Telomeres and human reproduction

Telomere shortening results from normal cell division, reactive oxygen species, genotoxic insults, and genetic predisposition. With every round of cell division, the process of DNA synthesis fails to replicate a small amount of DNA at the chromosome end. Telomeres serve as a disposable buffer that protects the gene-rich DNA on the interior of the chromosome from this end-replication problem. Reactive oxygen species produced from normal cellular metabolism and exogenous genotoxic insults also shorten telomeres by oxidizing the guanine-rich telomeric DNA and triggering a DNA damage response, which leads to excision of telomere repeats. Replication-linked telomere loss takes place only in dividing cells, but oxidative stress depletes telomeres even in nondividing cells such as oocytes ( 12 ). Telomere length is a highly heritable trait, and transmission of telomere length across generations arises from both genetic and epigenetic mechanisms. Genetic variation in loci involved in telomere length regulation, such as TERT and TERC, is associated with precocious aging and cancer ( 7 ). Exonic mutations in genes encoding critical components of shelterin and/or telomerase produce severe premature aging phenotypes, including dyskeratosis congenita, idiopathic pulmonary fibrosis, and acquired aplastic anemia ( 13 ). Even without deleterious mutations, human cells demonstrate progressive loss of telomere reserve over their life span. Critically short telomeres activate the senescence pathway, which results in p53-dependent cell cycle arrest and apoptosis ( 14 ). Rare cells compensate for telomere shortening by expressing telomerase, a reverse-transcriptase that restores a modest number of repeats to telomere ends during each S-phase of the cell cycle. In humans, however, telomerase is expressed only in male germ cells, stem cells, and cancer cells. Notably, oocytes, eggs, zygotes, and cleavage- and morula-stage embryos do not express appreciable levels of telomerase activity until the blastocyst stage. Moreover, because of the relative inefficiency of telomerase, even stem cells can eventually lose sufficient telomere reserve to lose their “stemness”—the stem cell theory of aging proposes that the body’s declining regenerative capacity arises from progressive shortening of telomeres and increasing cell senescence and death of stem cells ( 15 ).

Women universally experience marked loss of reproductive capacity well before other organ systems experience similar decline. The phenotype of oocyte aging in women includes decreased synapsis and chiasmata, increased meiotic and mitotic nondisjunction, spindle dysmorphologies, miscarriage, and embryo arrest, fragmentation, and apoptosis. Meiotic nondisjunction occurs at high rates throughout the life of women and further accelerates over the decade before menopause. Fertility in women begins to decline by their mid-30s, and conceptions that do occur carry increased risk of miscarriage and karyotypic abnormalities. Eggs donated from younger women completely abrogate the effects of reproductive aging, which highlights the central role of oocytes in reproductive aging. We have proposed a telomere theory of reproductive aging ( 16 , 17 ), which posits that age-related oocyte dysfunction in women results from progressive telomere shortening. Telomeres in oocytes begin to shorten during fetal oogenesis, when oocytes destined to ovulate late in life progress through more cell cycles ( 18 ), and continue to shorten in the adult ovary from the chronic effects of oxidative and genotoxic stress. Telomere shortening in eggs promotes genomic instability, apoptosis, and cell cycle arrest, the hallmarks of telomere-mediated cellular senescence. Telomere function is essential for meiosis. During the early prophase of meiosis in organisms as diverse as plants, yeast, mice, and humans, telomeres tether chromosomes to the nuclear membrane to facilitate homologous pairing and initiate synapsis to form chiasmata ( 19 , 20 ). Normal segregation of chromosomes during later meiosis in the adult depends on the chiasmata formed during fetal life. Chiasmata provide countertraction against spindle-pulling forces to allow all kinetochores to secure attachments to spindle fibers before the cell can progress to anaphase. Telomere shortening via genetic manipulation reduces synapsis and recombination in mice ( 21 ). We have tested the telomere theory of reproductive aging by experimentally shortening telomeres in mice, which normally do not exhibit significant age-related oocyte dysfunction, and have discovered that it recapitulates the phenotype of reproductive aging in women ( 21 , 22 ). As telomeres approach the length characteristic of telomeres in human oocytes, murine oocytes exhibit asymmetric spindles and abnormal chromosome congression. Subsequent embryos exhibit chromosome abnormalities, stop dividing, and tend to fragment. For more in-depth treatment of studies of animal models of reproductive senescence, we refer readers to our previous reviews ( 16 , 17 ). We also have demonstrated shorter telomeres in oocytes from women who did not conceive after in vitro fertilization (IVF) compared with those who did ( 23 ) and in oocytes from cycles that produced fragmented embryos ( 24 ). A more recent study demonstrated a direct relationship between short telomere length in polar bodies and aneuploid embryos from patients undergoing IVF ( 25 ). Another study found that women with diminished ovarian reserve had shorter telomeres and lower telomerase activity in their granulosa cells ( 26 ). Intriguingly, a number of studies report a relationship between reproductive aging and leukocyte telomere length, suggesting a relationship between germ line and somatic telomere lengths. Older mothers who give birth to children with Down syndrome have significantly shorter average leukocyte telomere length than age-matched mothers who gave birth to karyotypically normal children ( 27 ), although this does not seem to be the case for younger mothers of Down syndrome babies ( 27 , 28 ). Further, women with unexplained recurrent pregnancy loss also have shorter leukocyte telomere length compared with age-matched controls ( 29 ).

Cancers of the gynecologic tract affect over 80,000 women annually ( 61 ), and several of the most common gynecologic cancers and/or their treatments have been linked to the risk of infertility. Early studies showing high telomerase activity in cancers compared with healthy tissue led to the assumption that long telomeres would distinguish cancers ( 62 , 63 ). However, a consensus has emerged that progressive telomere shortening, from both age-related replicative attrition and oxidative stress, precedes the genomic instability that leads to transformation. Cancer patients generally appear to have shorter telomeres; a recent meta-analysis of 27 studies showed an association between short telomere length in leukocytes and the development of a variety of cancers ( 62 ), including ovarian cancer. Although this association in leukocytes is not consistent with all cancers studied ( 62 , 64 ), telomere length is consistently shorter when assayed in the tumor cells directly ( 65 , 66 ). The study of telomeres may be particularly relevant to gynecologic cancers due to the link between estrogen and telomerase activity. The human telomerase catalytic subunit (hTERT) is up-regulated by estrogen via direct and indirect effects on its promoter ( 67 , 68 ). Telomerase is not typically expressed in ovarian surface epithelium, although it is up-regulated in 95% of ovarian cancers ( 69 ). Similarly, advanced ovarian cancer staging typically correlates with an increased level of telomerase activity ( 63 ). Thus, hTERT appears to play a critical role in telomere length maintenance in cancerous ovarian tissue ( 70 ). As telomere length attrition occurs with age and cancer risk increases with age, it has been difficult to ascertain the causal role of telomere length in cancer pathogenesis. An elegant study in mother-daughter pairs affected by hereditary breast cancer recently demonstrated earlier disease onset and more aggressive disease coinciding with dramatically shorter telomere lengths in affected daughters ( 71 ). This relationship has not been explored in hereditary cancers affecting reproductive tissues. Because telomere shortening contributes both to female infertility and malignancy, a generalized predisposition to telomere shortening with age may explain the association between female infertility and some gynecologic cancers.

Telomeres, which are essential components of linear chromosomes, evolutionarily conserved from plants to man, protect against chromosome end-fusions and genomic instability. Telomeres erode with aging, both in dividing and nondividing cells, which triggers cell cycle arrest, senescence, and eventual cell death. The male germ line, replete with spermatogonia, maintains and even lengthens telomeres with age. In contrast, women’s ovaries lack germ line stem cells, so oocytes in women undergo profound aging that culminates in genomic instability, abnormal spindles, and in resulting abnormal embryos with cell cycle arrest, fragmentation, and apoptosis. Indeed, age provides the most robust predictor of fertility in women, regardless of other diagnoses, and the locus of reproductive aging rests almost exclusively in the oocyte. We propose that telomeres provide a timer of reproductive aging in the female. Experimental telomere shortening in female mice, which normally show almost no appreciable age-related oocyte dysfunction, recapitulates the reproductive-aging phenotype of women as the mouse’s telomere length approaches that of human telomeres. Experimental shortening of telomeres reduces synapsis and chiasmata, increases embryo fragmentation and arrest, disrupts meiotic spindles, and contributes to genomic instability. Telomeres are shorter in oocytes from women who fail IVF, and in oocytes that go on to produce fragmented ( 24 ) and aneuploid embryos ( 25 ). Telomere length in peripheral leukocytes also influences the risk of unexplained recurrent pregnancy loss and trisomic offspring. The telomere theory of reproductive aging, therefore, provides a parsimonious explanation for the otherwise disparate pathophysiologic changes in the eggs and embryos of older women. Germ line telomere shortening in women provides a striking contrast to the telomere lengthening in the male germ line. These differences in telomere dynamics across the reproductive life span may reflect the fundamentally different reproductive strategies followed by men and women. Men gain selective advantage whenever they reproduce, regardless of age. Late reproducing women, on the other hand, risk maternal death and rendering their heirs motherless and grand-motherless, thereby reducing their offspring’s reproductive fitness. Telomere-mediated oocyte aging may have protected women across the millions of years of protohuman evolution when grand multiparity and advanced maternal age made a catastrophic combination. Growing evidence also links endometriosis and gynecologic cancers, two pathologic conditions affecting the female reproductive system, with altered telomere biology. Cells from both endometriosis and cancer gain longevity by expressing telomerase and augmenting telomere reserve. Because infertility, endometriosis, and gynecologic cancers all involve alterations in telomeres, and because infertility has been associated with both endometriosis and gynecologic cancers, further studies should examine a common role for telomeres in these pathologies of the reproductive tract.

The gold standard method to measure telomere length, telomere restriction fragment (TRF), is based on the Southern blot assay, and remains the only method to determine absolute telomere length ( 72 , 73 ). This method, however, is severely limiting in studies of telomeres in eggs and embryos because it employs gel electrophoresis, hybridization with a radiolabeled probe, and therefore a large amount of DNA (0.5–5.0 μ g). Quantitative polymerase chain reaction (qPCR) substantially reduces the requirement of starting DNA material to 35 ng/reaction ( 74 ). The qPCR assay has been scaled to measure telomere length in single polar bodies and blastomeres ( 25 ). Quantitative PCR measures relative rather than absolute telomere length by generating a ratio between total telomere DNA (T) and DNA from amplification of a single copy gene (S), thereby providing a relative T/S ratio. With the advent of Sybr-green PCR technology, the time to measure telomere length by qPCR has been reduced from days to hours, thus facilitating application to large-scale human studies. Both TRF and qPCR methods have the limitation of measuring only average telomere length. Alternatively, quantitative fluorescence in situ hybridization (qFISH) of metaphase spreads reveals telomere length at the level of the individual telomere ( Fig. 3 ). Although qFISH returns a relative telomere length, it remains the most sensitive method for determining telomere length in samples limited to a small number of cells. Any of these methods may be complemented by a highly sensitive telomerase activity assay ( 47 , 75 ), which reveals the extent to which telomerase may or may not be contributing to telomere length maintenance in the tissue of interest.