Graphical Abstract Highlights d Declining NAD(P)H is associated with oocyte dysfunction during reproductive aging d Oocyte quality and fertility can be restored by NMN treatment in aged mice d Supplementation of embryo media with NMN improves developmental milestones d SIRT2 overexpression mimics benefits of NMN but is unlikely to mediate its effects SUMMARYReproductive 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.

Meiotic divisions in oocytes are extremely asymmetric and require pre-and post-anaphaseonset phases of spindle migration. The latter induces membrane protrusion that is moulded around the spindle thereby reducing cytoplasmic loss. Here, we find that depleting the NAD biosynthetic enzyme, nicotinamide phosphoribosyl-transferase (Nampt), in mouse oocytes results in markedly longer spindles and compromises asymmetry. By analysing spindle speed in live oocytes, we identify a striking and transient acceleration after anaphase-onset that is severely blunted following Nampt-depletion. Slow-moving midzones of elongated spindles induce cortical furrowing deep within the oocyte before protrusions can form, altogether resulting in larger oocyte fragments being cleaved off. Additionally, we find that Namptdepletion lowers NAD and ATP levels and that reducing NAD using small molecule Nampt inhibitors also compromises asymmetry. These data show that rapid midzone displacement is critical for extreme asymmetry by delaying furrowing to enable protrusions to form and link metabolic status to asymmetric division.

2Female infertility is a common and devastating condition with life-long health, emotional and 3 social consequences. There is currently no pharmacological therapy for preserving oocyte 4 quality during aging, which is the strongest risk factor for infertility. This leads to an age 5 dependent decline in natural conception and IVF success rates (1). Here, we show that this is 6 due in part to declining levels of the metabolic cofactor nicotinamide adenine dinucleotide 7 (NAD + ), and that restoring NAD + levels with its metabolic precursor nicotinamide 8 mononucleotide (NMN) rejuvenates oocyte quality and quantity in aged animals, leading to 9 improved fertility. These benefits extend to the developing embryo, where NMN 10 supplementation in embryo culture media following IVF enhances blastocyst formation in 11 older mice. The NAD + dependent deacylase SIRT2 is sufficient, but not essential, to 12 recapitulate the benefits of in vivo NMN treatment, and transgenic overexpression of SIRT2 13 maintains oocyte spindle assembly, accurate chromosome segregation, decreased oxidative 14 stress and overall fertility with ageing. Pharmacological elevation of NAD + may be an 15 effective, non-invasive strategy for restoring and maintaining female fertility during ageing, 16 and for improving the success of IVF.17 18 19 risks (4), is expensive and has a limited success rate. Repeated IVF failures are a substantial 1 source of emotional distress, and failure to conceive offspring is a substantial source of 2 relationship breakdown (5). 4The rate-limiting factors for successful pregnancies in IVF are oocyte quantity and quality, 5 both of which start to decline from the middle of the third decade of life in humans (1, 3). 6 Despite the enormous need, there are no clinically viable strategies to either preserve or 7 rejuvenate oocyte quantity or quality during ageing. There is a major need in reproductive 8 medicine for a non-invasive, pharmacological treatment to maintain or restore oocyte quantity 9 and/or quality during ageing. The effect of such a therapy would be to alleviate a rate-limiting 10 barrier to IVF success, or increase the chances of unaided conception, without having to resort 11 to IVF. 12 13The molecular basis for the decline in oocyte quality with advancing age is not clear but is 14 certainly multifactorial. The key factors thought to be involved include genome instability, 15 reduced mitochondrial bioenergetics, increased reactive oxygen species (ROS), and impaired 16 fidelity during meiotic chromosome segregation due to disrupted spindle assembly and 17 compromised function of the spindle assembly checkpoint (SAC) surveillance system (6). This 18 latter hypothesis is evidenced by an increased rate of aneuploidy in embryos with increased 19

Introduction Non‐tubal ectopic pregnancy (NTEP) is a rare but significant early pregnancy complication which can result in maternal morbidity and mortality. There is however a lack of evidence‐based guidelines for the management of NTEP. Purpose To evaluate the success rates of expectant, medical and surgical management in the treatment of NTEP at our tertiary centre. Methods Retrospective cohort study from 2010 to 2020. All NTEP were classified by ectopic sites. Primary management was classified by expectant, medical [systemic methotrexate (Sys‐MTX) and/or local ultrasound‐guided injection of MTX and/or KCl intra‐sac (L‐MTX, L‐MTX/KCl)] or surgical. Primary management was considered successful if no change in intervention was required. Treatment complications were compared. Results Twenty‐four NTEP were identified, which included 14 interstitial pregnancies (IP), 9 caesarean scar pregnancies (CSP) and 1 ovarian pregnancy (OP), which gave NTEP an incidence of 7.12% among all EP (4.15% for IP, 2.67% for CSP and 0.30% for OP). The success of primary surgical management was 100% (7/7), primary medical management was 76.9% (10/13) and primary expectant management was 33.3% (1/3). Primary medical management had a non‐statistically significant greater mean time to serum ß‐human Chorionic Gonadotrophin <5 IU/L, mean length of hospitalisation, mean number of follow‐up visits and hospital re‐presentation/readmissions compared to primary surgical management. There was no other difference in complication rates between the treatment management groups. Conclusion Surgery remains the most effective way to manage NTEP. However, medical management can be a safe and effective alternative option in carefully selected cases.