We conducted a direct test of an immunological explanation of the finding that gay men have a greater number of older brothers than do heterosexual men. This explanation posits that some mothers develop antibodies against a Y-linked protein important in male brain development, and that this effect becomes increasingly likely with each male gestation, altering brain structures underlying sexual orientation in their later-born sons. Immune assays targeting two Y-linked proteins important in brain development-protocadherin 11 Y-linked (PCDH11Y) and neuroligin 4 Y-linked (NLGN4Y; isoforms 1 and 2)-were developed. Plasma from mothers of sons, about half of whom had a gay son, along with additional controls (women with no sons, men) was analyzed for male protein-specific antibodies. Results indicated women had significantly higher anti-NLGN4Y levels than men. In addition, after statistically controlling for number of pregnancies, mothers of gay sons, particularly those with older brothers, had significantly higher anti-NLGN4Y levels than did the control samples of women, including mothers of heterosexual sons. The results suggest an association between a maternal immune response to NLGN4Y and subsequent sexual orientation in male offspring.

The fraternal birth order effect (FBOE) is the finding that older brothers increase the probability of homosexuality in later-born males, and the female fecundity effect (FFE) is the finding that the mothers of homosexual males produce more offspring than the mothers of heterosexual males. In a recent paper, Khovanova proposed a novel method for computing independent estimates of these effects on the same samples and expressing the magnitude and direction of the effects in the same metric. In her procedure, only families with one or two sons are examined, and daughters are ignored. The present study investigated the performance of Khovanova's method using archived data from 10 studies, comprising 14 samples totalling 5390 homosexual and heterosexual subjects. The effect estimate for the FBOE showed that an increase from zero older brothers to one older brother is associated with a 38% increase in the odds of homosexuality. By contrast, the effect estimate for the FFE showed that the increase from zero younger brothers to one younger brother is not associated with any increase in the odds of homosexuality. The former result supports the maternal immune hypothesis of male homosexuality; the latter result does not support the balancing selection hypothesis.