Infertility has become alarmingly common in otherwise healthy women and around 15% of healthy couples younger than 30 years old are unable to conceive within the first year of trying. High-stress levels are known to decrease short-term fertility in humans and other animals, which may serve to prevent pregnancy during times when food or other resources are in short supply.

However, it is not clear if exposure to stress has lasting effects on fertility. Previous studies have found that when male rats experience stress, they release a protein called RFRP3. This protein inhibits brain activity, leading to a reduction in the release of reproductive hormones.

Geraghty et al. took a closer look at how stress may cause lasting fertility problems in female rats. The researchers exposed female rats to stress by restricting their movements for 3 hr each day over the course of 18 days, which increased the levels of stress hormones in the animals. They allowed the rats to recover for one full reproductive cycle—equivalent to a month in humans—and found that while their stress hormone levels returned to normal, RFRP3 levels in the brain remained high. Even after the recovery period, the females were less likely to mate. Also, the females that did mate were less likely to become pregnant, and the ones that did were more likely to lose some of the embryos. Overall, the level of reproductive success in these rats was only 21%, down from 76% in the control group (who were not exposed to the stress).

Next, Geraghty et al. injected a genetically engineered virus into the brain of the stressed rats to switch off the gene that makes RFRP3 during the stress period. This reduced the levels of the RFRP3 protein and restored the mating, pregnancy, and embryo survival rates to the normal levels seen in unstressed rats.

These results suggest that increased levels of RFRP3 during stress can have lasting negative effects on fertility. In the future, developing therapies that lower RFRP3 levels may help individuals who experience fertility problems.

High psychological stress inhibits reproductive function when both occur concomitantly (Rivier and Rivest, 1991; Ferin, 1999; Tilbrook et al., 2000; Louis et al., 2011). From an evolutionary perspective, inhibition of reproductive function by acute stress may be adaptive, delaying reproduction in times of duress or resource scarcity (Wasser and Barash, 1983; Louis et al., 2011). Chronic stress, however can result in persistent, maladaptive sexual dysfunction and suppressed fertility (Young et al., 2006). Little is understood about the lasting effects of stress exposure. For example, after its cessation, can a prior, persistent stressor have long-term negative after-effects on reproductive health? In humans, a high-stress environment may be a significant barrier to sexual well-being and childbearing. In healthy couples under 30 years of age, 63–80% are unable to conceive within 3 months of attempting, and within 1 year of attempting pregnancy, 15% of couples remain unable to conceive (Practice Committee of the American Society for Reproductive Medicine in collaboration with the Society for Reproductive Endocrinology and Infertility, 2008). A molecular framework to understand the long-term effects of stress on female reproduction, and its implications for human health, is currently lacking.

The present series of studies sought to answer two main questions: 1) Do stressful events negatively impact female reproductive function even following recovery of the stressor, and, if so, 2) are the deficits observed mediated by stress-induced elevation of the inhibitory neuropeptide, RFamide-related peptide-3 (RFRP3)? To our knowledge, no study to date has elucidated the molecular mechanisms of stress-induced infertility nor has there been any investigation of long-lasting after-effects of pre-conception stress on reproductive success and pregnancy outcome. RFRP3, the mammalian ortholog of gonadotropin-inhibitory hormone (GnIH) first identified in Japanese quail (Tsutsui et al., 2000), is common across mammals, including rats and mice (Ukena and Tsutsui, 2001; Ukena et al., 2002; Kriegsfeld et al., 2006), hamsters (Ubuka et al., 2012), non-human primates (Ubuka et al., 2009a), and humans (Ubuka et al., 2009b), and is a hypothalamic hormone that directly inhibits the firing of kisspeptin-sensitive gonadotropin-releasing hormone (GnRH) neurons in the hypothalamus in mice (Ducret et al., 2009; Wu et al., 2009). It also reduces downstream luteinizing hormone (LH) secretion in rats (Johnson et al., 2007; Murakami et al., 2008), mice (Son et al., 2012), and hamsters (Kriegsfeld et al., 2006). There is some debate as to whether RFRP3 is a hypophysiotropic hormone (Murakami et al., 2008; Kirby et al., 2009; Pineda et al., 2010a, 2010b) or only centrally inhibits GnRH to elicit a response (Rizwan et al., 2009, 2012). Regardless of its mechanism of action, RFRP3 decreases the synthesis and release of pituitary gonadotropins, LH, and follicle stimulating hormone (FSH), in many species, including rats and mice (Ciccone et al., 2004; Johnson et al., 2007; Murakami et al., 2008; Sari et al., 2009; Kriegsfeld et al., 2010; Ubuka et al., 2011; Son et al., 2012). In females, RFRP was shown to be regulated throughout the ovulatory cycle in rats and hamsters, and it elicits a marked inhibitory effect on the pre-ovulatory LH surge through inhibition of GnRH activation in rats (Anderson et al., 2009). In male rats, RFRP3 expression is elevated 24 hr after a chronic stressor, suggesting that RFRP3 may mediate enduring changes in reproductive function (Kirby et al., 2009). Levels of the glucocorticoid stress hormone (in rodents, corticosterone) may mediate this effect; RFRP3 neurons in the rat hypothalamus were shown to express glucocorticoid receptor (GR) (Kirby et al., 2009), as well as RFRP-expressing neuronal cell line in vitro (Lee Son et al., 2014). Finally, the RFRP promoter region includes two glucocorticoid response elements (GREs), all together supporting the hypothesis that RFRP may be directly regulated by circulating glucocorticoid levels (Lee Son et al., 2014). Together, these findings provide support for the notion that stress-induced increases in RFRP3 might have long-lasting negative impact on female reproductive functioning. Despite knowledge of RFRP's responsiveness to stress and its role in regulating reproductive axis activity, no study to date has established a causal link between RFRP and fertility in any species. We set out to test the potential role of RFRP expression in stress-induced infertility in females.

In sum, we found that chronic stress led to elevated RFRP3 at all stages of the ovulatory cycle. This elevated level of expression persisted after a full cycle of recovery from stress, indicating that the impact of stress on RFRP3 lasts well beyond removal of the stressor. Stressed females exhibited fewer successful copulation events, fewer pregnancies in those that did successfully mate, and increased frequency of embryo resorption in the achieved pregnancies. These marked effects of stress on fertility were completely blocked by knockdown of RFRP3, even though RFRP3 function was restored following stress cessation. These findings indicate that stress has lingering negative consequences for female reproductive function that are mediated by a transient rise in RFRP3.

Female rats were subjected to an 18 day stress paradigm followed by quantification of hypothalamic markers of reproductive function either immediately after stress exposure or after one full estrous cycle (4 days) of recovery (Figure 1A). Serum levels of corticosterone (CORT) were measured on days 1, 4, 7, 11, and 18 of the 18 days immobilization stress paradigm, and on day 22, 4 days after the cessation of stressor. Baseline levels at the onset of stress exposure sessions were unchanged throughout the 18 days. However, CORT levels were significantly elevated in samples drawn on days 1, 4, 7, and 11 at the end of the 3-hr stress exposure.

Figure 1

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On day 22, after 4 days of recovery from the stressor, the stressed rats exhibited serum CORT concentrations indistinguishable from baseline values (Figure 1B). Rats exhibit a 4–5 days long estrous cycle, with rising estrogen concentrations triggering a surge of luteinizing hormone (LH) to initiate ovulation, and estrogen and progesterone driving sexual receptivity on the night of proestrus (Blaustein, 2008). Stress acutely inhibits the LH surge (Du Ruisseau et al., 1979) and subsequent sexual receptivity and fertility (Sirinathsinghji et al., 1983; Sato et al., 1996). However, it is unknown whether reproductive function continues to be negatively impacted even following recovery from stress (defined as exhibiting baseline levels of CORT after 4 days of no stress exposure). Rats were monitored daily by vaginal smear to determine whether estrous cyclicity was affected during application of the stressor and to allow separation of animals into different cycle stages (diestrus, proestrus, and estrus) at the termination of the stressor. Stress did not affect estrous cyclicity, with all animals exhibiting normal vaginal cytology throughout the stressor, and all animals exhibited a normal 4- to 5-day estrous cycle after the cessation of stress. At all estrous cycle stages, RFRP3 mRNA expression in the hypothalamus was significantly elevated both 0 and 4 days after the stressor was terminated (Figure 1C,D). Hypothalamic expression of the RFRP3 receptor, G-protein-coupled receptor-147 (GPR147), was also upregulated after stress during all stages of the cycle, and returned to baseline values after the cessation of stress (Figure 1E,F). We did not find significant differences in either GnRH or kisspeptin (KISS1) mRNA expression post-stress in any stage of the cycle (Figure 1G–J). However, hypothalamic samples were taken from whole hypothalamus, precluding the ability to differentiate between rostral and caudal kisspeptin cell populations, potentially masking subtle differences. Notably, the persistent increases in the expression of both RFRP3 and its receptor specifically in proestrus coincide with the cyclical onset of sexual receptivity, suggesting that RFRP3 provides a mechanistic basis for long-lasting suppression of reproductive behavior after stress.

To investigate whether the stress-induced increase in RFRP3 plays a causal role in prolonged sexual inhibition, we developed a conditional viral vector to knock down RFRP3 expression (tet-OFF lentivirus RFRP3 shRNA) in vivo during the strictly-defined time window of the chronic stressor. This lentiviral construct expresses RFRP3 shRNA from a constitutively active CMV promoter, driving both shRNA and blue fluorescent protein (BFP) marker expression. When exposed to doxycycline (DOX, via drinking water) (Figure 2A), the tet-Off element is prevented from driving TRE-initiated transcription and both shRNA and BFP production cease (location and extent of viral infection can be seen in Figure 2B,C). Stereotaxic infusion of RFRP3 shRNA lentivirus into the hypothalamus led to an 87% down-regulation of RFRP3 mRNA expression within 7 days relative to a control-scrambled shRNA (Figure 2D). Immunohistological labeling verified that the peptide level in the hypothalamus was similarly knocked down by 85% compared to scrambled control virus, measured 2 weeks following viral injection (Figure 2E, representative images of RFRP labeling with either scramble or RFRP-shRNA virus and pre- and post-DOX administration Figure 2F–I). Critically, administration of doxycycline in the drinking water restored RFRP3 mRNA to normal levels within 4 days (Figure 2D). This viral vector system permitted knocking down of RFRP3 expression during chronic stress and restoration of RFRP3 during the later stages of copulation, mating, and birthing, which may rely on RFRP3 function in unknown ways.

Figure 2

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A second group of female rats received dorsomedial hypothalamic injections of either RFRP3 shRNA or a scrambled control shRNA lentivirus 3 weeks before the 18 days of immobilization. Estrous cycles were monitored for each rat with immobilization timed to coincide with the onset of estrus, leaving most rats in proestrus 4 days after the end of stress. All rats were administered DOX on the final day of stress so that restoration of RFRP3 expression coincided with the onset of proestrus after the 4-day recovery period. (Figure 3A). After one full estrous cycle of recovery from stress, rats underwent a timed mating test on the night of proestrus and were monitored through gestation and birth, to assess the long-term effects of stress on reproductive success including successful copulation and pregnancy outcome.

Figure 3 with 1 supplement see all

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Tail vein serum samples taken at the onset and end of stress sessions on days 1, 11, and 18 revealed that post-stress circulating levels of CORT were elevated on days 1 and 11 of the immobilization period (Figure 3B). Moreover, RFRP3 knockdown during stress did not significantly alter CORT response during stress, indicating an intact hormonal stress response (Figure 3B). Stress exposure led to a profound decrease in total reproductive success in females that received the control virus: only 21% of stressed females became pregnant and carried to live birth, as compared to 76% of non-stressed females with control virus (Figure 3C). 80% of the females that received the RFRP-shRNA virus became pregnant and carried to live birth (Figure 3C). The stress-induced decline in reproductive success resulted from a cumulative decrease in mating success (from 88 and 90% in non-stressed groups to 43% in the stress-scrambled group, Figure 3D) and pregnancy rates in the females that mated (from 87 and 89% in non-stressed groups to 50% in the stress-scrambled group, Figure 3E). Interestingly, knockdown of RFRP3 expression in the hypothalamus during stress exposure prevented the stress-induced suppression of reproduction, leading to 79% copulation success, 82% pregnancy success, and overall reproductive success to 64%, a rate statistically equivalent to control (non-stress) levels (76%, Figure 3C).

Exposure to acute stress on the evening of the third day of pregnancy was reported to lead to reduced litter size, via inhibition of implantation which occurs normally 5 days after mating (Zhao et al., 2013). Therefore, we next assessed whether pre-copulation stress exposure affects pregnancy outcome. Stressed females that received control-scrambled shRNA had significantly smaller litter sizes (Figure 3F, 12.77 ± 0.91 vs 7.667 ± 2.60 pups/litter, p < 0.05) with no difference in placental scars (Figure 3G, 13.0 ± 0.91 vs10.667 ± 3.93, p > 0.05). Embryo survival was analyzed in the females that were successfully impregnated by determining the ratio of placental scars (indicative of successful implantation) to the number of live pups in the litter. These were first pregnancies for all females, so the number of placental scars is indicative of implantation events during this pregnancy. Embryo survival in stressed females that received scrambled shRNA was 78.8 ± 11.7% of fetuses, compared to 98.1 ± 0.95%, and 97.8 ± 1.5% survival in the control scrambled and control RFRP-shRNA groups, respectively (Figure 3F, p < 0.05). Most remarkably, RFRP3 shRNA administration suppressed stress-induced fetal resorption, showing a 93.4 ± 3.2% fetal survival rate (Figure 3H). These results demonstrate that stress-induced increases in RFRP3 expression has long-term detrimental effects on female reproductive fitness that persist long after the stressor has been removed and CORT levels have returned to baseline. In addition, knocking-down RFRP3 expression during stress eliminated the stress-induced decrease in sexual motivation, decrease in pregnancy success, and subsequent increase in embryo resorption.

We next examined plasma estradiol concentration in animals throughout the stress period in both scrambled and RFRP-shRNA groups. We found that animals with RFRP knocked down had significantly higher circulating estradiol in proestrus during the stress exposure than animals that received the scrambled virus (Figure 3—figure supplement 1A, p < 0.01), indicating that RFRP knockdown reverses the stress-induced blockade of the E2 rise that occurs during proestrus. Examining animals more closely during proestrus, we found that the RFRP-shRNA animals that successfully mated after the stressor had significantly higher circulating estradiol in their proestrus periods over the course of the stressor than both scrambled groups (Figure 3—figure supplement 1B, p < 0.05).

Finally, we investigated behavioral measures of female receptivity to test its potential contribution to the stress-induced reproductive deficits observed. Lordosis intensity is a rating (from 0-3) of the quality of all lordosis poses the female exhibits during the mating session, when 0 marks no lordosis and 3 is a fully mounted spinal flexion pose. In rats, a common index of relative sexual receptivity of a female in the presence of males, is the lordosis quotient (LQ), calculated by the number of times the female adopts a lordosis posture scored 2 or higher, divided by the number of times a male mounts her. All females included in the study exhibited lordosis when introduced to a male (indicating that they were in the correct stage of their cycle). Lordosis intensity did not differ within groups (Figure 3—figure supplement 1C) but a significant main effect of stress revealed that lordosis intensity was significantly suppressed by stress (F(1, 61) = 5.15, p = 0.0268). Furthermore, lordosis quotient measures revealed significantly lower ratio in the scrambled stress group compared to the non-stressed groups that received scrambled or RFRP-shRNA (0.30 ± 0.10 vs 0.73 ± 0.07 and 0.68 ± 0.07), indicating that stress exposure decreased the relative sexual receptivity of the females (Figure 3—figure supplement 1D), congruent with the stress-induced drop in mating success we found. Interestingly, LQ ratios in stressed females that received RFRP-shRNA were not significantly different from controls ratios (0.53 ± 0.10, Figure 3—figure supplement 1D), demonstrating that knock-down of RFRP reversed the stress-induced decrease in sexual receptivity, and congruent with the reversal of mating success found in this group.

In humans, high anxiety and psychological stress can lead to long-term impaired fertility, ranging from reduced libido, delayed pregnancy success to the extreme of complete reproductive axis suppression as in the case of hypothalamic amenorrhea (Ferin, 1999). In our studies, selective knock-down of hypothalamic RFRP3 during stress exposure preserved all aspects of reproductive function that were otherwise reduced in stress-exposed animals (summary schematic, Figure 4). The stress-induced spike in RFRP3 initiates a long-lasting suppression of reproduction, well after the removal of the stressor, perhaps via positive feedback that maintains elevated RFRP3 levels or engages downstream suppressive targets. These findings reveal a single molecular target that persistently underlies a range of different reproductive dysfunctions that may provide a novel translational framework for clinical study of human reproductive health.

Figure 4

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The stress-induced rise in RFRP may be acting on neural circuits influencing mating and pregnancy, potentially independently of sex steroids. RFRP projects to multiple brain regions responsible for successful reproduction and mating behavior, including the medial pre-optic area (mPOA) (where it is known to affect GnRH release) as well as the BNST, medial amygdala, anterior hypothalamus, and arcuate nucleus (Kriegsfeld et al., 2006). Piekarski et al. found that administering RFRP3 to hamsters reduced sexual motivation (as measured by percent of time spent with castrated vs intact males) and vaginal scent marking without effect on lordosis behavior, similar to our present findings. Additionally, RFRP3 administration altered cellular activation in regions of the brain implicated in female sexual motivation, including the mPOA, medial amygdala, and BNST—all regions that receive RFRP projections. These effects were independent of gonadal steroids and kisspeptin cellular activation (Piekarski et al., 2013). While we were unable to measure progesterone or prolactin in this study, it is possible that RFRP projections to the arcuate nucleus affect dopaminergic signaling required for prolactin release and maintenance of progesterone levels and pregnancy success. Future studies aimed at systematically examining each step in these processes is required to gain a full understanding of the neural circuits underlying the deleterious effects of stress on reproduction.

In humans, RFRP 1 and 3, and their cognate receptor are expressed in the hypothalamus (Ubuka et al., 2009b). It is possible that manipulation of RFRP3 signaling in humans may relieve stress-related reproductive dysfunction, including decreased sex drive, impaired fertility, and increased miscarriages. Likewise, if similar mechanisms of stress-induced reproductive suppression are common across species, such strategies may be similarly relevant to species bred in captivity that are susceptible to stress-induced infertility, in particular, endangered species whose preservation depends on captive breeding programs.

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Author details

1. Anna C Geraghty

   Department of Integrative Biology, University of California, Berkeley, Berkeley, United States

   Contribution

   ACG, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

2. Sandra E Muroy

   Department of Integrative Biology, University of California, Berkeley, Berkeley, United States

   Contribution

   SEM, Conception and design, Acquisition of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-4014-5189

3. Sheng Zhao

   Department of Psychology, University of California, Berkeley, Berkeley, United States

   Present address

   Department of Biochemistry and Molecular Biology, Southeast University, Jiangsu, China.

   Contribution

   SZ, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

4. George E Bentley

   1. Department of Integrative Biology, University of California, Berkeley, Berkeley, United States
   2. Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, United States

   Contribution

   GEB, Conception and design, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   Contributed equally with

   Lance J Kriegsfeld and Daniela Kaufer

   Competing interests

   The authors declare that no competing interests exist.

5. Lance J Kriegsfeld

   1. Department of Psychology, University of California, Berkeley, Berkeley, United States
   2. Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, United States

   Contribution

   LJK, Conception and design, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   Contributed equally with

   George E Bentley and Daniela Kaufer

   Competing interests

   The authors declare that no competing interests exist.

6. Daniela Kaufer

   1. Department of Integrative Biology, University of California, Berkeley, Berkeley, United States
   2. Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, United States
   3. Canadian Institute for Advanced Research, Toronto, Canada

   Contribution

   DK, Conception and design, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   Contributed equally with

   George E Bentley and Lance J Kriegsfeld

   For correspondence

   danielak@berkeley.edu

   Competing interests

   The authors declare that no competing interests exist.

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