Twin Fates

Kelly Klump is a curly-haired, compact woman who is fascinated by eating disorders. Her own habits are healthy, but as a high school “peer counselor” she found herself besieged by girls struggling with the addictive starvation of anorexia nervosa and the compulsive binge-and-purge of bulimia. Now a 37-year-old associate professor at MichiganStateUniversity in East Lansing, Klump has spent the past 10 years probing the genetic influences in such illnesses and pondering a stubborn question about why biology makes women more likely targets than men for eating disorders.

Lately she has revisited that frustrating question from a new angle. Working with graduate student Kristen Culbert and other colleagues, Klump published a paper in the March Archives of General Psychiatry focusing on a very specific group: females from a male-female twin pair.

A few years ago this would have seemed a rather narrow approach to a widespread problem. But several recent studies now suggest that the girl twin in a mixed pair offers provocative evidence concerning the way biology shapes people before birth.

Psychologists in both the United States and Europe have found that females from opposite-sex twin pairs tend to be more aggressive and adventurous, process spatial information more like men, and show more typically masculine left brain dominance during language tests. Across a range of research, these female co-twins seemed shifted toward the male end of the behavioral spectrum.

Such studies prompted Klump and Culbert to test a specific hypothesis: that girls with boy twins would also behave more like boys when it came to eating disorders. In other words, these girls’ risk would drop. Using an eating behavior survey, questioning several hundred twin-pairs from the Michigan twin registry, and looking at opposite-sex and same-sex, both male and female twins, the scientists found their prediction was absolutely on.

Their female co-twins had a lower level of eating disorders, tending toward the male range. By contrast, the same-sex female twins had the highest level of all the twin sets questioned. The MichiganState scientists suspect that the reason can be found in the prenatal environment: Sharing the space with a developing male can apparently alter female development in some small but interesting ways.

“Opposite-sex twins have not historically been a target for biological research,” says Dennis McFadden, a psychology professor at the University of Texas at Austin, who has been studying male-pattern hearing in female co-twins. “For a long time, people just thought that all the learning could be done in same-sex twins. So until recently they’ve been grossly understudied. And now, all at once, there’s awareness of this tantalizing potential.”

Testosterone tsunami

Developmental biologists have long known that exposure to hormones during gestation has a potent effect on fetal development, especially for males. Near the end of the first trimester of human pregnancy, at about 10 to 12 weeks, male fetuses begin producing a remarkable blast of the steroid hormone testosterone, “something like adult levels,” says psychology professor and primate researcher Kim Wallen of Emory University in Atlanta. A similar process exists for most mammals: The timing and the amount of androgens such as testosterone before birth are essential to normal male development.

Human fetuses begin life in a sex-neutral body, whether carrying the XX chromosome match that signals a tiny female or the XY pairing that means a male. But there’s another factor in play: Female is considered the “default” state for human development; without that extra testosterone, the body simply continues toward a female design. If XY males don’t get enough prenatal androgens, as happens with some genetic defects, those males develop looking like well-formed females.

The female fetuses, on the other hand, don’t need to crank up estrogen to turn into a girl. In fact, they tend to produce the hormone at mere trace levels throughout gestation. So, starting in the late 1970s, scientists began to wonder whether exposure to a brother’s testosterone before birth might affect the sister’s development.

Biologists studying mice turned up the first indications that this was a real possibility. Like humans, male mouse fetuses produce a critical testosterone boost, about midway through gestation. Unlike humans, mice routinely produce large mixed-sex litters. In fact, during their mother’s pregnancy, the fetal mice are packed in place, almost as neatly as a line of peas in a pod. The researchers discovered that for developing females, the sex of their pod-neighbor made a real difference.

Females who were surrounded by other females in utero (sometimes called 0M for their zero-male exposure) developed in the standard way. So mostly did those between a male and another female (1M). But females who were placed between two males (2M) could be identified, after birth, by their more-male body proportions. Further experiments confirmed that the physical difference could be traced to a prenatal diffusion of testosterone (steroid hormones slip fairly easily through cell membranes). In fact, as zoologist John Vandenbergh wrote, “the more males in proximity to a given female, the more masculine characteristics the female displays.”

Further studies showed that these changes in the body shape were indicators of other changes. For instance, a region of the brain called the hypothalamus, which is influential in mating behavior, is typically larger in male mice. It turned out that the 2M females also had larger hypothalamuses, had delayed puberty, had more irregular cycles, and were more independent-natured and aggressive. Notably, once the mice all reached mating age, if males were given a choice between a 0M and 2M female, they chose the more “female” mouse.

More like her twin brother

During the 1980s, Vandenbergh—now a professor emeritus of zoology at North CarolinaStateUniversity—decided to see what this meant in terms of how the mice interacted with others, including all important reproductive behaviors. With his colleagues, he let loose mice populations into the grassy cloverleafs of highways around Raleigh. Within each group, the scientists released about 20 0M and 20 2M females, marking them all for observation.

Over time, the zoologists discovered that the 2Ms were the most adventurous females; their home ranges were some 40 percent larger than the 0Ms, who tended to be homebodies. But being more assertive and adventurous didn’t seem to lend a reproductive advantage. The home-loving 0Ms tended to raise larger litters, although Vandenbergh points out that in a less confined, harsher environment, the exploring tendencies of the 2M mice would offer a greater advantage. In other words, the variety itself offers an advantage in overall species survival.

“It demonstrates the importance of changes during fetal development,” Vandenbergh says. “We all try to explain variability, why brains are different, and we’re all trying to find explanations for some of that variation. This is at least a reminder that a lot can happen in the fetal environment—that it’s a very sensitive time.”

Animal researchers can directly experiment with such ideas. Emory’s Wallen has demonstrated that by blocking testosterone at different times during gestation, he can alter both the physiology and behavior of monkeys, inducing little males, for instance, to vocalize like females. But as he and McFadden both note, comparable studies cannot be done with human research subjects.

“Animal research offers some really nice opportunities,” McFadden says. Human researchers are generally “forced to capitalize on the manipulations of nature,” mostly by studying people with genetic defects or variations that cause either an excess of androgens or an inability to absorb them.

Listening for differences

McFadden began his own work on female co-twins in the 1990s while running a series of tests on hearing, comparing different groups to try to sort out genetic influences. In particular, he was looking at a phenomenon called “otoacoustic emissions,” in which vibrations within the ear, as it responds to sounds, create faint popping noises that can be registered on scientific instruments. In general, the more sensitive the hearing is, the greater the frequency of such sounds. Overall, women’s ears pop more, men’s ears less often. But when McFadden tallied up his results across a range of population groups, he discovered that one group of women looked markedly different. Females with a twin brother tended toward the male range of the tests.

The finding has held up, with remarkable consistency, for more than a decade. McFadden, in the meantime, has developed a wide-ranging expertise on the association between prenatal testosterone and physical development in females, such as the heavy masculine bulk of female spotted hyenas, known to experience remarkably high androgen exposure before birth. But neither he, nor anyone else, reports such visible differences in humans. In fact, McFadden’s inner-ear finding, some controversial studies on finger length and one report on tooth sizes are the only results so far reported with measurable, physical differences between female co-twins and other women.

“I now argue that thinking about hormonal effects as always global is misleading,” McFadden says. “And that brings me back to a caveat about opposite-sex twins. There’s a whole array of things that definitely are not affected. It seems more sensible to think instead of relatively localized effects in body and in time. There are probably critical periods of development during which, if androgen levels are high, things end up being masculinized.”

Critical nurture

Such caveats, of course, provide an easy opening for critics who say that it would be a mistake to draw too many conclusions from existing opposite-sex twin research. After all, Wallen says, humans have not shown the easily observable effects found in other species. He speculates that human fetuses might be more buffered against each other’s hormone production: “The nature of the chorion [the protective membrane around each fetus] is so different in humans and rodents,” he says. “They have a much leakier system than ours. You have to think that would make a difference.”

Others complain that opposite-sex twin studies are usually done in relatively small groups, often of a hundred or less, and that few of them have been replicated.

Further, as CambridgeUniversity psychologist Melissa Hines emphasizes, the behavioral differences can also be explained by the simple fact of growing up with a twin brother.

Hines, author of the 2005 book Brain Gender, compared play styles across groups of children, including female co-twins. The opposite-sex twins did show a more boyish style of play, but, Hines says, no more so than most girls raised with brothers in the family. She concluded that the socializing effect of a brother was as important, or more, than prenatal exposure to androgens in such cases.

“Obviously, hormones have a prenatal effect,” Hines adds. “But we can’t say that’s the explanation for everything. A lot of these studies don’t control for the social effect of having an opposite-sex twin. Look, it could be one or the other and it could be both. So we deliberately set out to see if the effect of the twin was bigger than the effect of the brother. And it wasn’t. After that, I went on to other studies. I didn’t find that line of inquiry as rewarding.”

But Hines says that one study has encouraged her to reconsider. It appeared in the Proceedings of the National Academy of Sciences last June.

That research, published by evolutionary biologist Virpi Lummaa of the University of Sheffield in England, reviewed carefully kept Finnish birth, death and marriage records from the 18th and 19th centuries. In those pre-birth control, pro-marriage times, female co-twins were 25 percent less likely to have children than female-female twins, raised smaller families and were, in fact, less likely to marry at all. Further, Lummaa’s data showed that the result held steady across social classes and even if the male twin died within three months of birth, leaving the girl to be brought up as an only child.

“I definitely think that this is one of the major strengths of our paper,” Lummaa says. Most other studies using modern human data cannot rule out the fact that growing up and playing with a similar-aged brother affects your behavior and attitudes.

“The fact that twin mortality — especially during the few weeks after birth — was so high during our study period makes it possible for us to compare those females who were born with a brother or sister but subsequently raised as singletons. Given that we still see differences between them, this really has to be of prenatal origin,” she adds.

Pondering the prenatal

Michigan State’s Klump and Culbert also collected data suggesting that growing up with brothers was less influential than a shared womb. Opposite-sex twin girls consistently showed fewer signs of eating disorders than girls raised within a family with brothers. “In none of the groups could the eating patterns be accounted for by levels of anxiety or socialization,” Klump and Culbert wrote in the March paper. “Indeed, opposite-sex female twins exhibited lower levels of disordered eating compared to an independent sample of undergraduate women who were raised with one or more brothers.”

Culbert said they would still like to find out why prenatal androgens might affect eating disorders, as opposed to other behaviors. Like McFadden, she suggests that the scatter of human results hints at very specific effects of prenatal androgen exposure: “I can speculate that there are areas where this becomes highly concentrated,” she says. “But basically we just don’t know.” Klump adds that the next step is to replicate their finding and use that confirmation to argue for funding to search for the mechanism itself.

As Klump points out, the relevance is not that female opposite-sex twins are somehow different. What makes these studies worthwhile is the way they help reveal some of the fundamental processes involved in early human development. And, of course, Klump hopes that such understanding will also lead to improved options for those battling eating disorders. “The more we understand what factors are important,” she says, “the more we can move toward the right ways to treat them.”

Deborah Blum is a Pulitzer prize–winning science journalist on the faculty of the University of Wisconsin–Madison.