The relationship between hypothalamic activation and peripheral blood mononuclear cell responses to sexual arousal in homosexual men

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The relationship between hypothalamic activation and peripheral blood mononuclear cell
responses to sexual arousal in homosexual men
Cognitive Neuroscience: Vic Shao-Chih Chiang, 2019

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Specific Aims
Same-sex behavior is observed throughout nature and its expression has been coupled with the
intricate human concept of homosexuality (Bailey & Zuk, 2009). While the scientific conception
of homosexuality remains abstruse, there are emerging neuroimaging studies that have explored
the underlying neural correlates (Poeppl, Langguth, Rupprecht, Laird, & Eickhoff, 2016). The
elucidation of the mechanisms of homosexuality has proven difficult despite a myriad of theories
has been postulated (Poeppl et al., 2016). While neural correlates are useful to begin parsing out
the neuroscience angle of homosexuality, it lacks the molecular insight that facilitates deeper
comprehension. This lacuna is primarily due to the inaccessibility to molecular samples from the
human brain in vivo to dissect acute molecular changes to behavioral responses. As a preliminary
step towards future technologies that may facilitate this sample collection, some molecular insights
made be accessed through blood samples and highlights how peripheral immune systems can
communicate with the central nervous system to mediate behavior or as a consequence of it(Hodes,
Kana, Menard, Merad, & Russo, 2015). This is coupled with the immune component emerging to
be recognized as a potential driver of homosexuality (Bogaert et al., 2017). Therefore, the aim of
this study is as follows.
1. Determine hypothalamic activation patterns during sexual arousal in homosexual men.
2. Determine peripheral blood mononuclear cell heterogeneity during sexual arousal in
homosexual men.
3. Determine the correlation during sexual arousal in homosexual men, of peripheral blood
mononuclear cell heterogeneity with the level of hypothalamic activation or self-reported
level of sexual arousal.

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Background
Same-sex behavior has been observed for over 1500 animal species ranging from squash bugs to
dolphins (Bailey & Zuk, 2009). On the biological understanding that sexual behavior acts as a
biological mechanism to produce offspring, people have instigated queries as to why same-sex
behavior exist if it is obsolete for procreation. A panoply of theories has been posited that it is a
side effect of other genes for fitness, it strengthens the bond and the avuncular hypothesis that the
extra male increases survival of offspring within the family i.e. the survival of the fittest family
(Bailey & Zuk, 2009). A very recent theory postulates same-sex behavior to emerge very early in
time, and its persistence is on the grounds of it being not evolutionarily costly, partially due to the
concept that sex dimorphism is biologically complex (Monk, Giglio, Kamath, Lambert, &
Mcdonough, 2019).
Although sexuality cannot be assigned to animals, humans that display a penchant for same-sex
behavior are identified as homosexuals. This concept of homosexuality is however in itself
intricate, as it can be defined by sexual attraction, sexual behavior and self-identification, thus
results can vary when different operationalization is utilized (Fisher, Ristori, Morelli, & Maggi,
2018). Neural correlates under sexual arousal has been increasingly sought to engender progress
in the understanding of homosexuality. (Stoléru, Fonteille, Cornélis, Joyal, & Moulier, 2012)
provides a review of neural correlate studies and supports sexual arousal to use similar brain
regions in homosexual men with heterosexual men. In spite so, there appear to be fundamental
differences as reviewed by (Poeppl et al., 2016),where they expounded on studies such as the
interstitial nuclei of the anterior hypothalamus (INAH) being smaller in homosexual males,
differences of cerebral asymmetry and functional connections of the amygdala. They summarized
from their meta-analysis of 364 human subjects that sexuality can be controlled by the anterior and
preoptic area of the hypothalamus (Poeppl et al., 2016) and this region has been well-established
to play roles in rodent male sexual behavior, and has been further espoused by a recent optogenetic
study (Wei et al., 2018).
Aside from neural correlates, there have been other attempts to tease out mechanisms of
homosexuality. For example, the search for the “gay gene” as has been reported in the recent
genome-wide association study that profiled nearly 500,000 individuals (Ganna et al., 2019).

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Although they found differential genetic variants such as rs28371400-15q21.3, it was concluded
that at the genetic level, homosexuality cannot be predicted (Ganna et al., 2019). A slightly earlier
study has attempted to investigate this from the perspective of the mother which found the increase
in antibodies against neuroligin 4 Y-linked protein in mothers with subsequent pregnancies, and
this was correlated with the likelihood of attaining a homosexual son (Bogaert et al., 2017).
Although it can only explain ~10% of homosexuality, it bestows a step forward to learning
potential mechanisms (Bogaert et al., 2017). Other mechanisms have also been proposed such as
sexual conflict, which is when the trait of optimal fitness contradicts between the two sexes and
has been explained for homosexuality that what’s beneficial for the homosexual male’s mother
may divert masculine fetal programming down an alternative path (Gavrilets, Friberg, & Rice,
2018). Epigenetic canalization effect has by the same token, been proposed and alludes to the
ability to generate epigenetic trait no matter the variability of the environment or genotype (Fisher
et al., 2018).
In spite of sexual differentiation to be distinctive to sexual orientation, some insights can be
attained from the process. In particular, sex steroid hormones such as androgens, estrogen and
progesterone play an imperative role in sexual differentiation (Wright, Schwarz, Dean, &
McCarthy, 2010). At least in part through the peripheral circulation that communicates with the
brain, they can exert organizational effects on the rodent brain through influencing glutamate
receptors in key sexually dimorphic brain regions, or activation effects such as the onset of the
male sexual behavior repertoire (Wright et al., 2010). Some perspectives on humans have been
gained through gene mutations relating to these sex steroid hormones. Amongst others, congenital
adrenal hyperplasia results in an overproduction of androgen in girls, and complete androgen
insensitivity syndrome patients lack functional receptors to androgen (Fisher et al., 2018). Both
types of patients exhibit an increased proportion of homosexuality (Fisher et al., 2018). Added to
that are biological markers of the influence of prenatal sex steroid hormone that demonstrate a
correlation with sexual orientation, namely, finger digit size and otoacoustic emission (Fisher et
al., 2018). However, all of the above studies are inconsistent due to methodological limitations.
Needless to say though, the peripheral circulation do communicate with the brain to inform
behavior, as demonstrated through the gargantuan amount of work on how circulating interleukins
affects adolescent propensity to drug addiction (Brenhouse & Schwarz, 2016), peripheral TNFα to

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increase anhedonia (Hodes et al., 2015), and early life stress developed chronic low-grade
inflammation that instigates over-consumption of fat (Nusslock & Miller, 2016).
There have been recent surges in the past decade in the use of single-cell RNA sequencing ever
since the first study in 2009 on mice blastomere (Tang et al., 2009). This technology is crucial to
answering questions on cell heterogeneity and stochasticity of gene expression, which are lost with
bulk transcriptome technologies (Kolodziejczyk, Kim, Svensson, Marioni, & Teichmann, 2015).
This was able to reveal 47 molecularly distinct subclasses of cells in the mouse cortex and
hippocampus, with key marker genes that have widespread applications (Zeisel et al., 2015).
Following from that, this was escalated to humans using postmortem brains samples of the cerebral
cortex, which found 16 neuronal subtypes that were refined by known markers and cortical
cytoarchitecture (Lake et al., 2016).
Aside from the central nervous system, single-cell RNA-sequencing has also been carried out to
study blood samples. In particular, peripheral blood mononuclear cells (PBMC) are isolated from
blood samples and constitutes with an array of cell types including classical and non-classical
monocytes, dendritic cells, megakaryocytes, helper and cytotoxic T cells, and natural killer cells
(Villani et al., 2017). Single-cell RNA-sequencing enabled the identities and interrelationships of
dendritic cells to be disentangled and uncovered a new subset of dendritic cells, as well as
additional monocyte populations (Villani et al., 2017). Another study investigated CD4+ cytotoxic
T lymphocytes within PBMCs and unveiled further conception of the biology of their generation,
functional properties and heterogeneity (Patil et al., 2018).
The neural correlate of sexual arousal in homosexuality are beginning to be understood, but the
molecular mechanisms remain elusive. On the grounds that neuroimmune communications drive
behavior and that the fraternal birth order effect similarly involves immune systems, I propose to
study the related PBMC heterogeneity using single-cell RNA sequencing in response to sexual
stimuli and its correlation with hypothalamic activations using neuroimaging.

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Methods
Participants (A Manzouri & Savic, 2019; Amirhossein Manzouri & Savic, 2018)
Participants will be recruited through LGBTQ organizations, dating apps, and on-campus of 25 –
50 years old of 50 homosexual males. They will be healthy, ethnically diverse, unmedicated, and
right-handed as well as matched for age and education level. Sexual orientation will be scored
based on modified Kinsey scale (0 = maximally heterosexual, 6 = maximally homosexual) that
enquire about sexual fantasies, romantic attraction and sexual behavior in consecutive 5 year
historical time periods, from age 16 to the present. Subjects were excluded based on standard
questionnaires and clinical interviews with a practicing physician if they had a previous history of
psychosis, personality disorder, sexual dysfunction, gender dysphoria, hypogonadism, HIV
infection, paraphilia, or sexual offences, major or bipolar depression, alcohol or substance abuse,
chronic psychosocial stress, major life traumas, chronic fatigue, chronic pain, or systemic disease.
All methods will be approved by the institutional review board and ethics committee of the
University of Massachusetts Boston carried out in accordance with its guidelines. Informed
consent will be obtained from each participant for every portion of the study which they will
participate.
Stimuli (Hu et al., 2011; Ponseti et al., 2006; Sylva et al., 2013)
The sexual stimuli are pre-selected based on ratings from volunteers with similar demographics as
participants, who will not participate in the subsequent aspect of the study. They will rate full-
body photographs of a naked man or woman posed in a sexually provocative manner. These ratings
will be on basic emotional dimensions (valence and arousal) using the Self-Assessment Manikin
(SAM), and sexual arousing effect by a nine-point erotic rating scale. Nonsexual stimuli will be
taken from the International Affective Picture System such as landscape, humans in sports, animals
etc. Each participant will complete three randomized sessions 1 week apart with either female
sexual stimuli, male sexual stimuli, or non-sexual stimuli. Each session has 100 stimulus
presentations at the centre of the screen, with a fixation cross, for 3.5s in random order with no-
repeat, and in 1.5s intervals. To ensure attention, the participants are asked to click a button with
their right index finger when an oddball appears (six times each session). Immediately after, blood
is drawn from the participants and they will rate the stimuli during the session on basic emotional
dimensions and sexual arousal as above.

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Functional Magnetic Resonance Imaging (fMRI) (Amirhossein Manzouri & Savic, 2018)
The MRI scanner specifications should be on par with those described in previous homosexuality
fMRI publications on receiving coil, and tesla to perform echoplanar imaging on changes in blood
oxygen level-dependent (BOLD) signal as an index of regional neuronal activity. These require
optimization of echo time, repetition time, field of view, flip angle, and matrix to acquire images
with isotropic voxel size. Slices will be taken along the plane connecting the anterior and posterior
commissures, with a 1.72 mm × 1.72 mm × 3.99 mm resolution and finessed the number of axial
slices, slice thickness and inter-slice gaps.
Imaging Analysis (Burke, Manzouri, & Savic, 2017)
The MRI images will be pre-processed and analyzed with SPM and implemented in MatLab.
Firstly, spatial normalization will be carried out to the standard EPI template of the Montreal
Neurological Institute space and then smoothed with the appropriate Gaussian Kernel. Based on a
priori analysis, we are focusing on the hypothalamus, so ROI is defined from its activation. To
locate the hypothalamic clusters more precisely, the coordinates of Talairach’s atlas will be
translated to those of Schaltenbrant’s atlas, which visualizes the hypothalamic nuclei in detail. The
resulting regional cerebral blood flow is used for subsequent analysis.
Intelligent imaging activated cell sorting (ilACS) of peripheral blood mononuclear cells (PBMC)
(Kang et al., 2018; Nitta et al., 2018; Patil et al., 2018; Villani et al., 2017)
Whole blood is collected after each fMRI run, into EDTA coated tubes. PBMC is purified using
the Ficoll-Paque density gradient centrifugation and suspended as single cells into the fetal bovine
serum. The PBMC was then stained with antibodies for markers of different cell types: classical
and non-classical monocytes, dendritic cells, megakaryocytes, helper and cytotoxic T cells, and
natural killer cells. This method uses machine-intelligent technology to sort single cells beyond
one-dimensional signals to unique spatial, biochemical and morphological traits. The ilACS
machine consists of a liquid pump, a cell focuser, a microscope, a speed meter, an image processor
and a cell sorter. To characterize, count the sort, and execute sort-decision functions, ilACS is
equipped with ImageJ, R, NIS-Elements AR and C++. Cells are first gated based on propidium
iodide negative cells to obtain viable cells. Subsequent gating will be made based on area,

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perimeter and shape of bright field images. Final gating is made based on fluorescence signals of
different antibody markers.
Single Cell RNA Sequencing (Li et al., 2019; Moffitt et al., 2018)
Single cells are sorted into RNA extraction buffer followed by capturing on poly(T)
oligonucleotides that contain unique molecular identifier sequences, single-cell specific barcodes
and adaptor for subsequent amplification. After reverse transcription and second-strand synthesis
with hairpin primer, the transcriptome is amplified by polymerase chain reaction. The amplicons
are then fragmented by tagmentation and the quality assessed through the fragment analyzer and
concentration by Qubit. A final amplification step allows the sequencing adaptors to be attached,
and the resulting library is assessed with Bioanalyzer for size distribution and Qubit for
concentration. Illumina sequencing is next carried out to a depth of at least 1 million raw reads per
cell.
Bioinformatics (Lafzi, Moutinho, Picelli, & Heyn, 2018; Poirion, Zhu, Ching, & Garmire, 2016)
The raw data is presented in FastQ and pre-processed to trim the adaptor, normalized to the
housekeeping gene, and analyzed for different quality metrics. Alignment is then made to detect
alternative splicing and obtain quantities of the transcripts. Quality control measures are in place
to filter out transcripts with low quality. Normalization of batch effects and spike-in controls are
then made. Following from that, subpopulations of single cells are clustered with t-distributed
stochastic neighbor embedding. Differential gene expression between single cells is analyzed with
Find All Markers with Wilcoxon rank-sum tests. The data is subsequently reduced of its
dimensions for simplicity of visualization in two-dimensional formats. Next, gene ontology
analysis on the function and relation is carried out to determine relevant PBMC populations.
Correlation Analysis (Gaudillière et al., 2014)
The relative population of the top 20 PMBC subtypes are used for correlation analysis with the
level of hypothalamus BOLD, SAM valence and SAM arousal. Pearson correlation coefficient
between these comparisons is determined.

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Expected Results
The first aim is to determine the hypothalamic activation patterns during sexual arousal in
homosexual men. I expect the hypothalamus to be activated when the homosexual men are exposed
to male sexual stimuli as opposed to nonsexual and female sexual stimuli, similarly demonstrated
by Gizewski and her team (Paul et al., 2008). The use of photography in my study in lieu of videos
may present as a limitation to maximally stimulate arousal. However, most of the studies in the
homosexuality neuroimaging literature use photograph to decrease confounds from other variables
that may be present in videos. If the sexual arousal does not elicit activation of the hypothalamus,
it could be due to differences in participants and the stimuli. Under this circumstance, other brain
regions will be targeted that has demonstrated a proven history of activation in homosexual men
including the lateral occipital cortex, and the inferotemporal cortex (Stoléru et al., 2012).
The second aim is to determine PBMC heterogeneity during sexual arousal in homosexual men. I
expect in concert with changes in the endocrine system that instantaneously occurs with sexual
arousal, the differentiation of various PBMC subtypes will occur, that result in changes in their
population size. A strength of this is the ilACS allow better discrimination of different PBMC from
biochemical as well as morphological traits characterized from previous studies. Under the
condition that no changes in PBMC subtype population occur, it is possible that these cell subtype
populations are stable to sexual stimuli. In this case, I will shift my attention to the transcriptome
profile of major PBMC types.
The third aim is to determine the correlation during sexual arousal in homosexual men, of PBMC
heterogeneity with the level of hypothalamic activation or self-reported level of sexual arousal.
The basis that this correlation is possible is that the photographs will generate a variable response
in different participants based on their niche preferences and experiences. Therefore, I expect
differing levels of hypothalamic activation as well as different self-reported ranking scores of
sexual arousal. Coupled with variable changes in PBMC subtypes during sexual arousal, some of
these would correlate with levels of hypothalamic activation or self-reported ranking of sexual
arousal.

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Implications & Broader Importance
These results facilitate understanding of the neural correlate, the dynamics of PBMC subtypes of
sexual arousal in homosexual men, and their relationship with each other. On the condition that
the above aims prove successful, a panoply of subsequent opportunities is wide open to further the
neuroscience understanding of homosexuality. To name a few, future studies could perform
specific tasks to assess the pleasure aspect of the sexual arousal and to parse out the pleasure and
desire aspect of sexual arousal. Other future work can constitute examining the functional
connectivity with other brain regions, using longitudinal study design, investigating neuroimaging
epigenetics, conducting single-neuron studies (eg stereotaxic electroencephalography), and
incorporation of brain stimulation (eg transcranial magnetic stimulation).
Several mechanisms have been posited for homosexuality. In terms of the neuroscience
perspective, the organizational effects of prenatal hormones that induce irreversible changes on
the neuroanatomy such as the mPOA, exert influences to develop same sexual behavior as evident
in birds, mice, rats, hamsters, ferrets and pigs (Balthazart, 2011). Similar mechanisms have been
inferred in humans due to several sexually dimorphic traits to differ with homosexuals such as the
size of INAH, and auditory neurocircuits (Balthazart, 2011). However, the molecular mechanisms
of homosexuality such as genetics remain unknown (Ganna et al., 2019). The current proposal will
begin shed light on the grasp of homosexuality through the lens of sexual arousal.
Furthermore, there is increasing recognition and discussion around the globe surrounding sexual
diversity in terms of characteristics, gender identities, relationship paradigms and fetishes (Gupta,
2012). It is identically important to highlight current definitions of sexual diversity will also
continue to evolve to forgo the naïve assumption of homogeneity in homosexuality (Roselli, 2018).
Albeit, an important step forward is for future neuroscience studies to consider the sexuality
demographics, as this is an area that has long been neglected in neuroscience and helps address
challenges due to possible political misinterpretation. Science predominantly conceptualize human
existence from a heterosexual perspective, resulting in a parochial view of the world. The current
proposal hopes to offer baby steps towards understanding this labyrinth of human sexuality.

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