Severe COVID-19 linked to anxiety in offspring via altered sperm RNA

Male mice that recover from a severe COVID-19-like infection can pass anxiety-like traits to their offspring by altering the RNA molecules inside their sperm. These findings suggest that a paternal viral infection prior to conception might alter the developmental trajectory of the next generation. The research was published in the journal Nature Communications.

Biologists once thought that parents only passed down genetic information through the fixed sequence of DNA in their sperm and egg cells. Researchers now recognize that environmental factors, such as stress or diet, can alter traits in offspring without changing the underlying DNA code. This process is known as epigenetic inheritance. One major pathway for epigenetic inheritance involves small noncoding RNAs, which are tiny molecular messengers that control how and when other genes turn on or off without building proteins themselves.

Past experiments have shown that exposing male mice to bacterial or parasitic infections alters the small noncoding RNAs in their sperm. This internal shift can alter the brain development and behavior of their future offspring. A team of scientists led by Elizabeth Kleeman and Anthony Hannan at the Florey Institute of Neuroscience and Mental Health wanted to know if a respiratory virus could produce similar intergenerational outcomes. They chose to study SARS-CoV-2 because hundreds of millions of people have contracted the virus globally since the start of the pandemic.

The researchers utilized a small study design relying on an established mouse model of SARS-CoV-2. They infected adult male mice with the virus and gave a control group a harmless mock infection. The infected animals experienced moderate to severe illness, marked by a temporary drop in body weight. Four weeks later, after the mice had fully cleared the virus, the researchers mated both groups with healthy female mice that had never encountered the pathogen.

When the offspring of these pairings reached adulthood, the researchers evaluated their behavior. They placed the mice in an enclosure featuring a brightly lit area and a concealed dark zone. Mice naturally prefer dark spaces, and spending less time in the light indicates higher levels of anxiety. The offspring of the fathers infected with SARS-CoV-2 spent much less time exploring the bright zone compared to the uninfected control group.

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The male offspring in this group also hesitated much longer before entering the lit area at all. The researchers observed comparable results in an open field test, where the offspring of infected fathers avoided the exposed center of the testing arena. The researchers also subjected the mice to tests evaluating memory, sociability, and depression. The offspring of the infected fathers showed no differences in their ability to recognize novel objects or interact with unfamiliar mice, indicating that the primary behavioral shift centered on anxiety.

The scientists also examined the offspring’s brains, focusing on the hippocampus, a region involved in emotional regulation. They discovered altered gene expression profiles in the offspring of the infected mice. These alterations were particularly pronounced in the female offspring, who showed reduced activity in several genes linked to stress responses. Similar gene reductions frequently appear in rodent models of chronic stress.

To find out if these behavioral changes persisted across multiple generations, the team conducted a second breeding experiment. They took the male offspring from the first generation and mated them with a new group of healthy females. The resulting grand-offspring exhibited some early developmental differences, including slightly altered body weights. As these grand-offspring matured, they did not display the elevated anxiety traits seen in their parents, suggesting the behavioral effect faded after one generation.

The researchers then sought to uncover the biological mechanism driving the anxiety-like traits in the first generation. They collected sperm from the original groups of infected and healthy male mice four weeks after their initial exposure. An analysis of the sperm revealed modified levels of multiple small noncoding RNAs in the animals that had contracted SARS-CoV-2. Some clusters of these regulatory molecules were less abundant, while a few specific types were highly elevated.

Specifically, the researchers identified drops in the expression of PIWI-interacting RNAs, which are specialized molecules that protect the genome from mutations during sperm development. They also found elevated levels of certain microRNAs, which are known to influence early embryonic growth. To confirm that these specific RNA molecules caused the behavioral changes, the team designed a microinjection experiment. They extracted the small RNA cargo from the sperm of both the infected and the uninfected control mice.

Using microscopic needles, the researchers injected this extracted RNA directly into healthy, fertilized mouse eggs. They implanted these embryos into surrogate female mice and allowed the resulting offspring to grow into adulthood. The adult mice that developed from the eggs injected with the infected sperm RNA exhibited traits mimicking the naturally conceived offspring. In the light and dark box test, the male mice from this group showed heightened hesitation before entering the brightly lit zone.

While not every behavioral difference transferred perfectly, the presence of anxiety-like symptoms confirmed that the sperm RNA played a direct role in shaping the offspring’s brain development. The isolated molecular cargo was enough to recreate portions of the intergenerational effect. A few important caveats accompany these results. The study relied entirely on animal models, and biological responses in mice do not perfectly mirror human health outcomes.

The viral infection caused notable weight loss in the adult male mice, which presents a confounding variable. Severe metabolic stress and sudden weight loss can independently trigger epigenetic changes in sperm. It is difficult to separate the effects of the virus itself from the physical toll of a severe illness. Additionally, studying a dangerous pathogen required the researchers to conduct their behavioral assessments inside a highly restricted biosafety facility. Space limitations in this environment prevented the use of larger behavioral testing arenas.

The researchers noted that tracking human outcomes takes decades, making animal models a necessary starting point. Future studies will need to determine whether milder infections, antiviral treatments, or prior vaccinations modify the RNA content in sperm. Resolving these questions will help clarify if the global spread of SARS-CoV-2 might subtly influence the mental health of children conceived in the aftermath of the pandemic.

The study, “Paternal SARS-CoV-2 infection impacts sperm small noncoding RNAs and increases anxiety in offspring in a sex-dependent manner,” was authored by Elizabeth A. Kleeman, Carolina Gubert, Sonali N. Reisinger, Kathryn C. Davidson, Da Lu, Merle Dayton, Liana Mackiewicz, Bethany A. Masson, Pranav Adithya, Alexandra L. Garnham, Gemma Stathatos, Moira K. O’Bryan, Rikeish R. Muralitharan, Francine Z. Marques, Shanshan Li, Huan Liao, Shae McLaughlin, Emmet T. Keough, Michelle Y. Wheeler, Pamudika Kiridena, Marcel Doerflinger, Marc Pellegrini, and Anthony J. Hannan.