Reproductive aging in female mammals is an irreversible process associated with declining oocyte quality, which is the rate-limiting factor to fertility. Here, we show that this loss of oocyte quality with age accompanies declining levels of the prominent metabolic cofactor nicotinamide adenine dinucleotide (NAD+). Treatment with the NAD+ metabolic precursor nicotinamide mononucleotide (NMN) rejuvenates oocyte quality in aged animals, leading to restoration in fertility, and this can be recapitulated by transgenic overexpression of the NAD+-dependent deacylase SIRT2, though deletion of this enzyme does not impair oocyte quality. These benefits of NMN extend to the developing embryo, where supplementation reverses the adverse effect of maternal age on developmental milestones. These findings suggest that late-life restoration of NAD+ levels represents an opportunity to rescue female reproductive function in mammals.

Declining oocyte quality is considered an irreversible feature of aging and is rate limiting for human fertility. Bertoldo et al. show that reversing an age-dependent decline in NAD(P)H restores oocyte quality, embryo development, and functional fertility in aged mice. These findings may be relevant to reproductive medicine.

Increasing maternal age and subsequent infertility have rapidly become a significant challenge to family planning, as a result of the irreversible decline in female fertility in mammals. The rate-limiting factor for successful pregnancy is oocyte quality, which significantly declines from late in the third decade of life in humans (De Vos et al., 2014; Sauer, 2015). Despite the enormous demand, there are no clinically viable strategies to either preserve or rejuvenate oocyte quality during aging, which is defined by the capacity of the oocyte to support meiotic maturation, fertilization, and subsequent embryonic development. A non-invasive, pharmacological treatment to maintain or restore oocyte quality during aging would alleviate a rate-limiting barrier to pregnancy with increasing age that has driven demand for assisted reproduction technologies (ARTs) such as in vitro fertilization (IVF), which is invasive, carries health risks (Kumar et al., 2011), is expensive, and has a limited success rate.

Although somatic tissues undergo continual regeneration through turnover by a self-renewing population of resident precursor stem cells, oocytes in the ovary are laid down during in utero development in humans, where they form a finite pool that does not undergo self-renewal. Oocytes are therefore highly susceptible to age-related dysfunction. The molecular basis for the decline in oocyte quality with advancing age implicates genome instability, reduced mitochondrial bioenergetics, increased reactive oxygen species (ROS), and disturbances during meiotic chromosome segregation due to compromised function of the spindle assembly checkpoint (SAC) surveillance system (Franasiak et al., 2014; Greaney et al., 2018). The molecular cause of chromosome mis-segregation in oocytes with advancing age is still unknown, and as a result, there are no pharmacological strategies to correct this problem. Understanding the molecular or metabolic basis of this defect could lead to therapies that could maintain or even rescue female fertility with advancing age.

The metabolite nicotinamide adenine dinucleotide (NAD+/NADH) is a prominent redox cofactor and enzyme substrate that is essential to energy metabolism, DNA repair, and epigenetic homeostasis. Levels of this essential cofactor decline with age in somatic tissues (Massudi et al., 2012), and reversing this decline through treatment with metabolic precursors for NAD+ has gained attention as a treatment for maintaining late-life health (Mills et al., 2016; Rajman et al., 2018). Here, we demonstrate that autofluorescence of NADH and its phosphorylated form NADPH declines in oocytes with age, and we delineate a role for NAD+ and a potential role for the NAD+-consuming enzyme SIRT2 as mediators of fertility that are open to pharmacological intervention.

We sought to determine whether NAD+ declined in oocytes with age, contributing to infertility and declining oocyte quality, and whether this could be reversed through treatment with the NAD+ precursor nicotinamide mononucleotide (NMN) (Yoshino et al., 2011). To address these questions, we used mice, whose fertility starts to decline around 8 months of age due to oocyte defects that are similar to those in humans (Greaney et al., 2018). Because of the bioanalytical challenges of measuring NAD+ levels in individual oocytes, we used hyperspectral microscopy imaging techniques that exploit the autofluorescence of NADH and NADPH (Dong et al., 2019; Kolenc and Quinn, 2019). Twelve-month-old females were treated with NMN in drinking water (2 g/L) for 4 weeks, following which mature metaphase-II (MII) oocytes were recovered and subjected to multispectral microscopy imaging of autofluorescence to determine the relative abundances of native fluorophores (Figure 1A). Consistent with our hypothesis, we found that NAD(P)H levels declined in oocytes from aged animals, compared with young (4- to 5-week-old) animals, and NMN treatment increased NAD(P)H levels in oocytes from aged animals (Figure 1B). We next sought to determine whether this trend occurred across the entire ovary, rather than in oocytes alone. Using mass spectrometry, we did not observe a decline in whole ovary NAD(H) levels with age (Figure 1C), suggesting that the oocyte represents an especially vulnerable subunit of the ovary that is subject to an age-related decline in NAD+, in contrast to the surrounding stroma that makes up the bulk of ovarian tissue. Consistent with data from hyperspectral imaging of individual oocytes, NMN treatment increased NAD+ levels in the whole ovary, as would be expected following systemic delivery (Figure 1D).