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Link between stress and infertility can be broken

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Scientists from the University of California Berkeley have discovered that by knocking down a single gene, they can stop stress from causing female infertility and miscarriage - in rats.

Although stress has been linked to decreased sex drive, delayed pregnancy and an increase in miscarriages, this is the first time the molecular basis for the links has been explored.

The team, from left to right: Sandra Muroy, Lance Kriegsfeld, Anna Geraghty, George Bentley, and Daniela Kaufer.

Image CCBY 4.0 Daniela Kaufer.

The research, published in the open-access journal eLife, was carried out in rats. The gene encodes for a hormone that is common across all mammals and the findings could apply to humans and to endangered animals whose survival depends on captive breeding.

“Remarkably, genetic silencing of a single chemical compound, a peptide called RFRP3, restores mating and pregnancy success to a rate indistinguishable from non-stress controls,” says Professor Daniela Kaufer.

The team, from the Kaufer, Kriegsfeld and Bentley labs at Berkeley, also carried out the first investigation into the long-term impact of pre-conception stress on female reproductive fitness and pregnancy. They found marked and persistent reproductive dysfunction even after recovery from stress.

Stressed females were less motivated to mate, became pregnant less often and - in those that did copulate successfully - fewer live pups made it to term and were instead reabsorbed into the uterus. The findings show that the impact of stress lingers long after the stress has been removed. These marked effects were completely eliminated by knocking down RFRP3.

“A strikingly high proportion of healthy women struggle with fertility and our findings provide a new focus for the clinical study of human reproductive health,” says the graduate student lead

author Anna Geraghty.

RFRP3 is the mammalian equivalent of a hormone first identified in Japanese quail and is made in the hypothalamus portion of the brain. One way it exerts its effect on females is by decreasing the normal surge in hormones that accompanies ovulation. It may be directly regulated by the stress hormone corticosterone. However, even after recovery from stress, when corticosterone levels have returned to normal, RFRP3 was found to continue exerting its effect on reproduction.

Total reproductive success was determined by the percentage of females that successfully brought a litter to full term. This dropped from 80% in females not exposed to stress, to 20% in those previously exposed to stress. However, in those exposed to stress but with RFRP3 knocked down reproductive success was restored to 80%, and embryo survival was back up to 94%. In the future, developing therapies that lower RFRP3 levels may help individuals who experience stress-related infertility.

We spoke to George Bentley (associate professor of integrative biology), Anna Geraghty (graduate student), Daniela Kaufer (associate professor of integrative biology) and Lance Kriegsfeld (associate professor of psychology) about their research.

1. How might it be possible to lower RFRP3 in humans?

GB: Even though RFRP3 has been isolated from humans, we are still far from understanding the full extent of its actions and how it is regulated in the human brain and gonads. In principle, though, RFRP3 could be manipulated in a therapeutic manner. Before any such attempts were made, much more work (on human cell lines, for example) would be required. Potentially, a gene therapy approach could work – either on its own or in combination with a pharmacological approach.

2. How long might it take for such a therapy to become available?

GB: This would depend on a large number of variables: the number of people working on the problem, the identification of the full suite of actions of RFRP3 in humans and ways in which it is modulated by other hormones – along with the need for any therapeutic approach to be agreed upon by regulatory agencies before clinical trials could begin. Because we still do not understand the full extent of RFRP3’s actions in humans, I think we still have a way to go before considering such a therapy.

3. What most surprised you about your research findings?

AG: One of the most surprising aspects of our findings was the persistent effect of stress on embryo survival and the role RFRP3 may play in that. Though the stress ended four days prior to mating, stressed animals exhibited higher rates of embryo resorption, indicating that the effects of the chronic stress lasted at least two weeks past the cessation of the stressor. Knocking down RFRP3 prevented embryo resorption. What may be happening is that high stress is increasing RFRP3 levels. While we know RFRP3 levels remain high for four days, it could be that RFRP3 remains high for even longer than we tested, affecting not just mating and pregnancy rates, but also embryo survival rates - which are analogous to miscarriages in humans.

4. What are the potential implications for humans of the persistence of reproductive dysfunction beyond the removal of the stress?

DK: It is well documented that reproduction is suppressed during the stress response, and it makes a lot of sense. During times of stress our bodies are built to minimize all non-vital functions, and reproduction is not required for the survival of the individual organism. However, what our study shows is that the dramatic decrease of reproductive functions lingers well beyond the end of stress, and beyond the time when we see high levels of stress hormones in the blood. This can explain high incidence and long lasting effects of stress on human infertility. The study tells

us that stress effects are long-lasting, and that even when one does not see obvious clinical signs (like elevated stress hormones or cessation of menstrual cycling) stress will still lead to decreased reproductive success, through elevated levels of the RFRP3 peptide in the brain.

5. In rats, the stress concluded four days prior to mating – what is the equivalent in human terms?

DK: The equivalent in human terms is one full menstrual cycle, about 28 days.

6. What are the implications of courtship behaviour such as ear wiggling being unaffected? Does this show that, unlike in humans, libido was not affected?

LK: These findings suggest, that at least in rodents, fertility and the ability to carry a pregnancy to full term are more vulnerable than sexual motivation to the effects of prior stress. Unlike humans, when the stressor ceases, rodents presumably do not continue to ruminate about previous stressful life events, thereby prolonging the negative side effects. In humans, the restoration of libido likely requires both the avoidance of stressful stimuli and psychologically resolving the impact of previous negative life events.

7. There is some debate on the impact of stress on fertility but you say that it is well established that chronic stress induces female reproductive dysfunction. What is the evidence for this and, in your opinion, is there still any doubt about the link?

DK: There is a lot of compelling evidence to show that exposure to stress reduces fertility. I have no doubt about the link. However, It is harder to study the mechanisms in complex human populations by running correlative studies. For example, the study you are referring to correlated the levels of two blood hormones in two samples. It is very hard to use these findings to delineate a conclusive causative role. Moreover, our study shows that circulating levels of stress hormone are not a good predictor of the stress effect, whereas the brain levels of RFRP3 (GnIH) are. This is where animal models come to aid in clarifying causal relationship and molecular mechanisms, and many data collected from animal models point to a causative link. We were able in this study to pinpoint RFRP3 as the culprit underlying the stress response, and most importantly, were able to restore fertility to control levels by manipulating the expression of this single gene.

8. Is there anything else about the findings that you would like to emphasise?

LK: Our results identify RFRP3 as a neuropeptide that sits at the top of a neural hierarchy of control, responsible for communicating information about stressful circumstances to the reproductive system. Downstream of RFRP3, we find that increases in this peptide due to stress lead to deficits in sex steroid hormones. In previous work, we also found it leads to deficits in the neural structures that mediate female sexual behavior. We look forward to continuing to work together to apply similar approaches used in the present studies to further characterize the specific neuroendocrine systems impacted downstream of RFRP3 and to develop approaches to abrogate the effects of stress on female reproductive health.

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