Fertility decreases with advanced maternal age (Van Voorhis, 2007). This decrease is primarily due to poor egg quality, as successful pregnancies increase significantly in females of advanced maternal age when eggs from young, fertile donors are used (Van Voorhis, 2007). An aneuploid egg is known to be of poor quality, and in humans, egg aneuploidy is associated with advanced maternal age and occurs most often because of chromosome segregation errors at meiosis I (Hassold & Hunt, 2001).
Meiosis in females is particularly error prone, likely a consequence of being protracted and involving two cell cycle arrests. In humans, oocytes enter meiosis during fetal development and arrest in prophase of meiosis I (prophase I). This prophase arrest is maintained until ovulation when, in response to hormonal cues, meiotic resumption occurs. Homologous chromosomes are segregated with completion of meiosis I, and the oocyte arrests again at metaphase of meiosis II (MII) at which point it is called an egg. If the egg is fertilized, MII is completed with separation of sister chromatids. Because the physiologically relevant follicle pool is thought to be nonrenewable, a primordial follicle activated to grow in a woman of advanced maternal age contains an oocyte that has been arrested in prophase I for decades.
Several molecular mechanisms have been proposed to explain the meiotic origins of aneuploidy, including errors in recombination, improper spindle formation and microtubule–kinetochore interactions, and defects in the spindle assembly checkpoint (reviewed in Hunt & Hassold, 2008; Hassold & Hunt, 2009; Eichenlaub-Ritter et al., 2010; Jones & Lane, 2012). Recent findings in mouse suggest that deteriorating chromosome cohesion that occurs during the extended prophase I arrest is also likely a significant cause of age-associated aneuploidy (reviewed in Chiang et al., 2012; Jessberger, 2012; Jones & Lane, 2012). Chromosome cohesion, mediated by a multiprotein cohesin complex, is established along the chromosome arms and at the centromere and serves to keep homologous chromosomes and sister chromatids together until completion of meiosis I and II, respectively (Watanabe, 2005; Holt & Jones, 2009). Current evidence suggests that in the oocyte, cohesins load during S phase prior to recombination during fetal development and that little, if any, turnover occurs after this time (Revenkova et al., 2010; Tachibana-Konwalski et al., 2010). Thus, chromosome cohesion must remain functional for months in the mouse and years in the human to ensure faithful chromosome segregation. In several mouse strains, cohesion function is compromised in eggs from animals of advanced maternal age (Chiang et al., 2010; Lister et al., 2010; Chiang et al., 2011; Merriman et al., 2012). These eggs have reduced levels of chromosome-associated REC8, a meiotic-specific cohesin, resulting in increased inter-kinetochore distances (Chiang et al., 2010; Lister et al., 2010; Merriman et al., 2012). These cohesion changes precede and predict the most commonly observed chromosome segregation errors (Chiang et al., 2010). These findings are also consistent with functional gene deletion studies in which loss of the cohesin component, SMC1β, results in aneuploidy that is exacerbated with age (Revenkova et al., 2004; Hodges et al., 2005).
Although cohesin proteins are conserved between humans and mouse, it is not known whether a similar functional deterioration of these components occurs in the human with advanced reproductive age (Garcia-Cruz et al., 2010). Such studies have been hampered by the difficulty in obtaining mature gametes from reproductively young and older women. Here, we collected a total of 166 oocyte-cumulus complexes (OCCs) from 18 subjects who had their ovaries removed for medical indications (Fig. 1A,B and Data S1, Supporting information). The number of OCCs collected per subject decreased with age, and only one OCC was collected from subjects 40 years or older (Fig. 1B). In addition, fewer OCCs were collected from subjects who had prior cancer therapy compared to untreated subjects of similar age (Fig. 1B). We performed in vitro maturation (IVM) using 112 OCCs from ten subjects and observed that 28.2 ± 5.8% of the oocytes reached MII (Fig. 1C and Data S1). Maternal age did not impact meiotic competence as 29.2 ± 7.5% of oocytes within OCCs from subjects under 30 years resumed meiosis and reached MII compared to 27.2 ± 9.6% from subjects over 30 years (Fig. 1D; P > 0.05). The eggs derived from IVM had characteristic morphology with a small first polar body, a bipolar spindle asymmetrically positioned in the cortex, cortical actin microfilaments that were slightly enriched in the region adjacent to the spindle, and condensed chromosomes tightly aligned on the metaphase plate (Fig. 1E).
We used 18 of the eggs obtained through IVM from a subset of six subjects (ages 16.4, 19.3, 22.5, 27.5, 33.1, and 37.3 years) to assess how chromosome cohesion changes with maternal age. As a readout of chromosome cohesion, we measured the distance between kinetochores of sister chromatids, or the inter-kinetochore distance, in eggs using an in situ chromosome spreading technique (Figs 1F and 2A–F, and Data S1) (Duncan et al., 2009). We found that the average inter-kinetochore distances increased gradually and significantly with subject age (Fig. 2A–F). The average inter-kinetochore distance increased from 0.82 ± 0.03 μm in the youngest subject (16.4 years) to 1.1 ± 0.03 μm in the oldest subject (37.3 years) (Fig. 2E; P < 0.001). In the human, this absolute increase of 0.28 μm in inter-kinetochore distance between age extremes is consistent with data in two mouse strains in which increases of 0.13 and 0.44 μm were reported (Chiang et al., 2010; Merriman et al., 2012).
In addition to measuring inter-kinetochore distances within individual eggs, we were also able to accurately count total kinetochores in > 85% of the eggs examined (16/18) to assess chromosome status. Chromosome segregation errors were observed in 31% (5/16) of the eggs following IVM, and four of these eggs were from the two subjects over the age of 30 (Subject E and F; Fig. 2E–H). Subject F, who was 37.3 years old, had two eggs each with a set of unpaired sister chromatids indicative of a total loss of cohesion (F1 and F2; Fig. 2F,G). In addition to a set of unpaired sister chromatids, egg F1 also had an improperly segregated chromosome pair that was separated from the egg chromosomes and was not associated with the polar body DNA (Fig. 2H). This pair resided within the egg because, without including this pair, egg F1 would be hypoploid. Subject E, who was 33.1 years old, instead had two hyperploid eggs (E3 and E4; Fig. 2F). One egg had an extra chromosome pair (E3), whereas the other had an extra unpaired sister chromatid (E4) (data not shown). Subject B, who was 19.3 years old, had one egg (B1) that had an extra unpaired sister chromatid (Fig. 2F and data not shown). The five eggs with chromosome segregation errors were not restricted to those with the largest average inter-kinetochore distance (B1, E3, E4, F1, and F2; Fig. 2F).
Taken together, these results demonstrate for the first time that in human eggs, there is a maternal-age-associated deterioration of chromosome cohesion as indicated by increased inter-kinetochore distances between sister chromatids. In the oldest two subjects, this increased inter-kinetochore distance was also accompanied by chromosome segregation errors. Interestingly, the majority of the observed segregation errors involved the premature separation of sister chromatids, which is a clear manifestation of a complete cohesion defect. Although the majority of the observed aneuploidies occurred in subjects over the age of 30, there was one instance of aneuploidy in a reproductively young subject, suggesting that factors in addition to age contribute to aneuploidy. The eggs used for these studies were obtained from ovarian tissue, primarily from women with a cancer diagnosis, so future studies to investigate whether similar aging mechanisms are conserved in a noncancer population are warranted. Moreover, understanding whether and how cancer itself and cancer treatments affect chromosome dynamics during meiosis will be important for the field of fertility preservation where such eggs could be fertilized or cryopreserved for a patient’s future use (Fasano et al., 2011).
Deteriorating chromosome cohesion is likely a contributing factor in the age-associated meiotic origins of aneuploidy in the human. If chromosome cohesion in humans is only established during S phase (which happens one time in the development of an oocyte) and little turnover occurs after oogenesis as has been reported in model organisms, it is unlikely that supplementation of exogenous cohesin components will rescue the deterioration (Watanabe et al., 2001; Revenkova et al., 2010; Tachibana-Konwalski et al., 2010). Instead, methods that prevent the deterioration of chromosome cohesion are more likely have a greater impact on reducing the incidence of aneuploidy. The findings reported here thus provide important new opportunities for intervention in age-related infertility and are consistent with the progressive aging of an established oocyte pool.