Endometrial glands. (a) The superficial endometrium or functionalis has growth and degeneration cycles. Stem cells are located in the basalis and are common ancestors or progenitors of differentiated cells within a gland. (b) Endometrial gland fragments isolated free of underlying stroma (hematoxylin/eosin). (c) Thin gland tubes (patient 3). (d) Glands with closed ends (patient 10). (e) Wide gland tubes (patient 3).

Typical CSX methylation patterns. Each tag is arranged in a 5′ to 3′ horizontal order, with open circles representing unmethylated CpG sites and filled circles representing methylated sites. Eight tags are sampled from each gland. There are eight glands and a total of 64 tags per uterus. Tags are different within and between glands in the same uterus. Below is the 3′ region of the CSX gene, illustrating primer locations (underlined) and CpG sites in the CSX tag (red) and CSX6 tag (yellow). The CSX6 tag has a G/T polymorphism that distinguishes alleles in heterozygous individuals.

Endometrial gland vs. colon crypt CSX methylation. Colon crypt methylation (green circles, data from ref. 9) was linear with age and increased at a slower rate compared with endometrium, consistent with a relatively slower colon stem cell division rate. Tag diversity (unique tags per eight sampled tags) was lower in colon crypts compared with endometrial glands, consistent with a greater ability of endometrial stem cells to persist within a niche. Colon and endometrial diversity was stable with aging, suggesting niche stem cell numbers and turnover do not change with age.

Average uterine percent methylation increased during menstruation (ages 12-52 years) and was relatively stable after menopause. A regression analysis of these data was consistent with two lines (as opposed to one; F_1,27 = 10.55, P = 0.003), with methylation increasing up to age 52 years and no increase afterward (F_1,27 = 0.33, P = 0.57). Red circles indicate two or fewer children; blue circles, three or more children; plus marks indicate obesity (BMI >30) and solid circles lean women (BMI <24). Average uterine gland diversity (numbers of unique tags among eight sampled tags) was stable with age. More complete graphs that include individual gland values are illustrated in Fig. 7, which is published as supporting information on the PNAS web site.

CSX6 endometrial tags. (a) Comparisons of CSX vs. CSX6 tags sampled from the same glands (“X”) reveal little correlation (gray line, correlation coefficient of 0.27), consistent with independent errors between CpG sites. However, average CSX and CSX6 uterine values (circles) correlated (black line, correlation coefficient of 0.99), consistent with the hypothesis that different CpG sites overall record similar numbers of divisions. (b) CSX6 methylation in four uteri (Patients 1, 9, 10, and 30) heterozygous for a single-nucleotide polymorphism. Circles represent averages of 12 tags, and triangles represent averages of six T (red) or G (blue) alleles. More stochastic scatter is expected with the smaller number of sampled T or G alleles. Simulations of nulliparous women for either two alleles per cell (gray lines) or one allele per cell (dotted lines), adjusted for different numbers of sampled CSX6 alleles and a lower methylation error rate (2 × 10^-5 per CpG site per division), but with the same numbers of niche stem cells, mitotic rates, and niche turnover as the CSX simulations (Fig. 4), were consistent with the CSX6 experimental data. (c) Ratios of numbers of T to T+G alleles sampled from each gland. Skewed sampling bias (all one or the other allele) was not evident as approximately equal numbers of each allele were sampled.

Stem cell niche. Stem cells are defined by their location within the niche, and their numbers are constant. After division, cells that leave the niche differentiate and eventually die. Niche stem cell lineages may either expand or become extinct, because stem cell division may be asymmetric (one stem and one differentiated daughter) or symmetric (two stem or two differentiated daughter cells). The exact size of the niche and extent of symmetric divisions are uncertain, because a variety of combinations are consistent with the experimental data (see table). A large niche with frequent symmetric division is equivalent to a smaller niche with less frequent symmetric division. Eventually all current-day niche stem cell lineages are lost except one, which recurs with a median interval of ≈10,000 divisions. Below are CSX endometrial gland simulations that assume 64 stem cells, P₁ of 0.98, a methylation error rate of 4.5 × 10^-5, and a demethylation error rate of 5 × 10^-6 per CpG site per division. Smaller or larger niches as in the table have similar outcomes (see Supporting Text). The gray line simulates nulliparous women (no live births) with one division per week before menarche, one division per day between ages 12 and 52, and one division per week after menopause. Multiparity (blue line) is simulated with 0.75 divisions per day between menarche and menopause. Obesity (red line) is simulated with 1.25 divisions per day after menarche. Dotted lines include 95% of simulated gland outcomes indicating the scatter expected from stochastic errors and stem cell turnover.