Mechanisms Of Aneuploidy In Human Eggs

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Review

Mechanisms of Aneuploidy in Human Eggs Alexandre Webster1 and Melina Schuh1,* Eggs and sperm develop through a specialized cell division called meiosis. During meiosis, the number of chromosomes is reduced by two sequential divisions in preparation for fertilization. In human female meiosis, chromosomes frequently segregate incorrectly, resulting in eggs with an abnormal number of chromosomes. When fertilized, these eggs give rise to aneuploid embryos that usually fail to develop. As women become older, errors in meiosis occur more frequently, resulting in increased risks of infertility, miscarriage, and congenital syndromes, such as Down's syndrome. Here, we review recent studies that identify the mechanisms causing aneuploidy in female meiosis, with a particular emphasis on studies in humans. Aneuploidy in Human Oocytes: A Topic of Growing Relevance in Society Over the past century, greater medical knowledge and technological advances have improved the quality and duration of human lives. By contrast, the span of female fertility remains unchanged. A limited reserve of oocytes (see Glossary) is formed before birth, but gradually declines in quality with age, resulting in reduced fertility and an age-related increase in eggs with an abnormal number of chromosomes, termed ‘aneuploidy’. Despite this concern, women more frequently delay having their first child until later in life [1]. In 2013, nearly 43% of live births in the USA were delivered to mothers 30 years of age or older [2]. Consequently, women in their late 30s and 40s experience more miscarriages and pregnancies with fetuses [6_TD$IF]that have severe congenital abnormalities.

Trends Fertility steadily decreases as women age and, by mid-life, women fail to produce healthy eggs. Meiotic chromosomes experience age-related structural changes that may contribute to increasing rates of chromosome segregation errors. Novel error-causing pathways are reported in human oocytes that might explain how a previously undetected alternative segregation pattern arises. Emerging studies provide a better understanding of why oocytes are frequently defective and lead to agerelated infertility. Recent studies have found that meiosis in mammalian females is intrinsically error prone, causing high rates of aneuploidy and infertility. Cellular mechanisms responsible for segregating chromosomes are inefficient, affecting females of all ages.

Errors in chromosome segregation during meiosis occur frequently in human eggs and cause aneuploidy in embryos (Box 1). These errors increase dramatically in the eggs of older women. Here, we review recent work that has shed light on how the progressive deterioration of chromosome structures contributes to age-related aneuploidy. In addition, we examine several cellular pathways that cause aneuploidy in the oocytes of women of all ages. Evidence from mouse and human oocytes is discussed, with an emphasis on studies focusing on humans.

Meiosis in Human Oocytes Meiosis involves two successive[7_TD$IF] cell divisions, in which first homologous chromosomes (meiosis I) and then sister chromatids (meiosis II) separate. The segregation of homologous chromosomes during the first meiotic division requires that the homologous chromosomes become linked with each other. These links are established in the early stages of oocyte development during growth of the female fetus in a process called homologous recombination [3] (Figure 1). The maternal and paternal chromosomes are first zipped together by the synaptonemal complex and then undergo crossover (Figure 1A). After crossover, new sister chromatids are formed that comprise contiguous segments of maternal and paternal sister chromatids. The cohesin complexes that previously linked the sister chromatids of each homologous chromosome now link homologous chromosomes (Figure 1B) [4–6]: cohesin distal to crossover sites (distal cohesin) binds homologs together, while cohesin between crossover sites and centromeres (proximal cohesin) continues

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1 Department of Meiosis, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, D-37077, Göttingen, Germany

*Correspondence: [email protected] (M. Schuh).

http://dx.doi.org/10.1016/j.tcb.2016.09.002 © 2016 Elsevier Ltd. All rights reserved.

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Box 1. What Happens If [4_TD$IF]an Aneuploid Egg Is Fertilized? A healthy human embryo contains a maternal and paternal copy for each of the 22 autosomal (nonsex) chromosomes, in addition to a sex chromosome from each parent for a set of 46 chromosomes (females acquire two X chromosomes, while males inherit one X and one Y chromosome). Errors during chromosome segregation in meiosis lead to aneuploid eggs that carry an incorrect number of chromosomes [90]. If these eggs are fertilized, they give rise to aneuploid embryos. The frequency of chromosome segregation errors during meiosis increases as women age, so that older women more frequently conceive embryos with an incorrect number of chromosomes [96]. Most aneuploidies lead to severe cellular dysfunction since each chromosome encodes[5_TD$IF] up to thousands of genes. Consequently, aneuploid embryos often fail to develop into functional blastocysts and, thus, never implant [97]. In this case, pregnancy is never detected, since pregnancy tests screen for hormones produced only upon implantation. In less severe cases of aneuploidy, a functional blastocyst may form and implant [98]. However, aneuploidies of most chromosomes cause early embryonic lethality, and pregnancy usually terminates within the first trimester [41]. Only a few aneuploidies are viable. Trisomic embryos with three copies of chromosome 13, 18, or 21 can survive full-term pregnancies, although afflicted individuals typically suffer severe developmental syndromes. Down's syndrome caused by trisomy 21 is the most common fetal trisomy occurring in approximately 1 in 500 of pregnancies [99]. Several sex chromosome aneuploidies are also viable but cause developmental disorders, such as Turner's (XO) and Klinefelter's (XXY) syndromes. Females with X trisomy or XYY males are also born with less severe symptoms and often remain undiagnosed [100,101]. New methods are currently emerging to better study the early development of human embryos [102]. The recent introduction of non-invasive prenatal testing (NIPT) technology allows fetal aneuploidy to be routinely detected early in gestation [103–105]. Unlike amniocenteses or chorionic villus sampling, NIPT is a non-invasive technique that only requires a blood sample of the pregnant woman. The fetal DNA released into the maternal plasma can be used to determine the cytogenetics of a fetus. However, when parents learn that their fetus is afflicted with severe congenital disorders due to trisomy, a decision can be extremely difficult [106,107].

to link sister chromatids. The resulting chromosome configuration in which two homologous chromosomes are linked is called a bivalent (Figure 1C). During meiosis I, bivalents need to orient on the spindle so that the two sister chromatids of each homologous chromosome face towards the same spindle pole. The kinetochores of sister chromatids need to function as a single kinetochore. Coupling sister kinetochores into a functional unit is thought to facilitate this function [7]. Coupling has been suggested to involve different mechanisms in different species: the monopolin complex in budding yeast [8,9]; Moa1 in combination with cohesin in fission yeast [10]; and Meikin in conjunction with cohesin in mice (Figure 1D) [11]. There is a homolog of Meikin in humans, but it is unknown whether its function is conserved in human oocytes [11]. The oocytes then enter a state of cellular quiescence termed ‘dictyate arrest’, which may last several decades in humans. The functional unit of oocyte and somatic cells in the ovary is called the follicle. In their storage state, oocytes remain small and are surrounded by a single epithelial layer of flat somatic cells in a so-called ‘primordial follicle’. Periodically, some primordial follicles start to grow. Somatic cells feed the oocyte with macromolecular precursors via gap junctions and the oocyte volume increases dramatically. This enrichment of nutrients prepares the oocyte to mature into an egg that can give rise to an embryo after fertilization [12,13]. Oocytes exit from dictyate arrest after puberty. During the middle of the menstrual cycle, a surge of luteinizing hormone from the pituitary gland causes an oocyte to resume meiosis and mature into a fertilizable egg. First, the nucleus breaks down and a meiotic spindle assembles that aligns the chromosomes in metaphase of meiosis I (Figure 2A). The spindle migrates to the oocyte cortex, where it segregates homologous chromosomes. One set of homologous chromosomes remains in the oocyte, while the other is extruded into the first polar body (Figure 2B). At a molecular level, the segregation of chromosomes during meiosis I is triggered by cleavage of Rec8, a meiosis-specific subunit of the cohesin complex [6]. Rec8 is cleaved by Separase, which is activated during anaphase (Figure 1B) [14]. Only cohesin in the arm region is cleaved during anaphase I, so that the chromosomes can segregate from each other. Cohesin in centromeric

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Glossary Anaphase: the stage of cell division when the spindle segregates chromosomes by pulling them to opposite spindle poles. Blastocyst: a stage in early embryo development that forms before implantation into the uterus. Centromere: the region of a chromosome where the kinetochore is assembled and microtubules attach. Homologous recombination: an event specific to meiosis where DNA of homologous chromosomes is covalently exchanged to produce chromosomes with new allele combinations and that links homologous chromosomes with each other to form a bivalent. Kinetochore: a proteinaceous structure assembled on the centromeres of chromosomes that binds spindle microtubules responsible for pulling chromosomes apart during segregation. Merotelic attachment: a single kinetochore incorrectly bound to microtubules originating from opposite spindle poles. Metaphase: the stage of cell division when spindle microtubules align chromosomes at the metaphase plate between spindle poles before anaphase. Oocyte: a female germ cell that becomes a fertilizable egg after meiosis. Polar body: the product of asymmetric cell division during female meiosis containing discarded genetic material from the oocyte. Premature separation of sister chromatids (PSSC): a chromosome segregation error where cohesion between sister chromatids is lost, permitting them to randomly segregate during meiosis. Sister chromatids: two identical copies of a chromosome replicated during the S phase of the cell cycle. Sperm: a male germ cell produced by meiosis that can fertilize an oocyte. Spindle: a cytoskeletal network comprising microtubules formed between two spindle poles that segregates chromosomes during cell division. Univalent: a type of chromosome comprising a pair of sister chromatids that forms abnormally in meiosis I. Univalents can form by the premature splitting of a bivalent before anaphase I, or if chromosomes fail to undergo homologous recombination.

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Figure 1. Bivalent Chromosomes Are Formed after Homologous Recombination. (A) During fetal development, DNA replication produces two identical sister chromatids for both maternal and paternal homologous chromosomes in primary oocytes. Cohesin complexes are installed along the lengths of sister chromatids, binding them together. Homologous recombination between homologous chromatids covalently exchanges DNA strands to generate chromosomes with reshuffled allele combinations. Crossover sites appear where chromosomes have undergone homologous recombination. (B) Cohesin is a three-subunit protein complex that binds two strands of chromosomal DNA together. During anaphase, Separase cleaves the cohesin subunit, Rec8, allowing chromosomes to segregate. Shugoshin (Sgo) proteins prevent Separase from cleaving Rec8 in pericentromeric regions. (C) The bivalent is formed after homologous recombination. Exchange of DNA between homologous chromosomes causes cohesin distal to crossover sites (distal cohesin) to link homologous chromosomes together. Cohesin between centromeres and crossover sites (proximal cohesin) binds sister chromatids to tightly associate the kinetochores of sister chromatids. Sgo proteins localize to pericentromeric regions and protect proximal cohesin from Separase cleavage. (D) The centromeric regions of sister chromatids in meiosis I. In mouse, cohesin and the meiosis-specific kinetochore protein, Meikin, tightly associate kinetochores. Cohesin complexes are protected from removal by Sgo proteins. In meiosis II, the spindle reconfigures chromosomes, such that Sgo proteins no longer protect cohesin from Separase cleavage, allowing sister chromatids to segregate.

regions is protected from cleavage by Shugoshin proteins (Sgo), so that sister chromatids remain together during anaphase I (Figure 2A) [15–17]. In meiosis II, the second meiotic spindle is assembled. The now mature egg arrests in metaphase II and is transported to the oviduct during ovulation. The egg only completes the second meiotic division upon fertilization by sperm [18]. During the second meiotic division, Sgo proteins relocate to kinetochores, permitting cleavage of centromeric cohesin in anaphase II

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Figure 2. Two Cell Divisions in Meiosis Prepare Oocytes for Fertilization. (A) Oocytes generated during fetal development remain quiescent for decades in dictyate arrest within ovarian follicles. Only after puberty can they be reactivated to complete meiosis. During the menstrual cycle, hormonal signals release the oocyte from dictyate arrest to resume meiosis. Condensed chromosomes (purple) are released into the cytoplasm after nuclear envelope breakdown. Microtubules (green fibers) assemble the meiotic spindle and align bivalents in metaphase I. The spindle migrates to the oocyte cortex. Homologous chromosomes segregate at the onset of anaphase I. Meiosis I is completed when one set of homologous chromosomes is extruded into the first polar body (PB1), while the other remains in the oocyte. Sister chromatids remain bound by cohesin proximal to centromeres, which is protected from removal by Shugoshin (Sgo) proteins. This ensures that sister chromatids remain linked during meiosis II. (B) In meiosis II, a second meiotic spindle aligns sister chromatids and the oocyte arrests in metaphase II. Sgo proteins no longer protect cohesin from cleavage. At this stage, the oocyte is a mature egg and is released from the ovary during ovulation to be fertilized by a sperm.[3_TD$IF] (C) Fertilization triggers anaphase II, where sister chromatids are segregated into the egg and second polar body (PB2).[1_TD$IF] In the zygote, pronuclear envelopes form around the maternal chromosomes of the egg and paternal chromosomes contributed by the sperm. Pronuclei migrate towards each other to unite as the genome of the embryo. The embryo transitions from meiosis to mitosis and divides many times over several days to finally become a blastocyst.

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[16,17,19]. Sister chromatids of the remaining chromosomes segregate into the egg and the second polar body to complete meiosis (Figure 2B). Chromosomes from the egg and sperm become enclosed in pronuclear envelopes, which then coalesce in preparation for the first mitotic division of the embryo (Figure 2C) [18,20]. The embryo then divides into a multicellular blastocyst and implants into the uterus to develop further [18].

Types of Aneuploidy in Human Oocytes Recent technological developments have increased the ability to detect aneuploidy in eggs or during the early stages of embryonic development (See Outstanding Questions). During preimplantation genetic diagnostics, cells of an embryo can sometimes be biopsied and screened for genetic abnormalities to select healthy embryos for implantation [21]. Testing of oocytes can be helpful and may minimize the need to test embryos. In particular, polar bodies can be used to determine the cytology of an oocyte without causing it harm [22]. The use of polar bodies to diagnose aneuploidy for in vitro fertilization (IVF) treatments also improves embryo selection before implantation [23,24]. Analysis of both polar body genomes can correctly diagnose aneuploidy in mature eggs, because all but one set of chromosomal copies are extruded into polar bodies [25–27]. For example, a gain of an extra chromosome in the first polar body indicates a reciprocal loss of that chromosome in the oocyte after meiosis I, while an incorrect chromosome count in the second polar body implies a chromosome segregation error in meiosis II. However, the second polar body is extruded only after the egg is fertilized. Chromosomes from biopsied polar bodies were previously best analyzed by fluorescence in situ hybridization (FISH). Although still widely in use for embryo selection, clinical implementations of FISH only detect a subset of chromosomes and diagnosis is often inaccurate [28]. Newer, more sensitive methods, such as array Comparative Genome Hybridization (aCGH) [23,24,27,29–31] and next-generation sequencing (NGS) platforms [26,32,33], provide improved statistics for the prevalence of aneuploidy and better characterization of segregation errors. These findings have enhanced our understanding of how chromosome segregation errors arise in meiosis (Box 2). Two classical pathways 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 fail to segregate in meiosis I or meiosis II, respectively (Figure 3A). However, several studies have reported that many aneuploidies in meiosis I comprise gains or losses of single chromatids, but not pairs of chromatids that would instead indicate NDJ [34–36]. This finding helped establish PSSC as a model where sister chromatid pairs split from one another to independently (and often incorrectly) segregate during anaphase I (Figure 3A). Indeed, recent cytogenetic analyses of polar bodies suggest that errors caused by PSSC are more common than those caused by NDJ [27,37,38]. Segregation errors occur at similar rates between meiosis I and II, although meiosis II error rates are sometimes reported to be higher [27,38,39]. This could be explained by a fraction of meiosis I errors only appearing in meiosis II, since prematurely separated sister chromatids might segregate correctly in meiosis I, but experience errors later in meiosis II. Interestingly, PSSC errors in meiosis I can be rescued by a ‘balancing’ error in meiosis II: if both the first and second polar bodies share reciprocal errors (e.g., a loss in the first polar body followed by gain in the second polar body; or vice versa) the resulting oocyte will have a correct chromosome count [27,40]. Chromosomes 15, 16, 21, and 22 are among the most common contributors to human aneuploidy, although data on the contributions of individual chromosomes vary between studies, which may be due to limited statistics for each type of aneuploidy [27,30,37,41]. Frequently, an oocyte will experience simultaneous errors involving multiple chromosomes, suggesting that some oocytes are susceptible to global dysfunction [27,38]. This effect is also

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Box 2. How Can Scientists Study Meiosis in Human Oocytes? Human oocytes normally mature within the body where they cannot be accessed. However, human oocytes are routinely obtained and fertilized in vitro for assisted reproduction. To obtain these oocytes, women are most often treated with hormones that promote the growth of follicles and maturation of eggs. The oocytes are then collected from the ovaries by follicle aspiration. Oocytes that are obtained for in vitro fertilization are in different stages of meiosis. Typically, only those oocytes that have completed the second meiotic division with a metaphase II spindle, which are considered mature eggs, are used for IVF treatment. Oocytes that are immature are not suitable for fertilization because they still contain a full set of unsegregated chromosomes. Instead, they can be used to study how meiosis progresses in human oocytes and to investigate the causes of aneuploidy. There may be concerns that oocytes obtained from couples experiencing difficulty to conceive are more likely to be defective. However, this potential problem can be circumvented by focusing studies on oocytes that were obtained for treatment of male factor-related infertility. The quality of oocytes should not be affected for these couples. It is also difficult to assess how similar eggs obtained after hormonal stimulation are to eggs that have matured in vivo during a natural cycle [108]. Are the error rates in natural cycles different from the error rates in super-ovulated eggs? Studies in mouse oocytes have established that oocytes matured in vivo and in vitro are similar, but also reveal a few differences [109]. The large number of babies that are born from these eggs illustrates that they are capable of giving rise to viable embryos [110]. However, a detailed comparison for human oocytes is hindered by the fact that in vivo matured human oocytes are difficult to obtain since they are typically used for IVF treatments, and because oocytes cannot yet be studied as they mature within ovaries. For these reasons, oocytes obtained by hormonal stimulation for assisted reproduction are currently the best cells to study to learn more about aneuploidy in human eggs and the causes of the maternal age effect.

apparent in embryos, where up to 42% of detected aneuploidies involve multiple chromosomes [42,43]. However, the etiology of embryonic aneuploidy is more complex, since errors may also be derived from sperm [44–46] or during the rapid mitotic divisions in embryogenesis [47,48]. Recent advances in single-cell whole-genome amplification (WGA) allow for unprecedented characterization of genomic content within polar bodies [26,49]. Genome analyses of polar body-oocyte and polar body-embryo trios (i.e., an oocyte or embryo biopsy combined with first and second polar bodies) revealed an alternative segregation mechanism termed ‘reverse segregation’ (Figure 3A) [29]. Initially proposed more than 30 years ago as ‘balanced predivision of sister chromatids’ [50], reverse segregation had, until recently, remained undetected. Reverse segregation occurs when sister chromatids, but not homologous chromosome, segregate in meiosis I. While reverse segregation leads to a correct number of chromosomal copies inherited by the oocyte and first polar body, chromatid pairs have different parental origins and are heterozygous at their centromeres. They remain unlinked after meiosis I and may experience problems aligning to the spindle during metaphase II. In one study, reverse segregation was detected in less than 10% of analyzed trios, although it was the most represented error observed [29]. Interestingly, of the donors that participated in this study, all produced at least one oocyte or embryo that experienced reverse segregation [29]. Indeed, the oocytes included in this study were obtained from women between 33 and 41 years of age. A similar study examining oocytes of younger donors between 25 and 35 years of age did not report reverse segregation [26].

Age-Related Causes of Aneuploidy Women experience a gradual loss in the ability to become pregnant as they become older. Infertility is often reached from the age of 35 to about 10 years later. Meiotic chromosome segregation errors increase sharply in this age window. A comprehensive cytogenetic analysis examining more than 20 000 human oocytes by FISH reported that aneuploidy occurs in 20% of oocytes from 35-year-old women and increases to nearly 60% of oocytes from women over 43 years of age [39]. Recent studies utilizing aCGH confirmed that the rates of aneuploidy dramatically increase in oocytes from older women [23,27,30,37,38]. What are the pathways that might cause this age-related decline in female fertility?

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Figure 3. Chromosome Segregation Errors in Meiosis. (A) Three main classes of segregation error. In meiosis I, homologous chromosomes are normally segregated into the oocyte and first polar body (PB1). Nondisjunction (NDJ) errors occur when chromosome segregation fails. Premature separation of sister chromatids (PSSC) results in sister chromatids incorrectly segregating into the oocyte and PB1. The newly identified reverse segregation pattern occurs when sister chromatids (but not homologous chromosomes) are segregated in meiosis I. Note that a correct number of chromosome copies are inherited by the oocyte but are of different parental origins. However, the chromatids are no longer linked, which may hinder correct alignment and segregation in meiosis II. (B) The kinetochores of meiotic chromosomes can sometimes be incorrectly attached to spindle microtubules (green fibers). For correct alignment, amphitelic attachments are formed when a kinetochore binds spindle microtubules from a single spindle pole. Chromosomes to be segregated bind spindle microtubules from opposite poles. Incorrect merotelic attachments occur when a kinetochore binds microtubules from opposing spindle poles. (C) Meiotic spindles in human oocytes sometimes form multipolar intermediates that may lead to error-prone merotelic attachments. A kinetochore can become attached to opposing spindle poles (purple fibers), when the multipolar intermediate reforms into a bipolar spindle. Chromosomes with merotelic attachments fail to segregate correctly since they tend to lag behind while being pulled to both poles in anaphase. This may cause them to become trapped in the wrong cell after division. Vice versa, the separation of sister kinetochores in human oocytes may also facilitate the formation of merotelic attachments. This may contribute to spindle reorganization and instability, reflecting attempts of the spindle to correctly attach the chromosomes.

The integrity of bivalents is crucial for accurate chromosome segregation. However, recent work in human oocytes reveals that the structure of bivalents tends to be disintegrated in the oocytes of older women [51–54]. In both mice and humans, bivalents experience two major structural defects with age (Figure 4A). First, sister kinetochores separate by large distances, which correlate with the discordant and often incorrect attachment of sister kinetochores to the meiotic spindle. Second, bivalents from aged oocytes more frequently split into individual chromosomes, called univalents. Pairs of univalents can segregate in an uncoordinated manner and may also contribute to aneuploidy. Interestingly, it is possible for both defects to result in a reverse segregation pattern, as we discuss below. Sister Kinetochore Separation Promotes Incorrect Alignment to the Meiotic Spindle Sister chromatids in mouse and human oocytes lose cohesion with age, which may lead to incorrect alignment of bivalents in meiosis I [51–57]. The kinetochores of sister chromatids within

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Figure 4. Structural Changes and Alternative Alignments of Meiotic Chromosomes. (A) A model for the agerelated structural changes bivalent chromosomes in mammalian oocytes might experience. Left panel: a bivalent from the oocyte of a young female. Distal cohesin binds homologous chromosomes together and proximal cohesin associates the kinetochores of sister chromatids to orient together. Shugoshin (Sgo) proteins localize to both pericentromeric regions of sister chromatids and protect proximal cohesin from removal in anaphase I. Right panel: a bivalent in the oocyte of older females. Distal cohesin is lost and homologous chromosomes separate. Proximal cohesin deteriorates and sister kinetochores separate by large distances. Sgo is less able to protect cohesin in pericentromeric regions. (B) Bivalents take on unusual alignments in the meiotic spindle. For correctly aligned bivalents, each pair of sister chromatids align to the same spindle pole. Half-inverted bivalents form when one pair of sister chromatids is attached to microtubules from opposing spindle poles, leading to unbalanced segregation patterns. In fully inverted bivalents, both pairs of sister chromatids are bound to opposite spindle poles and may lead to a reverse segregation pattern. (C) Twisting of homologous chromosomes in the bivalent might strain cohesion between chromosomes. (D) In some bivalents, homologous chromosomes are separated by large gaps. This might indicate weak cohesion between homologous chromosomes. In extreme cases, bivalents can split into two univalents. (E) Univalents form when bivalents prematurely split into pairs of homologous chromosomes. Univalents in human oocytes often align on the meiotic spindle where sister chromatids bind microtubules from opposite spindle poles, which might lead to reverse segregation.

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bivalents separate by a mean distance of 0.2–0.4 mm in oocytes of young mice, and this distance can double in aged mice [54,56,58]. Recent observations found that, in women of all ages, sister kinetochores of bivalents can separate by as much as 2.0 mm, and the frequency of separated sister kinetochores greatly increases with maternal age [51–53]. Sister chromatids that are loosely associated may no longer function correctly when aligning on the meiotic spindle. In human oocytes, sister kinetochores that segregate tend to form more merotelic attachments to spindle microtubules (Figure 3B) [52]. In addition, other age-related pathways might promote defective kinetochore–microtubule attachments [59]. The extreme separation of sister kinetochores in human oocytes allows bivalents to take on unexpected alignments in the meiotic spindle. In a newly identified configuration of bivalents termed ‘inverted bivalents’ [52], the bivalents are rotated to the spindle axis: the sister chromatids of a homologous chromosome incorrectly orient as in mitosis, binding microtubules from opposing spindle poles instead of orienting towards the same spindle pole (Figure 4B). Both half- and fully inverted bivalents are observed. Only one pair of sister chromatids bind opposite spindle poles in half-inverted bivalents, while both pairs are bound to opposing poles in the fully inverted bivalents. Inverted bivalents are more frequently observed in oocytes from older women and correlate with increased distances between sister kinetochores [52]. Fully inverted bivalents could lead to a reverse segregation pattern, because the sister chromatids are oriented apart on the spindle, similar to mitosis (Figure 4B) [29,52]. Bivalents also sometimes appear twisted along their axis because homologous chromosomes rotate with respect to each other, which might exert further strain on already weakened cohesion (Figure 4C) [52]. Bivalents with Decreased Distal Cohesion Can Prematurely Split into Univalents Age-related cohesion loss within bivalents is not restricted to pericentromeric regions surrounding kinetochores. Cohesion linking homologous chromosomes is also compromised. Homologous chromosomes within bivalents are frequently separated by large gaps in the oocytes of older mouse and human females [52,54,60]. These structural defects indicate weak cohesion between homologous chromosomes in the bivalent. In more extreme cases, bivalents sometimes prematurely split into two individual chromosomes (univalents) before anaphase I (Figure 4D) [52,54,60]. The prevalence of univalents increases dramatically with age, occurring in 40% of oocytes from women older than 35 compared with 10% of oocytes from women 30–35 years of age [52]. In mouse oocytes, problems aligning univalents may contribute to chromosome segregation errors [60]. Univalents in mouse and human oocytes can also align on the first meiotic spindle with both sister kinetochores facing towards opposite spindle poles, in a similar manner to mitotic chromosomes (Figure 4E) [52,54,61–63]. This could lead to a segregation pattern similar to mitosis and result in reverse segregation: equal segregation of both univalents into sister chromatids will result in a correct number of chromosomes acquired by the oocyte and first polar body, but the chromatids will be of different parental origins (Figure 4E) [29,52,54]. However, the sister chromatids are already split and cannot align correctly to the spindle in metaphase II. The molecular mechanisms that might cause these dramatic age-related changes to chromosome architecture in human oocytes are still unknown (see Outstanding Questions). Studies in mice identified cohesin loss as a major contributor to age-related aneuploidy (Figure 4A) [56,64–66]. Rec8-containing cohesin complexes in mouse oocytes are already present during DNA replication in the early stages of meiosis. It is thought that they are only replenished after fertilization, when DNA is replicated again in the embryo [67–69]. Therefore, cohesin complexes must remain in place throughout the protracted period of dictyate arrest to ensure correct chromosome segregation in meiosis. The levels of Rec8 are markedly decreased on the bivalents of oocytes from naturally aged mice [55,56]. Oocytes experience a

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drastic increase in segregation errors when levels of Rec8 on bivalents become nearly undetectable by microscopy [56]. Pericentromeric Sgo2 is also decreased on the bivalents of oocytes from older female mice [55]. This might indicate that proximal cohesins are unprotected from Separase cleavage in meiosis I [60]. While many of the molecular details regarding human meiotic chromosomes are unknown [57], these chromosomes experience similar age-related structural changes to those observed in mouse [51–54]. It will be vital to understand how cohesin is depleted from chromosomes to prevent these mechanisms from acting (see Outstanding Questions) [70].

Age-Independent Causes of Aneuploidy Aneuploidy affects not only eggs from older women, but also those from younger women since eggs produced by young women in the prime years of reproductive potential are still frequently defective. Eggs donated by young women for IVF often produce embryos with aneuploidy [43,71] and many individuals with Down's syndrome are born to mothers under the age of 35 [72,73]. Depending on the study, 3–61% of oocytes from women younger than 30 years of age are affected by aneuploidy [74–76], suggesting that oocytes are intrinsically more prone to errors than are mitotic cells [77] or spermatocytes [45,46] (see Outstanding Questions). What primes human oocytes for aneuploidy? Spindle Assembly Checkpoint[2_TD$IF] in Oocytes Is Less Stringent than in Mitosis Accurate chromosome segregation depends on the correct attachment of spindle microtubules to kinetochores. In metaphase I, bivalent chromosomes align between the two spindle poles (Figure 2A). Each pair of sister kinetochores of a bivalent must be attached to the same spindle pole, while homologous chromosomes must be attached to opposing spindle poles. Kinetochores may form incorrect microtubule attachments, which are destabilized to allow for correct attachments to form [78]. During this process, kinetochores that are unattached to spindle microtubules are sensed by the spindle assembly checkpoint (SAC), which only licenses cells to progress into anaphase if all kinetochores are attached to spindle microtubules [79]. However, mammalian oocytes can proceed through meiosis despite misaligned chromosomes (see Outstanding Questions) [80]. In mouse oocytes, misaligned chromosomes merely delay the progression of meiosis [63,81–83], while correct spindle attachments are sometimes incorrectly destabilized [82,84]. Human oocytes with misaligned chromosomes also progress efficiently into anaphase [52]. Why is the SAC pathway permissive to misaligned chromosomes in mammalian oocytes? The alignment of chromosomes on the metaphase spindle takes several hours in mammalian oocytes. Evidence from mouse oocytes suggests that increasing activity of CDK1 in complex with Cyclin B stabilizes microtubule attachments and deactivates SAC arrest [82,84]. This may explain why oocytes are allowed to progress into anaphase despite chromosome alignment errors. Spindle Instability and Transient Multipolar Spindle Stages Impede Chromosome– Microtubule Attachments Recent work revealed that the spindle in human oocytes is frequently unstable, forming transient multipolar intermediates that correlate with segregation errors [85]. Somatic cells undergoing mitosis nucleate spindle microtubules from two centriole-containing centrosomes. Through a related mechanism, spindles assemble from acentriolar microtubule organizing centers (aMTOC) in mouse oocytes [86]. Human oocytes are devoid of prominent microtubule organizing centers and instead nucleate microtubules from chromosomes and kinetochores [85]. Microtubules project outward from chromosomes and undergo extensive reorganization as they assemble into a spindle (see Outstanding Questions). Spindles are often unstable and tend to fragment into multiple poles [85]. Interestingly, multipolar spindles have been implicated in the

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aneuploidy of cancer cells [87,88]. Transient multipolar stages are suggested to promote merotelic attachments, which occur when kinetochores bind to spindle microtubules from multiple spindle poles (Figure 3B). Indeed, merotelically attached chromosomes are common in human oocytes, and might form during the reorganization of transient multipolar spindles (Figure 3C) [85]. Spindle instability and merotelic attachments may also be favored by the specialized organization of sister kinetochores in human oocytes, which are often separated by large gaps instead of being closely associated, even in oocytes from young women [51–54]. Compared with bivalents in which each pair of sister kinetochores form single microtubule attachment sites, extensive error correction and spindle reorganization may be required to correctly align bivalents with four sister kinetochores interacting independently to spindle microtubules. Therefore, separated sister kinetochores may increase the probability of abnormal kinetochore microtubule attachments. Homologous Recombination Influences Chromosome Cohesion in the Bivalent Suboptimal positioning of crossover sites may cause weak association of chromosomes within the bivalent, making them prone to segregation errors [3,89,90]. The placement of crossover sites varies between species and genders. In human females, chromosomes tend to have more crossover sites than in males, and longer chromosomes form more crossovers than shorter ones [26,29,91]. When crossover sites form close to telomeres, reduced amounts of cohesin may link homologous chromosomes, while crossover sites within centromeres may compromise sister chromatid cohesion, or prevent the removal of cohesins, causing NDJ errors [92–95]. Two crossover sites that form in close proximity might also lead to weak cohesion between homologous chromosomes. However, crossover interference was reported in several studies where two crossover sites tended to form further away than what would be expected by chance [26,29,91]. This mechanism may help to decrease the probability that homologous chromosomes are only weakly associated during meiosis I.

Outstanding Questions What are the evolutionary pressures that have shaped mammalian meiosis to be intrinsically prone to chromosome mis-segregation? What are the mechanisms controlling the gradual deterioration of chromosome structures? What are the similarities and differences in the molecular changes observed in bivalents of oocytes from aged mice compared with those from aged humans? Why is the spindle assembly checkpoint less stringent in mammalian oocytes? Why are spindles in mammalian oocytes assembled by different mechanisms compared with mitotic cells? How will new technologies shape human reproduction in the near future?

Concluding Remarks Human oocytes frequently experience chromosome segregation errors in meiosis that result in aneuploidy. As women [8_TD$IF]become older, their oocytes lose the potential to give rise to viable embryos. Several pathways contribute to errors in meiosis. Age-related changes to the structure of chromosomes in oocytes promote incorrect attachments of chromosomes to the meiotic spindle and the premature splitting of bivalents before cell division. What is the cause of these age-related structural changes? In mice, cohesin complexes deteriorate from the chromosomes of oocytes during aging. Whether this is also the case for human oocytes remains to be confirmed. Nevertheless, is the age-related loss of [9_TD$IF]cohesin complexes sufficient to explain the dramatic increase in segregation errors that occurs in oocytes of older women? Indeed, several age-independent pathways also promote errors in meiosis, and insensitivity to meiotic checkpoint activation, instability of the meiotic spindle, in addition to defects in homologous recombination likely compound the risk of chromosome segregation errors in oocytes and aneuploidy in embryos. A greater understanding of aneuploidy in humans may provide opportunities to therapeutically counteract the age-related deterioration of chromosome structures in human oocytes. However, until a treatment is developed, biology obligates women to conceive before fertility is lost. Acknowledgments It was the authors’ intention to write an article accessible to a wide audience while also including as many new and exciting studies as possible. We would like to apologize for any omissions, because these were unintentional or due to space limitations imposed by the journal. We thank Agata Zielinska and other members of the Schuh lab for critical review of this article. M.S. and A.W. have received financial support from the Max Planck Society, the European Research Council under grant agreement no. 337415, and the Lister Institute for Preventive Medicine.

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