Mechanisms of Aneuploidy

Abstract

Aneuploidy is a very common occurrence in humans and occurs in an estimated 20–40% of all pregnancies. It is the most prominent cause of miscarriages and congenital defects in humans and is the main obstacle to infertility treatment. The vast majority of aneuploidies are caused by maternal meiotic non-disjunction errors. High levels of recombination errors were observed in studies on fetal oocytes. This suggests that some oocytes are more prone to not being separated due to events occurring before birth. Cell cycle checkpoints that work in the meiotic phase and metaphase-anaphase transition work more moderately in women than in men. As a result, while there are abnormal cells that have been sorted out in spermatogenesis, in females these cells can escape the actual control and ultimately give rise to aneuploid eggs.

1. Introduction

Although aneuploidy is a serious health problem, the reasons behind this phenomenon have not been fully confirmed. The development of a comprehensive set of tests is necessary for the evaluation and detection of aneugenic chemicals. The reliability of any aneuploidy analysis is always questioned by the fact that the mechanisms that cause aneuploidy are poorly understood, in part due to the multitude of factors involved in the occurrence of chromosome segregation and nondisjunction [1]. Errors in chromosome segregation during meiosis are usually seen in human oocytes and cause aneuploidy in embryos. These errors increase dramatically in the eggs of older women.

Here, we attempt to summarize recent studies commenting on how progressive disruption of chromosome structures contributes to age-related aneuploidy. In addition, various cellular pathways that cause aneuploidy in oocytes of women of all ages are being investigated. Data from mouse and human oocytes are discussed with emphasis on studies focusing on this phenomenon in humans [2].

2. Meiosis in human oocytes

Meiosis involves two sequential cell divisions in which homologous chromosomes (meiosis I) are separated in the first stage, followed by sister chromatids (meiosis II). In the first meiosis, the homologous chromosomes separate from each other, then the homologous chromosomes are joined together. These connections are established early in oocyte development during the growth of the female fetus in a process called homologous recombination. The maternal and paternal chromosomes are first compressed by the synaptonemal complex and then crossed over. After crossover, new sister chromatids are formed, containing adjacent portions of maternal and paternal sister chromatids. Cohesin complexes that previously linked sister chromatids of each homologous chromosome now link homologous chromosomes together: Cohesin (distal cohesin) distal to the crossover sites connects homologs, while cohesin (proximal cohesin) between crossover sites and centromeres continues to bind sister chromatids [3]. The chromosome configuration that turns out to be two homologous chromosomes is called bivalent. As meiosis I occurs, the bivalents must be oriented on the spindle so that the two sister chromatids contained in each homologous chromosome face the same spindle pole. The kinetochores of sister chromatids must behave as a single kinetochore. It is thought that adding sister kinetochores to a functional location will facilitate this function. Oocytes then enter a state of cellular stagnation called ‘interphase’ in processes spanning different periods of time. The functional units of oocyte and somatic cells in the ovary are called follicles [4, 5, 6]. During storage, the oocytes remain small and are surrounded by a single layer of squamous cell epithelium called the “primordial follicle”. Periodically, some primordial follicles begin to grow. Somatic cells supply the oocyte with macromolecular precursors through gap junctions, and oocyte volume increases significantly. This enrichment of nutrients prepares the oocyte [7, 8] to mature into an egg, which after fertilization can give rise to an embryo.

Oocytes emerge from dictyate arrest after puberty. In the middle of the menstrual cycle, the rise of luteinizing hormone from the pituitary gland causes the oocyte to continue meiosis and mature into a fertilizable egg. First, the nucleus disintegrates and sets of meiotic spindles are formed, which align the chromosomes in meiosis I metaphase. The spindle progresses to the oocyte cortex, where homologous chromosomes separate. One set of homologous chromosomes remains in the oocyte, while the other is extruded into the first polar body formed. Molecularly, the segregation of chromosomes during meiosis I is activated by the cleavage of Rec8, a meiosis-specific subunit of the cohesin complex [9]. Rec8 is cleaved by Separase, which is activated along with anaphase. During anaphase I, only the cohesin in the arm region is broken down so that the chromosomes can separate from each other. Cohesin in the centromeric regions is protected from cleavage by Shugoshin proteins (Sgo), so that sister chromatids stay together during anaphase I. As meiosis II occurs, the second meiotic spindle fuses [10, 11, 12, 13]. The maturing egg has transitioned to the quiescent phase in metaphase II and is transported to the fallopian tube during ovulation. The egg waits to complete its second meiosis until it is fertilized by the sperm. As the second meiosis continues, the Sgo proteins migrate to the kinetochores, and in anaphase II, the cleavage of the centromeric cohesin takes place [14, 15, 16, 17]. In order to complete meiosis, the sister chromatids of the remaining chromosomes, the oocyte and the second polar body, must be formed. Chromosomes from the oocyte and sperm separate as the pronuclear envelope and then stand ready for the first mitotic division of the embryo. The embryo then divides into a multicellular blastocyst and implants in the uterus to develop further [18, 19, 20].

3. Types of aneuploidy in oocytes

Recent technological advances have increased the chances of catching aneuploidy in eggs or in the early stages of embryonic development. In pre-implantation genetic diagnosis, an embryo may sometimes be biopsied and analyzed for genetic abnormalities to select healthy embryos for implantation. As an alternative to this technique, testing oocytes can minimize the need to test embryos. In particular, polar bodies can be used to determine the cytology of an oocyte without damaging it [21, 22]. The use of polar bodies for aneuploidy detection in IVF applications also facilitates embryo selection before implantation [23, 24]. Genetic analysis of both polar bodies can accurately detect aneuploidy in mature oocyte because all chromosomal copies are extruded into polar bodies [25, 26, 27]. For example, an excess chromosome in the first polar body indicates loss of the homolog of that chromosome in the oocyte after meiosis I, while an incorrect chromosome number in the second polar body indicates a chromosome segregation error in meiosis II. On the other hand, the second polar body is formed only after fertilization. Chromosomes from biopsied polar bodies are best previously analyzed by fluorescent in situ hybridization (FISH). Although widely used for embryo selection, clinical applications of FISH are only informative for a particular chromosome and results may be inaccurate [28]. New, more sensitive methods such as Sequence Comparative Genome Hybridization (aCGH) and next-generation sequencing (NGS) platforms provide improved statistics for aneuploidy prevalence and better characterization of segregation errors [29].

Two classical ways that have been suggested to account for chromosome segregation errors in meiosis are nondisjunction (NDJ) and premature separation of sister chromatids (PSSC). For NDJ, homologous chromosomes or sister chromatids cannot separate at meiosis I or meiosis II, respectively. Similar segregation errors are seen between meiosis I and II, although meiosis II error rates have sometimes been reported to be higher.

This can be explained by the fact that errors that can be seen in meiosis I occur in meiosis II, because early cleavage sister chromatids can separate correctly in meiosis I, while errors are observed later in meiosis II.

Surprisingly, PSSC mutations in meiosis I could be corrected by a ‘balance’ error during meiosis II: if both the first and second polar bodies share mutual errors (for example, a loss in the polar body first followed by a second gain in the polar body; or vice versa) the resulting oocyte will have the correct number of chromosomes [30, 31].

Chromosome pairs 15, 16, 21, and 22 are the chromosomes that most commonly contribute to human aneuploidies, but data on the contributions of other chromosomes are lacking due to limited statistical information for types of aneuploidy. Frequently, an oocyte will experience simultaneous errors involving more than one chromosome, suggesting that some oocytes are susceptible to global dysfunction. This effect is also evident in embryos where up to 42% of detected aneuploidies contain more than one chromosome [32, 33].

However, the etiology of embryonic aneuploidy is more complex, as errors can also occur from sperm or during rapid mitotic divisions in embryogenesis [34, 35]. Advances in single-cell whole genome amplification (WGA) allow unprecedented characterization of genomic content within polar bodies.

Analyzes of the genomes of polar body-oocyte and polar body-embryo triplets (i.e. a biopsy of an oocyte or embryo fused with first and second polar bodies) revealed an alternative mechanism of segregation, termed ‘reverse segregation’ [36].

Reverse segregation occurs when sister chromatids separate at meiosis I so that there are no homologous chromosomes.

Reverse segregation results in the correct number of chromosome cells. The chromatid pairs, the copies inherited by the oocyte and first pole body, have different parental origins and are heterozygous at the centromeres.

After meiosis I, their connection is broken and during metaphase II, alignment problems may occur in the spindle fibers. In one study; although it was the most observed error in number, reverse segregation was detected in less than 10% of the triples analyzed [36]. Interestingly, all of the donors participating in this study produced at least one oocyte or embryo that underwent reverse segregation. The oocytes included in this study were obtained from women aged 33–41 years. A similar study examining oocytes from younger donors aged 25–35 years reported that no reverse segregation was observed [26, 36].

4. Causes of aneuploidy increasing with age

Women experience a gradual decrease in their ability to get pregnant as they age. Loss of reproductive ability usually occurs approximately 10 years after the age of 35. Meiotic chromosome segregation errors increase very clearly in women of this age group. A large-scale cytogenetic analysis examining more than 20,000 human oocytes by FISH reported that aneuploidy occurred in 20% of oocytes retrieved from women aged 35 years, increasing to approximately 60% in women over 43 years of age [37].

Current studies with aCGH have confirmed that the rates of aneuploidy increase dramatically in oocytes from older women [23, 27, 38, 39, 40]. Conservation of bivalents is crucial for correct chromosome segregation. However, recent studies in human oocytes reveal that the structure of bivalents is prone to fragmentation in oocytes of older women.

In mice and humans, two major structural defects occur with increasing age in bivalents. First, sister kinetochores disperse over long distances, which is incompatible and often associated with incorrect attachment to the meiotic spindle. Second, the bivalents formed in senescent oocytes are more often separated into individual chromosomes, called univalents. Univalent pairs may split uncoordinatedly and may also contribute to aneuploidy. Interestingly, it is possible for both defects to result in an inverse decomposition pattern, as we will discuss below [41, 42, 43, 44].

Sister chromatids in mouse and human oocytes lose compatibility with age, which can cause misalignment of bivalents in meiosis I.

Loosely related sister chromatids may no longer function properly as they align on the meiotic spindle. In human oocytes, separated sister kinetochores tend to form more merotelic attachment to spindle microtubules.

In addition, other age-related factors may promote defective kinetochore-microtubule attachments.

Excessive segregation of sister kinetochores in human oocytes causes bivalents to take on unexpected alignments in the meiotic spindle. In a newly defined bivalent configuration called ‘inverted bivalents’, the bivalents are rotated to the spindle axis: the sister chromatids of a homologous chromosome misalign and misalign, linking microtubules at opposite spindle poles instead of orienting them to the same spindle pole, as in mitosis [45, 46, 47, 48].

Both half and fully inverted bivalents occur. Only one pair of sister chromatids is attached to opposite spindle poles in semi-inverted bivalents, while both pairs are attached to opposite poles in fully inverted bivalents. Reverse bivalents have been observed more frequently in oocytes from older females and are associated with increasing distances between sister kinetochores. Since sister chromatids are oriented separately on the spindle, similar to mitosis, fully inverted bivalents can lead to an inverse pattern of segregation. Bivalents also sometimes appear bent along their axis because homologous chromosomes rotate relative to each other, which can put more pressure on the already weakened cohesion.

The age-related loss of balance applicable to bivalents is not limited to the pericentromeric regions surrounding the kinetochores. There is also danger in the harmony that connects homologous chromosomes. Homologous chromosome pairs in bivalents often remain separated by large gaps in oocytes of aged mouse and human females. These and similar structural defects are indicative of decreased compatibility between bivalent homologous chromosomes. In more complex cases, bivalents sometimes divide earlier into two separate chromosomes (univalents) before anaphase I. The prevalence of univalents increases exponentially with age, occurring in 40% of oocytes in women older than 35 years and 10% of oocytes in women aged 30–35 years. In mouse oocytes, univalent alignment problems can cause chromosome separation errors. Univalents in mouse and human oocytes could also align on the first meiotic spindle, similar to mitotic chromosomes, with both sister kinetochores facing opposite spindle poles. This can create a mitosis-like pattern of segregation and result in reverse segregation: equal segregation of both univalents into sister chromatids will result in the correct chromosome number acquired by the oocyte and the first polar body, but the chromatids will originate from different parental origins. However, the sister chromatids have been divided much earlier and could not be properly aligned to the spindle at metaphase II.

The molecular mechanisms that may cause these dramatic developments in chromosomal organization in human oocytes, which change with advancing age, are still unresolved. However, studies in mice have clarified the loss of cohesin as a major contributor to age-related aneuploidy. Cohesin complexes containing Rec8 in mouse oocytes are already present during DNA replication in the early stages of meiosis. After fertilization, they are thought to be renewed when DNA is replicated again in the embryo. Therefore, the cohesin complexes must remain in place during the prolonged period of dictation arrest to ensure correct chromosome segregation in meiosis. Rec8 levels are severely reduced in bivalents of oocytes from naturally aged mice [48, 49, 50, 51].

5. Conclusions

Fertility declines gradually as women age, and by midlife women begin to lose their ability to produce healthy eggs. Meiotic chromosomes experience increased age-related structural changes that can result in increased Error rates in chromosome segregation. Newly described processes have been identified in human oocyte structures that may explain the emergence of an alternative form of segregation. Conducted studies will better reveal why oocytes are often defective, leading to age-related infertility. Recent studies have reported that meiosis in mammalian females is inherently error-prone, leading to high aneuploidy and sterility. The cellular pathways responsible for chromosome separations are prone to error and affect females of all ages.

Acknowledgments

I would like to thank my supervisors Hakan Gurkan for all his help and advice.

Conflict of interest

The authors declare no conflict of interest.

Acronyms and abbreviations

IVF	in-vitro fertilization
FISH	fluorescent in situ hybridization
a CGH	array Comparative Genome Hybridization
NDJ	nondisjunction
PSSC	premature separation of sister chromatids
WGA	whole genome amplification