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Since the 1990s, a large volume of research has demonstrated that each older biological brother a man has from the same mother increases his odds of being gay by 28–48%. This phenomenon is known as the fraternal birth order effect. The correlation is not found in those with older adoptive or step brothers, leading scientists to attribute this to a maternal immune response to developing male fetuses, rather than a social effect. It has been estimated that between 15% and 29% of gay men owe their orientation to this effect, although this may be higher when factoring in miscarriages of male fetuses. In 2017, biochemical evidence for the effect was found which demonstrated that mothers of sons, particularly those with gay sons, had significantly higher levels of antibodies to male NLGN4Y proteins than mothers with no sons. Biologist Jacques Balthazart said that the finding "adds a significant chapter to growing evidence indicating that sexual orientation is heavily influenced by prenatal biological mechanisms rather than by unidentified factors in socialization". The effect is an example of a non-genetic influence on male sexual orientation occurring in the prenatal environment. The effect does not mean that all or most sons will be gay after several male pregnancies, but rather, the odds of having a gay son increase from approximately 2% for the first born son, to 3% for the second, 5% for the third.
Between the 1960s and 2000, many newborn and infant boys were surgically reassigned as females if they were born with malformed penises, or if they lost their penises in accidents. Many surgeons believed such males would be happier being socially and surgically reassigned female. In all seven published cases that have provided sexual orientation information, the subjects grew up to be strongly attracted to females. In Psychological Science in the Public Interest, six scientists including J. Michael Bailey state this establishes a strong case that male sexual orientation is partly established before birth:
This is the result we would expect if male sexual orientation were entirely due to nature, and it is opposite of the result expected if it were due to nurture, in which case we would expect that none of these individuals would be predominantly attracted to women. They show how difficult it is to derail the development of male sexual orientation by psychosocial means.
They further argue that this raises questions about the significance of the social environment on sexual orientation, stating, "If one cannot reliably make a male human become attracted to other males by cutting off his penis in infancy and rearing him as a girl, then what other psychosocial intervention could plausibly have that effect?" It is further stated that neither cloacal exstrophy , nor surgical accidents, are associated with abnormalities of prenatal androgens, thus, the brains of these individuals were male-organized at birth. Six of the seven identified as heterosexual males at follow up, despite being surgically altered and reared as females, with researchers adding: "available evidence indicates that in such instances, parents are deeply committed to raising these children as girls and in as gender-typical a manner as possible." Bailey et al. describe the occurrence of these sex reassignments as "the near-perfect quasi-experiment" in measuring the impact of 'nature' versus 'nurture' with regards to male homosexuality.