Scientists from UNSW Sydney and the University of Queensland in Australia, along with David A. Sinclair from Harvard, demonstrate elevation of nicotinamide adenine dinucleotide (NAD+) levels may provide an effective and non-invasive strategy to restore and maintain female fertility during aging.  The scientists use the NAD+ precursor, nicotinamide mononucleotide (NMN), to restore NAD+ levels in female mice in their experiments.

A trend exists across the developing world where people delay pregnancy for later in life due to socio-economic reasons.1,2  Age-dependent barriers to pregnancy conflict with people waiting longer to conceive children.2,3,12  This situation leads to increasing demand for infertility treatment, including in vitro fertilization.  Resorting to in vitro fertilization has numerous disadvantages; because the procedure is invasive, has health risks,2,6 is expensive, and has limited rates of success.2  Emotional stress comes with repeated in vitro fertilization failures, and failure to conceive can also contribute to relationship breakdown.2,5

With in vitro fertilization, factors limiting success are quantity and quality of immature egg cells (oocytes) in women, which begin to decline when women reach their mid-30s.2,3,12  No strategies exist to rejuvenate or preserve quantity or quality of oocytes through aging currently, despite the major need for a non-invasive treatment option.2  Such a therapy could increase the chances of conception without medical intervention or reduce the effects of factors limiting success in in vitro fertilization.2

The molecular machinery involved in the age-related decline in oocyte quality remains unclear.  Factors thought to play key roles include chromosome instability (genome instability), decreased cellular metabolism (reduced mitochondrial bioenergetics), increased oxidative stress (increased reactive oxygen species) in cells, and impaired chromosome segregation during the production of egg cells (meiosis).  Impaired chromosome segregation during meiosis leads to abnormal numbers of chromosomes (aneuploidy) in embryos with increased age of the mother. This leads to increased incidence of children born with chromosomal abnormalities, including Trisomy 21,2,13 which causes Down’s Syndrome.2  The reason for chromosomal segregation abnormalities with increased age of mothers remains unclear.  As such, no pharmacological interventions currently exist to correct this problem. An understanding of the molecular underpinnings of this occurrence could lead to therapies which might improve female fertility during aging.

The authors of this study want to examine whether replenishing NAD+ levels improves age-related decline in fertility in females.  The scientists also investigate the role of an NAD+-consuming enzyme, SIRT2, in oocyte quality and with embryo development. The scientists examine NAD+ and enzymes consuming it, because levels of this essential coenzyme decline with age in other tissues of the body (somatic tissues).2,8  Reversing the declining NAD+ levels through supplementation with precursors for NAD+ has garnered attention as a disease treatment strategy for maintaining health later in life.2,9,10  The scientists want to examine whether supplementation with the NAD+ biosynthesis precursor, NMN, improves the integrity of oocytes which could improve female fertility during aging.

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Image from Bertoldo et al. (2020) illustrating declining NAD+ levels in ovary with age

The scientists find evidence suggesting NMN supplementation could be a strategy to partially replenish ovarian NAD+ levels from age-related decline.  To address the effects of replenishing NAD+ levels on fertility, the scientists examine mice. The scientists first want to find out whether NAD+ levels decline in reproductive tissue with age.  If so, the scientists seek to understand whether this contributes to infertility and diminished oocyte integrity with age. In mice, fertility begins to decline around eight months of age. Reduced fertility comes from oocyte defects which share many similarities to those in humans.2,4  In ovaries of normal (wild-type) mice, a sharp reduction of NAD+ levels begins at age four months (see figure ‘a’ above).  The scientists treat 10-month old mice with NMN, which significantly raises NAD+ levels in the ovaries.

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Image from Bertoldo et al. (2020) illustrating increased NAD(P)H levels in aged oocytes treated with NMN in ‘c’ and quantifying the increased NAD(P)H levels in ‘d’

The scientists find levels of a marker for NAD+ abundance, NAD(P)H, declining in oocytes from aged, 12-month old animals compared to young animals.  The group finds oral NMN administration to aged animals increases NAD(P)H levels (see figure above). The scientists seek to determine whether, in addition to ovaries, reductions in NAD+ levels occur in oocytes, the immature eggs, with age.  If so, the group examines whether oral NMN treatment can reverse the reduction in NAD+ levels. Using a technique called hyperspectral imaging of autofluorescence, the group examines oocytes extracted from 12-month old females. The scientists extract oocytes from the reproductive tracts of females treated with pregnant mare’s serum gonadotropin (PMSG) and human chorionic gonadotropic (hCG).

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Image and figure from Bertoldo et al. (2020) indicating improvements in chromosome structure of aged, 14-month old females with (above) and without (below) NMN treatment.  The graph on the right demonstrates improvements in proportion of normal oocytes in NMN-treated, aged females compared to aged females without NMN treatment.

The scientists then find NMN treatment of 14-month old females improves oocyte integrity compared to 14-month old females without NMN treatment.  The scientists treat 14-month old females with NMN in their drinking water (2 g/L) for four weeks. The scientists collect oocytes from ovaries of these animals and use a laboratory technique called ‘immunostaining’ to image the oocytes.  The scientists look at chromosome structure (spindle structure) and chromosome alignment (see figure ‘a’ above). The proportion of oocytes displaying ‘normal’ chromosome integrity improves significantly in the aged females treated with NMN compared to those not treated with NMN.  The scientists also produce data from this experiment indicating NMN increases oocyte yield in obese animals fed a high fat diet for five to six months; obesity presents a physiological challenge resulting in infertility2,11 and reduced NAD+ levels.2,14

The scientists generate data indicating NMN enhances oocyte quality for use in in vitro fertilization.  Inner cell mass highly predicts the success of implantation and pregnancy.2,7  The scientists use 12-month old mice consuming NMN in drinking water for 2, 7, 14, or 28 days, followed with oocyte extraction.  The scientists collect the oocytes (MII oocytes) and subject them to in vitro fertilization.  At day six, the scientists use a method called ‘staining’ to examine the inner cell mass under a microscope.  The results indicate inner cell mass size improves with NMN treatment. With longer NMN treatment in the animals, the scientists observe more significant improvements, also.

The scientists then find evidence supporting NAD+ enhances fertility in aged female mice.  The scientists treat mice with NMN (2 g/L in drinking water) from 10 weeks of age. At 18 weeks of age, the group introduces a male, with proven fertility, for mating.  The scientists repeat this process every seven to eight weeks until age 50 weeks. At 50 weeks of age, the scientists record the number of live pups born in each litter.  As previous experiments also indicate, NMN treatment increases litter size from the animals.

The group of scientists finds normal development in offspring whose mothers receive NMN exposure.  The data do indicate a small but consistent improvement in lean body mass of offspring whose mothers receive NMN treatment.  As the scientists say, “The reason for this change is unclear, and worthy of later investigation.”2

The scientists proceed to acquire data indicating when SIRT2, an NAD+-dependent sirtuin, has increased expression, it can provide benefits to oocyte integrity during aging.  The scientists use mice with increased SIRT2 expression (SirtTg/+ transgenic mice), which overexpresses SIRT2 in all tissues, including oocytes.  At age 14 months, the scientists assess oocyte quality. In normal mice (wild-type mice), approximately 70% of oocytes from aged animals have disordered and poorly aligned chromosomes.  In the mice overexpressing SIRT2, 80% of the oocytes exhibit normal, well-aligned chromosomes. In aged mice overexpressing SIRT2, percentage of oocytes displaying abnormal chromosome numbers (aneuploidy) was comparable to young female oocytes, even though aneuploidy rates typically increase with age.

To examine whether SIRT2 is necessary for fertility, the scientists use mice that do not express SIRT2 (Sirt2-/- knockout animals).  Young mice not expressing SIRT2 display normal chromosomes under the microscope.  As the authors say, “…at a younger age where NAD is replete, SIRT2 is not essential for accurate spindle assembly, or that there is redundancy in the role of SIRT2 with other yet to be identified factors.”2  The authors go on to say, “Together, these data suggest that SIRT2 is sufficient, but not required to improve oocyte quality during ageing.”2

The scientists provide data suggesting the addition of NMN can improve cell numbers in early embryos (blastocysts), indicating implantation success following in vitro fertilization.  The scientists perform in vitro fertilization using matured oocytes from 12-month old and 4-week old females.  Embryos grown (cultured) with NMN from the aged females have improved early embryo (blastocyst) formation compared to embryos grown without NMN from aged females.  The scientists observe no differences between embryos with and without NMN treatment in the embryos from young females.

The authors of this article demonstrate increasing NAD+ levels in aged, female mice can partially restore fertility.  The scientists use NMN supplementation to restore NAD+ levels. The data present interesting findings, because people in developed societies continue to postpone pregnancies.  Age-related barriers can impede pregnancies as people postpone conception. Finding a pharmacological method to maintain a degree of fertility in aged females provides an option other than in vitro fertilization for conception as emotional turmoil and relational breakdown associate with failed in vitro fertilization.  A pharmacological therapeutic could also improve the chances of embryo survival from an in vitro fertilization procedure.