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.

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 (), and reversing this decline through treatment with metabolic precursors for NADhas gained attention as a treatment for maintaining late-life health (). Here, we demonstrate that autofluorescence of NADH and its phosphorylated form NADPH declines in oocytes with age, and we delineate a role for NADand a potential role for the NAD-consuming enzyme SIRT2 as mediators of fertility that are open to pharmacological intervention.

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 (). 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.

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 (). 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 (), is expensive, and has a limited success rate.

Given the ability of sirtuin inhibitors to reduce blastocyst quality, we next sought to determine the pathway through which these inhibitors restrict embryo growth. The most prominently studied member of the sirtuins, SIRT1, deacetylates p53 to inhibit its activity and prevent apoptosis (). p53 activity is increased in embryos produced by IVF compared with in vivo-derived embryos, likely due to culture stress, and its genetic deletion overcomes the effects of culture on blastocyst development (). Activation of p53 is detrimental, because it inhibits proliferation and induces apoptosis of the inner cell mass, which goes on to form the developing fetus (). We observed that treatment with the p53 inhibitor pifithrin () overcame sirtinol-mediated reduction in cell number ( Figure 4 E) and blastocyst development ( Figure S5 H), suggesting that p53-dependent hypotrophy is sirtuin mediated ().

We next sought to determine whether NMN treatment mediated benefits via the sirtuins. The small-molecule sirtuin inhibitors sirtinol () and splitomicin () impaired embryo growth under simple defined media conditions ( Figure 4 C; Figure S5 G), and this decline in embryo cell number was partially reversed by NMN cotreatment ( Figure 4 D), suggesting that the sirtuins are not the primary target of NMN in enhancing preimplantation embryo development. This interpretation is clouded by the absence of data to confirm that sirtinol offered complete inhibition of its targets, because only partial inhibition could result in some of the target using increased NADto rescue embryo development.

Mammalian cells have the capacity to generate NADvia several biosynthetic routes and intermediate precursors ( Figure S6 ). To assess whether NADsynthesis via endogenous NMN production in the recycling pathway plays a role in embryo development, we cultured embryos in the presence of the NAMPT inhibitor FK866 (), which induced blastocyst degeneration on day 6 of culture ( Figure S5 D). This could be rescued by NMN cotreatment ( Figure 4 B), as well as other NADprecursors ( Figure 4 B). In contrast, treatment with the Priess Handler enzyme nicotinic acid phosphoribosyltransferase (NaPRT) inhibitor 2-hydroxynicotinic acid () did not affect embryo development ( Figures S5 E, S5F, and S6).

Next, we asked whether elevating NADmight also benefit pre-implantation embryo development. To test this, IVF was performed using in vivo matured oocytes from reproductively aged (12-month-old) or young (4-week-old) females. Following IVF, embryos were cultured in the presence or absence of NMN. Supplementation with NMN improved blastocyst formation in embryos derived from oocytes from aged females ( Figure 4 A), but not in embryos arising from oocytes from young females ( Figures S5 A and S5B). To further assess whether NMN could rescue embryo development under challenged conditions, embryos from young animals were maintained in a simple culture medium that accentuates culture stress and restricts embryo development (). Consistent with results from aged animals, the addition of NMN to simple media improved blastocyst cell number, an indicator of implantation success ( Figure S5 C).

(B) Treatment with the NAMPT inhibitor FK866 causes embryo death at day 6, which can be rescued by the NAD + precursors NMN, nicotinic acid mononucleotide (NaMN), nicotinic acid riboside (NaR), and NR (Kruskal-Wallis 50.65, p < 0.0001, ∗∗∗∗ p < 0.0001, ∗∗ p = 0.0048, ∗ p = 0.0197, ∗∗∗ p = 0.0004, ∗∗ p = 0.0014, ∗ p = 0.0156, n = 4–21 embryos per group as shown).

Although these data suggested that SIRT2 was sufficient to recapitulate the benefits of NMN to fertility, we next sought to determine whether SIRT2 was also obligatory for maintaining normal oocyte function through the use of Sirt2knockout animals. Oocytes from whole-body Sirt2knockout mice at the age of 5–6 months displayed normal spindle assembly and maturation ( Figure 3 I), indicating that at a younger age at which NADis replete, SIRT2 is not essential for accurate spindle assembly or that redundancy exists in the role of SIRT2 with other yet-to-be-identified factors. These in vivo results from Sirt2 knockout animals are in contrast to in vitro studies (). Altogether, these data suggest that SIRT2 is sufficient, but not required, to improve oocyte quality during aging.

Given the improved characteristics of oocytes from aged Sirt2animals, we sought to determine whether this translated into improved fertility. Starting at the age of 15 months, animals were subjected to breeding trials to determine pregnancy rates. Consistent with very low fertility at this age, only 25% of wild-type Sirt2females achieved a pregnancy over 5 mating rounds, whereas this rate tripled to 75% in Sirt2females ( Figure 3 H). These data demonstrate that the NAD-dependent deacylase SIRT2 is sufficient to maintain ovarian function and female fertility during aging.

To test whether SIRT2 could recapitulate the benefits of NMN treatment, we obtained a strain of Sirt2mice () that overexpress SIRT2 in all tissues, including oocytes ( Figure S4 A), and assessed oocyte quality at the age of 14 months. As with our previous experiments, oocytes were immunostained to assess spindle structure and chromosome alignment. As expected, more than 70% of oocytes from reproductively aged wild-type (Sirt2) animals had strikingly disordered spindles and poorly aligned chromosomes ( Figures 3 A and 3B ), whereas 80% of oocytes from SIRT2 transgenic (Sirt2) littermates exhibited normal barrel-shaped bipolar spindles and well-aligned chromosomes. Following stimulation with PMSG, twice as many COCs were obtained from aged Sirt2ovaries compared with Sirt2littermates ( Figure 3 C). Given the importance of spindle integrity and chromosome alignment for chromosome segregation, we next tested whether oocytes from aged Sirt2animals might be less prone to aneuploidy, a pathogenomic feature of poor-quality oocytes from aged females. Predictably, aneuploidy rates increased with age, from 15% at 3 months of age to 43% at 16 months of age in Sirt2wild-type oocytes, whereas in oocytes from aged Sirt2littermates, the incidence was 20%, comparable to young females ( Figure 3 D). Oxidative stress is a key driver of oocyte aging and female infertility (). SIRT2 deacetylates and maintains the activity of the pentose phosphate pathway enzyme G6PD (), which regenerates the antioxidant glutathione through its production of NADPH. Compared with wild-type oocytes, Sirt2oocytes from aged animals displayed markedly reduced levels of ROS, as determined by staining with the ROS-sensitive fluorescent dye HDCFDA under both young (5- to 6-month-old) unchallenged ( Figures 3 E and 3F) and H-challenged conditions ( Figures S4 D and S4E). Consistent with these data and our hypothesis, we observed increased G6PD enzyme activity in Sirt2oocytes ( Figure 3 G).

(E–G) Oocytes from Sirt2 Tg/+ mice had decreased ROS levels as determined by (E) H 2 DCFDA staining, quantified in (F) ( ∗∗∗∗ p < 0.0001, two-tailed t-test, n = 29 oocytes per group), with (G) increased G6PD enzyme activity ( ∗∗ p = 0.0019, two-tailed t-test, n = 11–13 using 5 pooled oocytes per sample from 4 animals per group).

(A and B) Oocytes from 14-month-old Sirt2 Tg/+ C57BL/6 mice were subjected to (A) immunostaining for spindle assembly (β-tubulin in green, Hoechst for DNA in blue, kinetochores in red). Disordered spindles with lagging chromosomes are indicated by arrows, and normal, barrel-shaped bipolar spindles with DNA aligned along the metaphase plate are indicated by arrowheads; this is quantified in (B) (p = 0.0131, n = 10–13 oocytes per group, Fisher’s exact test).

We next sought to determine the mechanism that mediated these benefits. One candidate that we hypothesized for this role is the NAD-dependent deacylase SIRT2. We previously showed that SIRT2 stabilizes the SAC protein BubR1 (), which is critical for meiotic progression (), kinetochore attachment, and chromosome segregation in oocytes (). Levels of BubR1 decline in mouse reproductive tissue () and human oocytes with advancing age (). SIRT2 maintains genome stability through deacetylation of Cdc20 and Cdh1 required for sustaining the activity of the anaphase-promoting complex (), which is an essential oocyte regulator ().

Given the potential for clinical translation of this work, it was important to assess whether this treatment would adversely affect the health or development of offspring following maternal NMN exposure. Offspring from a separate cohort of animals maintained on the higher dose of NMN (2 g/L) before mating, during pregnancy, and throughout lactation were maintained on normal chow or challenged with high-fat feeding to determine their health and development. Animals were assessed for physiological and behavioral changes ( Figure S3 ). There were no changes from maternal NMN exposure, except for a small increase in lean body mass from maternal NMN treatment ( Figure S3 D). These data are in line with recent work () suggesting that administration of the NADprecursor nicotinamide riboside (NR) during lactation is not harmful and may improve offspring health.

Next, we sought to determine whether late-life NMN treatment resulted in oocytes with improved developmental potential. Twelve-month-old animals were treated with NMN for 4 weeks (2 g/L, drinking water), and MII oocytes were collected from oviducts following PMSG and hCG stimulation. Oocytes from NMN-treated, aged (12-month-old) animals had a larger diameter ( Figure 2 F), which may be relevant given that oocytes with smaller diameters are associated with poorer outcomes following IVF (). A separate cohort of oocytes was subjected to IVF, and at day 6, the proportion of embryos that reached blastocyst formation was assessed ( Figure 2 G), with a trend toward improved blastocyst formation rates. We next sought to determine whether in vivo NMN treatment would alter subsequent inner cell mass development of blastocysts, which is highly predictive of pregnancy success (). Twelve-month-old mice were treated with NMN in drinking water for 2, 7, 14, or 28 days, and oocytes subjected to IVF. At day 6, embryos were subjected to differential staining of the inner cell mass. The length of NMN treatment in animals correlated with improvements in inner cell mass size ( Figure 2 H), and to confirm that this translated to improved fertility outcomes, we treated a cohort of 13-month-old animals with two different doses of NMN (drinking water, 0.5 and 2 g/L) for 4 weeks before the introduction of a male of proven fertility. Breeding performance as determined by pregnancy, live births, and litter size was then assessed for the following 9 weeks, from 14–16 months of age ( Figures 2 I–2M). NMN treatment improved the time to first live birth ( Figure 2 J) and the overall proportion of animals achieving live birth during the breeding trial ( Figure 2 K), though this surprisingly occurred at the lower dose of NMN (0.5 g/L), suggesting that previous experiments were performed at a dose that benefited oocyte quality but may have adversely affected other aspects of fertility. This could be related to an upper limit to NMN tolerability or the increased formation of the NMN degradation product nicotinamide, which is a sirtuin inhibitor (). These data from orthogonal pharmacological and genetic approaches show that increasing NADenhances ovulation rate, oocyte quality, and overall live birth rates in aged female mice, though they point to an optimum range of dosing.

To test whether addressing the age-related decline in oocyte NADlevels would alter oocyte quality, we treated 14-month-old females with NMN in drinking water (2 g/L) for 4 weeks, whereas control animals were maintained on the same drinking water in the absence of NMN. The effects of this intervention on oocyte quality were assessed in germinal vesicle (GV)-stage oocytes collected from the ovaries of pregnant mare’s serum gonadotropin (PMSG)-stimulated animals, which were matured in vitro to the MII stage, and immunostained to assess spindle structure and chromosome alignment. NMN treatment notably rescued spindle assembly ( Figure 2 A). Oocyte yield was also increased in aged animals following ovarian hyperstimulation of C57BL/6JAusb mice with PMSG and human chorionic gonadotrophin (hCG) to obtain MII oocytes from the oviduct and hyperstimulation of Swiss albino mice with PMSG alone to obtain GV-stage cumulus oocyte complexes (COCs) directly from punctured ovaries ( Figures 2 B and 2C). To further test the importance of NADbiosynthesis, we obtained transgenic mouse strains that constitutively overexpress the NADbiosynthetic enzymes NMNAT1 or NMNAT3 ( Figures 2 D and 2E; Figure S1 ), which are localized to the nucleus or the mitochondria, respectively (). These animals had increased levels of NMNAT1 and NMNAT3 in ovarian tissue, though these proteins could not be detected in oocytes by western blot, most likely because of the low protein concentrations that were obtained from oocytes relevant to other tissues. As with NMN treatment, 12- to 14-month-old Nmnat1animals, but not Nmnat3animals, yielded more oocytes than their wild-type littermates ( Figures 2 D and 2E), suggesting that the subcellular localization of NADbiosynthesis is important for follicular and oocyte function. To test the requirement for NADbiosynthesis in meiotic maturation, we treated GV-stage oocytes with FK866, an inhibitor of the NADbiosynthetic enzyme NAMPT (), and assessed meiotic progression ( Figure S2 ). Both germinal vesicle breakdown (GVBD) and polar body extrusion (PBE) were slowed by FK866 treatment, consistent with the idea that NADsynthesis is essential to oocyte function.

(I–M) Breeding performance was assessed by (I) cumulative time to pregnancy, (J) cumulative time to live birth (log-rank test, ∗∗ p = 0.0059), (K) overall proportion achieving live birth (Fisher’s exact test, ∗ p = 0.0253 ctrl versus 0.5 g/L NMN), (L) cumulative number of pups born over time (repeated-measures ANOVA, NMN F(2, 43) = 4.925, p = 0.0119, Dunnett’s multiple comparison ctrl versus NMN 0.5, ∗∗∗∗ p < 0.0001, error bars show SEM), and (M) overall number of pups per female (Kruskal-Wallis 9.220, p = 0.0100, Dunn’s multiple comparison, ∗ p = 0.0491 ctrl versus 0.5 g/L NMN).

(H) 12-month-old C57BL/6 females were treated for the indicated times with NMN in drinking water (2 g/L), and MII oocytes were subjected to IVF. At day 6, inner cell mass was assessed (Kruskal-Wallis 11.93, *p = 0.0337 by Dunn’s multiple comparison test, n = 8–26 oocytes per group). Previously untreated 13-month-old C57BL/6 females (n = 15–17 per group) were treated for 4 weeks with NMN in drinking water at two doses (0.5 and 2 g/L), following which a male was introduced and breeding performance was assessed over the next 9 weeks.

(G) In a parallel cohort, oocytes were used for IVF, with blastocyst formation at day 6 of embryo development (each datapoint is cumulative data from one independent experiment).

(A) NMN treatment (drinking water, 2 g/L, 4 weeks) with NMN from 14 months restores spindle assembly in immunostained oocytes (β-tubulin in green, Hoechst for DNA in blue, kinetochores in red, p = 0.0503 by Fisher’s exact test, n = 23–25 per group). Disordered spindles with lagging chromosomes are indicated by arrows, and normal, barrel-shaped bipolar spindles with DNA aligned along the metaphase plate are indicated by arrowheads.

We sought to determine whether NADdeclined in oocytes with age, contributing to infertility and declining oocyte quality, and whether this could be reversed through treatment with the NADprecursor nicotinamide mononucleotide (NMN) (). 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 (). Because of the bioanalytical challenges of measuring NADlevels in individual oocytes, we used hyperspectral microscopy imaging techniques that exploit the autofluorescence of NADH and NADPH (). 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 1 A). 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 1 B). 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 1 C), 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 NADlevels in the whole ovary, as would be expected following systemic delivery ( Figure 1 D).

(C and D) Mass spectrometry of the whole ovary (C) shows no change in NAD(H) levels between the ages of 1 and 14 months (n = 6 mice per group); however, (D) NMN increased ovarian NAD(H) in 14-month-old mice (1 h following 400 mg/kg oral gavage, ∗ p = 0.0123, ∗∗ p = 0.0072, two-tailed t test, n = 20 mice per group).

(A and B) Multispectral imaging (A) to determine NAD(P)H content in oocytes from young (4- to 5-week-old) or aged (12-month-old) mice treated with NMN (drinking water, 2 g/L, 4 weeks) (scale bar is 20 μm), quantified in (B) (one-way ANOVA 1.454 (2, 71), ∗ p = 0.0165, 0.0287 by Dunnett’s multiple comparison test, n = 21–27 oocytes per group).

Discussion

There is an ongoing trend across the developed world to defer pregnancy until later in life, leading to a steady increase in demand for ARTs. Despite maternal age being the greatest clinical challenge for reproductive medicine, there are no therapeutic treatments to improve oocyte quality. In the current study, we show that oocyte levels of NAD(P)H decline with age and demonstrate that NAD+ repletion using NMN restores oocyte quality and enhances ovulation rate and fertility. Furthermore, supplementation of NMN in embryo culture media reversed the adverse effects of age on development.

+ availability are a determinant of declining oocyte quality and female infertility and that pharmacological restoration of NAD+ opens a therapeutic window for the treatment of age-related infertility. Several questions remain, including how NMN treatment would restore oocyte quality in aged animals. One well-known consequence of poor oocyte quality with advancing age is chromosome segregation defects, which overwhelmingly affect the first meiotic division (MI). Indeed, 80%–90% of age-related embryonic aneuploidy is the consequence of female MI errors ( Greaney et al., 2018 Greaney J.

Wei Z.

Homer H. Regulation of chromosome segregation in oocytes and the cellular basis for female meiotic errors. Homer et al., 2005 Homer H.A.

McDougall A.

Levasseur M.

Yallop K.

Murdoch A.P.

Herbert M. Mad2 prevents aneuploidy and premature proteolysis of cyclin B and securin during meiosis I in mouse oocytes. Greaney et al., 2018 Greaney J.

Wei Z.

Homer H. Regulation of chromosome segregation in oocytes and the cellular basis for female meiotic errors. Dalton et al., 2014 Dalton C.M.

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Carroll J. Measurement of ATP in single oocytes: impact of maturation and cumulus cells on levels and consumption. + deficiency with age impedes energy metabolism through its requirement as an essential redox cofactor; therefore, treatment with NMN during this critical phase of susceptibility could lower rates of spindle assembly defects and aneuploidy. The present study supports the premise that age-related reductions in NADavailability are a determinant of declining oocyte quality and female infertility and that pharmacological restoration of NADopens a therapeutic window for the treatment of age-related infertility. Several questions remain, including how NMN treatment would restore oocyte quality in aged animals. One well-known consequence of poor oocyte quality with advancing age is chromosome segregation defects, which overwhelmingly affect the first meiotic division (MI). Indeed, 80%–90% of age-related embryonic aneuploidy is the consequence of female MI errors (). The ability to segregate chromosomes accurately depends upon multiple factors that critically include proper spindle assembly and surveillance of chromosome segregation by the SAC, which we and others have shown is critical for preventing aneuploidy in oocytes (). Adequate expression of key regulatory factors including SAC proteins is determined during a protracted growth phase that the oocyte undergoes concomitant with follicle development within the ovary; it is during this growth phase that the oocyte is transcriptionally and translationally active and stockpiles the reserves required for oocyte maturation and early embryonic divisions for when both transcription and translation have shut down (). This is a bioenergetically demanding process, and ATP consumption is maximal in mouse oocytes during the spindle assembly phase in MI (). In mouse oocytes, this growth phase lasts 2–3 weeks before ovulation, whereas we administered NMN for 4 weeks. Although one explanation for improved oocyte quality could be related to increased atresia of suboptimal oocytes before ovulation because of improved quality control mechanisms, we hypothesize that the length of the NMN treatment period improves overall oocyte quality during this critical phase of maturation, with no change in overall sorting or quality control mechanisms, and instead decreases atresia due to improved oocyte quality. It may be that NADdeficiency with age impedes energy metabolism through its requirement as an essential redox cofactor; therefore, treatment with NMN during this critical phase of susceptibility could lower rates of spindle assembly defects and aneuploidy.

Hyperspectral imaging of NADH and NADPH autofluorescence revealed a decline in oocyte NAD(P)H levels with age, whereas mass spectrometry of homogenized, whole ovary did not reveal an age-related decline in NAD(H) levels. This may be related to the increased age of oocytes, which in most mammals are formed during in utero development and do not undergo turnover in adult life, with these cells being older than surrounding somatic cells. It would be ideal to measure NAD(H) levels in individual oocytes by mass spectrometry as a complementary technique, because our inability to accurately assay NAD metabolites by mass spectrometry at the single-cell level is a limitation of this study.

One unexpected aspect of this study was that treatment with a lower dose of NMN resulted in improved functional fertility, as assessed by pregnancy and live birth in aged animals. This could suggest that there is an optimum range for dosing of NAD+ precursors beyond which other aspects of fertility could be adversely affected, lowering functional fertility. This will be an important concern to the clinical translation of this work, especially given that NAD+ precursors such as NR are freely available as supplements. Out of caution, we suggest that these supplements should not be taken by women wishing to become pregnant until further studies have been completed.

The present study implicates the NAD+ metabolome in regulating ovulation rate. We hypothesize that this change in ovulation results from decreased atresia of follicles during maturation, during which oocytes exhibit susceptibilities, as described earlier, rather than change to ovarian reserve. We do not exclude the possibility that NMN exerts benefits to follicular development through direct interactions with tissues other than the ovary, because we delivered NMN through systemic dosing. Furthermore, other tissues may also be susceptible to an age-related decline in NAD+.

+ is a prominent molecule used as a cofactor or substrate across a range of reactions, we sought to determine whether the benefits of NMN to oocyte integrity might be mediated partly by the NAD+-dependent deacylase SIRT2. The benefits of in vivo NMN treatment could largely be recapitulated by transgenic overexpression of Sirt2 in aged animals, although its constitutive deletion had no adverse impact on oocyte quality, at least in the younger animals studied here. SIRT2 plays a role in the maintenance of microtubule-kinetochore attachments through its deacetylation and stabilization of BubR1 ( North et al., 2014 North B.J.

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et al. SIRT2 induces the checkpoint kinase BubR1 to increase lifespan. Qiu et al., 2018 Qiu D.

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Wang Q. Sirt2-BubR1 acetylation pathway mediates the effects of advanced maternal age on oocyte quality. Tg/+-overexpressing animals, increases in kinetochore-microtubule stability may have contributed to augmented chromosome alignment and improved fertility. These observations are in agreement with separate in vitro studies in which chemical or morpholino-mediated Sirt2 knockdown in oocytes resulted in severe spindle defects and chromosome disorganization ( Qiu et al., 2018 Qiu D.

Hou X.

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Wang Q. Sirt2-BubR1 acetylation pathway mediates the effects of advanced maternal age on oocyte quality. Riepsamen et al., 2015 Riepsamen A.

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Wang S. Are sirtuins markers of ovarian aging?. + levels could instead be affecting oocytes by simply enhancing energy metabolism. Although NADis a prominent molecule used as a cofactor or substrate across a range of reactions, we sought to determine whether the benefits of NMN to oocyte integrity might be mediated partly by the NAD-dependent deacylase SIRT2. The benefits of in vivo NMN treatment could largely be recapitulated by transgenic overexpression of Sirt2 in aged animals, although its constitutive deletion had no adverse impact on oocyte quality, at least in the younger animals studied here. SIRT2 plays a role in the maintenance of microtubule-kinetochore attachments through its deacetylation and stabilization of BubR1 (), a process that safeguards fidelity in chromosome separation by ensuring the bipolar orientation of chromosomes to the spindle. Consequently, in oocytes from Sirt2-overexpressing animals, increases in kinetochore-microtubule stability may have contributed to augmented chromosome alignment and improved fertility. These observations are in agreement with separate in vitro studies in which chemical or morpholino-mediated Sirt2 knockdown in oocytes resulted in severe spindle defects and chromosome disorganization (). In contrast to those studies, we observed that oocytes from constitutive Sirt2 knockout mice maintained normal spindle assembly and chromosome organization, suggesting that SIRT2 may be dispensable for oocytes. This interpretation is limited by the young age of knockout animals in that experiment; the only way to definitively determine whether SIRT2 mediates the benefits of NMN in fertility would be to test NMN treatment in aged (12- to 14-month-old) Sirt2 knockout mice. Another limitation of this study was that we did not measure ROS or G6PD activity in oocytes from these knockout animals. These data suggest the existence of overlapping mechanisms that may compensate during development. Other members of the sirtuins family have been implicated in oocyte development (), and further studies could aim to investigate whether these members are involved in mediating the benefits of NMN treatment observed here. Overall, our data do not yet suggest a role for SIRT2 in mediating the benefits of increased NAD. Increasing NADlevels could instead be affecting oocytes by simply enhancing energy metabolism.

+ levels. This finding is highly relevant to the clinical practice of IVF. In addition to age-related issues of decreased oocyte numbers and oocyte quality, mitotic aneuploidy ( Munné et al., 2002 Munné S.

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Assisted Reproductive Technology in Australia and New Zealand 2016. Having demonstrated that in vivo NMN treatment in aged animals improved oocyte quality and increased ovulation rate and birth rates, we next showed that supplementing embryo culture media with NMN improved embryo development in embryos derived from oocytes from aged animals, but not young animals, supporting the idea that this intervention addresses an age-related deficit in oocyte NADlevels. This finding is highly relevant to the clinical practice of IVF. In addition to age-related issues of decreased oocyte numbers and oocyte quality, mitotic aneuploidy () and poor preimplantation embryo development () limit the number of euploid blastocysts available for transfer with increasing maternal age. The increasing preference for blastocyst-stage transfers in clinical IVF underscores the importance of reaching more advanced developmental milestones () and clinical demand for interventions that can improve embryo development.

+ precursor, alternative precursors from other pathways also raise NAD+, most notably NR ( Trammell et al., 2016 Trammell S.A.

Schmidt M.S.

Weidemann B.J.

Redpath P.

Jaksch F.

Dellinger R.W.

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Abel E.D.

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Brenner C. Nicotinamide riboside is uniquely and orally bioavailable in mice and humans. Although this study used NMN as an NADprecursor, alternative precursors from other pathways also raise NAD, most notably NR (). We anticipate that this and similar compounds will also exhibit efficacy in oocyte quality and fertility, and it is unlikely that these effects are unique to NMN.

This work represents a clinically tractable pharmacological intervention to non-invasively treat female infertility caused by a loss of oocyte viability in reproductively aged females, with important clinical implications. We envisage this work could lead to the development of orally delivered therapeutics that enhance oocyte quality for natural conception or IVF. Moreover, this work could enhance the success rates of existing IVF protocols by improving embryo culture conditions and developmental outcomes. Any intervention that improves fertility would lead to cost savings and lower the emotional stress of failed IVF rounds or infertility that can lead to long term psychological and social issues, including depression and relationship breakdown. This could represent an intervention that enables women with poor oocyte quality to have children with their own genetic makeup, because currently, these women have no alternative but to use donated oocytes. Future studies should aim to test NAD+-raising compounds in a clinical setting, both as an oral therapeutic and as an additive to embryo media, to assess the relevance of these findings to human infertility. While promising, we caution against the use of NAD+-raising supplements until these clinical studies have been completed.