A research team led by Brock University, which included researchers from Harvard and the University of Toronto, has further evidenced that men’s sexual orientation is likely determined in the womb.

The study, a culmination of two decades of research on the “older brother effect” further suggests a link between the number of older brothers and increased odds of being gay, at a biological level.

Brock said the study — called Male Homosexuality and Maternal Immune Responsivity to the Y-Linked Protein NLGN4Y — was “prompted by more than two decades of statistical data examining the “older brother effect” which shows that biological older brothers — but not older sisters — increase the odds of homosexuality in younger later-born males.”

The study was published Monday in the journal proceedings of the National Academy of Sciences and was the first-ever laboratory study of mothers of gay men.

Tony Bogaert, health sciences professor at Brock and lead researcher on the project, said the implications of the study, “especially if and when it is replicated by an independent team, are profound.”

“Along with more deeply understanding the exact origin of the older brother effect, it helps solidify the idea that, at least in men, there’s a strong biological basis to sexual orientation,” said Bogaert.

“The current study adds to the growing scientific consensus that homosexuality is not a choice, but rather an innate predisposition.”

Bogaert, an internationally recognized expert in human sexuality, said the study is groundbreaking for at two major reasons:

One, it supports the conclusion, suggested by previous studies, that genes alone do not completely account for homosexuality. And two, It suggests that immunological factors should be considered along with genetic and hormonal factors as possible biological influences on sexual orientation.

The team of psychologists and immunologists tested 16 women with no sons, 72 mothers with heterosexual sons, 31 mothers of gay sons with no older brothers, 23 mothers of gay sons with older brothers, and a control group of 12 men.

The women’s antibody reactivity was measured to two proteins (PCDH11Y and two forms of NLGN4Y) found only in males, both of which are expressed in the male fetal brain.

The team found that mothers of gay sons, especially those with older brothers, had significantly higher antibody levels to both forms of NLGN4Y than did the control samples of women, including mothers of heterosexual sons.

“It seems that some women during their first male pregnancy, or just after their first male birth, begin to detect this foreign substance (the NLGN4Y protein) and start to develop an immune response. And then later, with further male pregnancies, the high levels of antibodies directed toward this substance may change brain development in these later born males,” Bogaert said.

Bogaert said this implicates the older brother effect is very likely immunological in origin.

“We think it’s very important to understanding male sexual orientation … It speaks to the mechanisms of sexual orientation development,” said Bogaert.

However, he cautions that the effects are modest and the likelihood of a male child being born gay is still small even if they have multiple male siblings.

“The vast majority of men with older brothers are still heterosexual, but it says something very broad about sex and gender development.”

Bogaert says the NLGN4Y protein is likely very significant to male brain development, and will need to be examined further.

“NLGN4Y probably plays a role in how male nerve cells communicate with one another. So, it could potentially affect brain structures associated with attractions to others. And a maternal immune response might alter typical functioning of these structures in a male fetus.”

“We think it’s as important as any piece of sexual orientation research in the past 10 or 15 years, but it still needs to be replicated by an independent lab to verify it.”

Bogaert’s was joined by his assistant professor Adam MacNeil,  Brock PhD students José Gabrie, Malvina Skorska (now at University of Toronto) and Mark Hoffarth (now at New York University), Harvard’s Chao Wang and University of Toronto’s Doug VanderLaan, Ken Zucker and Ray Blanchard.