TESTO: Testosterone Effects on Short-Term Outcomes in Infants With XXY
Phase: PHASE4
Conditions: Klinefelter Syndrome
Sponsor: University of Colorado, Denver

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w 
COMIRB Protocol 
COLORADO MULTIPLE INSTITUTIONAL REVIEW BOARD 
CAMPUS BOX F-490    TELEPHONE:  303-724-1055  Fax:  303-724-0990 
 
 
 
Protocol #:  
 
17-1317 
Project Title: 
 
TESTO: Testosterone Effects on Short-Term Outcomes in Infants with XXY  
Principal Investigator:  Shanlee Davis, MD, MSCS 
Version Date:  
9/11/2020 
 
 
 
 
 
  
 
I. 
Hypotheses and Specific Aims:  
 
Karyotype 47,XXY, also known as Klinefelter syndrome, is the most common chromosomal abnormality in 
boys, affecting 1/600 male births or nearly 6 million individuals worldwide.1 XXY is associated with testos-
terone (T) deficiency from primary testicular failure, motor and neurocognitive deficits, and cardiometabol-
ic disorders.2 Men with XXY have higher mortality, with cardiovascular disease being the leading cause of 
death (standardized mortality ratio 5.8).3 An essential question in XXY is whether the observed phenotype 
is solely attributable to the gene dosage effect from the extra X chromosome, or whether T insufficiency 
contributes to the neurocognitive deficits and metabolic dysfunction and therefore could be mitigated by T 
treatment.  
 
The hypothalamic-pituitary-gonadal axis is activated twice in post-natal life: the mini-puberty period of in-
fancy and true puberty early in the second decade.4 Although understudied, the T surge of mini-puberty in 
the first few months of life may play a role in long-term metabolic programming of energy metabolism and 
neurocognition.5-7 Infants with XXY have a blunted T surge and giving T to infants with XXY has recently 
been proposed as an intervention.8-10 However, the only 
data to support this practice are retrospective reports of 
better cognitive scores in XXY boys who had received T 
in infancy.11,12 With the rapidly increasing rate of XXY 
diagnoses from non-invasive prenatal testing, it is crucial 
to study the efficacy and safety of infant T in XXY now.13 
Recognizing this clinical equipoise, we conducted a pilot 
study to evaluate body composition (a surrogate marker 
of cardiometabolic health) as a short-term outcome of T 
treatment. Building on these preliminary results, we pro-
pose further studying the short-term effects of T on car-
diometabolic and developmental outcomes. Our central 
hypothesis is that T treatment positively influences car-
diometabolic and neurodevelopmental outcomes in XXY 
infants (Figure 1). To test this hypothesis, we will under-
take the following specific aims.  
 
 
Specific Aim 1: To quantify the short-term effect of T in infants with XXY on cardiometabolic out-
comes.   
Hypothesis: Percent body fat (%BF) will follow a normal trajectory in treated infants, while infants not 
receiving T will have greater %BF. Approach: In the TESTO: Testosterone Effects on Short-Term Out-
comes trial, infants with XXY (n=75) age 4-12 weeks will be randomized 1:1 to receive 25mg intramus-
cular T injections every 4 weeks for 3 doses or placebo.. %BF will be assessed by air displacement ple-
thysmography at baseline, 12 weeks, and 24 weeks. The primary outcome is change in %BF z-scores 
at 12 weeks between groups (powered to detect a difference of 0.5SD). Infants initially receiving place-
bo will get T in the second half of the study (12-24 weeks). Secondary outcomes include change %BF 
z-scores from 12-24 weeks, growth parameters, and serum leptin..  
 
 
 
Figure 1. We hypothesize T treatment will 
improve outcomes in infants with XXY. 
BF=body fat. 

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Specific Aim 2: To quantify the short-term effects of T in infants with XXY on neurodevelopment, 
gonadal function and safety outcomes.  
Hypothesis: (1): XXY infants receiving T will have greater gains in standard scores on early motor skills, 
emerging language, and adaptive functioning. (2): T treatment will not impair gonadal function, as 
measured by gonadotropins and biomarkers of Sertoli cell function. (3): There will be no serious ad-
verse outcomes associated with T treatment in infants with XXY. Approach: Infants from Aim 1 will be 
given standardized neurodevelopment assessments at baseline, 12 and 24 weeks. Serum hormone bi-
omarkers of gonadal function will also be obtained at these times. Adverse events will be monitored 
throughout the study.  
 
Specific Aim 3 (exploratory): To measure metabolite changes induced by T in infants with XXY.  
Hypothesis: Circulating metabolites involved in energy metabolism, specifically long chain acyl-
carnitines (LCAC) and branched-chain amino acids (BCAA), will be lower due to improved mitochondri-
al function in T treated boys; T will favorably alter pathways involved in energy metabolism. Approach: 
LCAC and BCAA will be quantified at baseline and 12 weeks in the first 20 subjects. An unbiased plat-
form of over 4,000 metabolites will also be performed to identify pathways that are differentially regulat-
ed with T treatment.  
 
Assessing short-term safety and efficacy of T treatment in infants with XXY will provide the first evidence 
for establishing standard of care practices, with a planned extension for long-term outcomes. These re-
sults will generate preliminary data on mechanisms of the effects of T during the critical mini-puberty peri-
od for the development of future work, such as sex steroid-induced epigenetic modifications. Mentors with 
expertise in neurodevelopment, cardiometabolic outcomes, metabolomics, and clinical trials will optimize 
career development and foster expertise in gonadal and cardiometabolic function in children with sex 
chromosome aneuploidies.   
 
 
II. Background and Significance:  
 
XXY/Klinefelter syndrome is common and the diagnosis in childhood is increasing. An extra X 
chromosome is present in ~1/600 boys, therefore over 6 million boys and men worldwide are affected by 
this very common, but under-diagnosed, genetic condition known as XXY or Klinefelter syndrome (KS).1 
XXY is characterized by a high degree of phenotypic heterogeneity throughout the lifespan but nearly 
universal testicular insufficiency and infertility in adulthood.2,14 Our knowledge of the natural history of 
XXY in infancy and childhood is largely limited to birth cohort studies from the 1970’s and more recent 
cross-sectional studies limited by ascertainment bias. Historically, less than 10% of males with XXY are 
diagnosed before adolescence, however the rate of prenatal diagnosis is increasing exponentially as test-
ing of free-fetal DNA in maternal blood evolves to become standard screening in prenatal care.15,16 Fur-
thermore, there is evidence the actual incidence of XXY is increasing as well.17,18 With greater recognition 
in infancy and potentially increased prevalence of XXY, there is a high demand for evidence-based re-
search, particularly investigation of early determinants that contribute to increased morbidity and de-
creased quality of life in this population.19  
 
XXY results in universal testicular dysfunction and hypogonadism. While many features of the XXY 
phenotype are variable, testicular dysfunction is nearly universal.20 In adults, this manifests as small tes-
tes, infertility, and hypergonadotropic hypogonadism (elevated gonadotropins and low testicular hormone 
concentrations).2 The Leydig cells in the testes produce testosterone (T) and insulin-like hormone 3 
(INSL3), while the Sertoli cells produce inhibin B (INHB) and anti-mullerian hormone (AMH). All of these 
testicular hormones have been demonstrated to be lower in XXY when compared to typical males from 
mid-puberty through adulthood.21-23 Microscopically, the testes of men with XXY are fibrotic with rare germ 
cells and Leydig cell hypertrophy.20 
 

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Clinical evidence of testicular insufficiency in infants 
with XXY includes smaller testes size, reduced penile 
growth over the first months of life, mild hypotonia, 
and a passive temperament.10 Testicular biopsies 
have shown a lower number of germ cells in infants 
and children with XXY, however the histological ap-
pearance of Sertoli and Leydig cells is typically nor-
mal.24-28 Five studies (total N=83) of hormones during 
infancy in XXY conclude activation of the pituitary-
gonadal axis in the first months of life, known as the 
mini-puberty, does occur, however serum T is lower in 
XXY in the majority.20 The only study to quantify T 
concentrations with liquid chromatography/mass spec-
trometry (gold standard) was done by our collaborator 
Dr. Lahlou. She found 8/38 XXY infants (21%) had 
serum T below the normal range during the mini-
puberty period, and 83% were below the median (Fig-
ure 2).29 In summary, many XXY infants have evi-
dence of testosterone insufficiency. 
 
 
Testosterone insufficiency increases cardiometabolic risk, and cardiovascular disease is the lead-
ing cause of morbidity and mortality in men with XXY. Adult men with XXY have a high prevalence of 
type 2 diabetes, nonalcoholic fatty liver disease and cardiac dysfunction, with a standardized mortality 
ratio of 5.8 from these conditions.2,3,30-34 Many precursors of these conditions, such as insulin resistance, 
abdominal adiposity, and vascular dysfunction, have been shown to inversely correlate with T concentra-
tions, and it is presumed the increase in cardiovascular-related morbidity and mortality in XXY is second-
ary to chronic hypogonadism.32,35-39 In multiple populations including men with XXY, T has been shown to 
improve body composition – an early surrogate marker of altered metabolism associated with the devel-
opment of disorders of insulin resistance.30,40-42 However, there is a lack of randomized controlled trials of 
T in XXY and observational studies suggest that late treatment of hypogonadism does not reverse exist-
ing cardiovascular disease.38 Furthermore, two recent studies have found evidence of high body fat and 
serum markers of insulin resistance in children with XXY.43,44 Therefore, to reduce cardiovascular morbidi-
ty and mortality in XXY, we need to focus on prevention of early precursors. Body composition is a partic-
ularly intriguing outcome measure as it can be assessed throughout the lifespan, has been shown to be 
an early and modifiable risk marker of metabolic disease, and is directly altered by T.30,45 Body composi-
tion is also a clinically meaningful outcome measure in infancy as high body fat percentage early in life 
may contribute to the decreased strength, endurance, and athleticism seen in XXY. A gap exists in our 
knowledge of body composition in infants with XXY and the effect of T on this important outcome meas-
ure.  
 
The testosterone surge during the mini-puberty period in the first months of life likely plays a crit-
ical role in lifelong metabolic programming. We have known about the mini-puberty period for dec-
ades, however the purpose of this brief activation of the hypothalamic-pituitary-gonadal axis remains quite 
speculative. Many tissues are sexually dimorphic and exposure to T during sensitive time points is re-
quired for masculinization of these organs. Mouse models that manipulate exposure to sex steroids in the 
neonatal period have found that early T exposure induces epigenetic modifications and results in lifelong 
effects on neurocognition and energy metabolism5,46 Blocking the neonatal T surge in male mice results in 
higher leptin concentrations and greater fat to muscle ratio for life, while a single dose of T given to neo-
natal female rodents results in differential gene expression in the liver in a masculinized pattern.6,47 Expo-
sure to T in neonatal mice also leads to decreased DNA methylation in the brain, resulting in the sexually 
dimorphic differences observed in brain structure and function.48 In humans, several studies have report-
ed associations between T concentrations during the mini-puberty period and brain lateralization, lan-
guage organization, and gender identity.7,49,50 These findings are particularly relevant in XXY given the 
high risk of neurodevelopmental impairment and cardiometabolic disease, and suggest the mini-puberty 
period is a critical time for intervention.  
 
Figure 2. Serum testosterone in males with XXY 
(open circles) is low or low-normal during the mini-
puberty period. Normal ranges in gray with Upper and 
Lower Limit (UL, LL). Lahlou et al. 2004. 

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Studying tissue-specific gene expression and epigenetic modification in human infants is invasive and 
labor-intensive, however we can investigate circulating metabolites that reflect systemic pathway regula-
tion. Pathways involved in energy metabolism are of specific interest in XXY. Long chain acylcarnitines 
(LCAC) are intermediate metabolites of fatty acid oxidation within the mitochondria.51 Obesity and insulin 
resistance are strongly associated with mitochondrial dysfunction and accumulation of LCAC.52 Branched 
chain amino acids (BCAA) also build up in the presence of mitochondrial dysfunction and are regarded as 
an excellent biomarker for obesity and insulin resistance.53 Work by one of my mentors, Dr. Baker, has 
found that LCAC and BCAA are elevated in infants exposed to maternal obesity in utero, demonstrating 
abnormal metabolism from a very young age. We know maternal obesity is associated with insulin re-
sistance in adult offspring and these metabolite differences in infancy represent the early metabolic pro-
gramming that underlies childhood determinants of adult disease.54,55 This work and the work of others 
highlight the critical importance of timing of exposures on lifelong metabolic programming, and how me-
tabolites can serve as early biomarkers of this process.56,57  
 
Finally, T treatment for infants with XXY is becoming common despite a lack of scientific evidence 
to support this practice.8 Samango-Sprouse et al reported that a cohort of boys with XXY who had re-
ceived T in infancy had better neurodevelopment in early childhood.11,12 Largely because of this single 
study, many parents are requesting T for their XXY sons. These proactive parents are often successful in 
obtaining this treatment, reinforcing disparities in care without establishing if T is effective or safe in this 
population. A randomized and blinded trial of T is needed to fill these critical gaps and provide evidence 
for establishing standard of care. 
 
In summary, testicular dysfunction is universal in XXY and likely contributes to the clinical phenotype, in-
cluding abnormalities in metabolism and neurodevelopmental deficits. The mini-puberty period of infancy 
represents a biologically plausible time period for intervention that may not only have immediate effects, 
but play an important role for lifelong metabolic programming. A gap currently exists in our understanding 
of short and long-term outcomes of T treatment in infants with XXY. This study will investigate short-term 
outcome measures of T, including cardiometabolic, neurodevelopmental, and gonadal function, as well as 
explore altered regulation of energy pathway metabolites as a probable mechanism underlying metabolic 
programming in infancy. 
 
This study is novel and the time is now. T treatment in infants with XXY has emerged as an off-label 
treatment in the US in the past few years, however there is no evidence to support this practice and con-
siderable variability exists among providers. Our extensive experience through the eXtraordinarY Kids 
clinic, parent support groups, and ongoing research efforts in this population has proven this is a highly 
motivated group of parents who strongly desire T treatment for their sons. Despite the biologic plausibility, 
no study has rigorously investigated the efficacy or safety of T in infants with XXY. We have care-
fully designed this study with family engagement to allow us to answer important scientific questions on 
the short-term effects of T while ensuring successful recruitment and retention. Our research team has 
the expertise and ideal environment to successfully conduct this study and advance our understanding of 
the efficacy and safety of this practice. Recent changes in the clinical and research environments make 
now the opportune time to conduct this study:  
• 
Technological advances in non-invasive prenatal screening have resulted in exponentially more 
infants being diagnosed with XXY. The May 2016 American College of Obstetrics and Gynecolo-
gy Practice Bulletin recognized cell-free fetal DNA testing is a superior screening test that can be 
offered in all pregnancies.13 Implementation of this guideline means 10 times as many infants 
will be diagnosed with XXY. In addition, many proposed genomic-based newborn screening 
tools would incidentally diagnose sex chromosome aneuploidies. The demand for knowledge in 
infants with XXY is great.    
• 
At the 2nd International Workshop on Klinefelter Syndrome (Germany, March 2016) the lack of 
randomized controlled trials of T in XXY, and the need for these studies to be conducted with pa-
tient-centered outcomes, was emphasized and nearly unanimously agreed upon by experts in 
KS.58 
• 
The field of metabolomics offers an opportunity to investigate the mechanisms of these changes. 
The outcome measures chosen for this study are not only surrogate markers for future disease and disa-
bility, but are meaningful patient-centered outcomes in infancy and childhood.  

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• 
High body fat with poor muscle tone may limit an infant’s ability to engage with his environment.  
• 
Delays in motor, language, and social development affect 75% of boys with XXY and strongly 
correlate with future cognition, behavior, and academic performance.  
• 
This study will be the first to test the hypothetical mechanism demonstrated in animal models that 
T induces immediate metabolic and epigenetic modifications that in turn confer lifelong effects on 
metabolism and neurocognition.  
 
III. Preliminary Studies/Progress Report:   
 
Preliminary data to support the study concept: We have found 
testicular and cardiometabolic function are inversely related in 
pre-pubertal XXY boys, and androgen supplementation im-
proves cardiometabolic markers. In a study of 93 boys with XXY 
age 4-12 (NCT00348945), 20% had evidence of testicular failure (low 
INHB) at baseline, as well as elevated percent body fat (%BF, z-
score +1) and high prevalence of metabolic syndrome features.59,60 
Low INHB was associated with greater cardiometabolic risk including 
higher blood glucose, triglycerides, and low-density lipoprotein (LDL), 
and lower high-density lipoprotein (HDL), all p<0.05. This is evidence 
that both testicular and cardiometabolic function is impaired before 
puberty in boys with XXY. Furthermore, two years of treatment with a 
weak androgen improved body composition (Figure 3) and cardiomet-
abolic markers.61  
 
Preliminary data to support the study design: Our pilot study suggests this regimen of T is well 
tolerated and does affect body composition. In preparation for this study, I designed a feasibility study 
to evaluate the short-term effects of T on body composition (COMIRB 14-7240, NCT02408445) in infants. 
We enrolled 18 infants with XXY between 6-15 weeks of age and randomized subjects to receive testos-
terone cypionate 25mg intramuscularly every 4 
weeks x 3 or no treatment. I hold an Investiga-
tional New Drug (IND 124260) file for the use of 
T in infants with XXY, and this file will be updat-
ed with this protocol. There have been no seri-
ous adverse events and the only side effects 
were an increase in appetite (n=2) and acne 
(n=2). %BF at baseline was normal, however 
increased by a z-score of +0.86±0.65 over 12 
weeks in infants not receiving T, while the 
change in %BF z-scores for infants who re-
ceived T was -0.24±0.67 (Figure 4). XXY 
infants who did not receive T had 4% more 
body fat at 5 months of age than normal male 
infants in the local Healthy Start Study.62   
 
Preliminary data to support feasibility of recruitment: Our pilot study exceeded recruitment goals 
and allowed us to adapt our study design based on limitations. We had excellent recruitment with an 
average one new subject per month and 100% retention rate without funds for recruitment or travel reim-
bursement. Informal focus groups and surveys have been conducted with parents to identify their reasons 
for or against participation. The greatest limitations in recruitment/retention have been 1) costs incurred 
due to travel to Denver and 2) unwillingness to participate in a trial where they may potentially be random-
ized to not receive T (as all perceived high benefits and low risks). After designing the current study, we 
surveyed 34 parents of XXY infants anonymously using a link posted on targeted social media groups. 
67% reported they “definitely” would enroll their child if eligible in this study design, 21% said they “may-
be” would enroll, and only 12% indicated they would not enroll. These pilot findings were critical in design-
ing this study.  
Figure 3. %BF is high in XXY boys 4-
12 yrs old. Androgen therapy im-
proves body composition (p=0.04). 
Figure 4. Left: T shots favorably effect the change in %BF 
z-scores after 12 weeks in infants with XXY. Right: At 5 
months (visit 2), %BF is 4% higher in XXY boys not treated 
with T compared to 316 healthy boys.   
p=0.005
p=0.037

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IV. Research Methods 
 
A.  Outcome Measure(s):   
Primary for Specific Aim 1 
Change in %BF z-scores from baseline to 12 weeks (per PeaPod) 
Primary for Specific Aim 2 
Change in motor composite percentile on the AIMS assessment from baseline to 12 weeks 
Primary for Specific Aim 3 
Change in C14:1 long chain acylcarnitine (LCAC) from baseline  
 
Secondary outcomes: Change in %BF z-scores from 12 to 24 weeks; change in anthropometric meas-
urements from baseline to 12 and 24 weeks; change in gross and fine motor scaled scores from the 
PDMS; cognitive and language scores on the Bayley; adaptive functioning from the ABAS; serum hor-
mones, lipids, BCAA, LCAC, and pathway analysis of untargeted metabolomics; urine metabolites; and 
number and type of adverse events. 
 
B. Description of Population to be Enrolled:   
A total of 75 infants with XXY will be enrolled. We will include all races, ethnicities, and languages.   
 
Inclusion criteria: Male infants with 47,XXY karyotype identified prenatally who are 4-12 weeks old (31 to 
90 days of age). 47,XXY must be from a diagnostic test such as CVS, amniocentesis, or post-natal 
blood/tissue. Non-invasive prenatal screening results alone will not be accepted.  
 
Exclusion criteria:   
1) >20% mosaicism for a normal cell line  
2) Gestational age at birth <36 weeks  
3) Birth weight <2.5th%ile or >97.5%ile for age (small or large for gestational age) 
4) History of thrombosis in self or a first degree relative  
5) Exposure to androgen therapy outside the study protocol 
6) Use of medications known to affect body composition, such as growth hormone or insulin  
7) Known allergy to the testosterone cypionate solution components including benyl benzoate, 
benzyl alcohol, or cottonseed oil 
 
Involving children is imperative as boys <18 years of age with XXY are much a different population than 
adult men with XXY. Men with XXY have been shown to have a high prevalence of type 2 diabetes, met-
abolic syndrome, fatty liver disease, and psychosocial difficulties. Clearly these processes begin in child-
hood. The potential to intervene in a preventative fashion, prior to the onset of disease, is more promising 
and productive than trying to treat pre-existing disease. Infant boys are exposed to endogenous testos-
terone in the first few months of life known as the “mini-puberty period of infancy.” Animal and human 
studies suggest this is a critical period for biologic programming, and therefore cannot be replicated at 
any other point in life. Including children in this study design allows us to investigate testosterone as a 
targeted intervention at this critical time point with evaluation of early markers of disease and disability. 
The ultimate goal is to improve the lives of children and adults with this condition. 
 
The age of the infants at enrollment was chosen 1) to allow assessment of testicular function during the 
expected mini-puberty period of infancy, 2) to assure visits 2 and 3 will be completed before the infant is 
10kg, as this is the upper weight limit for the PeaPod (our body composition assessment tool), and 3) 
feedback from parents.  
 
 
C. Study Design and Research Methods   
 
Overall study design. TESTO is a randomized controlled trial assessing the efficacy and safety of T in-
jections in infants with a prenatal diagnosis of XXY. Body composition, neurodevelopment, serum hor-
mones, safety parameters, and metabolomics will be assessed at baseline, 12 and 24 weeks into the 

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study. Infants will be randomized 1:1 to receive T vs placebo for the first 12 weeks of the study with the 
primary outcomes assessed after 12 weeks (Figure 5). The second half of 
the study will cross the treatment groups allowing us to evaluate if timing 
makes a difference in response and if initial benefits are sustained. This 
plan will optimize enrollment, as many parents will only participate if their 
son will get treatment, and allow us to maintain the blinded design.  
 
Setting. The study will be conducted at Children’s Hospital Colorado 
(CHCO) in the outpatient Pediatric Clinical Translational Research Center 
(CTRC). CHCO is home to the eXtraordinarY Kids Clinic, the first com-
prehensive interdisciplinary clinic for sex chromosome aneuploidies.63 
The unique collaborative model and our expertise has led to national re-
ferrals and a high demand for clinical evaluations. We have successfully 
recruited hundreds of boys with XXY to participate in longitudinal and in-
terventional research studies through close collaboration with family sup-
port groups and national/international experts in XXY. Our investigative 
team has expertise in child development, infant body composition, child-
hood disorders of insulin resistance, metabolomics, clinical trial recruit-
ment, robust data acquisition and data analysis, making this the ideal set-
ting. 
 
Recruitment and Retention. Methods of recruitment will include 1) approaching new and existing pa-
tients seen at CHCO clinics including the eXtraordinarY Kids Clinic, Genetics Clinic, and Endocrinology 
Clinics, 2) outreach to the genetic counselors and obstetricians in the Denver metropolitan area who are 
ordering and interpreting prenatal testing results, 3) genetic counselors at CHCO who may receive 
calls/referrals for infants with XXY diagnoses, 4) Email and mailings to pediatric endocrinologists around 
the country (existing listserv) and through posting on the Pediatric Endocrinology Society active research 
website, 5) advertisements through the parent support group AXYS including websites, social media, 
emails, flyers, and mailings, 6) Email and mailings to prenatal genetic counselors throughout the country 
(existing listserv), and 7) study information on websites including Clinical Trials .gov, University of Colora-
do and Children’s Hospital of Colorado. To facilitate dissemination of information about our study, we will 
design study-specific webpage linked off the existing eXtraordinarY Kids website. 
 
The goal for recruitment will be 15 subjects per year in years 1-4. Based on our pilot study this goal is 
easily achievable. Modest travel expenses will be covered for out-of-state participants.  
 
In between study visits, families may receive newsletters from the eXtraordinarY Kids Clinic with updates 
from our ongoing research and periodic phone calls to stay in touch. Families will be invited to our eX-
traordinarY Party in conjunction with the bi-annual AXYS family conference.  
 
Enrollment in multiple clinical trials. Enrollment in this protocol does not exclude participation in other 
clinical studies, with the exception of studies involving an intervention of androgen and/or other treatment 
that may impact body composition. Our team is concurrently enrolling for other studies involving infants 
with XXY, including the eXtraorindarY Babies Study (COMIRB 17-0118). These studies are complemen-
tary and participants can enroll in either or both studies. If participants enroll in both studies, visits will be 
coordinated for convenience of the participant’s family and duplicate measures (ie blood draw, develop-
mental testing, etc) will be combined.   
 
 
Consent. When a child is identified through clinic or other method identified above, a member of the 
study team will contact the parent to inform them of the opportunity to participate in a study. In many cas-
es, the parents will contact the PI, PRA or genetic counselor of the eXtraordinarY Kids Clinic after hearing 
about the study from one of the above sources. Prior to any information being collected, verbal consent to 
ask questions pertaining to the inclusion/exclusion criteria will be obtained. A member of the study team 
will describe the study and will offer to email/mail the consent form to the parents so the parents have 
ample time to review prior to deciding whether to schedule the first visit and enroll in the study.  
Figure 5. Overall Study Design 

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Prior to any research procedures being performed, a study team member will review the consent form in 
person in a private location. Both verbal and written consent will be required from a parent or legal guard-
ian to enroll in the study. All of the subjects in this protocol are infants and therefore too young to as-
sent/consent to research participation themselves. To minimize coercion, parents will not be paid for en-
rolling their child in the study and only reimbursement for reasonable travel will be provided. We will em-
phasize to parents their child’s participation is entirely voluntary and can be withdrawn at any time. As 
part of the consent process, parents will be asked to summarize the study in their own words to ensure 
comprehension, and parents will have the opportunity to ask questions. If the consenting parent does not 
speak English, an interpreter will assist with the consent process and a short-form consent document in 
the parent’s preferred language will be completed. If the consenting parent is blind, illiterate, or with read-
ing limitations, the entire consent form will be read to the subject and witnessed by someone outside of 
the study team. 
 
Randomization. Subjects will be randomized 1:1 using block randomization in groups of 20 to 1) receive 
T treatment in the first 12 weeks followed by placebo, or 2) placebo for the first 12 weeks followed by T. 
Investigational Drug Services at CHCO will generate the automated randomization list and dispense the 
study drug to maintain allocation concealment. All investigators and parents will remain blind to the inter-
vention group.  
 
Study Procedures.  
1. Study Visits. There will be a total of three in-
person study visits:  
Visit 1, week 0 (4-12 weeks of age) 
Visit 2, week 12 ± 10 days (~5 months of age)  
Visit 3, week 24 ± 10 days (~8 months of age) 
 
Procedures during the study visits will take a total 
of approximately 3 hours and will usually be com-
pleted on the same day. If all procedures cannot 
be scheduled for the same day they will be sched-
uled for two days in a row. The study visits will in-
clude all of the following: 
 
• Infant and maternal history collection including 
medications, therapies, infant feeding method, 
and adverse reactions (~30 minutes) 
• Physical examination including length, weight, 
head circumference, arm span, waist circum-
ference, stretched penile length, Tanner staging, and testicular size by Prader orchidometer (~30 
minutes)  
• Quantified body composition assessment by the PeaPod (~30 minutes) 
• Standardized neurodevelopmental assessment (~30 minutes) 
• Morning venous blood draw following a minimum of 180 minutes fasting (~30 minutes)  
• Non-invasive urine and stool sample collection (~10 minutes) 
• Visit 1 only: intramuscular injection and teach  
 
2. Injections/Intervention. Participants in this study will receive a total of 6 injections. 25 mg testosterone 
cypionate (200 mg/mL) or placebo (saline) will be given via intramuscular injection every 4 weeks (study 
weeks 0, 4, 8, 12, 16, and 20). The total volume of each injection will be 0.125 mL. The first shot for all 
participants will be administered at CHCO before the end of study visit #1 and the infant will be observed 
for 15 minutes. Parents will elect to either have the 5 subsequent injections given by a nurse at CHCO or 
to give them at home themselves. If they elect to give them at home, a nurse in the Pediatric CTRC or in 
Endocrinology will teach the parents how to administer the shots. Parents will receive an automated email 
reminder 24 hours before the next injection is due. Parents will keep an administration log to document 
the injection was given.  
Figure 6. Overall study design, timeline and as-
sessments. T=testosterone, P=placebo 

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Although our intent is to give all injections intramuscularly, if the parent inadvertently gives an injection 
subcutaneously it should not alter our validity. A recent study found subcutaneous testosterone injections 
are well tolerated and effective at raising serum testosterone levels,64 although pharmacodynamics may 
differ from intramuscular injections therefore this method will not be encouraged.  
 
3. Interim surveys. A parent will complete a REDCap survey one week after each injection to capture per-
tinent information about the injection and potential side effects. One week out was chosen as most side 
effects attributable to the medication will be present by then and waiting longer may increase recall bias. 
This survey will serve as a drug administration log as well as interim safety monitoring. All survey results 
will be review by study staff and any potential unanticipated events or severe adverse events will be fol-
lowed up with a phone call to the parent.  
 
Safety Considerations. This testosterone dosing and duration is accepted practice for treatment of mi-
cropenis in this age group without reported side effects.65 We had no serious adverse events in our pilot 
study using this protocol. Intramuscular injections include the risk of inflammation and/or pain at the site 
of injection, sterile abscess, infection, and hypersensitivity reactions. Potential low-risk side effects of tes-
tosterone therapy include acne, pubarche, and increased erections. High doses and/or prolonged dura-
tion of androgen therapy in some populations has been associated with bone age advancement, venous 
thromboembolism, water retention, anaphalactoid reactions, suppression of clotting factors II, V, VII, and 
X, polycythemia, and priapism. Testosterone also suppresses spermatogenesis, however infants do not 
undergo spermatogenesis. The effect of early testosterone on later fertility is unknown.    
Likelihood of experiencing any risk: moderate (temporary pain at injection site)  
Likelihood of experiencing serious risk: very low  
Alternatives: There are multiple formulations of testosterone, however all deliver much higher 
doses than proposed in this protocol. Injections are routinely administered as accepted standard 
of care in infants with micropenis, and are the choice of individuals currently advocating for tes-
tosterone in XXY infants. We have experience with testosterone gel in older populations, however 
even if we were able to deliver gel in an appropriate dose, risks of gel include unintentional con-
tact, frequent application, skin reactions, and many of the possible side effects of testosterone 
therapy. Oxandrolone, an alternative androgen, is available as an oral formulation but has not 
been studied in this age group and appropriate dosing would be unknown. Therefore injections 
are the logical choice to study. 
Protection against risk: Standard procedure for reducing infection risk will be utilized including 
sterile single use needles and skin preparation. Parents will be present and/or giving the injec-
tions. Infants will be observed for at least 15 minutes after their first injection to insure there are 
no immediate adverse reactions. Any subjects with a known allergy to any ingredients in the 
study drug or with a first-degree relative with a history of a blood clot will not participate in the 
study. Testosterone can be stored at room temperature, therefore no special precautions are re-
quired to ensure the safety of the medication.  
Monitoring for reactions: One week after every testosterone injection, parents will be sent a brief 
REDCap survey via email to query for possible reactions. Parents will be given an optional log to 
record any side effects they notice at home to better help them complete the survey. At every 
study visit, a study team member will ask about all side effects, hospitalizations, surgeries, new 
diagnoses, and new medications. Parents will also be encouraged to report serious side effects to 
the study team immediately.   
 
The PI will discuss any serious adverse events with Drs. Tartaglia and Zeitler and all these events will be 
reported to regulatory bodies as required. A Data and Safety Monitoring Board (DSMB) will be comprised 
of a minimum of three clinicians/scientists with expertise in clinical trial conduct and methodology, XXY 
syndrome/development, and biostatistics. The DSMB will meet after every 20 subjects are enrolled. 
Members will review data on all adverse events, changes in growth parameters, and testicular function. 
There will not be any predetermined criteria for altering the study protocol or discontinuing the study, 
therefore these options will be at the discretion of the DSMB. 
 
Study Timeline. The timeline for major study activities are in Table 1.        

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Table 1. Study Timeline 
Study Year   
Year 1 
Year 2 
Year 3 
Year 4 
Year 5 
  
Year 
2017 
2018 
2019 
2020 
2021 
2022 
Quarter 
1   2   3   4 
1   2   3   4 
1   2   3   4 
1   2   3   4 
1   2   3   4 
1   2   3   4 
IRB/IND/startup   
  
  
  
  
  
  
  
  
  
  
  
Recruitment & Enrollment   
  
  
  
  
  
  
  
  
  
  
  
Final Study Visits   
  
  
  
  
  
  
  
  
  
  
  
Metabolomics analysis   
  
  
  
  
  
  
  
  
  
  
  
DSMB Meetings   
  
  
  
  
  
  
  
  
  
  
  
Data analysis   
  
  
  
  
  
  
  
  
  
  
  
R03 (metabolomics); R01   
  
  
  
  
  
  
  
  
  
  
  
Manuscripts   
  
  
  
  
  
  
  
  
  
  
  
 
 
 
D. Description, Risks and Justification of Procedures and Data Collection Tools: 
 
1. 
BODY COMPOSITION ASSESSMENT: The PeaPod provides a reliable, non-invasive, quantita-
tive assessment of fat mass and non-fat mass utilizing air-displacement plethysmograph technology 
in infants weighing less than 10 kilograms.66 This instrument has been validated with other methods 
of body composition assessment, such as deuterium dilution and dual-energy X-ray absorptiometry, 
and normative values for healthy, term, average-gestational age males are published from birth 
through 6 months.67 The PeaPod is safe and comfortable for infants and takes less than 10 minutes 
to perform, with the actual test less than 2 minutes. The chamber is heated and has white noise that 
distracts most infants. The infant can move, cry, and urinate during the assessment, therefore it is 
very “baby friendly”. However, the PeaPod may cause temporary distress to the infant as they are 
unclothed and not swaddled. A blinded study investigator will perform this assessment at all study 
visits. Age-specific Z-scores for %BF will be calculated from published data.67 
Likelihood of experiencing any risk: low  
Likelihood of experiencing serious risk: very low  
Alternatives: Body composition can be measured by dual-energy x-ray absorptiometry (DXA), 
however DXA does involve low amounts of radiation exposure in addition to laying on a flat sur-
face similar to the PeaPod. DXA also is best when the subjects lie still, which is challenging for 
young infants. Body fat can also be estimated with skin fold calipers, however this is generally not 
as accurate as the PeaPod and does have the risk of temporary discomfort as well. Therefore, we 
have determined the PeaPod has the least risk to obtain our outcome of interest.  
Protections against risk: When possible, we will perform the PeaPod assessment when the infant 
is awake and content having recently eaten. We will encourage the parents to bring a familiar toy 
that may distract the infant if he becomes distressed. We would encourage comforting/consoling 
after the procedure if temporary distress was experienced. Parents will be present during the 
PeaPod. 
   
2. 
NEURODEVELOPMENTAL ASSESSMENT: The Alberta Infant Motor Scale (AIMS), Peabody 
Developmental Motor Scales -2 (PDMS-2), and the Bayley Scales of Infant Development – 3rd edi-
tion will be performed by a psychometrist trained to administer these standardized neurodevelop-
mental assessments. The AIMS is a performance-based and norm-referenced measure of infant 
gross motor maturation from birth to 18 months. It is intended to evaluate gross motor development 
over repeated assessments and has good reliability and predictive validity for later gross motor de-
velopment.68,69 In a systematic review of neurodevelopmental tests, the AIMS had the best psycho-
metric properties and clinical utility.70 The PDMS-2 is a standardized assessment for infant develop-
ment and includes six subtests to assess gross and fine motor scales from birth to 5 years. It is one 

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of the most common tools for discriminating normal from abnormal infant motor development.71 This 
instrument was chosen as a broad assessment of motor development with normative reference data, 
and strongly correlates with other motor assessments. The Bayley 3 is a comprehensive standard-
ized assessment for infant development containing three domains: Cognitive, Language, and 
Motor.72 The Bayley-3 is widely used to diagnosis developmental delays up to 3.5 years of age. Fi-
nally, a parent will complete the Adaptive Behavior Assessment System, 3rd edition (ABAS 3), a val-
idated measure of adaptive skills across the lifespan, including infancy.73 The ABAS incorporates 
early developmental skills in areas such as feeding, sleep, play, self-regulation, and social develop-
ment. The primary risk of the developmental evaluation is identification of developmental delays that 
may warrant further evaluation and/or intervention. This may cause psychological distress to the 
parent.  
Likelihood of experiencing any risk: low  
Likelihood of experiencing serious risk: very low  
Alternatives: None identified.  
Protections against risk: Parents will be made aware of the high prevalence of at least mild de-
velopmental delays in XXY. Parents will be present during the evaluation. If delays are identified, 
the study team will disclose this to the parent(s) in a private area with specific recommendations 
for next-steps, potentially including referrals and/or resources.   
 
3. VENIPUNCTURE. Venous blood samples will be drawn in the morning a minimum of 180 minutes 
after eating (fasting for an infant.) The collection of blood samples may result in temporary discomfort, 
bruising, bleeding, and on rare occasions, infection. Standard hospital precautions for venipuncture will 
be followed. Anemia is also a risk; the routine guidelines in our Pediatric CTRC are 2.5 ml/kg per blood 
draw. We will draw 8 ml of blood per visit. If the subject is <3.2kg on the first visit we will reduce the 
amount of blood to be drawn.   
Likelihood of experiencing any risk: moderate (temporary discomfort).  
Likelihood of experiencing serious risk: very low  
 
Hormones. Serum will be processed and stored at -80 degrees until batch analysis for quantification 
of LH, FSH, total and free T by liquid chromatography tandem mass spectroscopy (LC/MS), AMH, 
INHB, estradiol and leptin. Serum testosterone, inhibin B, AMH, estradiol and gonadotropins at 
baseline will allow us to assess endogenous testicular function. Hormone concentrations after tes-
tosterone exposure will serve as a short-term safety measure of testicular function. Exploratory aims 
may include looking at whether baseline serum testosterone predicts change in our outcomes after 
testosterone exposure. Leptin is biomarker of adiposity and will be used as a secondary measure to 
support our Peapod findings. Our previous work has shown that leptin concentrations are higher in 
boys with Klinefelter syndrome and this study will be the first to assess this in infants and explore 
how testosterone supplementation alters leptin in this population. Finally, we will measure serum li-
pids (triglycerides and HDL) and insulin as markers of insulin resistance using Trig/HDL ratio and 
1/insulin as estimates. There are very limited validated assessments of cardiometabolic health in in-
fants, therefore these data will largely be exploratory. If blood volume is limited, the order of priority 
for serum measurements will be as follows: Testosterone > inhibin B > leptin > gonadotropins > 
AMH > lipids > insulin > estradiol.  Additional serum may be saved for future analyses.  
Metabolomics. Plasma will be processed and stored until batch analysis using electrospray tandem 
mass spectroscopy per standard protocols in the Reisdorph Lab.74,75 Concentrations of each metab-
olite of interest will be quantified: acylcarnitines (short, medium, and long-chain) and BCAA (leu-
cine/isoleucine and valine). Non-targeted measurements of metabolites using liquid chromatog-
raphy/mass spectroscopy will be analyzed using a well-established platform for comprehensive un-
biased metabolomics resulting in the relative quantification of over 4,000 metabolites, including ami-
no and organic acids, glycerolipids, sphingolipids, and sterols.76,77 Compound identification will be 
done through an in-house database. Principal components analysis will be used to consolidate the 
raw metabolite variables into clusters. We will use these data to determine pathways that are up or 
down regulated with exogenous testosterone. Additional plasma may be saved for future analyses.  
DNA and RNA storage. Samples will be collected, processed, and stored at -80 degrees for future 
analyses.  

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4.  
URINE AND STOOL COLLECTION. Urine will be collected by placing cotton balls in the diaper 
immediately after arriving at the study visits. Cotton balls become saturated when the infant urinates, 
and the urine will be squeezed out into a cryovial for storage to later measure hormones and metabo-
lites. If the infant stools during the study visit, a stool sample will be collected from the diaper and stored 
for future microbiome analysis. Both urine and stool samples will be stored as part of this protocol, with 
future plans to use these stored specimens to measure hormones and metabolomics (urine) and assess 
the microbiome (stool) both to look for differences unique to XXY infants and to assess acute changes 
with testosterone exposure on these measures. No invasive methods for obtaining urine or stool will be 
used in this protocol, therefore no risk to the participant is identified.  
Likelihood of experiencing any risk: very low  
Likelihood of experiencing serious risk: very low  
Alternatives: There are no less-invasive alternatives to assess the microbiome or metabolites.    
 
5. 
HISTORY AND PHYSICAL EXAM. A medical history for the participant including intrauterine 
history, birth history, medical and developmental history, and feeding history will be obtained from the 
parent at the first study visit by a member of the study team. Subsequent study visits will collect inter-
im/updates to these histories as well as any potential study adverse events. A physical exam will be 
completed by the PI (in her absence, one of the other physicians on the protocol). Weight will be rec-
orded to the nearest 0.01 kg. Length measurements will be recorded to the nearest 0.1 cm. Testicular 
volume to the nearest mL will be assessed using a Prader orchiometer. If testes are significantly less 
than 1mL, an estimate of 0.5mL will be used.    
    
E.   Potential Scientific Problems:   
Recruitment and retention of subjects with rarer pediatric conditions can be problematic, however with our 
successful feasibility study and accommodation for participant travel in this budget, we do not anticipate 
enrollment as an obstacle. Utilizing a cross-over-type design reduces our major parental reservation of 
the study, which is the possibility of being randomized to not receive T, and still allows us to evaluate 
short-term effects of T and observe the metabolic pathways that have been altered. As with all clinical 
research, we acknowledge there will be ascertainment bias and strict inclusion criteria that may limit the 
generalizability of our results. Our proposal only includes prenatally diagnosed infants, however our pre-
vious work demonstrated no differences in cardiometabolic or gonadal function between pre- or postnatal-
ly diagnosed XXY boys therefore results from these outcomes should be generalizable.78. Finally, to fur-
ther assess the external validity of our study we will collect and compare basic demographic data from 
families who decline study participation, and will also compare our study cohort to our historic data of in-
fants who are seen in our eXtraordinarY Kids Clinic.    
 
Results and interpretation from the second half of the participant protocol (weeks 12-24) does require cer-
tain assumptions, including that this larger study finds similar differences in body composition between 
treated and untreated infants as our pilot study did. There will not be a control group as part of this study 
for the second half of the study, but importantly these outcomes are all secondary. In addition, we are 
concurrently recruiting for a natural history study in infants with X&Y chromosome variations (eXtraordi-
narY Babies) that includes 100 infants with XXY who will have body composition assessments (PeaPod 
and later DEXA). Some of these will be infants participating in this study, but presumably an important 
subset will not be in the TESTO study and will not have received testosterone clinically. Finally, data from 
the Health Start Study of healthy local infants can serve as a non-XXY control group for normal adipose 
accumulation in infancy.        
    
Our study does not address the large phenotypic variability in XXY. It is possible we will fail to find a dif-
ference in outcomes with T treatment because some boys with XXY have adequate gonadal function or 
have polymorphisms in their androgen receptor gene.79,80 If we fail to find a difference in our primary out 
comes, we will perform post-hoc sub-analyses controlling for age and baseline testosterone concentra-
tions. In addition, DNA will be extracted and saved for future genomic analysis, including sequencing of 
the androgen receptor gene and other polymorphisms that have been associated with phenotypic variabil-
ity in XXY.79-82    
 

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Finally, we acknowledge there are many factors that influence body composition. These potential con-
founders will be minimized due to our randomized design and inclusion/exclusion criteria. We will collect 
data on variables known to contribute to body composition, including race, maternal BMI, maternal weight 
gain during pregnancy, presence of gestational diabetes, birth weight and gestational age, infant feeding 
method, and physical therapy interventions. We anticipate our randomized design will minimize differ-
ences in these potential confounding variables, however if significant differences are found we will adjust 
for them in the analyses. 
 
F.   Data Analysis Plan:   
The primary outcome for specific aim 1 is the change in %BF z-scores between baseline and 12 weeks 
between treatment groups. Z-scores will be calculated using published norms for age,67 and the differ-
ence between groups will be assessed with a two-sided t-test using an alpha of 0.05. Allowing for a 10% 
drop-out rate, a sample size of 27 in each group gives us 80% power to detect a difference down to 0.5 z-
score change between groups, and >99% power to detect the change score we observed in our pilot 
study (1.0 z-score change) assuming a variance of 0.65 per our pilot data. We will next use regression 
models with treatment group, baseline %BF, age of enrollment and feeding method as explanatory varia-
bles to assess absolute %BF at 12 weeks. Secondary analyses will include change in %BF z-scores from 
12 to 24 weeks 1) in the group receiving T first (are the effects of T maintained or lost?) and 2) in the 
group receiving T second (does later T have the same effect?). We will analyze anthropometric meas-
urements and serum biomarkers similarly.  
 
For specific aim 2, the change in standard scores on developmental assessments will be analyzed in a 
similar fashion as in specific aim 1. Based on our pilot data, our sample size will yield 80% power to de-
tect a difference between groups of 23 percentile points on the AIMS composite. Secondary analysis will 
include the changes in scaled scores for the individual Peabody domains and AIMS subtests, the Bayley 
composite scores, and the ABAS composite at 12 and 24 weeks. Gonadal function and safety outcomes 
will be compared between subjects on T and placebo using descriptive statistics. 
 
For specific aim 3 (exploratory), we will compare C14:1 concentrations (an easily identified LCAC) be-
tween treatment groups at 12 weeks using two-sided t-tests, hypothesizing C14:1 will be lower in the T 
treated group (representing improvement in fatty acid oxidation and energy metabolism). This aim is ex-
ploratory; with 10 subjects per group we will have 80% power to detect a difference of 1.3 SD with a two-
sided alpha of 0.05. Then, paired t-tests will be used to compare C14:1 before and after T treatment (5 
paired samples), with similar power. We will analyze secondary outcomes including other LCAC and 
BCAA (leucine, isoleucine, and valine) in a similar fashion. Next, we will use principle component analysis 
on the entire dataset from the comprehensive untargeted analysis to identify patterns and pathways al-
tered with T treatment. The spectra data file from the non-targeted metabolomics panel will be uploaded 
to MetaboAnalyst, and the software will be used for Metabolite Set Enrichment Analysis as well as Path-
way Enrichment Analysis. We will have >80% power to detect at least 2-fold changes between T treated 
and untreated using a significance level of 0.0001 (less than two false positives based on the conserva-
tive Bonferroni correction for multiple comparisons). We will also input the analytes into known KEGG 
pathways of energy metabolism to look for pathway enrichment.  
 
Table 2. Summary of primary and secondary outcome measures for each Specific Aim (SA). 
SA 1: 
Cardiometabolic 
Primary Outcome 
Weeks 
Measurement Tool 
12 24 
Change %BF z-scores from baseline 
X 
 
PeaPod 
Secondary Outcomes 
 
 
 
Change %BF z-scores from baseline 
 
X 
PeaPod 
Change in height, weight, weight-for-
length and waist circumference z-scores 
from baseline 
X 
X 
Physical exam measurements 
Serum leptin, lipids, and insulin  
X 
X 
Immunoassays in CCTSI core laborato-
ry 
S
A
 
2
Primary Outcome 
 
 
 

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Change in motor composite from baseline 
X 
 
AIMS percentile composite score (all 
domains)  
Secondary Outcomes 
 
 
 
Serum LH, FSH, INHB, AMH, E2 and To-
tal T 
X 
X 
Immunoassay and LC/MS (Lahlou lab) 
Change in gross and fine motor scaled 
scores   
X 
X 
Scaled scores on all PDMS-2 domains, 
AIMS 
Cognitive 
and 
Language 
composite 
scores 
X 
X 
Bayley III: cognitive and language do-
mains 
Adaptive functioning 
X 
X 
ABAS III standard scores 
Number of adverse events 
X 
X 
Verbal questionnaire, parent-report 
SA 3: 
Metabo-
lomics 
Primary Outcomes 
 
 
 
Change in C14:1 (LCAC) from baseline 
X 
X 
Targeted metabolomics 
Secondary Outcomes 
 
 
 
BCAA, other LCAC 
X 
X 
Targeted metabolomics 
Pathway analysis 
X 
 
Unbiased metabolomics 
 
 
G.  Summarize Knowledge to be Gained:   
This will be the largest assessment of cardiometabolic health, neurodevelopment and testicular function 
biomarkers in infants with XXY to date and the only longitudinal or interventional study. Management of 
KS in infancy and childhood is particularly important as screening for fetal aneuploidy is becoming stand-
ard of care in all pregnancies through non-invasive prenatal testing. Thousands more infants will be diag-
nosed with KS with very limited evidence-based management guidelines currently available. Providers 
and parents alike are seeking more information and options to prevent complications and co-morbidities 
in this condition. Our results will inform the clinical management of testosterone therapy in infants with 
XXY, with the promise to provide an early intervention during a critical period that may prevent cardi-
ometabolic and neurodevelopment deficits. If testosterone therapy positively effects body composition 
and development in infants and does not have safety issues, there would be support for giving this thera-
py as well as doing long-term follow-up studies. If no short-term benefits are shown following testosterone 
administration, this would provide evidence for not treating these infants. This study also sets up a unique 
opportunity to evaluate changes in the metabolome in response to exogenous testosterone, revealing 
underlying mechanisms involved in the regulation of metabolism and neurodevelopmental pathways dur-
ing a critical period of infancy. 
 
Furthermore, results of this work may be generalizable beyond KS in understanding the understudied re-
lationship of hypogonadism, body composition, and insulin resistance characteristic of obese boys, a 
problem of increasing significance secondary to the pediatric obesity epidemic. Other hypogonadal popu-
lations including Turner syndrome, Prader-Willi syndrome, and Down syndrome, will also benefit from the 
results of this study as they too have a blunted mini-puberty period of infancy. 
 
Future Directions. Based on the metabolomics results from Aim 3, I will seek funding for the targeted 
and untargeted metabolomics analysis on stored specimens from all subjects in this study and potentially 
be able to compare to normal control infants and infants with other high risk factors for cardiometabolic 
dysfunction, such as maternal obesity and diabetes. We will consent for future contact to be able to follow 
this cohort longitudinally and compare their long-term neurodevelopmental outcomes to infants who do 
not receive T but have identical services through the eXtraordinarY Kids Clinic. Depending on our results, 
my future R01 proposals may include long term follow up of this cohort and using additional –omic studies 
to understand the programming mechanisms occurring during the mini-puberty period or other critical 
times in development. Another future direction is long-term longitudinal assessment of boys with XXY 
compared to XY controls, evaluating the many covariates (genetic, epigenetic, environment, T treatment, 
and other interventions) that influence phenotypic variability in XXY. My work will contribute to the devel-
opment and refinement of evidence-based guidelines to ultimately improve cardiometabolic and neu-
rocognitive outcomes in this population. 

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