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

Elizabeth Schaeffer, Leonardo Porchia, Almena López-Luna, Dinorah Hernández-Melchor and Esther López-Bayghen

## Abstract

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.

Keywords: aneuploidy, preimplantation genetic testing, embryo implantation, in vitro fertilization, karyotype

## 1. 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.

## 2. 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

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

Case reports of live births with complete or partial chromosomal abnormalities (mosaic or multiple aberrations

Chr Case report References

[29] [30]

[31] [32]

[42] [43]

8 Trisomy 8: report of four cases [28]

11 Partial 11q trisomy syndrome: two cases [33, 34] 12 Clinical report of a patient with de novo trisomy 12q23.1q24.33 [35] 13 Trisomy 13, Patau's syndrome: reports of three cases [36–38] 14 Partial proximal trisomy 14 [39] 15 Duplication of distal 15q: reports of 14 cases [40, 41]

17 A 790 kb chromosome 17p13.3 microduplication: case report/literature review [44, 45] 18 Trisomy 18, Edward's syndrome: reports of five cases and discussion [46–48] 19 Three cases of trisomy 19 [49, 50] 20 20q11.2 duplication syndrome and pure trisomy 20p [51, 52] 21 Cardiovascular and general health status of adults with trisomy 21 [53] 22 Trisomy 22 syndrome: a report of four cases in newborns and literature review [54–56] X Fragile X syndrome: a case report/review of clinical and molecular diagnoses [57, 58] Y Morphology and pathogenesis of 47,XYY/47,XY patients super male syndrome [59]

9 Pure 9p trisomy derived from a terminal balanced unreciprocal translocation

Proximal 10q duplication in a child with severe central hypotonia

Aneuploidy Rates Inversely Correlate with Implantation during In Vitro…

16 Partial trisomy/long arm of chromosome 16: case report/review of literature

10 Distal 10q trisomy with copy number gain in chromosome region 10q23.1–10q25.1

Trisomy 9: review and report of two new cases

DOI: http://dx.doi.org/10.5772/intechopen.81884

Complete trisomy 16: a case report

Although about 65% of chromosomally abnormal embryos culminate in sponta-

neous miscarriages, there is still evidence of live births harboring crucial aneu-

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

and sex-chromosome trisomies 47,XXX, 47,XXY, and 47,XYY [5, 8].

ploidies. Table 1 describes cases that are well documented.

3. Impact of aneuploidy on the efficiency of ART

2.1 Aneuploidies and live births

Chr, chromosome number.

are not considered).

Table 1.

39


Aneuploidy Rates Inversely Correlate with Implantation during In Vitro… DOI: http://dx.doi.org/10.5772/intechopen.81884


#### Table 1.

of 46 chromosomes. Human gametes (or sex cells), sperm and oocytes, are haploid

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

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

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

Chr Case report References

3 Duplication 3p syndrome: reports of three cases and review of the literature [15–17]

5 Trisomy 5p: reports of four cases report and review of the literature [21–23]

Partial duplication and duplication region 4q28.3-qter in monozygotic twins with

[9] [10, 11]

[12, 13]; [14]

[18] [19, 20]

[24] [25]

[26] [27]

1 Pure duplication 1q41-qter: further delineation of trisomy 1q syndromes Partial duplication 1q: reports of five cases and review of the literature

6 De novo "pure" partial trisomy (6)(p22.3!pter): case report/review

Familial trisomy 6p in mother and daughter

7 Interstitial de novo tandem duplication of 7 (q31.1-q35) New case of pure partial 7q duplication

missegregation levels range from 1/1000 to 1/10,000 in human cells [4].

selection due to a positive correlation with fertilization rates.

2. Incidence of aneuploidy

2 Duplication 2q2.1-q3.1

4 Patient with trisomy 4p

38

discordant phenotypes

Partial trisomy 2q: two cases

and contain only one set of 23 chromosomes.

Modern Medical Genetics and Genomics

Case reports of live births with complete or partial chromosomal abnormalities (mosaic or multiple aberrations are not considered).

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].

#### 2.1 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. Table 1 describes cases that are well documented.

### 3. 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 disabilities and mental retardation [5]. Table 2 describes data from different infertility centers predominantly showing that aneuploidy rates are similar.

N samples (country)

150 (Japan)

5879 (USA)

240 (Mexico, Center A)

210 (Mexico, Center B)

404 (Mexico)

15,169 (USA)

2204 (UK)

21 sets (Italy)

1025 (Mexico)

\*

#

+

Table 2.

41

chromosomes are listed.

No data

No data

3 47.6+ 5 or 6 80++

Percentage of the total number of samples.

Workshop on Embryo Assessment [78].

Percentage of the total number of aneuploid samples.

Rate from the previous stage of development, PBs to blastomere.

Day of biopsy Aneuploidy rate

DOI: http://dx.doi.org/10.5772/intechopen.81884

5 47.8

Trisomy %

Aneuploidy Rates Inversely Correlate with Implantation during In Vitro…

52 (UK) 5 40.4 51.3# 48.7# 22, 16, 15, 18,

6 61.1 5.55\* 10\*

0/1 97.4 Chromosomal loss three

195 (USA) 0/1 65.5 39.86# 60.14# 22, 13, 15, 16,

Monosomy %

5 40.6 18\* 21.3\* 15, 22, 21, 16, 18 4, 12 [63]

3 70.6 No data No data No data No data [66]

87 (India) 3 54 14.9\* 42.5\* 22, 18 No data [62]

12 (UK) 3 75 22\* 11\* 20, 21, 22 6 [65]

759 (UK) 3 64.6 40# 60# 16, 22, 21, 4, 5 4, 6 [67] 274 (US) 3 72.3 39.8\* 44.5\* 22, 16, 7 6, 9, 19 [68] 192 (Italy) 5 55.2 37.9\* 42.7\* No data No data [69]

5 36.3 5.34\* 5\* 16, 22, XXX, 9 3, 7, 8, 10, 12,

5 48.9 15.96\* <sup>4</sup>\* 15, 16, 21, 4 1, 2, 3, 5, 8, 9,

17, 20, 22, X, Y 6 43.1 14.65\* 5\*

No data No data No data 13, 15, 16, 18,

3 83 49# 51# 22, 16, 19, 21, 13 5 58 47# 53# 22, 16, 15, 21, 19

> times more frequent than gain

5/6 45.2 52# 48# 22, X, 16, 18, 21

3/5 43.9 59.3# 40.7# 16, 21, 22, 19,

++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

Aneuploidy rates of different IVF clinics around the world; when mentioned, the most commonly affected

60.89 No data No data 4, 15, 22, 16 No data [71]

0 74 56# 44# 16, 21, 22, 15, 19 No data [73]

19, 21, 22

19, 21

15, 20

Most affected chromosomes

21, X

Less affected chromosomes

1, 2, 5, 10, 17, 19

18, 20

10, 11, 12, 14,

22, 15, 16, 17 No data [74]

1, 12, 3 [72]

6, 5, Y, 3 [75]

8, 4, 3, 2, 7, 1 Current

study

Ref.

[64]

[70]

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.

#### 3.1 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.

### 3.2 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].

#### 3.3 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


#### Aneuploidy Rates Inversely Correlate with Implantation during In Vitro… DOI: http://dx.doi.org/10.5772/intechopen.81884

\* Percentage of the total number of samples.
