Years of research have shown that time in the womb is crucial for brain development; so is early childhood, when experiences and nutrition alike can shape the growing nervous system.

But what about the day that separates the two?

Birth may play a larger role in brain development than previously believed, a new and ongoing study reveals. The long-term implications for humans are still fuzzy, but if the findings hold, they'll position the brain as one of many organs influenced by the birth experience.

"We know these major organs — the heart, the lungs — are being prepared prenatally for life outside the mother," said Alexandra Castillo-Ruiz, a postdoctoral researcher at Georgia State University. "We know little about the brain."

And we don't know whether changing the method of birth will affect how the brain develops — and if so, how. The research is still in its early stages, but scientists are finding evidence that cesarean delivery may influence health in the long run by, for example, increasing the child's risk for juvenile arthritis and inflammatory bowel disease, among other autoimmune conditions. Is there a similar effect for the brain as well?

Birth, bacteria and natural neuronal death

One of the links between birth and health has been traced to the microbiome, the mass of bacterial colonies that live on our skin and in our gut. During labor and delivery babies are colonized with their mothers' vaginal microbiota, primarily Lactobacillus, according to a 2012 review of research. Babies born via cesarean, by contrast, are mostly colonized by skin-dwelling species such as staphylococcus and Aceinetobacter. Scientists are increasingly converging on the idea that early bacterial exposure influences the immune system for life.

These immune changes — or perhaps something else about the experience of birth — may affect the brain, too. Castillo-Ruiz and her advisor, neuroscientist Nancy Forger, had noticed a strange spike in cell death right around the time of birth in baby mice. That sounds alarming, but it's not: As the nervous system grows, it overproduces brain cells, only to kill off the ones it doesn't need.

"We were struck by this peak of cell death right at birth," Forger said. It happens across the brain, she said, in the hippocampus, the prefrontal cortex, the hypothalamus and more.

Forger and Castillo-Ruiz wondered if birth itself was kick-starting this process. Some recent work suggests that the immune system plays a role in this cell destruction, and birth is an immunological experience. Not only are babies introduced to mom's microbiome during delivery, but are also exposed to inflammatory signals in the uterus as the process of labor begins.

The researchers bred mice and watched them like hawks 19 days after conception, when delivery was imminent. Forger's team made hourly, round-the-clock checks on the pregnant mothers. When one began to give birth, she selected another mouse at the same point in gestation that wasn't yet in labor. Then she conducted a mouse-y cesarean to deliver that mother's litter. In this way, the researchers could compare the effects of delivery method without any confounding data on gestational length.

The project is still ongoing, but the researchers presented some preliminary results at the annual meeting of the Society for Neuroscience in October. Early analysis of the mice brains suggests that the delivery method mattered. Three hours after birth, baby mice born by C-section show decreased cell death compared with mice born vaginally, she said.

The team has also measured physical development of the offspring. The method of birth didn't seem to affect initial body weight or time of eye-opening in juvenile mice. But later on at weaning age, the researchers observed an increase in body weight in cesarean born mice, which is in line with reports of higher body mass index and risk of adult obesity in humans born by cesarean section, the researchers said.

Looking at the behavior of baby mice, the researchers found that cesarean-born mice made softer cries when separated from their mothers at 9 days old than mice born vaginally. This could be meaningful for a little mouse's life and survival, Forger said, because other research has found that moms pay more attention to the mouse babies when they're louder.

As for the change in the rate of naturally occurring neuronal death, the researchers are planning to get to the bottom of the underlying mechanism. They are now investigating whether the microglia, the immune cells of the brain, have a role in neuronal cell death and whether their function is altered depending on how a baby mouse is born. That focus on a potential mechanism for the changes is a strength of the study, said John Cryan, a neuroscientist at University College Cork who was not involved in the research but who has studied the long-term neurobehavioral effects of cesarean sections and found changes in anxiety and social behavior in mice.

"Early-life manipulations can have long-term effects in sculpting behavior later on, and this is a very important area both in basic science, but also in clinical medicine," Cryan said.

Compensating for cesareans

The effects of birth aren't all immunological, either — babies about to be born are exposed to a massive flood of hormones during labor. If labor never starts, that hormonal influence never happens. One of these hormones is oxytocin, a poorly understood compound, which nevertheless seems to have a variety of effects in the brain. For example, it has been found to switch the function of the neurotransmitter GABA in mice—In fetal mice brain, GABA has an excitatory influence on neurons. Shortly before delivery, oxytocin triggers a chain of events that causes GABA to have inhibitory actions in the mature brain. This shift doesn't happen in some rodent models of autism, Aix-Marseille University researcher Yehezkel Ben-Ari and colleagues found in a 2014 paper published in the journal Science. Cryan and his colleagues, too, have also looked at the effects of oxytocin exposure on the newborn brain. They found that a post-delivery dose of oxytocin can reverse some of the behavioral effects seen in mice born by C-section, they reported in October at the Society for Neuroscience as well.

In the face all these potential effects just coming to light, other researchers have started to look into ways to make cesareans seem a little bit more like vaginal birth. The World Health Organization estimates that about 10 percent to 15 percent of births need to end in C-section in order to prevent the deaths of both moms and babies. Researcher Maria Dominguez-Bello of the New York University School of Medicine and her colleagues have been conducting an experiment in which a piece of gauze is placed in the vagina of a mother about to undergo a cesarean, and then used to swab the baby's skin after birth. Her research suggests that this strategy can partially restore the baby's skin microbiome to what it would look like after a vaginal delivery.

There are caveats, of course. We still know too little about the long-term, real-world neurological effects of birth in humans, Cryan said. In fact, some mothers may not have a vaginal microbiome you'd want a baby introduced to. In Dominquez-Bello's work, the team tries to control for such unknowns by testing mothers to ensure their vaginal microbiome is Lactobacillus-dominated, strep-B negative and otherwise healthy.

And there are mothers' needs to consider. Choosing the method of giving birth is not just a medical but also a personal decision, which makes it all the more important to continue investigating the true effects of birth methods, so moms can make informed birth choices.

"The last thing we want to do is go blaming moms," Cryan said. But in some places, medical policy has pushed cesarean rates sky-high. In the United States, the C-section rate is about 30 percent. Although this is considered a high rate by medical societies such as The American College of Obstetricians and Gynecologists, rates are even higher elsewhere. In Brazil, for example, 85 percent of private hospital births are done by cesarean, as are 45 percent in public hospitals. Those high numbers make a close look at consequences necessary, Forger said.

Cryan agreed.

"When you have up to 70 percent, you know there are other factors playing a role [in the C-section rate]," Cryan said. "If we appreciated what the long-term consequences are, then we may change our obstetric practice a little bit."