Things that Increase Female Reproductive Lifespan

Staying fertile longer -- lessons from mice

Are there interventions that keep female animals fertile longer into old age? Can any of them be applied to humans?

One thing to note is that animals vary a lot in reproductive aging.  Most mammals don’t even go through menopause! So we should expect, even more than usual, that we need to be suspicious of generalizing from mice (or other lab animals) to humans regarding menopause.

 

Like most domesticated animals, lab mice are bred for fast reproductive maturation, which tends to correlate with early age-related infertility. So if we extend the fertile lifespan of lab mice, we should be suspicious that we’re working on “easy mode”, adjusting from an unusually low baseline. 

In fact, it has been observed that inbred lab mice, the kind most experiments are done on, are much less fertile in old age than genetically heterogeneous mice.[1]

On the other hand, mouse experiments are a good place to start looking for candidate interventions, and there are a lot of them, so we’re going to summarize the results here.

Reproductive Aging in Female Mammals

The C57Bl/6 strain of mice, the most common strain used in lab experiments, typically produces 3-7 pups per litter, starting at around 2 months. Their fertility starts to decline around 8 months and almost all are sterile above 16 months.[2] The mean lifespan for female C57Bl/6 is about 26 months.[3]

Wait a minute, that’s a pretty long period between infertility and death. So why isn’t this an example of mouse menopause?

Well, in humans, menopause is defined by the cessation of menstruation.  And mice don’t menstruate.

Menstruation is confined to primates and a few other species such as bats, elephant shrews, and the Cairo spiny mouse. 

Most mammals have estrus cycles instead of menstrual cycles. After ovulation, if the egg is not fertilized, the endometrium is reabsorbed rather than expelled through the vagina as menses. Mammals can have one, two, or many estrus cycles a year. Rats and mice are polyestrous with a cycle length of 4-5 days.

A few mammals, including rabbits and cats, are induced ovulators, who have no estrus cycle, but rather release eggs in response to the act of copulation. 

All mammals have broadly similar female reproductive organs and hormones. The ovaries contain follicles (proto-eggs) which develop into oocytes (eggs). During each ovulatory period, oocytes are released into the uterus. The development and release of follicles, oocytes, and the lining of the uterus, as well as the physical changes of pregnancy, are governed by the same female sex hormones in all mammals: gonadotropin-releasing hormone (GNRH) from the hypothalamus, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from the pituitary, and estrogen and progesterone from the ovary.

Loss of fertility with age, in all mammals, occurs when there is diminished “ovarian reserve”, i.e. fewer and lower-quality follicles remaining in the ovaries.  In many species, there is also a decline in sex hormone levels. Over time, ovulation becomes more irregular and eventually stops altogether.

Most wild mammals decline in fertility as they age. There are a few counterexamples, such as the naked mole rat and the tundra vole, whose fertility stays steady or even increases with age. Other exceptions include hyenas, mongooses, and bats.  Interestingly, naked mole rats and bats are unusually long-lived for their size, and mongooses and naked mole rats are both “cooperative breeders” in which non-reproducing adult animals care for their relatives’ offspring.[4][5]

Birds and reptiles, by contrast, often have no decline in fertility with age.[4]

Many species of mammals have a significant post-reproductive lifespan, including badgers, elephants, whales, and primates.[4]

Across 101 species of mammals, 69 had evidence of reproductive senescence.[5] 

 Humans have the longest period of female post-reproductive lifespan as a percent of total lifespan, followed by false killer whales, orcas, and pilot whales. The latter are all toothed whales: long-lived social carnivores who hunt together in pods led by matriarchs.

Extending Reproductive Lifespan in Mice

For the purposes of this review, we’re only looking at interventions that affect the number of offspring in older female mice. We’re not looking at proxy measures like the number of oocytes or follicles, since it’s possible to improve these without improving fertility itself. We’re also not looking at mouse models of premature reproductive aging, since those may not be as realistic as the actual decline in fertility of healthy older mice.

I’ve ranked the below table in order of the effect size, from largest to smallest. The background colors are as follows:

• Red: claims of fraud in the paper, results suspect

• Yellow: some reason to doubt this will be a good fertility-extending therapy in humans

• Blue: actionable option that people can safely try today

• Green: not actionable but looks promising as a future therapy

Discussion and Context

Dietary Omega-3 Fatty Acids

Can this really be the biggest effect size in the literature? Just diet?

In the experiment, all mice were fed a chow consisting of equal amounts of casein (whey protein), sugar, corn starch, vitamins and minerals, and varying oil sources: all soybean oil (which contains mostly omega-6 fatty acids), all hydrogenated coconut oil (which contains no fatty acids at all), or a mix of hydrogenated coconut oil and omega-3 fatty acids found in fish oil, including DHA (docosahaexonoic acid) and AA (arachidonic acid).  

The relevance to humans here may be limited, because we do not normally eat a diet consisting exclusively of protein powder, sugar, corn starch, and vegetable oil. (If you do, please stop!) 

But the results really are that stark. The all-soybean-oil mice became completely infertile by 10 months; the all-hydrogenated-coconut-oil mice were still much worse off than the omega-3 mice.  And standard mouse chow is even lower in omega-3 fatty acids than the specially prepared soybean-oil chow, and results in even faster loss of viable oocytes.

Can women extend their fertile lifespan to superhumanly high ages by eating lots of DHA? Probably not. The “control” mice had an abnormally deficient diet compared to anything a human would willingly eat. But DHA is safe and beneficial for health overall, so there’s little downside to trying to get it in your diet.  DHA is found mostly in fatty fish (salmon, mackerel, herring, anchovy), and can also be found as a supplement derived from fish oil or algae.

SIRT1 Knock-In

In this experiment, mice were genetically engineered to overexpress the gene SIRT1 in the ovaries alone. 

SIRT1 is a sirtuin, a transcriptional regulatory molecule; it silences other genes by causing DNA to wrap more tightly around the histones. In yeast, its homolog Sir2 is the “active ingredient” of the lifespan-extending effect of caloric restriction. The contention that sirtuins are longevity genes (or that increasing/enhancing/activating sirtuins is a good anti-aging intervention) has a long history of failure to reproduce results and even research misconduct (in the form of doctored data and images), so I start out with a pretty skeptical prior about anything in the sirtuin world.

The effects reported in this particular SIRT1 knock-in paper are also not particularly promising as a fertility intervention. The genetic modification caused caloric-restriction-like effects, including delayed sexual maturity; even if it worked in humans exactly as it worked in mice, you wouldn’t necessarily like the side effects.

MSC-derived Exosomes

This paper, unfortunately, has some apparent doctored images, which calls into question the rest of the results.

The authors take mesenchymal stem cells from human umbilical cords, and remove exosomes, small lipid-covered blobs intended to export chemicals out of a cell, and then inject them into the ovaries of old mice.  This stimulated follicle development and increased fertility. 

Could something like this work? Maybe so; there’s often something in stem cells that can reprogram old cells or tissues to be more like young ones. But, of course, this paper is suspicious.

Bax-/- Deficiency

BAX is a protein that, together with BCL2, induces apoptosis, or cell death. 

It makes sense that Bax deficiency in mice would result in slower ovarian aging and less of other aging symptoms (like hair loss and skin wrinkling) whose proximate cause is cell death.

Genetically engineering humans from birth isn’t particularly actionable, but could drugs be developed to inhibit BAX or otherwise use this pathway to delay reproductive aging? Looks like yes.[21] There are also peptide inhibitors of BAX-mediated apoptosis.

Of course, if you do something to block apoptosis, you should worry about whether you’re increasing cancer risk. But yeah, this could be the beginning of a viable research direction.

Brown Fat Transplant

Brown fat is especially common in newborn babies and hibernating animals. Its purpose is thermogenesis -- creating heat to warm up the body.  In brown fat, mitochondria are signaled to to “uncouple”, or allow “proton leak” -- the potential energy stored in the electron transport chain is allowed to dissipate as heat rather than being directed to produce ATP as in ordinary cellular respiration.

For this reason, brown fat and mitochondrial uncoupling is particularly intriguing to researchers looking for ways to treat obesity, since a cellular mechanism to “waste” energy will prevent it from being stored as fat. The scary old weight-loss drug DNP is a mitochondrial uncoupler and some new startups like Equator Therapeutics are based around less-toxic ways to treat obesity by stimulating mitochondrial uncoupling and thermogenesis through the same mechanisms found in brown fat.

Brown fat also has intriguing associations with longevity. The longest-lived small mammals, bats and naked mole rats, have unusually high levels of brown fat.

The mice transplanted with brown fat, in addition to keeping ovarian follicles longer and being more fertile in old age, had signs of increased thermogenesis (higher body temperature, particularly when exposed to cold). Treated mice also had increased levels of adiponectin, a protein which is secreted by fat cells, is higher in non-obese than obese individuals, is elevated during fasting and mitochondrial uncoupling, and promotes insulin sensitivity. All of this makes sense -- the brown fat was indeed doing what brown fat usually does.

A transplant of newborn brown fat is, obviously, not an immediately actionable intervention for people to try today. (Step 1, find a baby; step 2, get a knife…) Someone would have to develop a therapy that replicates the desirable effects, perhaps by isolating a particular compound found in brown fat.  But I have a basically hopeful intuition about this as a direction of research.

IL-1 Deficiency

IL-1a is an interleukin, a signaling molecule in the immune system, produced mainly by activated macrophages. It is involved in promoting inflammation, fever, and sepsis.

It’s not surprising that mice lower in an inflammatory signaling molecule would have a slower decline in fertility -- inflammation is involved in many of the diseases of aging.

While genetically engineered IL-1 deficiency in humans is not immediately actionable, there are plenty of drugs that inhibit IL-1 or block its receptor to treat autoinflammatory diseases, such as anakinra and rilonacept.  These drugs increase the risk of infection, so the risk-benefit tradeoff may not work out for healthy people who are just looking to extend their fertility, but it’s certainly a viable area of research.

Short-Term Rapamycin

Rapamycin is one of the best-studied drugs in the aging biology world. 

It is an immunosuppressant, used medically to prevent transplant rejection; it also reliably extends lifespan in worms, flies, and mice. The Dog Aging Project is doing a trial of rapamycin to see if it can extend lifespan in companion dogs.

The big concern with rapamycin is safety. In human transplant contexts, it causes diabetes-like symptoms, lung toxicity, and vulnerability to infection.  Could lower doses be safer while still having the same anti-aging effects?

Well, one study in healthy older adults aged 75-90 [22] randomized to 1 mg/day rapamycin or placebo for 16 weeks, found no adverse effects that were more common in the treatment than placebo group, only a mild tendency towards anemia.

In the mouse study, rapamycin treatment temporarily impaired fertility in female mice, but two weeks of rapamycin treatment, while it initially shrank the ovaries significantly, produced a recovery in fertility 2 months later, and resulted in preserving ovarian function and fertility later in life.

Could you try this at home? Well, people do take rapamycin off-label, so if you’re convinced by the preliminary clinical evidence that it’s safe at low doses, you could give it a try. 

Hdac6 Overexpression

HDAC6 is a histone deacetylase gene, which enables DNA to wrap a histone more tightly, silencing the gene. (HDACs have a similar function to sirtuins in blocking the transcription of other genes.)

HDAC6 overexpression, in the mouse experiment, was associated with lower levels of markers for DNA damage. HDAC proteins have been associated with DNA damage repair, so this makes sense. Perhaps HDAC6 overexpression prevents an accumulation of DNA damage that would otherwise cause loss of ovarian follicles and lower fertility with age.

Could you go from genetically modified HDAC6-overexpressing mice to some kind of HDAC6-enhancing therapy in humans? HDAC6 inhibitor drugs exist, usually for anti-cancer applications; they encourage cell death via allowing misfolded proteins or DNA damage to accumulate. I don’t know of a direct way to enhance HDAC6 levels or promote its damage repair function via small molecules, but I don’t know of any a priori reason why one couldn’t do it either.

Melatonin

Melatonin is frequently called the “sleep hormone”, though it’s probably better described as the “circadian rhythm hormone” or the “darkness hormone.” It sets the “body clock” of wakefulness and sleepiness, advancing the subjective “bedtime” when it starts getting dark earlier in the autumn evenings, and pushing subjective “bedtime” back when it starts staying light in the spring evenings.

Chronic melatonin exposure might make mice live longer -- it’s controversial. Prolonged periods of darkness and melatonin consistently have “anti-gonadal” effects -- smaller sex organs, lower levels of sex hormones -- which you’d generally expect to preserve fertility for longer if it doesn’t destroy fertility altogether.

In the mouse study, melatonin preserved ovarian follicles and fertility, and also reduced aging-related oxidative stress in the ovaries and increased the expression of the mitochondrial antioxidant SOD2. This makes sense -- reactive oxygen species can lead to cell death, and effectively suppressing them in the ovaries can preserve ovarian cells. And melatonin is itself an antioxidant.

In humans, melatonin has some evidence of improving fertility, for instance, nearly doubling the pregnancy rate in women with PCOS treated for infertility with intrauterine insemination.[25]

Melatonin is easily available as an over-the-counter supplement, and has extensive evidence of being safe at low dose. On the other hand, in this study, melatonin was administered at 10mg/kg at night for 6-12 months. An equivalent dose for a human would be more than half a gram a day! (A typical over-the-counter melatonin pill is 10 mg. So, imagine taking 60 pills a day…)  We don’t have a lot of studies on what happens to humans who take extremely high melatonin doses, though e.g. in 12 healthy volunteers, 100 mg of IV melatonin doesn’t seem to have immediately harmed them.[26] 

Coenzyme Q10

Coenzyme Q10, also known as ubiquinone, is an antioxidant and readily available as a supplement. It is present in the mitochondria of all cells, where it catalyzes a step in cellular respiration. 

There’s also some human evidence that coenzyme Q10 helps with fertility: in a randomized trial of 186 women with poor ovarian response undergoing IVF, treatment with coenzyme Q10 increased the number of oocytes and high-quality embryos in the treated women.[27]

In the mouse study, CoQ10 increased the size of aged ovaries and increased the number of follicles, as well as increasing the number of respiring mitochondria and mitochondrial membrane potential (a measure of intact vs. damaged mitochondria.) This is consistent with CoQ10 improving mitochondrial function.

Mild Calorie Restriction with Refeeding

Calorie restriction is the most-studied life-extending intervention. It extends lifespan and healthspan in many animals, from yeast, worms, and flies to mice, rats, and even monkeys, although the magnitude of the life-extension effect declines with body size and is negligible in primates.

Calorie restriction in female mammals, of course, will impair fertility, because even brief periods of fasting will temporarily prevent the release of sex hormones that trigger ovulation. Dietary restriction suppresses menstruation in humans, and underweight women have more problems with infertility and miscarriage. 

On the other hand, in this experiment, mild calorie restriction (by 10%) followed by a two-week period of ad-lib refeeding, extended fertility in mice. 

Something like this feasible as an intervention for humans -- reducing food intake followed by a period of higher-calorie “refeeding” when trying to conceive can be practical and safe.

Bmf Deficiency

The Bmf gene, or Bcl2-modifying factor, is part of a family of proteins that regulate apoptosis. Like Bax, Bmf promotes apoptosis, so it makes sense that a genetic deficiency in Bmf would reduce the depletion of ovarian cells including follicles and oocytes.

I couldn’t find any molecules that inhibit or block the function of Bmf, but I also don’t know of any a priori reason why they couldn’t be developed. 

Alpha-Ketoglutarate

Alpha-ketoglutarate is an intermediate in the Krebs cycle of cellular respiration. It is an over-the-counter supplement, and supplementation has been found to extend life in mice.  It has antioxidant properties, and reduces frailty and inflammation in old animals.[28]

Like CoQ10, alpha-ketoglutarate is an antioxidant involved in mitochondrial cellular respiration, hypothesized to work via preventing the accumulation of oxidative or inflammatory damage to tissues.  

Bin2 deficiency

Bin2 is a gene that is heavily expressed in ovaries. Mostly, its function seems unknown, but in the paper they found that it activates RPS6, a ribosomal protein involved in protein production and cell growth, and a step on the mTOR pathway. Speculatively, you could group Bin2 -/- mice with rapamycin and caloric restriction as interventions that work along the mTOR inhibition/nutrient-sensing pathway .

I couldn’t find any known compounds that inhibit Bin2, but I also don’t know of any reason why they couldn’t be developed.

Constituitively Active Foxo3

Foxo3 is a transcription factor which is involved in reducing oxidative stress by upregulating mitochondrial antioxidants like catalase and Sod. Foxo3 deficient mice have premature infertility due to loss of ovarian reserve.

In general, Foxo genes inhibit the mTOR pathway, and are activated by caloric restriction.[29] 

Thus, one would expect a genetically modified mouse with permanently activated Foxo3 to have less oxidative damage to its ovaries.

It’s unclear to me how you would go about designing a therapy that worked along this mechanism -- how could you permanently keep Foxo3 activated in a human without germline genetic modification? 

Stuff that Doesn’t Work

Ten weeks of every-other-day rapamycin injections, 5 mg/kg, did not enable female rats to be more fertile late in life; they kept their primordial follicles (proto-eggs) longer than control rats, but they were also much leaner and smaller, had irregular estrous cycles, and failed to reproduce.[30] You do not want high-dose rapamycin if you’re trying to conceive.

Notable Results Besides Fertility

BGP-15, an insulin sensitizer and PARP inhibitor which reduces mitochondrial reactive oxygen species production, can reverse ovarian fibrosis and induce ovulation in aged female mice. It also reduces age-related markers of inflammation and mitochondrial stress.[31]  I have a good feeling about this one despite the fact that this experiment didn’t directly measure fertility, because fibrosis is usually a permanent state of tissue dysfunction and reversing it is a big deal.

References

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[2]Franks, L. M., and JOAN PAYNE. "The influence of age on reproductive capacity in C57BL mice." Reproduction 21.3 (1970): 563-565.

[3]Kunstyr, Ivo, and Hans-georg W. Leuenberger. "Gerontological data of C57BL/6J mice. I. Sex differences in survival curves." Journal of gerontology 30.2 (1975): 157-162.

[4]Comizzoli, Pierre, and Mary Ann Ottinger. "Understanding reproductive aging in wildlife to improve animal conservation and human reproductive health." Frontiers in Cell and Developmental Biology 9 (2021): 680471.

[5]Lemaître, Jean-François, Victor Ronget, and Jean-Michel Gaillard. "Female reproductive senescence across mammals: a high diversity of patterns modulated by life history and mating traits." Mechanisms of Ageing and Development 192 (2020): 111377.

[6]Nehra, Deepika, et al. "Prolonging the female reproductive lifespan and improving egg quality with dietary omega‐3 fatty acids." Aging cell 11.6 (2012): 1046-1054.

[7]Long, Guan-Yun, et al. "SIRT1 knock-in mice preserve ovarian reserve resembling caloric restriction." Gene 686 (2019): 194-202.

[8]Yang, Weijie, et al. "HucMSC-derived exosomes mitigate the age-related retardation of fertility in female mice." Molecular Therapy 28.4 (2020): 1200-1213.

[9]Perez, Gloria I., et al. "Absence of the proapoptotic Bax protein extends fertility and alleviates age-related health complications in female mice." Proceedings of the National Academy of Sciences 104.12 (2007): 5229-5234.

[10]Chen, Liang‐Jian, et al. "Single xenotransplant of rat brown adipose tissue prolonged the ovarian lifespan of aging mice by improving follicle survival." Aging cell 18.6 (2019): e13024.

[11]Uri-Belapolsky, Shiri, et al. "Interleukin-1 deficiency prolongs ovarian lifespan in mice." Proceedings of the National Academy of Sciences 111.34 (2014): 12492-12497.

[12]Dou, Xiaowei, et al. "Short‐term rapamycin treatment increases ovarian lifespan in young and middle‐aged female mice." Aging cell 16.4 (2017): 825-836.

[13]Zhang, Xiaoxi, et al. "Overexpression of Hdac6 extends reproductive lifespan in mice." Protein & cell 8.5 (2017): 360-364.

[14]Song, Chao, et al. "Melatonin improves age-induced fertility decline and attenuates ovarian mitochondrial oxidative stress in mice." Scientific reports 6.1 (2016): 1-15.

[15]Ben‐Meir, Assaf, et al. "Coenzyme Q10 restores oocyte mitochondrial function and fertility during reproductive aging." Aging cell 14.5 (2015): 887-895.

[16]Isola, José VV, et al. "Mild calorie restriction, but not 17α-estradiol, extends ovarian reserve and fertility in female mice." Experimental Gerontology 159 (2022): 111669.

[17]Liew, Seng H., et al. "Loss of the proapoptotic BH3-only protein BCL-2 modifying factor prolongs the fertile life span in female mice." Biology of reproduction 90.4 (2014): 77-1.

[18]Zhang, Zhenzhen, et al. "α‐ketoglutarate delays age‐related fertility decline in mammals." Aging cell 20.2 (2021): e13291.

[19]Zhu, Feng-Yu, et al. "Bridge Integrator 2, a New Specific Target for Improving Reproductive Life Span through RPS6 and NNT." (2021).

[20]Pelosi, Emanuele, et al. "Constitutively active Foxo3 in oocytes preserves ovarian reserve in mice." Nature communications 4.1 (2013): 1-7.

[21]Xu, Yangying, et al. "Pretreatment with coenzyme Q10 improves ovarian response and embryo quality in low-prognosis young women with decreased ovarian reserve: a randomized controlled trial." Reproductive Biology and Endocrinology 16.1 (2018): 1-11.

[22]Garner, Thomas P., et al. "Small-molecule allosteric inhibitors of BAX." Nature chemical biology 15.4 (2019): 322-330.

[23]Kraig, Ellen, et al. "A randomized control trial to establish the feasibility and safety of rapamycin treatment in an older human cohort: Immunological, physical performance, and cognitive effects." Experimental gerontology 105 (2018): 53-69.

[24]Lee, Joo‐Yong, et al. "HDAC6 controls autophagosome maturation essential for ubiquitin‐selective quality‐control autophagy." The EMBO journal 29.5 (2010): 969-980.

[25]Andersen, Lars PH, et al. "Pharmacokinetics of high‐dose intravenous melatonin in humans." The Journal of Clinical Pharmacology 56.3 (2016): 324-329.

[26]Mokhtari, Fataneh, et al. "Effects of melatonin administration on chemical pregnancy rates of polycystic ovary syndrome patients undergoing intrauterine insemination: a randomized clinical trial." International journal of fertility & sterility 13.3 (2019): 225.

[27]Zhang, Zhenzhen, et al. "α‐ketoglutarate delays age‐related fertility decline in mammals." Aging cell 20.2 (2021): e13291.

[28]Shahmirzadi, Azar Asadi, et al. "Alpha-ketoglutarate, an endogenous metabolite, extends lifespan and compresses morbidity in aging mice." Cell metabolism 32.3 (2020): 447-456.

[30]Luo, Li-li, Jin-jie Xu, and Yu-cai Fu. "Rapamycin prolongs female reproductive lifespan." Cell Cycle 12.21 (2013): 3353-3354.

[31]Kriebs, Anna. "Reducing ovarian fibrosis extends female reproductive lifespan." Nature Aging (2022): 1-1.