Editing Biology and sexual orientation (section)

==Scientific research and studies==

=== Fetal development and hormones ===
{{Main|Prenatal hormones and sexual orientation}}
The influence of hormones on the developing fetus has been the most influential causal hypothesis of the development of sexual orientation.<ref name="Bailey_2016" /><ref name="Balthazart_2011">{{cite journal | vauthors = Balthazart J | title = Minireview: Hormones and human sexual orientation | journal = Endocrinology | volume = 152 | issue = 8 | pages = 2937–47 | date = August 2011 | pmid = 21693676 | pmc = 3138231 | doi = 10.1210/en.2011-0277 | author-link = Jacques Balthazart }}</ref> In simple terms, the developing fetal brain begins in a "female" state. Both [[INAH3]] (third interstitial nucleus of the anterior hypothalamus) area on the left side of the hypothalamus, which stores gender preference, and the center area of the bed stria terminalis (BSTc) area on the right side of the hypothalamus, which stores gender identity, are undeveloped and function as female. The action of the SRY gene in the Y-chromosome in the fetus prompts the development of testes, which release testosterone, the primary androgen receptor-activating hormone, to allow testosterone to enter the cells and masculinize the fetus and fetal brain. If a sufficient amount of testosterone is received by INAH3 at 12 weeks following conception, the testosterone stimulates the enlargement of INAH3, which is known to be involved in directing typical male sex behavior, such as attraction to females. If INAH3 does not receive enough testosterone to override the circulating estrogen, it may not grow to the size typically observed in males. Subsequently, INAH3 may function as female or partially female, potentially causing same-sex attraction to males. Although the ''size'' of INAH3 in homosexual men compared to heterosexual men may not be statistically different, homosexual men may have a greater cell ''density'' per unit volume than heterosexual men, though a similar total number of INAH3 neurons.<ref name="Balthazart_2011" />

Studies have shown that INAH3 in [[gay men]] has likely been exposed to lower levels of testosterone in the brain compared to straight men, or had different levels of receptivity to its masculinizing effects, or experienced hormone fluctuations at critical times during fetal development. In women, if INAH3 receives more testosterone than is normal for females, INAH3 may enlarge somewhat or even to the size that is normal for males, increasing the likelihood of same sex attraction.<ref name="Bailey_2016"/> Supporting this are studies of the finger [[digit ratio]] of the right hand, which is a robust marker of prenatal testosterone exposure.{{Disputed inline|date=March 2023}} Lesbians tend to have significantly more masculine digit ratios, a finding which has been replicated in numerous cross-cultural studies.<ref name="Breedlove_2017">{{cite journal | vauthors = Breedlove SM | title = Prenatal Influences on Human Sexual Orientation: Expectations versus Data | journal = Archives of Sexual Behavior | volume = 46 | issue = 6 | pages = 1583–1592 | date = August 2017 | pmid = 28176027 | pmc = 5786378 | doi = 10.1007/s10508-016-0904-2 }}</ref> Controlled experiments in animals, where scientists manipulate exposure to sex hormones during gestation, can also induce lifelong male-typical behavior and mounting in females, and female-typical behavior in males.<ref name="Bailey_2016"/><ref name="Breedlove_2017"/><ref name="Balthazart_2011" /><ref name="Roselli_2018"/>

Maternal immune responses during fetal development are strongly demonstrated as causing male homosexuality and bisexuality.<ref name="Balthazart_2018">{{cite journal | vauthors = Balthazart J | title = Fraternal birth order effect on sexual orientation explained | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 115 | issue = 2 | pages = 234–236 | date = January 2018 | pmid = 29259109 | pmc = 5777082 | doi = 10.1073/pnas.1719534115 | bibcode = 2018PNAS..115..234B | doi-access = free }}</ref> Research since the 1990s has demonstrated that as a woman has more sons, there is a higher chance of later born sons having same-sex attraction. During pregnancy, male cells enter a mother's bloodstream, which provoke an immune response to neutralize them. These antibodies are then released on future male fetuses and may neutralize Y-linked antigens, which play a role in brain masculinization, leaving areas of the brain responsible for sexual attraction in the female-typical default position, i.e. expressing attraction to men. Each subsequent son will increase the levels of these antibodies, creating the observed [[fraternal birth order effect]]. Biochemical evidence to support this effect was confirmed in a lab study in 2017, finding that mothers with a gay son, particularly those with older brothers, had heightened levels of antibodies to the NLGN4Y Y-protein than mothers with heterosexual sons.<ref name="Balthazart_2018"/><ref name="Bogaert_2018">{{cite journal | vauthors = Bogaert AF, Skorska MN, Wang C, Gabrie J, MacNeil AJ, Hoffarth MR, VanderLaan DP, Zucker KJ, Blanchard R | display-authors = 6 | title = Male homosexuality and maternal immune responsivity to the Y-linked protein NLGN4Y | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 115 | issue = 2 | pages = 302–306 | date = January 2018 | pmid = 29229842 | pmc = 5777026 | doi = 10.1073/pnas.1705895114 | bibcode = 2018PNAS..115..302B | doi-access = free }}</ref> This effect is estimated to account for between 15 and 29% of gay men, while other gay and bisexual men are thought to owe sexual orientation to genetic and hormonal interactions.<ref>{{cite journal | vauthors = Blanchard R | title = Fraternal Birth Order, Family Size, and Male Homosexuality: Meta-Analysis of Studies Spanning 25 Years | journal = Archives of Sexual Behavior | volume = 47 | issue = 1 | pages = 1–15 | date = January 2018 | pmid = 28608293 | doi = 10.1007/s10508-017-1007-4 | s2cid = 10517373}}</ref><ref name="Balthazart_2018"/>

Socialization theories, which were dominant in the 1900s, favored the idea that children were born "undifferentiated" and were socialized into gender roles and sexual orientation. This led to medical experiments in which newborn and infant boys were surgically reassigned into girls after accidents such as botched circumcisions. These males were then reared and raised as females without telling them, but contrary to expectations, this did not make them feminine nor attracted to men. All published cases providing sexual orientation grew up to be strongly attracted to women. The failure of these experiments demonstrate that socialization effects do not induce feminine sexual behavior or psychology in males, and that the organizational effects of hormones on the fetal brain prior to birth have permanent effects. These indicate the primary role of nature, not post-birth nurture, in the development of male sexual orientation.<ref name="Bailey_2016"/>
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|caption=Average volumes of INAH3 in straight and gay men and in women.<ref name="LeVay_2016"/>
{{legend|#1f77b4|Heterosexual (straight) men}}
{{legend|#9467bd|Homosexual (gay) men}}
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In the brain, the sexually dimorphic nucleus of the preoptic area (SDN-POA) is a key region which differs between males and females in humans and a number of mammals (e.g., sheep/rams, mice, rats), and is caused by sex differences in hormone exposure.<ref name="Bailey_2016" /><ref name="Breedlove_2017"/> Also, the [[INAH 3|INAH-3]] region is bigger in males than in females, and is known to be critical for sexual behavior. Dissection studies found that gay men had significantly smaller INAH-3 than heterosexual men, a shift in the female direction, as first demonstrated by neuroscientist [[Simon LeVay]], which has been replicated.<ref name="Breedlove_2017" /> Dissection studies are rare, however, due to lack of funding and brain samples.<ref name="Bailey_2016" />
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|caption=Average volumes of the equivalent cell group in sheep (oSDN) for heterosexual and homosexual rams and for ewes.<ref name="LeVay_2016" /> Sex differences are formed under the influence of prenatal hormones in utero, rather than acquired after birth.<ref name="Roselli_2018" />
{{legend|#1f77b4|Heterosexually oriented rams}}
{{legend|#9467bd|Homosexually oriented rams}}
{{legend|#e377c2|Ewes (females)}}
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Long-term studies of [[Homosexual behavior in sheep|homosexual behavior in domesticated sheep]] led by Charles Roselli have found that 6-8% of rams have a homosexual preference through their life. Dissection of ram brains also found a similar smaller (feminized) structure in homosexually oriented rams in the brain region equivalent to human SDN, the ovine sexually dimorphic nucleus (oSDN).<ref name="LeVay_2016">{{cite book |url=https://archive.org/details/gaystraightreaso0000leva_e5c2/mode/1up?view=theater |title=Gay, Straight, and the Reason Why: The Science of Sexual Orientation |vauthors=LeVay S |date=2017 |publisher=Oxford University Press |isbn=978-0-19-029739-8 |language=en |chapter= |ol=26246092M |chapter-url= |ol-access=free}}</ref>{{rp|107–110}} The size of the sheep oSDN has also been demonstrated to be formed in utero, rather than postnatally, underscoring the role of prenatal hormones in masculinization of the brain for sexual attraction.<ref name="Roselli_2018">{{cite journal | vauthors = Roselli CE | title = Neurobiology of gender identity and sexual orientation | journal = Journal of Neuroendocrinology | volume = 30 | issue = 7 | pages = e12562 | date = July 2018 | pmid = 29211317 | pmc = 6677266 | doi = 10.1111/jne.12562 }}</ref><ref name="Bailey_2016" />

Other studies in humans have relied on brain imaging technology, such as research led by [[Ivanka Savic]] which compared hemispheres of the brain. This research found that straight men had right hemispheres 2% larger than the left, described as modest but "highly significant difference" by LeVay. In heterosexual women, the two hemispheres were the same size. In gay men, the two hemispheres were also the same size, or sex atypical, while in lesbians, the right hemispheres were slightly larger than the left, indicating a small shift in the male direction.<ref name="LeVay_2016" />{{rp|112}}

A model proposed by evolutionary geneticist William R. Rice argues that a misexpressed epigenetic modifier of testosterone sensitivity or insensitivity that affected development of the brain can explain homosexuality, and can best explain twin discordance.<ref name=":4" /> Rice et al. propose that these epimarks normally canalize sexual development, preventing [[intersex]] conditions in most of the population, but sometimes failing to erase across generations and causing reversed sexual preference.<ref name=":4" /> On grounds of evolutionary plausibility, Gavrilets, Friberg and Rice argue that all mechanisms for exclusive homosexual orientations likely trace back to their epigenetic model.<ref>{{cite journal | vauthors = Gavrilets S, Friberg U, Rice WR | title = Understanding Homosexuality: Moving on from Patterns to Mechanisms | journal = Archives of Sexual Behavior | volume = 47 | issue = 1 | pages = 27–31 | date = January 2018 | pmid = 28986707 | doi = 10.1007/s10508-017-1092-4 | s2cid = 33422845 | url = https://www.gwern.net/docs/psychology/2017-gavrilets.pdf }}</ref> Testing this hypothesis is possible with current stem cell technology.<ref>{{cite journal | vauthors = Rice WR, Friberg U, Gavrilets S | title = Homosexuality via canalized sexual development: a testing protocol for a new epigenetic model | journal = BioEssays | volume = 35 | issue = 9 | pages = 764–70 | date = September 2013 | pmid = 23868698 | pmc = 3840696 | doi = 10.1002/bies.201300033 }}</ref>

=== Prenatal thyroid theory ===
[[File:Thyroid PCOS Orientation.jpg|thumb]]
Prenatal thyroid theory of same-sex attraction/gender dysphoria has been based on clinical and developmental observations of youngsters presenting to child psychiatry clinics in Istanbul/Turkey. The report of 12 cases with same-sex attraction/gender dysphoria born to mothers with thyroid diseases was first presented in EPA Congress, Vienna (2015) and published as an article in the same year.<ref>{{cite journal | vauthors = Sabuncuoglu O |date= March 2015 |title=Maternal Thyroid Dysfunction During Pregnancy May Lead to Same-sex Attraction/gender Nonconformity in the Offspring: Proposal of Prenatal Thyroid Model |journal=European Psychiatry |volume=30 |pages=374 |doi=10.1016/s0924-9338(15)30294-7 |s2cid= 143359069 |issn=0924-9338}}</ref><ref>{{cite journal | vauthors = Sabuncuoglu O | title = High Rates of Same-Sex Attraction/Gender Nonconformity in the Offspring of Mothers with Thyroid Dysfunction During Pregnancy: Proposal of Prenatal Thyroid Model | journal = Mental Illness | volume = 7 | issue = 2 | pages = 5810 | date = September 2015 | pmid = 26605033 | pmc = 4620281 | doi = 10.4081/mi.2015.5810 }}</ref> The extremely significant relationship between the two conditions suggested an independent model, named as ''Prenatal Thyroid Model of Homosexuality''. According to Turkish child & adolescent psychiatrist Osman Sabuncuoglu, who generated the theory, maternal thyroid dysfunction may lead to abnormal deviations from gender-specific development in the offspring. Autoimmune destructive process as seen in Hashimoto's thyroiditis, diminished supply of thyroid hormones, and impacts on prenatal androgen system were all considered as contributing mechanisms. In a follow-up theoretical paper,<ref>{{cite journal | vauthors = Sabuncuoglu O | title = Towards a further understanding of prenatal thyroid theory of homosexuality: Autoimmune thyroiditis, polycystic ovary syndrome, autism and low birth weight | journal = Mental Illness | volume = 9 | issue = 2 | pages = 7325 | date = October 2017 | pmid = 29142667 | pmc = 5661141 | doi = 10.4081/mi.2017.7325 }}</ref> previous research findings indicating higher rates of polycystic ovary syndrome (PCOS) in female-to-male transsexuals and lesbian women were conceived as an indication of ''Prenatal Thyroid Model'' since PCOS and autoimmune thyroiditis are frequently comorbid diseases. Likewise, increased rates of autism spectrum disorder in children born to mothers with thyroid dysfunction and overrepresentation of ASD individuals in gender dysphoria populations suggest such an association. A second group of young children with this pattern were presented in IACAPAP Congress,<ref>{{cite web |date=30 August 2022 |title=A second group of youngsters with gender nonconformity/same-sex attraction born to mothers with thyroid dysfunction in pregnancy |url=https://www.researchgate.net/publication/326723698 |vauthors=Sabuncuoglu O}}</ref> Prague (2018). Furthermore, 9 additional cases were reported in IACAPAP Congress,[https://www.researchgate.net/publication/380934726_Maternal_and_familial_thyroid_dysfunction_associated_with_gender_dysphoriasame-sex_attraction_in_the_offspring_The_cases_identified_between_2018_and_2023] Rio de Janeiro (Sabuncuoglu, 2024).

The findings from previous research in LGBT populations had called for attention to be paid to thyroid system.<ref>{{cite journal | vauthors = Ellis L, Hellberg J |date= January 2005 |title=Fetal exposure to prescription drugs and adult sexual orientation |journal=Personality and Individual Differences |volume=38 |issue=1 |pages=225–236 |doi=10.1016/j.paid.2004.04.004 |issn=0191-8869}}</ref><ref name="nielsen2013"/> A commentary by Jeffrey Mullen, published shortly after the 2015 article, underlined the importance of ''Prenatal Thyroid Model'' and supported developments in this field.<ref>{{cite journal | vauthors = Mullen J | title = A Link Between Maternal Thyroid Hormone and Sexual Orientation? | journal = Mental Illness | volume = 8 | issue = 1 | pages = 6591 | date = May 2016 | pmid = 27403279 | doi = 10.4081/mi.2016.6591 | pmc = 4926038 }}</ref> Afterwards, several authors have emphasized the role of thyroid system in sexuality while citing the ''Prenatal Thyroid Model.''<ref name="Carosa_2018">{{cite journal | vauthors = Carosa E, Lenzi A, Jannini EA | title = Thyroid hormone receptors and ligands, tissue distribution and sexual behavior | journal = Molecular and Cellular Endocrinology | volume = 467 | pages = 49–59 | date = May 2018 | pmid = 29175529 | doi = 10.1016/j.mce.2017.11.006 | hdl = 11573/1132156 | s2cid = 36883213 | hdl-access = free }}</ref><ref>{{cite journal | vauthors = Basavanhally T, Fonseca R, Uversky VN | title = Born This Way: Using Intrinsic Disorder to Map the Connections between SLITRKs, TSHR, and Male Sexual Orientation | journal = Proteomics | volume = 18 | issue = 21–22 | pages = e1800307 | date = November 2018 | pmid = 30156382 | doi = 10.1002/pmic.201800307 | s2cid = 52115603 }}</ref><ref>{{cite journal | vauthors = Wang Y, Wu H, Sun ZS | title = The biological basis of sexual orientation: How hormonal, genetic, and environmental factors influence to whom we are sexually attracted | journal = Frontiers in Neuroendocrinology | volume = 55 | pages = 100798 | date = October 2019 | pmid = 31593707 | doi = 10.1016/j.yfrne.2019.100798 | s2cid = 203667616 }}</ref><ref>{{cite journal | vauthors = Castello R, Caputo M | date = September 2019 |title=Thyroid diseases and gender |journal=Italian Journal of Gender-Specific Medicine | volume = 5 |issue = September–December | pages = 136–141 |doi=10.1723/3245.32148}}</ref> Among them, Carosa et al. concluded that thyroid hormones, affecting the human sexual function strongly, the thyroid gland must be considered, along with the genitals and the brain, a sexual organ.<ref name="Carosa_2018" /> As a tertiary source, an authoritative book on the subject of interplay between endocrinology, brain and behavior has also cited the thyroid-homosexuality proposal article in the latest edition.<ref>{{cite book | vauthors = Castellanos-Cruz L, Bao AM, Swaab DF  | chapter = Sexual Identity and Sexual Orientation |date=2017 | veditors = Pfaff DW, Joels M | title = Hormones, Brain and Behavior | edition = Third |pages=279–290 |publisher=Elsevier | oclc = 971456116 | isbn = 978-1-78684-205-3 }}</ref>

=== Genetic influences ===
Multiple genes have been found to play a role in sexual orientation. Scientists caution that many people misconstrue the meanings of ''genetic'' and ''environmental''.<ref name="Bailey_2016"/> ''[[Environment and sexual orientation|Environmental influence]]'' does not automatically imply that the social environment influences or contributes to the development of sexual orientation. Hypotheses for the impact of the post-natal social environment on sexual orientation are weak, especially for males.<ref name="Bailey_2016"/> There is, however, a vast non-social environment that is non-genetic, such as [[prenatal development]], which remains poorly understood.<ref name="Bailey_2016"/>{{rp|76}}

====Twin studies====
Twin studies are one method of testing genetic and environmental influences, although they cannot reveal what kind of environmental influence this may include (social or non-social).{{Sfn|Bailey|2003|p=110–111}} Identical or monozygotic twins share their genes, while fraternal or dizygotic twins are only as genetically similar as any other sibling pair. When twins both share a trait, they are ''concordant'' for this trait; and when they differ they are [[Discordant Twins|''discordant'']]. If identical twins have a higher rate of concordance for a trait than fraternal twins, it indicates that genes may contribute to the trait.<ref name="Bailey_2016" />{{rp|74–76}}

The 2016 Bailey et al. meta-analysis of all twin studies on sexual orientation found that the [[median]] concordance for homosexual or non-heterosexual orientation in twins in unbiased probability samples is 24% for monozygotic identical twins, and 15% for dizygotic twins.<ref name="Bailey_2016" />{{rp|74–76}} According to Rice et al. the identical twin concordance for homosexuality is similar to identical twin concordance for two other traits influenced by prenatal androgens: [[cryptorchidism]] and [[hypospadias]] (feminized male gonads) which have an identical twin concordance around 25%, despite twins sharing genes and prenatal environments.<ref name=":4">{{Cite journal |last1=Rice |first1=William R. |last2=Friberg |first2=Urban |last3=Gavrilets |first3=Sergey |date=2012 |title=Homosexuality as a Consequence of Epigenetically Canalized Sexual Development |url=http://scottbarrykaufman.com/wp-content/uploads/2012/12/Rice-et-al.-2012.pdf |journal=The Quarterly Review of Biology |volume=87 |issue=4 |pages=343–368 |doi=10.1086/668167 |pmid=23397798 |issn=0033-5770}}</ref>{{rp|356}}[[File:Gary_and_Larry_Lane_photo.jpg|alt=|thumb|250x250px|Identical twin studies are a useful mechanism for assessing the role of genes and environment.]]Twin studies have also found that among twins with differing sexual orientations, homosexual twins were significantly more [[gender nonconforming]] than their heterosexual co-twin, and that this was noticeable from a young age.{{Sfn|Bailey|2003|p=110–111}} Bailey states:
{{Quote|text=What kind of environmental factor can cause genetically identical twins reared in the same family from birth—often dressed alike and given the same toys—to differ in their sexual orientations? It is a fascinating question that we haven’t begun to answer well. One hint comes from the childhood behavior findings. When identical twins differed in their sexual orientation, the gay one tended to recall being much more feminine than the straight one. This means that the environmental factors that cause the twin differences are there early on, by childhood. Based on other things we know, such as studies of children with cloacal exstrophy, I suspect that these factors operate in the womb.{{Sfn|Bailey|2003|p=110–111}}}}
Identical twins reared apart from birth are another method of studying the origin of psychological traits. Unfortunately, such twin pairs are rare. Three sets of male twin pairs exist in the literature. In the first pair found by Thomas Bouchard, both male twins reared apart from birth were homosexual. In a second pair found by Whitham, both males were also homosexual. In the third pair found by Bouchard, the male twins were neither definitively concordant nor discordant, as both had relations with males and females, thus this pair may be concordant for bisexuality. Among the female twin pairs; four female twin pairs were all discordant, although the small number of cases prevent any strong conclusions.{{Sfn|LeVay|2017|p=89–90}}

According to William Rice and colleagues, the concordance of homosexuality among twins raises the possibility that homosexuality is not caused by genes nor atypical levels of hormones, but an epigenetic mechanism controlling how sensitive fetuses are to prenatal hormones.<ref name=":4" /><ref>{{Cite journal |last=Tasos |first=Emmanouil |date=2022 |title=To What Extent are Prenatal Androgens Involved in the Development of Male Homosexuality in Humans? |url=https://www.researchgate.net/publication/352125966 |journal=Journal of Homosexuality |language=en |volume=69 |issue=11 |pages=1928–1963 |doi=10.1080/00918369.2021.1933792 |pmid=34080960 |issn=0091-8369 |via=ResearchGate}}</ref>{{Rp|page=22}}

====Chromosome linkage studies====
{{gay gene}}
Chromosome linkage studies of sexual orientation have indicated the presence of multiple contributing genetic factors throughout the genome. In 1993, [[Dean Hamer]] and colleagues published findings from a linkage analysis of a sample of 76 gay brothers and their families.<ref>{{cite journal | vauthors = Hamer DH, Hu S, Magnuson VL, Hu N, Pattatucci AM | title = A linkage between DNA markers on the X chromosome and male sexual orientation | journal = Science | volume = 261 | issue = 5119 | pages = 321–7 | date = July 1993 | pmid = 8332896 | doi = 10.1126/science.8332896 | url = https://zenodo.org/record/1231257 | bibcode = 1993Sci...261..321H }}</ref> Hamer et al. found that the gay men had more gay male uncles and cousins on the maternal side of the family than on the paternal side. Gay brothers who showed this maternal pedigree were then tested for X chromosome linkage, using twenty-two markers on the X chromosome to test for similar alleles. In another finding, thirty-three of the forty sibling pairs tested were found to have similar alleles in the distal region of [[Xq28]], which was significantly higher than the expected rates of 50% for fraternal brothers. This was popularly dubbed the "'''gay gene'''" in the media, causing significant controversy. In 1998, Sanders et al.{{citation needed|date=June 2022}} reported on their similar study, in which they found that 13% of uncles of gay brothers on the maternal side were homosexual, compared with 6% on the paternal side.<ref name="Wilson_2008">{{cite book | vauthors = Wilson G, Rahman Q  |title=Born Gay: The Psychobiology of Sex Orientation |date=2008 |publisher=Peter Owen Publishers |isbn=9780720613094 |edition= 2nd |language=en}}</ref>

A later analysis by Hu et al. replicated and refined the earlier findings. This study revealed that 67% of gay brothers in a new saturated sample shared a marker on the X chromosome at Xq28.<ref name="Hu_1995">{{cite journal | vauthors = Hu S, Pattatucci AM, Patterson C, Li L, Fulker DW, Cherny SS, Kruglyak L, Hamer DH | display-authors = 6 | title = Linkage between sexual orientation and chromosome Xq28 in males but not in females | journal = Nature Genetics | volume = 11 | issue = 3 | pages = 248–56 | date = November 1995 | pmid = 7581447 | doi = 10.1038/ng1195-248 | url = https://zenodo.org/record/1233407 | type = Submitted manuscript | s2cid = 721490 }}</ref> Two other studies (Bailey et al., 1999; McKnight and Malcolm, 2000) failed to find a preponderance of gay relatives in the maternal line of homosexual men.<ref name="Wilson_2008"/> One study by Rice et al. in 1999 failed to replicate the Xq28 linkage results.<ref>{{cite journal | vauthors = Vilain E | title = Genetics of sexual development | journal = Annual Review of Sex Research | volume = 11 | pages = 1–25 | year = 2000 | doi = 10.1080/10532528.2000.10559783 | pmid = 11351829 }}</ref> Meta-analysis of all available linkage data indicates a significant link to Xq28, but also indicates that additional genes must be present to account for the full heritability of sexual orientation.<ref name="Hamer_1999">{{cite journal | vauthors = Hamer DH, Rice G, Risch N, Ebers G |title=Genetics and Male Sexual Orientation |journal=Science |date=1999 |volume=285 |issue=5429 |page=803 |doi=10.1126/science.285.5429.803a |doi-access=free}}</ref>

Mustanski et al. (2005) performed a full-genome scan (instead of just an X chromosome scan) on individuals and families previously reported on in Hamer et al. (1993) and Hu et al. (1995), as well as additional new subjects. In the full sample they did not find linkage to Xq28.<ref>{{cite journal | vauthors = Mustanski BS, Dupree MG, Nievergelt CM, Bocklandt S, Schork NJ, Hamer DH | title = A genomewide scan of male sexual orientation | journal = Human Genetics | volume = 116 | issue = 4 | pages = 272–8 | date = March 2005 | pmid = 15645181 | doi = 10.1007/s00439-004-1241-4 | url = http://mypage.iu.edu/~bmustans/Mustanski_etal_2005.pdf | url-status = dead | s2cid = 206989147 | archive-url = https://web.archive.org/web/20050415233637/http://mypage.iu.edu/~bmustans/Mustanski_etal_2005.pdf | archive-date = 2005-04-15 }}</ref>

Results from the first large, comprehensive multi-center genetic linkage study of male sexual orientation were reported by an independent group of researchers at the American Society of Human Genetics in 2012.<ref name="Sanders_2014">{{cite journal | vauthors = Sanders AR, Martin ER, Beecham GW, Guo S, Dawood K, Rieger G, Badner JA, Gershon ES, Krishnappa RS, Kolundzija AB, Duan J, Gejman PV, Bailey JM | display-authors = 6 | title = Genome-wide scan demonstrates significant linkage for male sexual orientation | journal = Psychological Medicine | volume = 45 | issue = 7 | pages = 1379–88 | date = May 2015 | pmid = 25399360 | doi = 10.1017/S0033291714002451 | s2cid = 4027333 }}</ref> The study population included 409 independent pairs of gay brothers, who were analyzed with over 300,000 [[single-nucleotide polymorphism]] markers. The data strongly replicated Hamer's Xq28 findings as determined by both two-point and multipoint (MERLIN) LOD score mapping. Significant linkage was also detected in the pericentromeric region of chromosome 8, overlapping with one of the regions detected in the Hamer lab's previous genomewide study. The authors concluded that "our findings, taken in context with previous work, suggest that genetic variation in each of these regions contributes to development of the important psychological trait of male sexual orientation". Female sexual orientation does not seem to be linked to Xq28,<ref name="Hu_1995"/><ref name="Ngun_2014">{{cite book | vauthors = Ngun TC, Vilain E |title=The Biological Basis of Human Sexual Orientation: Is There a Role for Epigenetics? |chapter=The Biological Basis of Human Sexual Orientation |date=2014 |volume=86 |pages=167–84 |doi=10.1016/B978-0-12-800222-3.00008-5 |pmid=25172350 |url=http://gaystudies.genetics.ucla.edu/UCLA%20Twin%20Sexual%20Orientation%20Study/Read%20More_files/Ngun.pdf |access-date=20 March 2016 |issn=0065-2660 |series=Advances in Genetics |isbn=9780128002223 |archive-url=https://web.archive.org/web/20160331225443/http://gaystudies.genetics.ucla.edu/UCLA%20Twin%20Sexual%20Orientation%20Study/Read%20More_files/Ngun.pdf |archive-date=2016-03-31 |url-status=dead}}</ref> though it does appear moderately heritable.<ref name="Sanders_2014"/>

In addition to [[allosome|sex chromosomal]] contribution, a potential [[autosomal]] genetic contribution to the development of homosexual orientation has also been suggested. In a study population composed of more than 7000 participants, Ellis et al. (2008) found a statistically significant difference in the frequency of blood type A between homosexuals and heterosexuals. They also found that "unusually high" proportions of homosexual males and homosexual females were [[Rh factor|Rh negative]] in comparison to heterosexuals. As both blood type and Rh factor are genetically inherited traits controlled by [[alleles]] located on chromosome 9 and chromosome 1 respectively, the study indicates a potential link between genes on autosomes and homosexuality.<ref>{{cite journal | vauthors = Ellis L, Ficek C, Burke D, Das S | title = Eye color, hair color, blood type, and the rhesus factor: exploring possible genetic links to sexual orientation | url = https://archive.org/details/sim_archives-of-sexual-behavior_2008-02_37_1/page/145 | journal = Archives of Sexual Behavior | volume = 37 | issue = 1 | pages = 145–9 | date = February 2008 | pmid = 18074215 | doi = 10.1007/s10508-007-9274-0 | s2cid = 8303331 }}</ref><ref name="AnHomGenetics">{{cite book | vauthors = Poiani A |title=Animal Homosexuality: A Biosocial Perspective |date=2010 |publisher=Cambridge University Press |isbn=978-1139490382 |pages=55–96|url=https://books.google.com/books?id=EftT_1bsPOAC}}</ref>

The biology of sexual orientation has been studied in detail in several animal model systems. In the common fruit fly ''[[Drosophila melanogaster]]'', the complete pathway of sexual differentiation of the brain and the behaviors it controls is well established in both males and females, providing a concise model of biologically controlled courtship.<ref>{{cite journal | vauthors = Pavlou HJ, Goodwin SF | title = Courtship behavior in Drosophila melanogaster: towards a 'courtship connectome' | journal = Current Opinion in Neurobiology | volume = 23 | issue = 1 | pages = 76–83 | date = February 2013 | pmid = 23021897 | pmc = 3563961 | doi = 10.1016/j.conb.2012.09.002 }}</ref> In mammals, a group of geneticists at the Korea Advanced Institute of Science and Technology bred female mice specifically lacking a particular gene related to sexual behavior. Without the gene, the mice exhibited masculine sexual behavior and attraction toward urine of other female mice. Those mice who retained the gene fucose mutarotase (FucM) were attracted to male mice.<ref>{{cite journal | vauthors = Park D, Choi D, Lee J, Lim DS, Park C | title = Male-like sexual behavior of female mouse lacking fucose mutarotase | journal = BMC Genetics | volume = 11 | pages = 62 | date = July 2010 | pmid = 20609214 | pmc = 2912782 | doi = 10.1186/1471-2156-11-62 | doi-access = free }}</ref>

In interviews to the press, researchers have pointed that the evidence of genetic influences should not be equated with genetic determinism. According to Dean Hamer and Michael Bailey, genetic aspects are only one of the multiple causes of homosexuality.<ref>{{cite web |url=https://www.independent.co.uk/news/the-gay-gene-is-back-on-the-scene-1536770.html |title=The 'gay gene' is back on the scene | vauthors = Connor S |date=31 October 1995 |website=[[The Independent]]}}</ref><ref>{{cite web |url=https://www.telegraph.co.uk/science/science-news/10637532/Being-homosexual-is-only-partly-due-to-gay-gene-research-finds.html |archive-url=https://web.archive.org/web/20140214023529/http://www.telegraph.co.uk/science/science-news/10637532/Being-homosexual-is-only-partly-due-to-gay-gene-research-finds.html |url-status=dead |archive-date=February 14, 2014 |title=Being homosexual is only partly due to gay gene, research finds | vauthors = Knapton S |date=13 February 2014 |website=The Telegraph |publisher=[[Telegraph Media Group]]}}</ref>

In 2017, ''[[Scientific Reports]]'' published an article with a [[genome wide association study]] on male sexual orientation. The research consisted of 1,077 homosexual men and 1,231 heterosexual men. A gene named ''[[SLITRK6]]'' on [[chromosome 13]] was identified.<ref name="Sanders_2017">{{cite journal | vauthors = Sanders AR, Beecham GW, Guo S, Dawood K, Rieger G, Badner JA, Gershon ES, Krishnappa RS, Kolundzija AB, Duan J, Gejman PV, Bailey JM, Martin ER | display-authors = 6 | title = Genome-Wide Association Study of Male Sexual Orientation | language = En | journal = Scientific Reports | volume = 7 | issue = 1 | pages = 16950 | date = December 2017 | pmid = 29217827 | pmc = 5721098 | doi = 10.1038/s41598-017-15736-4 | bibcode = 2017NatSR...716950S }}</ref> The research supports another study which had been done by the neuroscientist [[Simon LeVay]]. LeVay's research suggested that the [[hypothalamus]] of gay men is different from straight men.<ref name="LeVay_1991"/> The SLITRK6 is active in the mid-brain where the hypothalamus is. The researchers found that the [[Thyrotropin receptor|thyroid stimulating hormone receptor]] (TSHR) on chromosome 14 shows sequence differences between gay and straight men.<ref name="Sanders_2017" /> [[Graves' disease]] is associated with TSHR abnormalities, with previous research indicating that Graves' disease is more common in gay men than in straight men.<ref name=nielsen2013>{{cite journal | vauthors = Frisch M, Nielsen NM, Pedersen BV | title = Same-sex marriage, autoimmune thyroid gland dysfunction and other autoimmune diseases in Denmark 1989-2008 | journal = European Journal of Epidemiology | volume = 29 | issue = 1 | pages = 63–71 | date = January 2014 | pmid = 24306355 | doi = 10.1007/s10654-013-9869-9 | s2cid = 11819672 }}</ref> Research indicated that gay people have lower body weight than straight people. It had been suggested that the overactive TSHR hormone lowered body weight in gay people, though this remains unproven.<ref>{{cite journal | vauthors = Deputy NP, Boehmer U | title = Determinants of body weight among men of different sexual orientation | journal = Preventive Medicine | volume = 51 | issue = 2 | pages = 129–31 | date = August 2010 | pmid = 20510272 | doi = 10.1016/j.ypmed.2010.05.010 }}</ref><ref>{{cite journal | vauthors = Blanchard R, Bogaert AF | title = Biodemographic comparisons of homosexual and heterosexual men in the Kinsey Interview Data | url = https://archive.org/details/sim_archives-of-sexual-behavior_1996-12_25_6/page/551 | journal = Archives of Sexual Behavior | volume = 25 | issue = 6 | pages = 551–79 | date = December 1996 | pmid = 8931880 | doi = 10.1007/BF02437839 | s2cid = 23951518 }}</ref>

In 2018, Ganna et al. performed another [[genome-wide association study]] on sexual orientation of men and women with data from 26,890 people who had at least one same-sex partner and 450,939 controls. The data in the study was meta-analyzed and obtained from the [[UK Biobank]] study and [[23andMe]]. The researchers identified four variants more common in people who reported at least one same-sex experience on chromosomes 7, 11, 12, and 15. The variants on chromosomes 11 and 15 were specific to men, with the variant on chromosome 11 located in an olfactory gene and the variant on chromosome 15 having previously been linked to male-pattern baldness. The four variants were also correlated with mood and mental health disorders; major depressive disorder and schizophrenia in men and women, and bipolar disorder in women. However, none of the four variants could reliably predict sexual orientation.<ref>{{cite web|url=https://www.science.org/content/article/giant-study-links-dna-variants-same-sex-behavior|title=Giant study links DNA variants to same-sex behavior| vauthors = Price M |date=2018-10-19|website=Science | publisher = AAAS |language=en|access-date=2019-01-21}}</ref>

In August 2019, a genome-wide association study of 493,001 individuals concluded that hundreds or thousands of genetic variants underlie homosexual behavior in both sexes, with 5 variants in particular being significantly associated. Some of these variants had sex-specific effects, and two of these variants suggested links to biological pathways that involve [[sex hormone]] regulation and [[olfaction]]. All the variants together captured between 8 and 25% of the variation in individual differences in homosexual behavior. These genes partly overlap with those for several other traits, including openness to experience and risk-taking behavior. Additional analyses suggested that sexual behavior, attraction, identity, and fantasies are influenced by a similar set of genetic variants. They also found that the genetic effects that differentiate heterosexual from homosexual behavior are not the same as those that differ among nonheterosexuals with lower versus higher proportions of same-sex partners, which suggests that there is no single continuum from heterosexual to homosexual preference, as suggested by the [[Kinsey scale]].<ref name="Science 2019">{{cite journal | vauthors = Ganna A, Verweij KJ, Nivard MG, Maier R, Wedow R, Busch AS, Abdellaoui A, Guo S, Sathirapongsasuti JF, Lichtenstein P, Lundström S, Långström N, Auton A, Harris KM, Beecham GW, Martin ER, Sanders AR, Perry JR, Neale BM, Zietsch BP | display-authors = 6 | title = Large-scale GWAS reveals insights into the genetic architecture of same-sex sexual behavior | journal = Science | volume = 365 | issue = 6456 | pages = eaat7693 | date = August 2019 | pmid = 31467194 | pmc = 7082777 | doi = 10.1126/science.aat7693 }}</ref><ref>{{cite web |title=Genetics of Sexual Behavior |url=https://geneticsexbehavior.info/what-we-found/ |website=Genetics of Sexual Behavior |publisher=geneticsexbehavior.info |access-date=30 August 2019 |date=28 February 2018}}</ref>

In October 2021, another research paper reported that genetic factors influence the development of same-sex sexual behavior. A two-stage genome-wide association study (GWAS) with a total sample of 1478 homosexual males and 3313 heterosexual males in [[Han Chinese]] populations identified two genetic loci (FMR1NB and ZNF536) showing consistent association with male sexual orientation.<ref name="Hu_2021">{{cite journal | vauthors = Hu SH, Li HM, Yu H, Liu Y, Liu CX, Zuo XB, Lu J, Jiang JJ, Xi CX, Huang BC, Xu HJ, Hu JB, Lai JB, Huang ML, Liu JN, Xu DG, Guo XC, Wu W, Wu X, Jiang L, Li M, Zhang GP, Huang JW, Wei N, Lv W, Duan JF, Qi HL, Hu CC, Chen JK, Zhou WH, Xu WJ, Liu CF, Liang HY, Du J, Zheng SF, Lu QL, Zheng L, Hu XW, Chen FX, Chen P, Zhu B, Xu LJ, Ni ZM, Fang YZ, Yang ZK, Shan XR, Zheng ED, Zhang F, Zhou QQ, Rao Y, Swaab D, Yue WH, Xu Y | display-authors = 6 | title = Discovery of new genetic loci for male sexual orientation in Han population | journal = Cell Discovery | volume = 7 | issue = 1 | pages = 103 | date = October 2021 | pmid = 34719679 | pmc = 8558329 | doi = 10.1038/s41421-021-00341-7 }}</ref>

===Epigenetics studies===
{{Main|Epigenetic theories of homosexuality}}
A study suggests linkage between a mother's genetic make-up and homosexuality of her sons. Women have two X chromosomes, one of which is "switched off". The inactivation of the X chromosome occurs randomly throughout the embryo, resulting in cells that are mosaic with respect to which chromosome is active. In some cases though, it appears that this switching off can occur in a non-random fashion. Bocklandt et al. (2006) reported that, in mothers of homosexual men, the number of women with extreme skewing of X chromosome inactivation is significantly higher than in mothers without gay sons. In a study of 94 participants, 13% of mothers with one gay son, and 23% of mothers with two gay sons, showed extreme skewing, compared to 4% of mothers without gay sons.<ref name="Bocklandt_2006">{{cite journal | vauthors = Bocklandt S, Horvath S, Vilain E, Hamer DH | title = Extreme skewing of X chromosome inactivation in mothers of homosexual men | journal = Human Genetics | volume = 118 | issue = 6 | pages = 691–4 | date = February 2006 | pmid = 16369763 | doi = 10.1007/s00439-005-0119-4 | url = http://repositories.cdlib.org/postprints/1413 | url-status = dead | s2cid = 1370892 | citeseerx = 10.1.1.533.4517 | archive-url = https://web.archive.org/web/20070609140924/http://repositories.cdlib.org/postprints/1413/ | archive-date = 2007-06-09 }}</ref>

===Birth order===
{{Main|Fraternal birth order and sexual orientation}}
Blanchard and Klassen (1997) reported that each additional older brother increases the odds of a man being gay by 33%.<ref name="Blanchard_1997">{{cite journal | vauthors = Blanchard R, Klassen P | title = H-Y antigen and homosexuality in men | journal = Journal of Theoretical Biology | volume = 185 | issue = 3 | pages = 373–8 | date = April 1997 | pmid = 9156085 | doi = 10.1006/jtbi.1996.0315 | bibcode = 1997JThBi.185..373B | url = http://faculty.bennington.edu/~sherman/sex/H-Y%20anitgen.pdf | url-status = dead | citeseerx = 10.1.1.602.8423 | archive-url = https://web.archive.org/web/20120915214732/http://faculty.bennington.edu/~sherman/sex/H-Y%20anitgen.pdf | archive-date = 2012-09-15 }}</ref><ref>{{cite news |url=https://www.nytimes.com/2007/04/10/health/10gene.html |title=Pas de Deux of Sexuality Is Written in the Genes |date=10 April 2007 |newspaper=The New York Times| vauthors = Wade N }}</ref> This is now "one of the most reliable epidemiological variables ever identified in the study of sexual orientation".<ref>{{cite journal | vauthors = Blanchard R | title = Birth order and sibling sex ratio in homosexual versus heterosexual males and females | journal = Annual Review of Sex Research | volume = 8 | pages = 27–67 | year = 1997 | doi = 10.1080/10532528.1997.10559918 | pmid = 10051890 }}</ref> To explain this finding, it has been proposed that male fetuses provoke a maternal immune reaction that becomes stronger with each successive male fetus. This maternal immunization hypothesis (MIH) begins when cells from a male fetus enter the mother's circulation during pregnancy or while giving birth.<ref name="Anthony_2011">{{cite journal |author-link=Anthony Bogaert |name-list-style=amp |vauthors=Bogaert AF, Skorska M |date=April 2011 |title=Sexual orientation, fraternal birth order, and the maternal immune hypothesis: a review |journal=Frontiers in Neuroendocrinology |volume=32 |issue=2 |pages=247–54 |doi=10.1016/j.yfrne.2011.02.004 |pmid=21315103 |s2cid=45446175}}</ref> Male fetuses produce H-Y antigens which are "almost certainly involved in the sexual differentiation of vertebrates". These Y-linked proteins would not be recognized in the mother's immune system because she is female, causing her to develop antibodies which would travel through the placental barrier into the fetal compartment. From here, the anti-male bodies would then cross the blood/brain barrier (BBB) of the developing fetal brain, altering sex-dimorphic brain structures relative to sexual orientation, increasing the likelihood that the exposed son will be more attracted to men than women.<ref name="Anthony_2011"/> It is this antigen which maternal H-Y antibodies are proposed to both react to and 'remember'. Successive male fetuses are then attacked by H-Y antibodies which somehow decrease the ability of H-Y antigens to perform their usual function in brain masculinization.<ref name="Blanchard_1997"/>

In 2017, researchers discovered a biological mechanism of gay people who tend to have older brothers. They believed the [[Neuroligin 4 Y-linked]] protein was responsible for a later son being gay. They found that women had significantly higher anti-NLGN4Y levels than men. In addition, 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.<ref name="Bogaert_2018"/>

The fraternal birth order effect, however, does not apply to instances where a firstborn is homosexual.<ref name="Cantor_2002">{{cite journal | vauthors = Cantor JM, Blanchard R, Paterson AD, Bogaert AF | title = How many gay men owe their sexual orientation to fraternal birth order? | journal = Archives of Sexual Behavior | volume = 31 | issue = 1 | pages = 63–71 | date = February 2002 | pmid = 11910793 | doi = 10.1023/a:1014031201935 | s2cid = 203129 }}</ref><ref name="Blanchard_2004">{{cite journal | vauthors = Blanchard R, Bogaert AF | title = Proportion of homosexual men who owe their sexual orientation to fraternal birth order: An estimate based on two national probability samples | journal = American Journal of Human Biology | volume = 16 | issue = 2 | pages = 151–7 | date = 2004 | pmid = 14994314 | doi = 10.1002/ajhb.20006 | s2cid = 21108939 }}</ref>

===Pheromone studies===
Research conducted in Sweden<ref name="Savic_2005">{{cite journal | vauthors = Savic I, Berglund H, Lindström P | title = Brain response to putative pheromones in homosexual men | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 102 | issue = 20 | pages = 7356–61 | date = May 2005 | pmid = 15883379 | pmc = 1129091 | doi = 10.1073/pnas.0407998102 | bibcode = 2005PNAS..102.7356S | doi-access = free }}</ref> has suggested that gay and straight men respond differently to two odors that are believed to be involved in [[sexual arousal]]. The research showed that when both heterosexual women and gay men are exposed to a testosterone derivative found in men's sweat, a region in the hypothalamus is activated. Heterosexual men, on the other hand, have a similar response to an estrogen-like compound found in women's urine.<ref>{{cite web | vauthors = Wade N | date = 9 May 2005 | url = https://www.nytimes.com/2005/05/09/science/09cnd-smell.html | title = Gay Men Are Found to Have Different Scent of Attraction | work = [[The New York Times]] }}</ref> The conclusion is that sexual attraction, whether same-sex or opposite-sex oriented, operates similarly on a biological level. Researchers have suggested that this possibility could be further explored by studying young subjects to see if similar responses in the hypothalamus are found and then correlating these data with adult sexual orientation.{{Citation needed|date=March 2007}}

===Studies of brain structure===
A number of sections of the [[Human brain|brain]] have been reported to be sexually dimorphic; that is, they vary between men and women. There have also been reports of variations in brain structure corresponding to sexual orientation. In 1990, [[Dick Swaab]] and Michel A. Hofman reported a difference in the size of the [[suprachiasmatic nucleus]] between homosexual and heterosexual men.<ref>{{cite journal | vauthors = Swaab DF, Hofman MA | title = An enlarged suprachiasmatic nucleus in homosexual men | journal = Brain Research | volume = 537 | issue = 1–2 | pages = 141–8 | date = December 1990 | pmid = 2085769 | doi = 10.1016/0006-8993(90)90350-K | url = https://pure.knaw.nl/portal/files/488706/14928_285_swaab.pdf | s2cid = 13403716 }}</ref> In 1992, Allen and Gorski reported a difference related to sexual orientation in the size of the [[anterior commissure]],<ref name="Allen_1992">{{cite journal | vauthors = Allen LS, Gorski RA | title = Sexual orientation and the size of the anterior commissure in the human brain | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 89 | issue = 15 | pages = 7199–202 | date = August 1992 | pmid = 1496013 | pmc = 49673 | doi = 10.1073/pnas.89.15.7199 | bibcode = 1992PNAS...89.7199A | doi-access = free }}</ref> but this research was refuted by numerous studies, one of which found that the entirety of the variation was caused by a single outlier.<ref>{{cite journal | vauthors = Byne W, Parsons B | title = Human sexual orientation. The biologic theories reappraised | journal = Archives of General Psychiatry | volume = 50 | issue = 3 | pages = 228–39 | date = March 1993 | pmid = 8439245 | doi = 10.1001/archpsyc.1993.01820150078009 }}</ref><ref name="Byne_2001"/><ref>{{cite journal | vauthors = Lasco MS, Jordan TJ, Edgar MA, Petito CK, Byne W | title = A lack of dimorphism of sex or sexual orientation in the human anterior commissure | journal = Brain Research | volume = 936 | issue = 1–2 | pages = 95–8 | date = May 2002 | pmid = 11988236 | doi = 10.1016/s0006-8993(02)02590-8 | s2cid = 7774361 }}</ref>

Research on the physiologic differences between male and female brains are based on the idea that people have male or a female brain, and this mirrors the behavioral differences between the two sexes. Some researchers state that solid scientific support for this is lacking. Although consistent differences have been identified, including the size of the brain and of specific brain regions, male and female brains are very similar.<ref name="Cahill_2006">{{cite journal | vauthors = Cahill L | title = Why sex matters for neuroscience | journal = Nature Reviews. Neuroscience | volume = 7 | issue = 6 | pages = 477–84 | date = June 2006 | pmid = 16688123 | doi = 10.1038/nrn1909 | s2cid = 10847255 }}</ref><ref name="Lenroot_2010">{{cite journal | vauthors = Lenroot RK, Giedd JN | title = Sex differences in the adolescent brain | journal = Brain and Cognition | volume = 72 | issue = 1 | pages = 46–55 | date = February 2010 | pmid = 19913969 | pmc = 2818549 | doi = 10.1016/j.bandc.2009.10.008 }}</ref><ref name="McCarthy_2009">{{cite journal | vauthors = McCarthy MM, Wright CL, Schwarz JM | title = New tricks by an old dogma: mechanisms of the Organizational/Activational Hypothesis of steroid-mediated sexual differentiation of brain and behavior | journal = Hormones and Behavior | volume = 55 | issue = 5 | pages = 655–65 | date = May 2009 | pmid = 19682425 | pmc = 2742630 | doi = 10.1016/j.yhbeh.2009.02.012 }}</ref><ref name="Sakuma_2009">{{cite journal | vauthors = Sakuma Y | title = Gonadal steroid action and brain sex differentiation in the rat | journal = Journal of Neuroendocrinology | volume = 21 | issue = 4 | pages = 410–4 | date = March 2009 | pmid = 19226349 | doi = 10.1111/j.1365-2826.2009.01856.x | s2cid = 5558045 }}</ref><ref name="Jäncke_2013">{{cite journal | vauthors = Jäncke L, Mérillat S, Liem F, Hänggi J | title = Brain size, sex, and the aging brain | journal = Human Brain Mapping | volume = 36 | issue = 1 | pages = 150–69 | date = January 2015 | pmid = 25161056 | pmc = 6869393 | doi = 10.1002/hbm.22619 }}</ref><ref name="Berenbaum_2016">{{cite journal | vauthors = Berenbaum SA, Beltz AM | title = How Early Hormones Shape Gender Development | journal = Current Opinion in Behavioral Sciences | volume = 7 | issue = | pages = 53–60 | date = February 2016 | pmid = 26688827 | pmc = 4681519 | doi = 10.1016/j.cobeha.2015.11.011 }}</ref>

====Sexually dimorphic nuclei in the anterior hypothalamus====
LeVay also conducted some of these early researches. He studied four groups of [[neuron]]s in the [[hypothalamus]] called INAH1, INAH2, INAH3 and INAH4. This was a relevant area of the brain to study, because of evidence that it played a role in the regulation of [[sexual behaviour in animals]], and because INAH2 and INAH3 had previously been reported to differ in size between men and women.<ref name="LeVay_1991">{{cite journal | vauthors = LeVay S | title = A difference in hypothalamic structure between heterosexual and homosexual men | journal = Science | volume = 253 | issue = 5023 | pages = 1034–7 | date = August 1991 | pmid = 1887219 | doi = 10.1126/science.1887219 | s2cid = 1674111 | bibcode = 1991Sci...253.1034L }}</ref>

He obtained brains from 41 deceased hospital patients. The subjects were classified into three groups. The first group comprised 19 gay men who had died of [[AIDS]]-related illnesses. The second group comprised 16 men whose sexual orientation was unknown, but whom the researchers presumed to be heterosexual. Six of these men had died of AIDS-related illnesses. The third group was of six women whom the researchers presumed to be heterosexual. One of the women had died of an AIDS-related illness.<ref name="LeVay_1991"/>

The [[HIV-positive people]] in the presumably heterosexual patient groups were all identified from medical records as either [[intravenous drug abuse]]rs or recipients of [[blood transfusion]]s. Two of the men who identified as heterosexual specifically denied ever engaging in a homosexual sex act. The records of the remaining heterosexual subjects contained no information about their sexual orientation; they were assumed to have been primarily or exclusively heterosexual "on the basis of the numerical preponderance of heterosexual men in the population".<ref name="LeVay_1991"/>

LeVay found no evidence for a difference between the groups in the size of INAH1, INAH2 or INAH4. However, the INAH3 group appeared to be twice as big in the heterosexual male group as in the gay male group; the difference was highly significant, and remained significant when only the six AIDS patients were included in the heterosexual group. The size of INAH3 in the homosexual men's brains was comparable to the size of INAH3 in the heterosexual women's brains.{{Citation needed|date=March 2019}}

William Byne and colleagues attempted to identify the size differences reported in INAH 1–4 by replicating the experiment using brain sample from other subjects: 14 HIV-positive homosexual males, 34 presumed heterosexual males (10 HIV-positive), and 34 presumed heterosexual females (9 HIV-positive). The researchers found a significant difference in INAH3 size between heterosexual men and heterosexual women. The INAH3 size of the homosexual men was apparently smaller than that of the heterosexual men, and larger than that of the heterosexual women, though neither difference quite reached statistical significance.<ref name="Byne_2001">{{cite journal | vauthors = Byne W, Tobet S, Mattiace LA, Lasco MS, Kemether E, Edgar MA, Morgello S, Buchsbaum MS, Jones LB | display-authors = 6 | title = The interstitial nuclei of the human anterior hypothalamus: an investigation of variation with sex, sexual orientation, and HIV status | journal = Hormones and Behavior | volume = 40 | issue = 2 | pages = 86–92 | date = September 2001 | pmid = 11534967 | doi = 10.1006/hbeh.2001.1680 | s2cid = 3175414 }} {{dead link|date=March 2020 |bot=InternetArchiveBot |fix-attempted=yes}}</ref>

Byne and colleagues also weighed and counted numbers of neurons in INAH3 tests not carried out by LeVay. The results for INAH3 weight were similar to those for INAH3 size; that is, the INAH3 weight for the heterosexual male brains was significantly larger than for the heterosexual female brains, while the results for the gay male group were between those of the other two groups but not quite significantly different from either. The neuron count also found a male-female difference in INAH3, but found no trend related to sexual orientation.<ref name="Byne_2001" />

LeVay has said that Byne replicated his work, but that he employed a two-tailed statistical analysis, which is typically reserved for when no previous findings had employed the difference. LeVay has said that "given that my study had already reported a INAH3 to be smaller in gay men, a one tailed approach would have been more appropriate, and it would have yielded a significant difference [between heterosexual and homosexual men]".<ref name="LeVay_2016" />{{rp|110}}

[[J. Michael Bailey]] has criticized LeVay's critics—describing the claim that the INAH-3 difference could be attributable to AIDS as "aggravating", since the "INAH-3 did not differ between the brains of straight men who died of AIDS and those who did not have the disease".<ref name="Bailey_2003">{{cite book |url=https://www.researchgate.net/publication/281747420 |title=The Man Who Would Be Queen |vauthors=Bailey J |date=2003-03-10 |publisher=Joseph Henry Press |isbn=978-0-309-08418-5 |format=PDF}}</ref>{{rp|120}} Bailey has further criticized the second objection that was raised, that being gay might have somehow caused the difference in INAH-3, and not vice-versa, saying "the problem with this idea is that the hypothalamus appears to develop early. Not a single expert I have ever asked about LeVay's study thought it was plausible that sexual behavior caused the INAH-3 differences."<ref name="Bailey_2003" />{{rp|120}}

The SCN of homosexual males has been demonstrated to be larger (both the volume and the number of neurons are twice as many as in heterosexual males). These areas of the hypothalamus have not yet been explored in homosexual females nor bisexual males nor females. Although the functional implications of such findings still have not been examined in detail, they cast serious doubt over the widely accepted Dörner hypothesis that homosexual males have a "female hypothalamus" and that the key mechanism of differentiating the "male brain from originally female brain" is the epigenetic influence of testosterone during prenatal development.<ref>{{cite journal | vauthors = Swaab DF, Gooren LJ, Hofman MA | title = Gender and sexual orientation in relation to hypothalamic structures | journal = Hormone Research | volume = 38 | issue = Suppl 2 | pages = 51–61 | year = 1992 | pmid = 1292983 | doi = 10.1159/000182597 | doi-broken-date = 1 November 2024 | url = https://pure.knaw.nl/portal/en/publications/gender-and-sexual-orientation-in-relation-to-hypothalamic-structures(7cb8b769-4329-407a-b0ee-13e011017f68).html | hdl-access = free | hdl = 20.500.11755/7cb8b769-4329-407a-b0ee-13e011017f68 | type = Submitted manuscript }}</ref>

A 2010 study by Garcia-Falgueras and Swaab stated that "the fetal brain develops during the intrauterine period in the male direction through a direct action of testosterone on the developing nerve cells, or in the female direction through the absence of this hormone surge. In this way, our gender identity (the conviction of belonging to the male or female gender) and sexual orientation are programmed or organized into our brain structures when we are still in the womb. There is no indication that social environment after birth has an effect on gender identity or sexual orientation."<ref>{{Citation |last1=Garcia-Falgueras |first1=Alicia |title=Sexual Hormones and the Brain: An Essential Alliance for Sexual Identity and Sexual Orientation |date=2010 |journal=Endocrine Development |volume=17 |pages=22–35 |editor-last=Loche |editor-first=S. |url=https://karger.com/books/book/2680/chapter/5758416 |access-date=2024-06-13 |publisher=S. Karger AG |language=en |doi=10.1159/000262525 |isbn=978-3-8055-9302-1 |last2=Swaab |first2=Dick F. |pmid=19955753 |editor2-last=Cappa |editor2-first=M. |editor3-last=Ghizzoni |editor3-first=L. |editor4-last=Maghnie |editor4-first=M. |url-access=subscription}}</ref>

====Ovine model====
The [[domestic ram]] is used as an experimental model to study early programming of the neural mechanisms which underlie homosexuality, developing from the observation that approximately 8% of domestic rams are sexually attracted to other rams (male-oriented) when compared to the majority of rams which are female-oriented. In many species, a prominent feature of sexual differentiation is the presence of a sexually dimorphic nucleus (SDN) in the preoptic hypothalamus, which is larger in males than in females.

Roselli et al. discovered an ovine SDN (oSDN) in the preoptic hypothalamus that is smaller in male-oriented rams than in female-oriented rams, but similar in size to the oSDN of females. Neurons of the oSDN show [[aromatase]] expression which is also smaller in male-oriented rams versus female-oriented rams, suggesting that sexual orientation is neurologically hard-wired and may be influenced by hormones. However, results failed to associate the role of neural aromatase in the sexual differentiation of brain and behavior in the sheep, due to the lack of defeminization of adult sexual partner preference or oSDN volume as a result of aromatase activity in the brain of the fetuses during the critical period. Having said this, it is more likely that oSDN morphology and homosexuality may be programmed through an androgen receptor that does not involve [[aromatisation]]. Most of the data suggests that homosexual rams, like female-oriented rams, are masculinized and defeminized with respect to mounting, receptivity, and gonadotrophin secretion, but are not defeminized for sexual partner preferences, also suggesting that such behaviors may be programmed differently. Although the exact function of the oSDN is not fully known, its volume, length, and cell number seem to correlate with sexual orientation, and a dimorphism in its volume and of cells could bias the processing cues involved in partner selection. More research is needed in order to understand the requirements and timing of the development of the oSDN and how prenatal programming effects the expression of mate choice in adulthood.<ref>{{cite journal | vauthors = Roselli CE, Stormshak F | title = Prenatal programming of sexual partner preference: the ram model | journal = Journal of Neuroendocrinology | volume = 21 | issue = 4 | pages = 359–64 | date = March 2009 | pmid = 19207819 | pmc = 2668810 | doi = 10.1111/j.1365-2826.2009.01828.x | author2-link = F. Stormshak | name-list-style = amp | author-link = C. E. Roselli }}</ref>

=== Childhood gender nonconformity ===
[[Childhood gender nonconformity]] is a strong predictor of adult sexual orientation that has been consistently replicated in research, and is thought to be strong evidence of a biological difference between heterosexual and non-heterosexuals. A review authored by [[J. Michael Bailey]] states: "childhood gender nonconformity comprises the following phenomena among boys: cross-dressing, desiring to have long hair, playing with dolls, disliking competitive sports and rough play, preferring girls as playmates, exhibiting elevated separation anxiety, and desiring to be—or believing that one is—a girl. In girls, gender nonconformity comprises dressing like and playing with boys, showing interest in competitive sports and rough play, lacking interest in conventionally female toys such as dolls and makeup, and desiring to be a boy". This gender nonconformist behavior typically emerges at preschool age, although is often evident as early as age 2. Children are only considered gender nonconforming if they persistently engage in a variety of these behaviors, as opposed to engaging in a behavior on a few times or on occasion. It is also not a one-dimensional trait, but rather has varying degrees.<ref name="Bailey_2016" />

Children who grow up to be non-heterosexual were, on average, substantially more gender nonconforming in childhood. This is confirmed in both retrospective studies where homosexuals, bisexuals and heterosexuals are asked about their gender typical behavior in childhood, and in prospective studies, where highly gender nonconforming children are followed from childhood into adulthood to find out their sexual orientation. A review of retrospective studies that measured gender nonconforming traits estimated that 89% of homosexual men exceeded heterosexual males level of gender nonconformity, whereas just 2% of heterosexual men exceeded the homosexual median. For female sexual orientation, the figures were 81% and 12% respectively. A variety of other assessments such as childhood home videos, photos and reports of parents also confirm this finding.<ref name="Bailey_2016" /> Critics of this research see this as confirming stereotypes; however, no study has ever demonstrated that this research has exaggerated childhood gender nonconformity. J. Michael Bailey argues that gay men often deny that they were gender nonconforming in childhood because they may have been bullied or maltreated by peers and parents for it, and because they often do not find femininity attractive in other gay males and thus would not want to acknowledge it in themselves.<ref name="Bailey_2003" />{{Rp|page=80}} Additional research in Western cultures and non-Western cultures including Latin America, Asia, Polynesia, and the Middle East supports the validity of childhood gender nonconformity as a predictor of adult non-heterosexuality.<ref name="Bailey_2016" />

This research does not mean that all non-heterosexuals were gender nonconforming, but rather indicates that long before sexual attraction is known, non-heterosexuals, on average, are noticeably different from other children. There is little evidence that gender nonconforming children have been encouraged or taught to behave that way; rather, childhood gender nonconformity typically emerges despite conventional socialization.<ref name="Bailey_2016" /> Medical experiments in which infant boys were sex reassigned and reared as girls did not make them feminine nor attracted to males.<ref name="Bailey_2016"/>

=== Boys who were surgically reassigned female ===
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.<ref name="Bailey_2016"/>{{rp|72–73}} 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 attracted to females. Six cases were exclusively attracted to females, with one case 'predominantly' attracted to females. In a review article in the journal ''[[Psychological Science in the Public Interest]],'' six researchers including [[J. Michael Bailey]] state this establishes a strong case that male sexual orientation is partly established before birth:
{{Blockquote|text=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.|author=|title=|source=}}
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]] (resulting in a malformed penis), 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 these sex reassignments as 'the near-perfect quasi-experiment' in measuring the impact of 'nature' versus 'nurture' with regards to male homosexuality.<ref name="Bailey_2016" />