oogenesis ($20%), especially during the first meiotic division; this incidence of meiotic errors in oocytes is more elevated in women with advanced maternal age and may be due to the prolonged time that oocytes spend arrested at meiosis I stage, before ovulation [6]. However, some patterns of nondisjunction appear to be chromosomespecific; almost all cases of trisomy 16 are linked to errors at maternal MI, while MII errors are surprisingly common in trisomy 18. Oppositely, in sperm the incidence of aneuploidy is only 2%. Another considerable percentage of errors ($20%) arise during the first mitosis after fertilization. Among clinically recognized spontaneous abortions (fetal deaths occurring between 6 and 8 weeks and 20 weeks gestation), the incidence increases to $50% [7]; the most common specific abnormalities are sex-chromosome monosomy (45,X), accounting for nearly 10% of all spontaneous abortions, and trisomies 16, 21, and 22, which together constitute 50% of all trisomies identified in spontaneous abortions. The incidence among stillbirths (fetal 1 Pure duplication 1q41-qter: further delineation of trisomy 1q syndromes Partial duplication 1q: reports of five cases and review of the literature  [27] deaths occurring between $20 weeks gestation and term) is $4% with the types of abnormality being similar to those identified in newborns, and $0.3% of live-born are aneuploid with the most common abnormalities being trisomies 21, 18, and 13 and sex-chromosome trisomies 47,XXX, 47,XXY, and 47,XYY [5,8].

Aneuploidies and live births
Although about 65% of chromosomally abnormal embryos culminate in spontaneous miscarriages, there is still evidence of live births harboring crucial aneuploidies.

Impact of aneuploidy on the efficiency of ART
Assisted reproduction is a solution in many of the growing cases of infertile couples worldwide. A high rate of embryos produced in vitro presents chromosomal aneuploidy ($50%), and such aneuploid embryos have reduced the potential for achieving a viable pregnancy. Such abnormalities are recognized as the leading cause of implantation failure and spontaneous miscarriage [60]. Among conceptions that survive to term, aneuploidy is the leading genetic cause of developmental Chr, chromosome number. Table 1.
Case reports of live births with complete or partial chromosomal abnormalities (mosaic or multiple aberrations are not considered).
disabilities and mental retardation [5]. Table 2 describes data from different infertility centers predominantly showing that aneuploidy rates are similar. The relatively high aneuploidy rate observed in human embryos after an IVF/ ICSI cycle has been attributed to the technique itself since this prevalence seems to be lower in natural conceptions [61]. Many hypotheses have been proposed that may explain these findings: (1) controlled ovarian stimulation treatments, (2) factors related to the ICSI technique and (3) lab conditions as embryo culture.

Ovarian stimulation and the incidence of embryo aneuploidy
To increase the number of oocytes that can be retrieved for IVF, gonadotrophins are commonly used for superovulation in humans. Exogenous administration of gonadotrophins results in higher concentrations of steroids that may affect oocyte and embryo quality. Ovarian stimulation effects have been well characterized mainly in the murine model and have shown that aggressive stimulation leads to a poorer embryo development potential that could increase the chromosomal abnormality rate [79]. In humans, studies are scarce and less conclusive. A recent study in a population of young normovulatory women showed that a high ovarian response after controlled ovarian stimulation with moderate gonadotropin doses did not increase the embryo aneuploidy rate. Indeed, the higher the ovarian response, the more the euploid embryos obtained [80]; the remaining question is whether this can also be extrapolated to infertile patients with good ovarian reserve.

Intracytoplasmic sperm injection (ICSI) technique and the incidence of embryo aneuploidy
ICSI has become critical for the treatment of severe male infertility. The principal feature of ICSI is the direct injection of spermatozoa into an oocyte, which facilitates the production of fertilized embryos regardless of semen characteristics, such as sperm concentration and motility. However, the chromosomal integrity of ICSI zygotes is degraded compared to zygotes obtained from an in vitro fertilization [81,82]. During the ICSI procedure, a sperm pretreatment is performed to mimic the conditions of natural fertilization and support the progression of fertilization effects. Studies on mouse models revealed that the chromosomal integrity of zygotes derived from ICSI without any pretreatment of spermatozoa was impaired in comparison with zygotes derived from conventional IVF [83]; even the culture sperm conditions may affect the chromosomal stability of the embryo [84]. Chromosomal damage may occur due to the injection of non-capacitated, acrosomeintact spermatozoa, so to reduce the risk of chromosomal aberrations during the ICSI procedure, it is crucial that sperm capacitation and the acrosome reaction be appropriately artificially induced in the proper medium before use [85].

Embryo culture and the incidence of embryo aneuploidy
Fertilization and embryo development in vitro have the potential to introduce (often inadvertently) stress which cannot only impair embryo development in the laboratory but also have downstream effects after transfer.
In vivo, the developing preimplantation embryo is exposed to gradients of nutrients, hormones, cytokines, and growth factors as it progresses through the fallopian tube to the uterus. Within the lumen of the female tract, the embryo resides in a few 100 nanoliters of a complex viscous fluid characterized by high levels of mucins, albumin, and glycosaminoglycans and by reduced levels of oxygen Rate from the previous stage of development blastomere to TE, PBs = polar bodies, TE = trophectoderm. For the current study, infertile patients who underwent ART at the Ingenes Institute were included. The patients were clinically evaluated according to a standardized protocol that includes family and personal clinical history. The protocol was approved by the Ethics Committee of the Ingenes Institute, and a signed informed consent was obtained from all patients. IVF, embryo biopsy, and mCGH were performed according to the standard protocols of the Institute Ingenes as previously described [76,77]. Only optimal morphological embryos were considered for this study. Selection and embryo transfer were done on Day 3 or Day 5 of development according to the embryo morphological assessment, using the criteria established by the Istanbul consensus Workshop on Embryo Assessment [78]. Table 2. Aneuploidy rates of different IVF clinics around the world; when mentioned, the most commonly affected chromosomes are listed.
(typically 2-8%). The embryo is in constant motion, moved by gentle ciliated and muscular action of the female tract [86]. This scenario is in stark contrast to the laboratory environment, where typical gametes and embryos are exposed to relatively large volumes of culture medium, remain static during culture while resting on a polystyrene substrate, and create unstirred layers where the end products of metabolism concentrate and nutrients become limited [87].
Embryos are sensitive to both chemical and physical signals within their microenvironment. Factors within the laboratory as oxygen level, ammonium released from amino acids into the culture, poor laboratory air quality, temperature and pH, oil overlay, embryo culture volume/density, the static nature of culture, light, or even mechanical factors as pipetting, can negatively impact gametes and embryos and generate stress. When more than one stress factor is present in the laboratory, more negative synergies can result, and these factors play a significant role in influencing the development and events post transfer [88]. For example, recent studies have reported that a decrease in temperature has the potential to affect the stability of the oocyte's meiotic spindle, reducing fertilization rates, delaying embryo development, and decreasing clinical pregnancy rates [89]. However, more studies are needed to demonstrate the impact of embryo culture on aneuploidy rates.

Aneuploidy detection: techniques for PGT
PGT is the genetic diagnosis analysis performed to identify euploid embryos before uterine transfer [90]. PGT determines the numeric chromosomal constitution of a cellular biopsy sample obtained from a cultured embryo to determine its competence [91,92].
PGT was first described in 1990 by Handyside et al. [93] when the sex of the sixto eight-cell stage embryos from two couples with a known risk of transmitting Xlinked diseases was assessed by DNA amplification of a Y chromosome-specific repeat sequence. The earliest PGT studies in the 2000s were based on the fluorescence in situ hybridization (FISH) technique where 3-12 chromosomes can be analyzed on the cleavage stage or polar body biopsies [90]. Those studies had disappointing results in clinical practice since it had no beneficial effect on live birth rate after IVF [94]. The major drawback of FISH-based PGT is the limited number of chromosomes that can be analyzed considering that aneuploidy can affect any of the 22 autosomes and both sex chromosomes [95]; consequently, there have been dramatic improvements in PGT technology to make it valuable for clinical practice.
Nowadays, several methodologies for 24-chromosome analysis are available for clinical use that aim to increase implantation rates and decrease miscarriage rates associated with IVF [90]: microarray comparative genomic hybridization (mCGH), single-nucleotide polymorphism (SNP) microarray, real-time polymerase chain reaction (qPCR), and next-generation sequencing (NGS) [96,97]. This review will focus on the relevant aspects of the PGT techniques used in our laboratory.

Microarray comparative genomic hybridization (mCGH)
mCGH is a ratio labeling protocol to compare the DNA product of a clinical sample to a healthy control. For PGT, biopsied embryonic cells must be lysed to extract the sample's DNA, which will be further amplified by a protocol that provides whole genome coverage [90,95,98]. The resulting DNA products are co-hybridized with a standard DNA control sample (46,XY and 46,XX) with a series of site-specific fluorophores on a microarray chip with approximately 4000 markers spaced throughout the genome [90]. Then, a confocal laser platform detects the relative color intensity, and a bioinformatics compares the intensity of each fluorophore in the sample versus the control to identify any bias and determine the ploidy status of the sample [90,95,98].
The mCGH analysis reports the ratio of sample DNA to a reference DNA, as a chromosomic profile where the molecular karyotype is represented. Usually, the sample DNA is labeled with a green fluorescent dye, while the reference DNA sample is tagged red [99]. Thus, diploid embryos will have a relatively equal ratio of green-to-red fluorescence in every pair of chromosomes, represented as a continuous horizontal plot line. Monosomy will be represented as a clear downward deviation in the plotted line, indicating a relative lack of green-to-red signal intensity; in contrary, a trisomy will be displayed as an upward deviation in the plotted line due to a relative increase in the green-to-red signal intensity.
The specificity rate of mCGH-based PGT is about 99% [90]. The test results can be available within 12-15 h, considering that the entire analysis can be performed during this short time frame [90,91]. Additionally, brand-specific features are offered by each manufacturer: Agilent's GenetiSure Pre-Screen Microarray offers a detection rate of 100% for aberrations >10 Mb and 89% for >5.3 Mb [100]; KaryoLite BoBs Kit from Perkin Elmer uses an alternative BACs-on-Beads technology and results are interpreted by the BoBsoft™ analysis software [99]; and RHS's EmbryoCellect Kit is the only mCGH-based PGT validated for mosaicism detection [101]. Recently, Illumina's 24sure PGS Microarray had been discontinued, and the NGS-based VeriSeq PGS is now offered as an alternative solution [102]. mCGH entails some disadvantages: first, the embryo sample requires a previous whole genome amplification (WGA) process to support single-cell diagnostics by mCGH [95], raising the possibility of introducing errors during the amplification [91]; second, mCGH is a semiquantitative technique that only reports the ratio of sample DNA to a reference DNA; it is to say that only imbalances in DNA content can be identified. Therefore, mCGH is unable to detect uniparental disomic or triploid embryos as it cannot discriminate between 46,XX from 69,XXX, and 46, XY from 69,XXY [90,91,95]. Last, the mCGH used for PGS cannot identify structural chromosome aberrations or diagnose mosaicism in a trophectoderm sample [90].

Next-generation sequencing (NGS)
NGS refers to the emerging technology of non-Sanger-based DNA sequencing that allows the sequence in parallel millions of DNA strands with high-throughput yield. In the field of ART, this powerful tool is being applied for PGT to replace cytogenetic microarrays [98,102].
Different platforms are commercially available for NGS with different technological approaches. Illumina's MiSeq NGS platform applies a sequencing-bysynthesis method, where DNA is attached and amplified in situ to be subsequently used as a template for synthetic sequencing with fluorescent-labeled reversibleterminator nucleotides [103]. Ion Torrent NGS technology, commercialized by ThermoFisher Scientific, is based on collecting data by sensing the hydrogen ions that are released as by-products when nucleotides are incorporated by a templatedirected DNA polymerase synthesis on an ion chip [104].
Despite the dissimilarities between platforms, the common basis of chromosome copy number analysis by NGS is the fragmentation of the amplified DNA sample into small segments of 100-200 base pairs that are further sequenced in parallel until the number of reads covering a determined position in the genome is attained, in general, a 30Â coverage (sequencing each base pair 30 times) ensures sufficient accuracy. The sequence data obtained are then compared with a reference genome and counted by bioinformatics software. The copy number of a specific chromosome should be proportional to the number of counted sequences; therefore, an increase or reduction in the number of reads will, respectively, represent a trisomy or monosomy [97,99].
NGS allows to simultaneously perform both qualitative and quantitative analyses of multiple embryos with high-resolution data for chromosomal analysis [96,97]. The higher sensitivity and precision offered by NGS [96,105,106] makes possible to exclude embryos with mosaicism [105,106] and partial aneuploidies or triploidies [106], improving pregnancy outcomes due to its enhanced capability for detecting those challenging abnormalities.
PGT by NGS can predict not only chromosome copy number for the diagnosis of whole chromosome aneuploidy with 99.98% assignment consistency [97] but also single-gene disorders [107], abnormalities of the mitochondrial genome [108], and segmental chromosome imbalances [97,99]. Balanced chromosomal rearrangements cannot be detected by NGS [97].
The increasing demand and accelerated development are continuously reducing the cost of NGS technology [109]. Also, potential cost-benefit ratios can be achieved when the full sequencing capacity of the apparatus is exploited [96,97,99]. Furthermore, molecular tools, like barcoding, are being implemented to allow multiplex high-throughput sequencing [110]; this promising strategy will reduce the diagnosis' cost per patient by performing simultaneous analysis of multiple embryos from different patients [97].

Aneuploidy and women age
In our study, by analyzing the mCGH data, the total number of aneuploidies was found to be 734, and from these, 641 (87.3%) were derived from patients and 93 (12.7%) from donors. Overall, this study displayed similar rates of monosomies, trisomies, double aneuploidies, and multiple aneuploidies. The total number of monosomies (191) was similar to the number of trisomies (194), accounting for 26 and 26.4% of the total aneuploidies, correspondingly. Furthermore, the total number of double (165) and multiple (184) aneuploidies was also very similar, accounting for 22.5 and 25.1% of the total aneuploidies, correspondingly. Nevertheless, it is worth noticing that when considering only the donor group, monosomies seem to be more prevalent: 38.7% of the total donors' aneuploidies were monosomies vs. 24.7% of trisomies, 16.1% of double aneuploidies, and 20.4% of multiple aneuploidies; what is more, the percentage of monosomies in the donor group is higher than that of the monosomies of the patient group (38.7 vs. 24.3%). The most common monosomies affected chromosomes 15, 16, and 22, whereas the most common trisomy affected chromosomes 16, 19, and 21 ( Table 3).
It has been shown that the lowest risk for embryonic aneuploidy is between ages 26 and 30, with aneuploidy rates steadily increasing with maternal age after 26 years of age [111] and leaping significantly from the age of 39 [112]. For this reason, women of advanced maternal age are encouraged to favor oocyte donation to yield high-quality viable embryos.
Interestingly, some studies have identified that women of younger ages possess an increased prevalence of aneuploidy, with >40% of aneuploidy in women of 23 years and under [111] and 58% of aneuploidy in women of <31 years of age. In the current study, both the donor (≤29 years) and the patient group of ≤29 years displayed high aneuploid rates, 28.5 and 27.4%, respectively ( Table 4). Given the high rates of aneuploidy in younger women, attention should be paid in detecting aneuploidy in embryos from women of young maternal age, especially since this group of patients is not routinely encouraged to perform a PGT. Still, whether there is a difference between the distribution of aneuploidies between donors and patients remains uncertain.
When stratifying our analysis in age groups (a, ≤29; b, 30-34; c, 35-37; d, 38-40; e, 41-43; and f, ≥44 years of age), a visible continuous increase in aneuploidy rate can be observed as maternal age increases (Table 4); furthermore, this increase in aneuploidy goes hand in hand with a continuous decrease in implantation, as it can be observed in the decrease of positive beta-human chorionic gonadotropin (β-hCG) values as age increases (β-hCG values ≥10 mUI/ml from Day 14 after transference were considered positive).   Table 4.
Comparative of aneuploidy and pregnancy rates between age groups.

Remarks
One of the most critical reasons for unsuccessful IVF procedures is implantation failure due to aneuploid embryos. Aneuploidies are the primary cause of perinatal death and genetic abnormalities; consequently, the detection of chromosomal disorders constitutes the most frequent indication for PGT. Here, we report on the aneuploidy rates found in IVF procedures in Mexico. Even though there are studies that assert that PGT does not improve pregnancy rates, we show that aneuploidy rates inversely correlate with implantation and that levels of aneuploidy among high morphological quality embryos are still an important issue to be faced in everyday ART practice, and this evidence works in favor of continuing to use PGT analysis.