Bayesian Pragmatic Trial for Pubertal Induction in Turner Syndrome: TRansformation Initiative For Efficient Clinical TriAl Design Advancement in RarE Diseases (TRIFECTA-DARED Framework)
Phase: PHASE2
Conditions: Turner Syndrome
Sponsor: Universiti Kebangsaan Malaysia Medical Centre

 
 
Title: 
Bayesian Pragmatic Trial for Pubertal Induction in Turner Syndrome: TRansformation 
Initiative For Efficient Clinical TriAl Design Advancement in RarE Diseases (TRIFECTA-
DARED framework) 
Version 5 (Date: 4 / 01 / 2026) 
Researchers 
Dr Muhammad Irfan Abdul Jalal (UKM Medical Molecular Biology Institute (UMBI), UKM) – 
Principal Investigator and Trial Methodologist 
Prof Dr Ani Amelia Dato’ Zainuddin (PAG unit, Dept of O&G, Faculty of Medicine, UKM) – 
Main Investigator (Clinical) 
Prof Dr Nur Azurah Abdul Ghani ( PAG unit, Dept of O&G, Faculty of Medicine, UKM) 
Dr Esther Loh Sweet Yi ( PAG unit, Dept of O&G, Faculty of Medicine, UKM) 
                        Industrial Collaborator 
Malaysian Rare Disease Society (0064-07-WKL) 
No.7, 2nd Floor, Bangunan Sultan Salahuddin Abdul Aziz Shah, No. 16 Jalan Utara, 46000 
Petaling Jaya, Selangor 
                           Research Locations:  
1) UKM Medical Molecular Biology Institute (UMBI), UKM 
2) Pediatrics and Adolescent Gynecology Unit, Department of Obstetrics and Gynecology, 
Faculty of Medicine, UKM         
 

1.0 PROTOCOL SYNOPSIS 
Abbreviated Title  
Bayesian Pragmatic Trial for Pubertal Induction in Turner 
Syndrome: Transformation Initiative For Efficient Clinical TriAl 
Design Advancement in RarE Diseases (TRIFECTA-DARED 
framework) 
Trial Phase  
 Phase II 
Clinical Indication 
Pubertal Induction in Adolescents and young women with Turner 
syndrome as evidenced by normal breast, uterine development 
and healthy bone mineral profile  
Trial Type 
Interventional 
Type of Control  
Active control (Oral Estradiol Valerate (Progynova)) without 
placebo 
Route of administration Oral 
(Estradiol 
Valerate 
(Progynova)), 
Transdermal 
gel 
application (17β Estradiol, Oestrogel) 
Trial Masking  
No Blinding /Masking 
Treatment Groups   
17β Estradiol  group (transdermal gel): Transdermal 17β 
Estradiol gel (Oestrogel) administered 0.75 mg twice a week for 
month 1, then 0.75 mg daily for the next 6 months, 1.5 mg daily 
for another 6 months and 3.0 mg daily for another 6 months 
Estradiol Valerate group (oral tablets): Oral tablets of Estradiol 
Valerate administered at 1.0 mg  twice a week for 1 month then 
1.0 mg daily for 6 months then 2.0 mg daily for 6 months followed 
by 4.0 mg daily for another 6 months 
Number 
of 
trial 
subjects  
At least 12 concurrent trial participants per Progynova and 
Oestrogel groups (ntotal=24 participants) based on Bayesian 
sample size calculation 

Estimated Duration of 
Trial 
2 years (24 months), starting from the date the first subject signs 
the informed consent until the date the last subject completes 
the last follow-up  
Duration 
of 
Participation 
 The participants will be enrolled for 19 months (1 ½  years). The 
enrolment date will be Day 1 (baseline) of the trial, 1st follow-up 
at Month 7, 2nd follow-up at Month 13, and final follow-up at 
Month 19. 
Randomization Ratio  
The participants will be randomised to oral Estradiol Valerate 
(Progynova) or transdermal 17β estradiol gel (oestrogel) at a 1:1 
ratio based on stratified blocked randomisation of varying block 
sizes.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

SECTION 1 
INTRODUCTION 
Turner syndrome (TS) is a condition that occurs in females who have either a complete 
or partial absence of the second X chromosome, resulting in only one X chromosome. This 
condition can result in various physical characteristics such as a distinct facial appearance, 
neck webbing, short stature, and lymphedema, as well as medical issues like ovarian 
insufficiency, sensorineural hearing loss, congenital cardiovascular disease, renal anomalies, 
some neurodevelopmental disorders, and an increased risk of thyroid and celiac disease. TS 
affects around 25 to 50 out of every 100,000 females, with a wide range of possible 
symptoms. The karyotype (number and appearance of chromosomes) in TS can range from 
complete 45 X to various forms of mosaicism where the individual has a normal cell line (46,XX 
or 46,XY) and an abnormal second or third cell line.(1) 
The objective of pubertal induction is to replicate the natural pubertal process and 
achieve normal timing and extent of breast and uterine development, as well as adolescent 
growth spurts. This is accomplished by using low initial doses of oestrogen which are gradually 
increased. The patient's clinical response, particularly linear growth, breast development and 
uterine development, is closely monitored throughout the process. The standard treatment 
for patients with TS involves using estrogen replacement therapy, often combined with 
growth hormone. The primary goal of estrogen therapy is to initiate puberty, promote healthy 
secondary sexual development, and provide other significant physiological benefits of 
estrogen, such as uterine maturation and bone mineralization. (1,2) 
Low-dose estrogen can be used over approximately three years, similar to the normal 
pubertal process. Ideally, the dosage should mimic the physiological level of the hormones as 
produced by normal ovaries. The dosage can be increased at six months intervals to mimic 
the normal pubertal tempo until the adult dosing is reached. The choices of estrogen are 17ß-
estradiol, ethinylestradiol, and conjugated equine estrogens. Estrogen can be administered 
via oral or transdermal routes. Both routes provide normal plasma concentrations of estradiol 
and have similar effects in suppressing gonadotropin concentrations. However, transdermal 
estrogen has fewer systemic complications because it avoids first-pass metabolism by the 

liver. It can achieve higher plasma levels of circulating estradiol with a lower treatment dose, 
thus resulting in fewer circulating estrogen metabolites. Both regimes effectively prevent 
bone loss and produce favourable lipid and lipoprotein changes. (2,3,4) 
According to a randomised controlled trial, oral and transdermal 17ß-estradiol in 
gradual doses can produce oestrogen concentrations similar to those seen in a normal 
oestrogen milieu. However, there were notable differences in bio-oestrogen and total 
estrone and estrone sulphate concentrations between the two administration routes, with 
transdermal 17ß-estradiol resulting in much higher levels of these compounds. This suggests 
that transdermal administration may lead to a more physiological oestrogen environment 
than the oral route. Moreover, because transdermal oestrogen avoids first-pass hepatic 
metabolism, it is more effective and better tolerated, with fewer side effects such as breast 
tenderness and erratic vaginal bleeding. In addition, transdermal oestradiol replacement can 
restore mean femoral neck bone mineral density (BMD) to normal levels in young women 
with spontaneous primary ovarian deficiency. (3,4,5) 
Currently, there is limited research on the effectiveness of various hormone 
replacement therapy (HRT) regimes in treating estrogen deficiency in adolescents and young 
adults, specifically those with Turner Syndrome. A pilot study in Indianapolis found that 
transdermal HRT resulted in faster spine bone accrual and increased uterine growth 
compared to orally administered conjugated estrogen. Another study indicated that both oral 
and transdermal 17ß-estradiol resulted in comparable increases in uterine size for those with 
Turner Syndrome, which was similar to normal women. (6,7) 
The research gap is the significant need for more knowledge and understanding of 
Turner syndrome patient care. Specifically, there is a notable absence of studies comparing 
the efficacy of oral estradiol valerate and transdermal 17ß-estradiol in managing this 
population, as well as a need for clear references for the ideal dosage of estrogen 
replacement therapy and pubertal induction. Importantly, this gap is further compounded 
within the context of Turner syndrome patients in Malaysia, as no specific research has been 
conducted in this population. Addressing these research gaps is crucial for improving the care 
for individuals with Turner syndrome, not just in Malaysia, but globally and ensuring that 
appropriate, evidence-based treatment protocols are developed and implemented. 

SECTION 2 
LITERATURE REVIEW 
2.1 Hormone Replacement Therapy (HRT)  
Hormonal replacement therapy (HRT) is vital in managing young females with ovarian 
insufficiency. The rationale for hormonal treatment, specifically estrogen, in this population 
is for the attainment of secondary sexual characteristics, menstrual induction, pubertal 
growth spurt generation, acquisition of adequate bone mineral mass, and promotion of 
sufficient uterine development. Low-dose estrogen can be used over approximately three 
years, similar to the normal pubertal process. Ideally, the dosage should mimic the 
physiological level of the hormones as produced by normal ovaries. The dosage can be 
increased at six months intervals to mimic the normal pubertal tempo until the adult dosing 
is reached (2).  
Continuous estrogen replacement is required to avoid symptoms of estrogen 
deficiency. The choices of estrogen are 17ß-estradiol, ethinylestradiol, and conjugated equine 
estrogens. Estrogen can be administered via oral or transdermal routes (3). Both routes 
provide normal plasma concentrations of estradiol and have similar effects in suppressing 
gonadotrophin concentrations (4). However, transdermal estrogen has fewer systemic 
complications because it avoids first-pass metabolism by the liver (5). It can achieve higher 
plasma levels of circulating estradiol with a lower treatment dose, thus resulting in fewer 
circulating estrogen metabolites (6). Both regimes effectively prevent bone loss and produce 
favourable lipid and lipoprotein changes (7). Conjugated equine estrogen (CEE) is no longer 
recommended due to its higher incidence of thromboembolic and cardiovascular events, as 
documented in post-menopausal females (8). Besides, Wakatsuki et al demonstrated a higher 
plasma level of triglycerides and smaller LDL particles that are less impervious to oxidation in 
CEE recipients than transdermal 17β estradiol recipients (9). This is further supported by 
Zaiem et al who showed in their small-scale meta analysis (n=4) that CEE is associated with a 
higher LDL-C level than transdermal and oral 17β estradiol in Turner Syndrome individuals 
(10). Again, these findings are in agreement with Ho and coworkers who showed a reduction 
in atherosclerotic vascular disease (ASVD) risk in the transdermal 17β estradiol recipients 
compared to the CEE group (11). Furthermore, Shah and colleagues showed increased level 
of estradiol and a higher rate of successful feminization in adolescent girls with hypogonadism 
who received transdermal 17β estradiol than the CEE recipients in their small RCT (n=20, age 

range: 12-18 years)(12). As a result, CEE is now considered inferior to either transdermal or 
oral 17β estradiol in terms of its safety profile. 
 
A randomised controlled trial has shown that both oral and transdermal 17ß-estradiol 
in titrating doses produce close-to-normal estrogen concentrations. However, despite similar 
estrogen concentrations, bio-estrogen and total estrone and estrone sulfate concentrations 
were much higher after oral 17ß-estradiol, proposing that the transdermal route leads to a 
more physiological estrogen milieu than the oral formulation (4). In addition, by avoiding first-
pass hepatic metabolism, transdermal estrogen maintains its effectiveness with a better 
tolerability profile due to fewer side effects like breast tenderness and erratic vaginal bleeding 
(6). In addition, transdermal estradiol replacement restores mean femoral neck bone mineral 
density (BMD) to normal in young women with spontaneous primary ovarian deficiency (13). 
Furthermore, transdermal 17β-estradiol is associated with a significant reduction in the 
thromboembolic event risk compared to oral estradiol preparation in post-menopausal 
females (8). This is further corroborated by a small-scale meta-analysis (n= 4 studies) by Zaiem 
et al. which showed the superiority of transdermal 17β estradiol over oral estradiol in 
reducing fasting blood glucose and total cholesterol and in elevating bone mineral density (9). 
Besides, Taboada et al. showed that transdermal estradiol administration resulted in 
estradiol, estrone, and bioestrogen concentrations that were closer to the physiological levels 
of those three compounds than when estradiol was administered orally in Turner Syndrome 
(14). All this evidence demonstrates the possible superiority of transdermal 17β estradiol 
over oral estradiol. However, a definitive conclusion is thwarted by the small sample size 
employed by each study. Besides, the only available systematic review and meta-analyses 
conducted by Zaiem et al. on this topic also suffered from many methodological weaknesses, 
such as: 
 
1) combining trials that have different methodological qualities (no blinding of the 
participants and outcome assessors and no allocation concealment performed in all trials, 
one trial (Gravholt et al.) is a non-randomized trial, and one trial by Shah et al. has severe 
participant attrition) based on the GRADE assessment system for quality of the assessed trials 
included in the meta-analysis;  

2) the included trial used different regimens for the oral estradiol route (3 out of 4 trials 
combined oral estradiol with conjugated estrogen or norethisterone acetate);  
3) the included trials utilised different doses for oral and transdermal estradiol routes;  
4) the included trials have different follow-up times (in 3 out of 4 trials, the follow-up 
durations were too short (12 months or less));  
5) the included trials’ participants have different age distributions at trial recruitment (one 
trial (Gravholt et al. 1998) recruited mid-age TS adults (30 years and above), whilst, for the 
other  trials, the mean ages of participants at baseline in both oral and transdermal routes 
were less than 18 years old); 
6) No metaregression technique was performed to control the confounding effects of these 
different trial qualities (as evidenced by high I2 statistics indicating high trial heterogeneity, 
which may compromise the reliability of the pooled results) and other potential confounding 
variables (for instance, the concurrent use of growth hormone, which may impact the bone 
mineral density (BMD) of the TS patients treated with either the transdermal or oral form of 
estrogen); 
7) the authors did not report the differential efficacy of these two routes of estradiol 
administration on pubertal induction parameters (uterine size, time to withdrawal bleeding 
occurrence, and breast development as assessed by the Turner staging system). 
8) Overreliance on surrogate markers such as bone mineral density (BMD), fasting glucose 
and lipid profiles as outcome measures. No direct comparisons of hard clinical outcome 
measures between the oral and transdermal forms of oestrogen replacement therapy were 
performed, such as breast development (Tanner stage B4 and above), time to the first 
menstrual initiation (defined as time to first withdrawal bleeding occurrence) and 
normalization of uterine dimensions (the uterine length and fundal AP diameter).   
9) The presence of publication bias was not assessed. As a result, the exclusion of trials with 
negative findings could not be ruled out; 
10) No pooled results for the safety profiles of both transdermal and oral routes of oestrogen 
replacement were provided (e.g., rates of local skin reaction for transdermal oestrogen and 
liver function abnormalities for both forms of oestrogen replacement therapies). 
 
In fact, Zaiem et al. concluded at the end of their report that a well-designed RCT that 
satisfactorily addresses all the aforementioned methodological shortcomings is warranted to 

provide more concrete evidence of the differential efficacy between the two different routes 
of estrogen replacement therapy in Turner syndrome. Consequently, it can be surmised that 
there is still clinical equipoise on the best routes of administration for estrogen replacement 
therapy that may optimize the rates of successful pubertal induction (as defined by overt 
clinical endpoints such as normalization of uterine dimension, shorter time to withdrawal 
bleeding occurrence, and successful breast development (Tanner Stage B4 and above)) in TS 
patients. 
 
Patients with a uterus need progestogen supplementation. After 1 to 2 years of 
estrogen replacement therapy, progestin should be added to prevent endometrial 
hyperplasia. (15) There are several types of progestin, and each has different effects on 
various receptors like progesterone, androgen, glucocorticoid, and mineralocorticoid 
receptors. Therefore, some progestin will have not only progestational effects but also anti-
androgenic, anti-mineralocorticoid, and/or glucocorticoid-agonistic action (2). Once 
progestin is added, many women prefer the ease of use of a pill containing both estrogen and 
progestin. Thus, another accepted option is to use combined oral contraceptive pills (COCP) 
(2). 
 
Few studies compare the efficacy of different HRT regimes in treating adolescents and 
young adults with estrogen deficiency and mainly concentrate on Turner Syndrome (TS) only. 
A pilot study in girls with TS in Indianapolis demonstrated that there is faster bone accrual at 
the spine and increases in uterine growth in those who received transdermal preparation 
compared to conjugated oral estrogen recipients (16). Another study looking at the increment 
of uterine size after oral vs transdermal 17ß-estradiol in TS showed that both groups achieved 
similar increases in uterine size comparable to normal women(17).  
 
Most studies have enlightened more on the effect of HRT regimes in postmenopausal 
women. Hillard et al. found no significant difference between oral and transdermal estrogen 
on bone density and bone turnover (18). Both were equally effective in preventing 
osteoporosis in postmenopausal women (7). Moreover, a randomised control trial found no 
difference in reducing hot flushes for both oral and transdermal estrogen (19).  
 

The aim of this study is to compare the efficacy of different estrogen replacement 
regimes in adolescents and young women with estrogen deficiency in induction of puberty 
(breast and uterine development), attainment of withdrawal bleeding, improvement in bone 
mineral density and also the patients’ satisfaction regarding the treatment received.   
 
2.2 Safety profile of estrogen-only replacement therapy 
The safety profile of estrogen-only hormone replacement therapy was primarily 
investigated among post-menopausal women. The details from various study sources (meta-
analysis, RCTs, observational studies and case series) are summarized as follows.  
2.2.1 General safety information 
In a Bayesian meta-analysis by Derzko and colleagues, the use of 1.5 mg estrogel was 
associated with statistically-significant relative risk of adverse events (occurrence of any of 
these AEs: post-menopausal bleeding, headache, breast pain, infection, nausea, rash and 
vaginitis) than placebo (RR: 1.58 (95% CI 1.19, 2.00)) (20). However, the use of the lower-dose 
0.75 mg oestrogel was not significantly associated with AEs compared to placebo (RR:1.21 
(95% CI 0.87, 1.61). Besides, both 0.75 and 1.5 mg oestrogel were not significantly associated 
with AEs-associated treatment discontinuation compared to placebo (RR0.75 mg oestrogel: 2.21 
(95% CI:0.59, 5.89); RR1.5 mg oestrogel: 2.27 (95% CI:0.61, 6.09)) (20). Hence, oestrogel use does 
not result in a higher probability of treatment discontinuation secondary to AEs occurrence 
than placebo. 
In another meta-analysis by Canonico et al., oral estrogen was significantly associated 
with venous thromboembolism (VTE) (OR: 2.5 (95% CI 1.9, 3.4)), but not transdermal estrogen 
(OR: 1.2 (95% CI 0.9, 1.7)) (21). The findings were further corroborated by a large UK 
population-based  cohort study based on a cohort of 955 582 post-menopausal females aged 
50 to 79 years old from the United Kingdom’s General Practice Database (period of 
recruitment 1st January 1987 until 1st March 2008) (22). The authors found similar findings 
where oral estrogen was significantly associated with VTE (RR: 1.49 (95% CI: 1.37, 1.63)). 
However, the risk of VTE was not elevated in transdermal estrogen users (RR: 1.01 (95% CI 
0.89–1.16)). Hence, transdermal estrogen is deemed safer than oral estrogen in terms of VTE 
occurrence. 
2.2.2 Cardiovascular Disorders 

An elevated probability of cardiovascular accident (CVA) and DVT has been 
documented with estrogen-only therapy. Besides, an increased risk of venous 
thromboembolism (VTE) (RR: 1.63 (95% CI 1.40–1.90)), DVT (RR: 2.09 (95% CI 1.35,3.23)), 
stroke (RR: 1.24 (95% CI 1.03, 1.48), but not MI (RR: 1.17 (95% CI 0.80, 1.71)), has been 
documented in oral estrogen users relative to transdermal oestrogen recipients (23). Should 
any of these occur or be suspected, estrogen with or without progestin therapy should be 
withheld promptly. 
Other concomitant arterial vascular disease risk factors such as hypertension, diabetes 
mellitus, tobacco use, hypercholesterolemia, and obesity and/or (VTE) should be managed 
accordingly. 
Cerebrovascular Accident (CVA) / Stroke 
In the Women’s Health Initiative (WHI) estrogen-only sub-trial, a statistically 
significant increased risk of CVA was documented in females aged 50 to 79 years old receiving 
0.625 mg conjugated estrogen (CE) daily compared to similar-age-group females who were in 
the placebo arm (incidence rate: 45 versus 33 per 10,000 women-years, HR: 1.33 (95% CI 1.05, 
1.68), Absolute excess risk per 10000 women-years: 10.9 (95% CI 1.6, 10.3)) (24). The 
heightened risk was shown in year 1 and remained increased throughout the study. Based on 
a subgroup analysis of women aged 50-59 years old, no elevated risk of CVA was found in 
0.625 mg CE-alone recipients compared to similar age women administered with placebo (18 
versus 21 per 10,000 women-years, HR: 0.89 (95% CI 0.47, 1.69), Absolute excess risk per 
10,000 women-years: 2.0) (24). However, the risk of CVA was increased in 0.625 mg CE 
recipients aged 60-69 years old compared to placebo recipients of similar age (84 versus 54 
per 10,000 women-years; HR: 1.62 (95% CI 1.15, 2.27); Absolute excess risk per 10,000 
women-years: 20.0), but not in women aged 70-79 years old (66 versus 52 per 10,000 women-
years; HR: 1.21 (95% CI 0.84, 1.75); Absolute excess risk per 10,000 women-years: 17.0). 
In the WHI combined estrogen-progestin sub-trial, a statistically significant increased 
risk of CVA was documented in women aged 50 to 79 years old receiving 0.625 mg CE daily 
plus 2.5 mg MPA compared to 50-to-79-year-old women administered with placebo (33 
versus 25 per 10,000 women-years; HR: 1.31 (95% CI 1.03, 1.68); Absolute excess risk per 
10,000 women-years: 7.8 (95% CI 0.8, 14.2)) (24). The heightened risk was demonstrated after 
the first year of observation and remained elevated throughout the study (24). Based on these 
results, estrogen plus progestin therapy should be promptly halted if CVA is suspected or 

occurs in the post-menopausal women. Nevertheless, CVA occurrence in young children or 
adolescents with TS receiving estrogen-based pubertal induction therapy is perhaps low due 
to the young age factor. Nonetheless, further well-designed studies are required to prove this 
assumption. 
Coronary Heart Disease 
In the WHI estrogen-sole sub-trial, no statistically-significant overall effect on 
coronary heart disease (CHD) events (defined as nonfatal MI, silent MI, or CHD death) was 
reported in women receiving estrogen-alone compared to placebo (HR 0.95 (95% CI 0.79, 
1.16)) (25). Subgroup analyses of women 50 to 59 years of age suggest a statistically non-
significant reduction in CHD events (daily 0.625 mg CE-alone recipients compared to the 
placebo group) in women with less than 10 years since menopause (8 versus 16 per 10,000 
women-years; HR 0.48 (95% CI 0.20, 1.17)) (24). 
In the WHI estrogen plus progestin sub-study, there was a statistically non-significant 
increased risk of CHD events reported in women receiving daily CE (0.625 mg) plus MPA (2.5 
mg) compared to women receiving placebo (41 versus 34 per 10,000 women-years; HR 1.23 
(95% CI 0.99, 1.23)) (24). An increase in relative risk was demonstrated in year 1, and a trend 
toward decreasing relative risk was reported in years 2 through. 
In postmenopausal women with documented heart disease (nCE+MPA = 1380, nplacebo = 
1383) average 66.7 years (SD 6.7 years) of age), in the Heart and Estrogen/Progestin 
Replacement Study [HERS] trial, treatment with daily combination of 0.625 mg CE and 2.5 mg 
MPA did not confer statistically-significant benefits against non-fatal MI or CHD-associated 
mortality compared to placebo (HR: 0.99 (95% CI 0.80, 1.22)) (26).  During an average follow-
up of 4.1 years, treatment with CE plus MPA did not also improve other cardiovascular 
outcomes (revascularization rate, coronary artery bypass graft surgery rate, hospital 
admission rates due to angina or congestive heart failure and others) in postmenopausal 
women with established coronary heart disease (p>0.05). There were more CHD events in the 
CE plus MPA-treated group than in the placebo group in year 1, but not during the subsequent 
years. Around 2,321 women from the original HERS trial then consented to take part in an 
open-label extension of HERS, HERS II that has an additional average follow-up period of 2.7 
years, resulting in a mean total follow-up of 6.8 years overall. The CHD event rates were also 
found to be similar among women in the CE plus MPA group and the placebo group in this 
HERS II trial (27). 

Venous Thromboembolism 
In the WHI estrogen-alone sub-study involving 10739 women of  50-79 years of age, 
the VTE (DVT and PE) risk was increased for women receiving daily 0.625 mg CE-alone 
compared to placebo (30 versus 22 per 10,000 women-years; HR 1.32 (95% CI 0.99, 1.75)) 
(28). However, the effect was marginally insignificant. Based on a more in-depth analysis, the 
risk of DVT attained statistical significance (23 versus 15 per 10,000 women-years; HR 1.47 
(95% CI 1.06, 2.06)) whilst the risk of PE remained statistically insignificant (14 versus 10 per 
10,000 women-years; HR 1.37 (95% CI 0.90, 2.07)) (28). The increased VTE risk was the 
greatest during the first 2 years of follow-up. Statistically-significant interactions between 
estrogen use and other VTE risk factors (e.g. age, BMI and others) were absent. 
In another WHI sub-trial investigating the effects of estrogen plus progestin on VTE 
development in 16608 women aged 50-79 years old, a statistically significant 2.06-fold 
greater hazard of VTE was reported in women assigned to 0.625 mg CE plus 2.5 mg MPA daily 
than the placebo recipients (35 against 17 per 10,000 women-years; HR 2.06 (95% CI 1.57, 
2.70)) (29). They also found statistically significant higher risks of VTE with increasing age and 
being overweight (HR 3.80 (95% CI 2.08, 6.94)) or obese (HR 5.61 (95% CI 3.12, 10.11)). 
Besides, the presence of Factor V Leiden further raised venous thrombosis risk (HR 6.69 (95% 
CI 3.09, 14.49)). The increased VTE risk was shown during the first year of the study and 
remained elevated throughout the whole study period (29). These findings were further 
supported by the results from the HERS trials, which showed higher incidence of venous 
thromboembolic events in the combined CE-MA group compared to placebo recipients (HR: 
2.89 (95% CI 1.50, 5.58)) (26). Estrogen plus progestin therapy should be promptly halted if 
VTE occurs or is suspected. If possible, estrogen should be halted for a minimum of 4 to 6 
weeks prior to any surgical types associated with a raised thromboembolic risk, or during the 
extended period of immobility. 
Apart from those, the presence of familial hereditary thrombophilia should also be 
screened for before initiating estrogen replacement therapy. In the Malaysian context, 
Abdullah and co-workers demonstrated 5.6% heterozygous FVL mutation  in 71 healthy 
Malaysian Indians (30). Besides, they did not find any homozygous FVL or  G2021A 
prothrombin mutations in their samples. However, Mohd Yusoff and colleagues did not find 
any factor V Leiden mutation in 46 Kelantanese Malays experiencing recurrent spontaneous 
abortions (31). Nevertheless, Ayadurai, Muniandy and Omar (2009) found FVL prevalence of 

about 8 out of 87 (9.2%) women with active protein C resistant (APC-R) recurrent pregnancy 
losses (32). Following racial stratification, the investigators found FVL prevalence rates of 1% 
(3/287) in Malays, 6.1% (5/82) in Indians and none in Chinese. The majority of the rest of the 
APC-R cases were due to protein S deficiency (6.9%), protein C deficiency (51.7%), combined 
proteins C and S deficiencies (6.9%) and 1.1% with anti-thrombin deficiency (32). These 
findings underscore the importance of familial thrombophilia screening in our TS population 
prior to formal inclusion into the trial and subsequent estrogen replacement therapy 
initiation. 
2.2.3 Malignant Neoplasms 
2.2.3.1 Endometrial Cancer 
In a meta-analysis by Grady et al. unopposed estrogen usage was associated  with 2.3-
fold (95% CI 2.1, 25) increased risk of endometrial carcinoma among females with functioning 
uterus and this is dependent upon treatment duration  (a) <1 year: RR= 1.4 (95% CI 1.0, 1.8); 
b) 1-5 years: RR= 2.8 (95% CI 2.3, 3.5); c) 5-10 years: RR= 5.9 (95% CI 4.7, 7.5); d) >10 years: 
RR= 9.5 (95% CI 7.4, 12.3)) and the dose of estrogen (a) 0.625 mg: RR= 3.4 (95% CI 2.0, 5.6); 
b) 1.25 mg and above: RR= 5.8 (95% CI 4.5, 7.5)) (33). The findings were further supported by 
the large nationwide Million-Women Study which confirmed the association between 
unopposed estrogen use and endometrial cancer in post-menopausal women without prior 
history of cancer or hysterectomy (RR: 1.45 (95% CI 1.02, 2.45), p=0.04) (34). These 
observations were further scrutinised and verified by Sjogren, Morch and Lokkegaard in their 
systematic review where they identified unopposed estrogen use as one of the risk factors of 
endometrial cancer development (35). Hence, clinical surveillance, which includes directed or 
random endometrial sampling when indicated, in all women using estrogen-alone is 
imperative.  
So far, there is no evidence that the use of natural estrogen leads to a different 
endometrial risk profile than synthetic estrogen of equivalent estrogen dose. Adding a 
progestin to estrogen therapy in postmenopausal women has been shown by Grady and 
colleagues and in the Million-Women study to decrease the risk of endometrial carcinoma. 
Breast Cancer 
The most pivotal randomized clinical trial providing information about breast cancer 
in estrogen-alone users is the WHI sub-trial of daily 0.625 mg CE only. In the WHI estrogen-
alone sub-study, after an average follow-up of 7.1 years (SD 1.6 years), daily CE-only regime 

was not significantly associated with a raise in invasive breast cancer risk (HR: 0.80 (95% CI 
0.62, 1.04), p = 0.09) or overall breast cancer (HR: 0.82 (95% CI 0.65, 1.04), p = 0.10)  (36). The 
authors concluded that the use of unopposed estrogen was not associated with raised breast 
cancer cases in post-menopausal women with a prior history of hysterectomy. 
On the hand, information about breast cancer incidence in combined estrogen-
progestin users was provided by the WHI sub-study of daily 0.625 mg CE combined with 2.5 
mg MPA (37). After a mean follow-up of 5.6 years (SD 1.3 years), a significant increased risk 
of invasive breast cancer was found in women who was allotted to the combined daily CE-
MPA group (Unweighted HR: 1.24 (95% CI 1.01, 1.54), p=0.003), as well as overall breast 
cancer cases (Unweighted HR: 1.24 (95% CI 1.02, 1.50), p<0.001). Among women who 
reported 5 or more years of prior use of hormone therapy, the hazard ratio of invasive breast 
cancer was 2.27 (95% CI 1.00, 5.15), and the absolute risk was 54 versus 24 cases per 10,000 
women-years for combined CE-MA recipients and placebo users, respectively. Among women 
who reported no prior use of hormone therapy, the hazard ratio of invasive breast cancer was 
1.09 (95% CI 0.86, 1.39), and the absolute risk was 40 per 10,000 women-years in the 
combined CE-MPA group versus 36 cases per 10,000 women-years in the placebo recipients. 
In the same sub-trial, invasive breast cancers were larger (mean tumor size in cm (CE+MPA vs 
placebo): 1.7 (SD 1.1) vs 1.5 (SD 0.9), p=0.04), were diagnosed at a more advanced stage 
(metastatic / regional tumours:  25.4% vs 16.0%, p=0.04) and with greater proportions of 
being node positive (25.9%  versus 15.8%, p = 0.03) in the combined CE-MPA group than the 
placebo group. No significant differences were found with respect to other prognostic factors 
such as histological characteristics, tumour grades, ER and PR status between the CE+MPA 
and placebo groups. However, the risks are assumed to be much more reduced if estrogen 
replacement therapy is used in young women such as TS patients requiring pubertal induction 
therapy. 
 
Ovarian Cancer 
The WHI estrogen plus progestin sub-study reported a statistically non-significant 
increase in ovarian cancer risk. After an average monitoring duration of 5.6 years, the relative 
risk for ovarian cancer in the combined CE and MPA group with respect to  placebo was 1.58 
(95% CI 0.77, 3.24) (38). The absolute risk for CE plus MPA versus placebo was 4 versus 3 cases 
per 10,000 women-years. Besides,  the investigators also demonstrated that current or recent 

use of estrogen therapy was significantly associated with serous (OR=1.63, 95% CI 1.27, 2.09) 
or endometroid (OR=2.00, 95% CI 1.17, 3.41) ovarian cancer subtypes in their case-control 
study. In addition, the use of estrogen plus progestin and estrogen-only products, in particular 
for 5 or more years, has been linked with an increased in ovarian cancer risk in some 
epidemiologic studies. Nonetheless, the associative effect of the duration of estrogen with 
ovarian cancer risk is inconsistent across all epidemiologic studies since several studies report 
no significant association. 
 
2.2.4 Probable Dementia 
In the WHIMS estrogen-only ancillary study of WHI, a sample of 2,947 
hysterectomized women aged 65 to 79 years old was randomly allotted to either daily 0.625 
mg CE-only or placebo (39). Following a mean follow-up of 5.2 years, 28 women in the 
estrogen-alone group and 19 women in the placebo group were diagnosed with probable 
dementia. The relative risk of probable dementia for CE-alone versus placebo was 1.49 (95% 
CI 0.83, 2.66). The absolute risk of probable dementia for CE-alone versus placebo was 37 
versus 25 cases per 10,000 women-years. 
In the WHIMS estrogen plus progestin auxiliary study of WHI, a sample of 4,532 
postmenopausal females aged 65 to 79 years old was randomly assigned to daily 0.625 mg CE 
in combination with 2.5 mg MPA or placebo (39). After a mean duration of follow-up of 4 
years, 40 women in the CE and MPA group and 21 women in the placebo group had been 
diagnosed with probable dementia. The relative risk of probable dementia for CE plus MPA 
versus placebo was 2.05 (95% CI 1.21,3.48). The absolute risk of probable dementia for CE 
plus MPA versus placebo was 45 versus 22 cases per 10,000 women-years (39). The 
detrimental effects of both estrogen-only  and combined estrogen-progestin replacement 
therapies were further verified by Espeland and colleagues who showed statistically 
significant worse global cognitive function, measured by the Modified Mini-Mental State 
Examination (3MSE) scores, in CE users (mean difference (SE): 0.26 points lower (0.13), 
p=0.04) and combined CE-MPA users (mean difference (SE): 0.21 points lower (0.08), 
p=0.006) compared to placebo in 2808 post-menopausal women aged 65-79 years old (40). 
After combining the data from the two populations in the WHIMS estrogen-only and 
estrogen plus progestin ancillary studies as pre-specified in the original WHIMS protocol, the 
reported overall relative risk for probable dementia was 1.76 (95 %CI, 1.19-2.60). Since both 

auxiliary analyses were carried out in females aged 65 to 79 years old, it is thus uncertain 
whether such findings are relevant to younger postmenopausal females. However, Pourhadi 
and co-workers recently showed an increased dementia rate among estrogen-progestin users 
(HR: 1.24 (95% CI 1.09, 1.35)) compared to age-matched controls that include younger post-
menopausal females (41). The effects persisted irrespective of regimen types (cyclic or 
continuous) or the onset of dementia (late or early). Nevertheless, only those who received 
estrogen-progestin combination at the age 55 years or less exhibited persistent association 
between estrogen progestin use and dementia. 
 
2.2.5 Gallbladder Disease 
In an analysis of the WHI trial data by Cirillo and co-workers, increased hazard of 
gallbladder disease and surgery was observed in estrogen-only (CEE) (HR: 1.67 (95% CI, 1.35-
2.06)) or combined estrogen-progestin (HR: 1.59 (95% CI, 1.28-1.97)) users. Besides, estrogen 
users had high risks of developing cholecystitis and gallstones (CEE: HR: 1.86 (95% CI 
1.48,2.35); combined estrogen-progestin regime: HR: 1.68 (95% CI 1.34-2.11)) (42). These 
findings were further corroborated by HERS trial results which a higher hazard of gallbladder 
disease in women receiving the combined estrogen and progestin regimen (HR 1.38 (95% CI 
1.00, 1.92)) (26). Therefore, caution should be exercised when prescribing estrogen-
containing HRT, especially in those with a high risk of gallbladder diseases. 
 
2.2.6 Hypercalcemia 
Estrogen administration may potentially cause severe hypercalcemia in individuals 
with stage IV breast cancer with bone deposits. This was firstly observed by Beckett in 1969 
who showed 7 out of 36 post-menopausal breast cancer patients with bone metastasis 
experienced hypercalcemia following estrogen administration (43). A more recent review by 
Coelingh Bennink and colleagues summarised the evidence for estrogen use in breast cancer 
and they demonstrated hypercalcemia as one of the common adverse events occurred in 
diethylstilbesterol (DES) recipients (44). Hence, extra considerations must be borne in mind 
when prescribing estrogen to those with heightened risks of hypercalcemia. 
 
2.2.7 Visual Abnormalities 

Retinal vascular occlusion has been reported among estrogen recipients. Several case 
reports have previously documented the possible link between oral estrogen use and retinal 
vein occlusion (RVO) or retinal artery occlusion (RAO) (45-46). However, a multiple cohort 
study (205 304 estrogen-containing female hormone therapy (e-FHT) users vs 755 462 
matched controls) showed no significant elevation of hazards of either RVO (HR: 1.07; 95% 
CI, 0.82-1.39; P = 0.65) or RAO (HR: 1.17; 95% CI:  0.83,1.65; P = 0.36) among estrogen users 
compared to the age-and-race-matched controls (47). Hence, we can fairly assume that 
estrogen use does not cause elevated risk of retinal vascular thrombosis. 
 
2.2.8 Addition of a Progestin in non-hysterectomised females 
Previous research in 1970s demonstrated an elevated risk of endometrial hyperplasia 
and endometrial cancer in estrogen-only HRT recipients (48-49). Therefore, progestin 
addition for 10 or more days within a cycle of estrogen administration, or daily if a continuous 
regimen of combined progestin-estrogen is used, is recommended since it reduces 
endometrial hyperplasia incidence (50-51). Nevertheless, the risk of breast cancer may be 
elevated when estrogen is combined with progestin in an HRT regimen, especially in the 
presence of prolonged duration of use (52). Hence, caution should exercised so that the 
benefits will outweigh the risks when prescribing combined estrogen-progestin therapy in 
non-hysterectomised females. 
 
2.2.9 Elevated Blood Pressure 
Previous research documented that significant blood pressure elevation can be 
ascribed to the differences in the modes of estrogen delivery (53). This is further supported 
by a recently-published population-based cohort study (112 240 Canadian females aged 45 
years and above who used estrogen-only HRT postmenopausal symptoms alleviation) which 
showed a higher risk of hypertension in oral estrogen users than the transdermal (HR: 1.14 
(95% CI 1.08, 1.20)) and vaginal estrogen (HR: 1.19 (95% CI 1.13, 1.25)) users (54). Besides, 
the length of estrogen use and the cumulative estrogen dose were significantly predictive of 
hypertension. Based on these findings, the mode of estrogen delivery is thus associated with 
the development of hypertension. 
 
2.2.10 Hypertriglyceridemia 

In females with antecedent elevated triglyceride level, estrogen use may be associated 
with severe hypertriglyceridemia which may result in pancreatitis (55). Estrogen treatment 
should be interrupted if hypertriglyceridemia-induced pancreatitis occurs. 
 
2.2.11 Liver Derangement and/or Prior Episode of Cholestatic Jaundice 
Estrogen has long been known to be metabolised by the liver (56). Hence, females 
with hepatic impairment may inadequately metabolise estrogen, resulting in an increased 
serum estrogen concentration level. Besides, women with a previous history of cholestatic 
jaundice due to prior use of estrogen or pregnancy, extra care is warranted. In the presence 
of recurrence, estrogen must be promptly withheld. 
 
2.2.12 Hypothyroidism 
Estrogen use may result in inflated thyroid-binding globulin (TBG) levels (57). Those 
without abnormal thyroid function may offset the increased TBG by producing more thyroid 
hormone, thus keeping the free T4 and T3 serum levels within the normal range. Individuals 
who are reliant on thyroid hormone replacement therapy and are also recipients of estrogen 
may need additional doses of their thyroid-replacement medication. Their thyroid function 
should be regularly examined so that the thyroid function parameters can be maintained 
within an optimal range. 
 
2.2.13 Fluid Retention 
Estrogens may lead to significant edema via transcapillary retention of albumin, 
resulting in the extracellular fluid volume expansion (58). Women with illnesses that might be 
impacted by fluid retention, for instance cardiac or renal impairment, require cautious 
monitoring when estrogen-only regimen is administered. 
2.2.14 Hypocalcemia 
Estrogen replacement therapy should be judiciously and cautiously administered in 
patients with hypoparathyroidism since estrogen-induced low serum calcium concentration 
may transpire. It was first demonstrated in a case report in 1975 where a 35-year-old female 
experienced tetanic episodes after being treated with estrogen-containing oral 
contraceptives (59). Therefore, serum parathyroid hormone should be measured prior to 
starting estrogen replacement therapy. 

2.3 Study Rationale 
2.3.1 Trial Rationale from the Clinical and Biological Perspectives 
Adequate ovarian function is vital to promote secondary sexual characteristics and 
uterine development. However, in patients with premature ovarian insufficiency (POI) of 
various causes, including genetic disorders like Turner syndrome, Complete gonadal 
dysgenesis, and Swyer syndrome, the ovaries are impaired thus, they have insufficient 
endogenous estrogen production to promote breast and pubic hair growth, uterine 
development and initiation and normal regulation of menses. Lack of estrogen can lead to 
vasomotor symptoms and psycho-affective, urogynecology, and sexual changes. Estrogen is 
important for bone health and cardiovascular risk reduction in women. There are no proven, 
effectively established regimes of hormone replacement therapy (HRT) for pubertal induction 
and maintenance in these affected young women.  
 
2.3.2 Rationale for Chosen Subject Population  
Turner Syndrome patients are selected as the trial participants since TS is the most 
prevalent condition (1 in 2000) among those with gonadal dysgenesis. The inclusion of 
oestrogen-deficient participants afflicted with other conditions, such as Swyer Syndrome or 
Premature Ovarian Insufficiency, will make the data analyses more complex since we need to 
conduct multiple subgroup analyses to identify the effects within each specific disease entity. 
This procedure will be made more complicated if the number of successfully-recruited 
participants is small due to the high attrition rate. Hence, we plan to recruit only TS patients 
for this trial to ensure sample homogeneity. 
 
We will also include mosaic TS patients since excluding such a subpopulation of TS 
patients will make our sample size smaller. However, we will perform a subgroup analysis for 
this particular TS subpopulation. We believe a single subgroup analysis will not affect the 
study power much since we are confident that we will be able to recruit a sufficient number 
of mosaic TS patients as our trial participants. 
 
2.3.3 Rationale for Hormone Replacement Therapy (HRT) Choices 
Based on our literature review, there is still a knowledge gap with respect to whether 
estradiol valerate (Progynova) or 17ß-estradiol (Oestrogel) is the best HRT regime for pubertal 

induction in Turner Syndrome. Conjugated equine estrogen (CEE) will not be investigated in 
this trial since a number of previous research have conclusively shown the superiority of 
estradiol over CEE (see Section 2.1). Hence, this trial is designed to provide a conclusive 
answer to this unanswered research gap.  
 
2.3.4 Rationale for the Trial Design and Statistical Analysis  
A hybrid real-world (RWD) and concurrent trial design was chosen due to the rarity of 
Turner Syndrome. The double-dummy design could not be opted due to inavailability of 
dummy (placebo) gel or tablets (60). As a result, the ascertainment bias could not be ruled 
out, particularly during trial outcome assessment. 
We will also employ the Bayesian statistical paradigm and Bayesian Hierarchical Model 
(BHM) for statistical analysis to reduce the impacts of possible small sample sizes for this 
study. By using the Bayesian paradigm, the valuable findings from the previously published 
small-size trials can be represented by a prior distribution. This will be combined with our 
trial’s data that are summarised in the form of likelihood. Using Bayes’ theorem, posterior 
updates of the estimates of each trial endpoint can be obtained by multiplying the likelihood 
(i.e. information in our trial’s data) with the prior distribution (valuable information/findings 
about the trial endpoint from prior research). In this case, we “borrow” the strength of 
information from previous research to make our estimates of the trial endpoint parameters 
(i.e. mean difference in uterine length (measured in cm) between the Progynova and 
Oestrogel groups at 2 years) more precise (61). 
 
2.3.5 Rationale for the Choices of Efficacy Endpoints and Safety Parameters 
The Tanner Staging for breast development was chosen since this is a primary 
endpoint of interest that is frequently monitored during clinical management for TS patients. 
Withdrawal bleeding status and time to the first withdrawal bleeding were selected since no 
previous research has investigated the distinctive effects of different HRT regimes on these 
two endpoints. More frequent measurement points (six-monthly follow-ups instead of yearly 
or end-of-trial measurements) are employed to obtain more granular information about the 
impacts of different HRT regimes on each trial endpoint. 
Besides, the safety profiles of both estrogen-only preparations have not been 
extensively investigated in female TS adolescents and young women requiring long-term HRT 

for pubertal induction. As previously elaborated, the safety features of both estrogen-only 
preparations were characterized in post-menopausal females aged 50 to 79 years old in the 
majority of studies.  Hence, we believe this trial will provide further evidence on the safety 
characteristics of both estrogen preparations in female TS adolescents and young women. 
Besides, a composite safety outcome could also be constructed and this can be analysed using 
the win-ratio method (62), resulting in increased statistical power (63). However, the 
individual component of the composite safety outcome may not have similar clinical 
importance to the trial participants and the individual component of the composite safety 
outcome might not be similarly affected by the interventions, two chief pre-requisites for the 
validity of a composite outcome (64-65). Therefore, we choose to analyse each safety 
parameter separately. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

2.4 Conceptual Framework  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 1: The biological framework elucidating the impacts of different HRT regimes (oral 
estradiol valerate versus transdermal 17β estradiol) on pubertal induction in Turner 
Syndrome patients 
 
 
 
 
 
 
 
EEOestrogen-deficient Turner 
Syndrome requiring pubertal 
induction
EEOral Estradiol valerate 
EE Transdermal 17β estradiol 
EE Avoids hepatic first pass 
metabolism 
EE Higher level of bio-
estrogen, more physiological 
oestrogen milieu
EE Better efficacy (higher 
feminization rate, higher BMD) 
and better safety profiles (less 
breast tenderness, erratic 
vaginal bleeding)
EEExperience hepatic first pass 
metabolism 
EELower level of bio-estrogen, 
less physiological oestrogen 
milieu
EEPoorer efficacy (lower 
feminization rate and BMD) 
and poorer safety profiles 
(more breast tenderness, 
erratic vaginal bleeding)

SECTION 3 
TRIAL OBJECTIVES  
 
 
3.1 General Objective 
To compare the efficacy and safety of different estrogen replacement regimes in 
pubertal induction in adolescents and young women with Turner Syndrome  
3.2 Specific Objectives 
1. To compare the proportions of complete breast development in adolescents and young 
women with Turner Syndrome who receive two different HRT regimes (Oral estradiol valerate 
(Progynova) or Transdermal 17β estradiol (Oestrogel)). 
2. To compare the mean differences in uterine dimensions in adolescents and young women 
with Turner Syndrome receiving two different HRT regimes (Oral estradiol valerate 
(Progynova) or Transdermal 17β estradiol (Oestrogel)). 
3. To compare the proportions of adolescents and young women with Turner Syndrome who 
attain uterine withdrawal bleeding after receiving two different HRT regimes(Oral estradiol 
valerate (Progynova) or Transdermal 17β estradiol (Oestrogel)).  
4. To compare the timing of uterine withdrawal bleeding in adolescents and young women with 
Turner Syndrome who received two different HRT regimes (Oral estradiol valerate 
(Progynova) or Transdermal 17β estradiol (Oestrogel)). 
5. To compare the differences in the proportions of adverse events (nausea, vomiting, 
hypersensitivity, hypertension, new-onset headache, lower abdominal pain, vaginal bleeding 
and discharge) or mean adverse event parameters (serum triglyceride)  in adolescents and 
young women with Turner Syndrome who receive two different HRT (Oral estradiol valerate 
(Progynova) or Transdermal 17β estradiol (Oestrogel)).  
3.3 Research Questions 
1) Is there a difference in the proportions of complete breast development in adolescents and 
young women with Turner Syndrome who receive two different HRT regimes (Oestrogel or 
Progynova)? 
2) Is there a difference with regard to the mean uterine dimensions in Turner Syndrome 
adolescents and young women receiving two different HRT regimes (Oestrogel or 
Progynova)? 

3) Is there a difference with respect to the proportions of Turner Syndrome adolescents and 
young women who attain withdrawal bleeding after receiving two different HRT regimes 
(Oestrogel or Progynova)? 
 
4) Is there a difference in the timing of withdrawal bleeding since the first day of HRT in 
adolescents and young women with Turner Syndrome who receive two different HRT regimes 
(Oestrogel or Progynova)? 
5) Are there differences in the proportions of adverse events (nausea, vomiting, 
hypersensitivity, hypertension, new-onset headache, lower abdominal pain, vaginal bleeding 
and discharge) or mean adverse event parameters (serum triglyceride)  in adolescents and 
young women with Turner Syndrome who receive two different HRT regimes (Oestrogel or 
Progynova)? 
3.4 Research Hypotheses  
In the Bayesian statistical framework, H1 and H2 are known as hypotheses 1 and 2, 
respectively, not the null and alternative hypotheses. The Bayesian paradigm is a separate 
school of thought in statistics and, therefore, does not adhere to the null hypothesis 
significant testing (NHST) methodology subscribed by the p-value-based frequentist 
paradigm. 
 
1) H1: There is no significant difference between the two estrogen replacement therapy 
regimes (Oestrogel or Progynova) with respect to the proportions of complete breast 
development in adolescents and young women with Turner Syndrome. 
 
H2: There is a significant difference between the two estrogen replacement therapy regimes 
(Oestrogel or Progynova) with respect to the proportions of complete breast development in 
adolescents and young women with Turner Syndrome. 
 
2. H1: There is no significant difference between the two estrogen replacement therapy 
regimes (Oestrogel or Progynova) in terms of the mean uterine dimensions in adolescents 
and young women with Turner Syndrome. 
 

H2: There is a significant difference between the two estrogen replacement therapy 
(Oestrogel or Progynova) regimes in terms of the mean uterine dimensions in adolescents 
and young women with Turner Syndrome. 
 
3. H1: There is no significant difference in terms of the proportions of adolescents and young 
women with Turner Syndrome who attain withdrawal bleeding in the two estrogen 
replacement therapy regime groups (Oestrogel or Progynova). 
 
H2: There is a significant difference in terms of the proportions of adolescents and young 
women with Turner Syndrome who attain withdrawal bleeding in the two estrogen 
replacement therapy regime groups (Oestrogel or Progynova). 
 
4. H1: There is no significant difference between the two estrogen replacement therapy 
regimes (Oestrogel or Progynova) in terms of the time to withdrawal bleeding in adolescents 
and young women with Turner Syndrome. 
 
H2: There is a significant difference between the two estrogen replacement therapy regimes 
(Oestrogel or Progynova) in terms of the time to withdrawal bleeding in adolescents and 
young women with Turner Syndrome. 
 
5. H1: There are no significant differences in the proportions of adverse events (nausea, 
vomiting, hypersensitivity, hypertension, new-onset headache, lower abdominal pain, vaginal 
bleeding and discharge) or mean adverse event parameters (serum triglyceride) in 
adolescents and young women with Turner Syndrome receiving two different estrogen 
replacement therapy regimes (Oestrogel or Progynova). 
 
H2: There are significant differences in the proportions of adverse events (nausea, vomiting, 
hypersensitivity, hypertension, new-onset headache, lower abdominal pain, vaginal bleeding 
and discharge) or mean adverse event parameters (serum triglyceride) in adolescents and 
young women with Turner Syndrome receiving two different estrogen replacement therapy 
regimes (Oestrogel or Progynova). 
 

SECTION 4 
METHODOLOGY 
 
4.1 Study Design  
 
This is a  Phase II Hybrid Concurrent + Historical/Synthetic Active Controlled Randomized, 
Parallel-arm, Open-Label Exploratory Clinical Trials with a 1:1 control-to-intervention ratio for 
label expansion since both Oestrogel and Progynova have received the US Food and Drug 
Authority (FDA) and Malaysian National Pharmaceutical Regulatory Agency (NPRA)’s approval 
for hormone replacement therapy in post-menopausal women (NPRA 2022; 2023). 
 
4.2 Study Locations 
 
1. Pediatric and Adolescent Gynecology (PAG)  Clinic, Dept of Obstetrics & Gynaecology (O&G) 
at Hospital Canselor Tuanku Muhriz (HCTM). 
 
2. Paediatric and Adolescent Gynaecology (PAG) Clinic, Hospital Tunku Ampuan Besar Tuanku 
Aishah Rohani (HTABTAR),  Hospital Pakar Kanak-Kanak (HPKK), UKM. 
 
4.3 Study Duration. 
 
1st November 2024 – 31st October 2027 (36 months) 
 
4.4 Reference population  
 
The population of Turner Syndrome adolescents and young women aged 11 to 30 
years in Malaysia who have not received pubertal induction therapy. 
 
4.5 Source population 
 
Turner Syndrome adolescents and young women aged 11 to 30 years who have not 
received pubertal induction therapy and are attending clinical follow-up at HCTM and HPKK. 
 
4.6 Sampling frame 
 
The source population and those who fulfil the trial’s eligibility criteria.  
 
4.7 Sampling method 
 
A purposive sampling method will be employed due to the scarcity of the number of 
potential Turner Syndrome patients fulfilling the eligibility criteria. 
  
4.8 Study subjects  
 
Those who fulfil 4.6 and also consent to trial participation. 
 
 

4.9 Eligibility criteria  
 
Inclusion criteria 
1. Females aged 11-30 years old with karyotype-verified (45, X or other similar 
karyotypes) and clinically confirmed Turner’s syndrome prior to the time of pubertal 
induction 
2. Confirmed estrogen deficiency with primary ovarian failure (high level of follicular-
stimulating hormone (FSH > 25 IU/L)) 
3. Patients who have not undergone pubertal development (no breast development and 
an underdeveloped uterus). 
4. HRT-naive TS patients 
5. Breast Tanner Stage of 2 or less. 
6. Patients on Growth Hormone (GH) will be allowed entry into the study. 
7. Consented to trial participation (from individual TS patients (if aged 18 and above) or 
the parents or guardians (for under-18 TS patients) with the individual’s assent) 
 
Exclusion Criteria 
1. Patients with signs of spontaneous puberty 
2. Contraindications to trial products (e.g., hypersensitivity to any components of the 
HRT) based on the most recent version of the British National Formulary (BNF 85)  
3. Previous history of exposure to estrogen treatment. 
4. Concomitant use of other drugs that affect the bone mineral density (BMD) of the 
participants (e.g. Bisphosphonates or prolonged use of systemic corticosteroids). 
Vitamin D supplementation and short corticosteroid usage are allowed. 
5. Acute or chronic hepatic disease 
6. Patients with untreated hypothyroidism 
7. Inflammatory bowel disease (Ulcerative Colitis, Crohn’s disease) and coeliac disease 
8. Cigarette smoking patients 
9. Severely obese patients based on the following criteria: 
a) For TS patients aged 11-17 years old: Based on the WHO chart with BMI > 95th 
percentile  
b) For TS patients aged 18 years until 30 years: BMI of 37.5 or above based on the 
Malaysian Clinical Practice Guideline for the Management of Obesity (66)) 
10. Unknown abnormal genital bleeding 
11. Porphyria 
12. Recent involvement with clinical research studies (previous 6 months) investigating 
new HRT formulations 
 
4.10 Outcome Measures 
 
4.10.1 Primary Outcomes 
There are four primary domains that will be measured at 4 separate time points: 
 
4.10.1.1: Breast Development 
 
There are two main outcomes that will be measured for this domain:  
 

i) proportions of Turner Syndrome patients achieving Tanner Stage B3 and above at months 
1, 7, 13 and 19 after Oestrogel or Progynova initiation;  
 
ii) Median time to achieving Tanner Stage B4 and above (in days) 
 
4.10.1.2 Uterine Development 
 
Two main outcomes will be measured for this domain: 
 
i) Uterine length (in cm) measured at month 1, 7, 13 and 19 by ultrasound (USS) after 
Oestrogel or Progynova initiation. 
 
ii) Anteroposterior fundal diameter (in cm) measured at month 1, 7, 13, and 19 by ultrasound 
(USS) after Oestrogel or Progynova initiation. 
 
4.10.1.3 Withdrawal Bleeding 
 
Two main outcomes will be measured for this domain: 
 
i)Proportions of TS subjects achieving self-reported withdrawal bleeding (defined as spotting 
or bleeding episodes that occur at least for one day during the intake of the estrogen regimen; 
average duration of withdrawal bleeding: 3-5 days) (67-68) at month 1, 7, 13 and 19 after 
Oestrogel or Progynova initiation. 
 
ii) Median time to withdrawal bleeding (in days) after the initiation of Oestrogel or Progynova 
initiation 
 
4.10.1.4 Endocrinological Profile (Safety Parameter) 
 
1. Serum Triglyceride levels at month 1, 7, 13 and 19 after Oestrogel or Progynova initiation 
(safety parameter) 
 
 
 
4.11 Sample size calculation (Frequentist) 
 
1) General Information and Assumptions 
The sample size was calculated using PS Software Version 3.1.6 (Plummer and Dupont, 
2018). All sample size calculations were performed with α = 0.05, 1-β = 0.80 and the ratio of 
controls to cases (m) =1. The dropout rate is considered 5% for all endpoints. We also assume 
that 17β-estradiol and estradiol valerate dosages are equivalent. 

Table 1: Sample size calculation for the HRT in Turner Syndrome Trial Efficacy Endpoint Parameters 
Objectives 
Endpoints 
Parameters 
(reference) 
Assumed Effect Size* 
Sample 
size 
Final sample size, including drop out rate 
(ntotal+) 
1: Breast 
development 
i) 
Proportions 
of subjects 
achieving at 
least Tanner 
Stage B3 at 
1 year 
Estradiol 
Valerate 
group:0.56 
based on rough 
extrapolation of 
graph 4a (page 
331 of 
Schonhoff et al) 
(69) 
 
In the Estrogel group, 
we assume a higher 
proportion: 0.80 
 
Hence: 
P1-P0 = 0.24 
66 
subjects 
per groupa 
70 subjects per group (140 subjects) 
ii) Time to 
Tanner 
Stage ≥B4 
(in days) 
Median  time to 
Tanner Stage 
≥B4  in 17β 
estradiol group: 
733 days 
(Labarta et al) 
(70) 
 
Median time to Tanner 
Stage ≥B4: 365 days 
 
Additional parameters: 
 
1) Accrual time (A) for 
patient recruitment: 
1095 days (3 years) 
 
2) Additional follow-up 
time: 730 days (2 years) 
 
41 
subjects 
per groupb 
44 per group (88 subjects) 
2. Uterine 
development 
i) Uterine 
length (cm) 
 
Uterine length 
(SD): 0.96 cm 
(Kim et al) (71). 
 
A moderate 
standardised effect size 
was chosen based on 
Cohen’s d = 0.50 
64 
subjects 
per groupc 
68 per group (136 subjects) 
 

 
Mean difference (δ) = 
0.96 x 0.50 = 0.480 
 
ii) AP Fundal 
Parameter 
(cm) 
Uterine Fundal 
AP Diameter 
(SD): 0.54 cm 
(Kim et al) (71) 
A moderate 
standardised effect size 
was chosen based on 
Cohen’s d = 0.50 
 
Mean difference (δ)= 
0.54 x 0.50 = 0.270 
64 
subjects 
per groupc 
68 per group (136 subjects) 
3. Proportion of TS 
achieving 
withdrawal 
bleedingd 
 
- 
- 
- 
- 
- 
4. Time to 
withdrawal 
bleedingd 
 
- 
- 
- 
- 
- 
aBased on two-proportion formula, Fisher’s exact test; bBased on the log-rank test procedure (73) cBased on t-test; dCannot be calculated due to 
the absence of estimates from prior studies;*Based on clinically important difference 
 
 
 
 
 

Table 2: Sample size calculation for the HRT in Turner Syndrome Trial Safety Endpoint Parameters 
Safety 
Parameters 
Endpoints 
Parameters 
(reference) 
Assumed Effect Size* 
Sample 
size 
Final sample size, including drop out rate 
(ntotal+) 
1: Lipid profile 
Serum 
Triglyceride 
Level 
(mmol/L)  
SE: 5.5 (Shah et 
al, in mg/dL)  
(72) 
 
SD was obtained 
using this 
formula = sqrt 
(5.52 x 5)  = 
12.298 
 
Conversion of 
SD to SI unit 
(mmol/L) = 
12.298 
mg/dL*0.0113 = 
0.1390 mmol/L 
 
Assumed mean 
difference (δ) = 0.36 
mmol/L (assumed to be 
half the effect size  that 
Shah et al reported) (74)  
 
4 subjects 
per group 
5 subjects per group (10 subjects) 
*Based on clinically important difference

2) Methodology for determining standardised effect size (Cohen’s d) 
Cohen’s d (74) is given by 
d = 
𝑥1 − 𝑥2
√(𝑛1−1)𝑠12+(𝑛2−1)𝑠22
𝑛1+𝑛2−2
2
 
where n1 = sample size for group 1, n2 = sample size for group 2, 𝑠1
2 is the variance for group 
1 and 𝑠2
2 is the variance for group 2. 𝑥1 is the sample mean for group 1 , and 𝑥2 is the 
sample mean for group 2, and both can be considered as unbiased estimators for the 
population means of groups 1 and 2 (μ1 and μ2, respectively). 
However, if we assume n1 and n2 are the same and 1 and 2 are relatively very small 
compared to n1 and n2, the denominator of the formula for Cohen’s d (pooled SD) can be 
reduced to 
𝜎𝑝𝑜𝑜𝑙𝑒𝑑≈√𝑠1
2 + 𝑠2
2
2
2
 
Since we only have an estimate of standard deviation from one group only from previous 
literature, we have to assume 𝑠1
2 = 𝑠2
2. Hence, 
𝜎𝑝𝑜𝑜𝑙𝑒𝑑≈√2𝑠12
2
2
 = √𝑠1
2
2
  =𝑠1  
Hence, the SD used for calculating sample size is from one group only. Based on Cohen’s d, 
the magnitude of effect size can be classified as follows: 
d = 0.20 (small effect size) 
d = 0.50 (moderate effect size) 
d = 0.80 (large effect size) 
In our study, we shall assume a moderate effect size (d=0.50). However, according to 
Thompson (75), the effect size should not be rigidly interpreted and adhered to. In our case, 
we use Cohen’s d just to facilitate the calculation of the mean difference (δ) between the 
groups after consulting the clinicians involved in this study to ensure that the effect size also 
represents a clinically significant difference between the two intervention groups. 
3) Sample Size Calculation Based on the Minimum Detectable Alternative (δ) 
The sample size can also be estimated based on the minimum detectable alternative 
(δ). In this case, we assumed, at the most, we could only obtain n=30 subjects per group (ntotal 
= 60, m=1). The study power and type I error rate are fixed at 0.80 and 0.05, respectively. 
Hence, the minimum detectable alternative (δ) for each parameter for each trial objective is 
as follows: 

Table 3: The minimum detectable alternative (δ) for each efficacy endpoint parameter of the HRT in the Turner Syndrome Trial when n=30 
per group (ntotal = 60, without 5% drop-out rate included) 
Objectives 
Endpoints 
Parameters (reference) 
Minimum detectable alternative (δ) 
1: Breast 
development 
i) 
Proportions 
of 
subjects achieving at 
least Tanner Stage B3 
at 1 year 
Estradiol Valerate group:0.56 
based on rough 
extrapolation of graph 4a 
(page 331 of Schonhoff et al. 
2011) 
 
The proportion of subjects achieving Tanner stage ≥ B3 at 1 year 
should be ≤0.185 or ≥ 0.906.  
 
Minimum lower limit difference: 0.56-0.185 = 0.375 
Minimum upper limit difference: 0.906-0.56 = 0.346 
 
ii) Time to Tanner 
Stage ≥B4 (in days) 
Median  time to Tanner 
Stage ≥B4  in 17β estradiol 
(oral) group: 733 days  
 
Additional parameters: 
 
1) Accrual time (A) for 
patient recruitment: 1095 
days (3 years) 
 
2) Additional follow-up time: 
730 days (2 years) 
 
All estimates are taken from 
(Labarta et al. 2012) 
The median time to Tanner Stage ≥B4 in the estrogel group should be 
325 days or less.  
 
It means that the estrogel should demonstrate a median time of 
around 10.8 months to show there is a significant difference between 
the intervention groups in terms of this parameter if the median time 
to Tanner Stage ≥B4 for the 17β estradiol (oral) is around 24.4 
months. 
2. Uterine 
development 
i) Uterine length (cm) 
 
Uterine length (SD): 0.96 cm 
(Kim et al. 2012). 
The MDA (δ) should be ≥+0.706 or ≤-0.706 for the difference in the 
mean uterine length between the 2 intervention groups to be 
significant 
 

ii) AP Fundal 
Parameter (cm) 
Uterine Fundal AP Diameter 
(SD): 0.54 cm (Kim et al. 
2012) 
The MDA (δ) should be ≥+0.397 or ≤-0.397 for the difference in the 
mean AP Fundal Parameter between the 2 intervention groups to be 
significant  
3. Proportion 
of TS 
achieving 
withdrawal 
bleedingd 
 
- 
- 
- 
4. Time to 
withdrawal 
bleedingd 
 
- 
- 
- 
aBased on two-proportion formula, Fisher’s exact test; bBased on the log-rank test procedure (Schoenfeld & Richter (1982)) (73);cBased on t-test; 
dCannot be calculated due to the absence of estimates from prior studies;*Based on clinically important difference 
 
 
 
 
 
 
 
 

Table 4: The minimum detectable alternative (δ) for each safety endpoint parameter of the HRT in the Turner SyndromeTrial when n=30 per 
group (ntotal = 60, without 5% drop-out rate included) 
Objectives 
Endpoints 
Parameters (reference) 
Minimum detectable alternative (δ) 
1: Lipid profile 
Serum 
Triglyceride 
Level (mmol/L)  
SE: 5.5 (Shah et al, in mg/dL)  
(74) 
SD was obtained using this 
formula = sqrt (5.52 x 5)  = 
12.298 
Conversion of SD to SI unit 
(mmol/L) 
= 
12.298 
mg/dL*0.0113 
= 
0.1390 
mmol/L 
 
The MDA (δ) should be ≥+0.102 or ≤-0.102 for the difference in the 
mean serum triglyceride level between the 2 intervention groups to be 
significant  

Based on the MDA (δ), the greatest threat to achieving statistical significance is for both 
parameters for objective 1. This verdict can be explained as such: the difference in the 
proportions of subjects achieving Tanner stage B3 and above at 1 year should be very large 
to achieve statistical significance. For instance, if transdermal 17β estradiol (Oestrogel) is 
found to be inferior to oral estradiol valerate (Progynova), the proportion of trial participants 
achieving Tanner stage B3 and above at 1 year in the transdermal 17β estradiol (Oestrogel) 
group should be 0.185 or less (a proportional difference of 0.375). However, if transdermal 
17β estradiol (Oestrogel) is superior to oral estradiol valerate, the proportion of trial 
participants achieving Tanner stage B3 and above at 1 year in the Oestrogel group should be 
at least 0.906 or more (a proportional difference of 0.346).  
4) Final Verdict based on Frequentist sample size calculation 
Without considering the attrition rate, the largest sample size required for this trial is 
66 subjects per group (ntotal, without attrtition= 132). After considering a 5% dropout rate, ntotal is 
140 subjects. Hence, the final sample size for this trial is 70 Turner Syndrome subjects per 
group. 
However, based on the MDA (δ), if we could only achieve a sample size of n=30 per 
group (ntotal=60), then differences between the groups need to be larger. The greatest 
possibility of not achieving statistical significance is thus for objective 1 (parameter) which is 
detecting the difference in the proportions of TS participants achieving Tanner Stage B3 and 
above. 
5) Sample size calculation based on Bayesian paradigm (presentation slides) 
The sample size calculation under the Bayesian paradigm was performed based on the 
methods outlined in the previous work by Kuzmann and colleagues (76) and Lindley (77). 
Besides, the concept of study power was substituted with assurance (78) and the sample size 
was based on maximizing the utility function (79). However, based on Stallard and colleagues, 
the optimal sample size for phase II clinical trials should be at least nphase II trial = nphase III trial  * 
0.03 (multiplier) (80). However, it was deemed that the multiplier was too small, leading to 
an underpowered trial. Hence, the multiplier value was increased to 0.20.  
The sample size calculation was based on the uterine development parameter and the 
procedure is outlined in the slides below: 

 
 

 
List of abbreviations:  
a) SE: Standard error 
b) SD: Standard deviation 
 
Since our trial is based on the Bayesian design and analysis, the final sample size required for 
the concurrent Progynova and Oestrogel trial participants is 12 subjects per group (n=24). The 
sample size will be augmented by the historical Progynova and Oestrogel users identified 
from our TS patient registry through the HCTM Electronic Medical Records. Thus far, the 
number of identified TS patients already on Progynova-only or Oestrogel-only regimes for 
pubertal induction is 28 and 11, respectively 
 
 
4.12 Recruitment process  
The overall process of participant recruitment at each trial site is represented by Figure 1 
below: 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 2: The overall process for participant recruitment at each trial site 
 
 
4.13 Participant Screening and Randomization  
 
After the patients consented to trial participation, they will be screened for trial eligibility. 
Each patient will be assigned a unique screening number before randomisation and 
intervention assignment. Each screening number must be used once for each patient, even in 
patients screened multiple times.  
  
Eligible study participants will be randomised in a 1:1 fashion to one of the two 
intervention groups (oral estradiol valerate (Progynova) or transdermal 17β estradiol 
(Oestrogel)) using a  block randomisation scheme of varying block  
Karyotype and clinically-proven new TS patients  aged 11-30 
years old diagnosed at HCTM PAG Unit and HPKK 
FSH level quantification 
FSH equals to or less 
than 25 IU/L 
FSH exceeds 25 IU/L, pubertal 
induction warranted 
FSH re-quantification after 6  
months 
FSH equals to or 
less than 25 IU/L 
FSH exceeds 25 
IU/L 
Excluded from further 
consideration for trial 
entry 
Provision of PIS to the TS 
patients/parents/.legal guardians. Invitation to 
trial participation 
Consent from the TS patients/parents/legal guardians. Further screening 
against the remaining eligibility criteria. Participant refused: excluded. 
Consented participants who fulfil all eligibility 
criteria: Blocked Randomisation 
Oral estradiol valerate 
(Progynova) 
Transdermal 17β 
estradiol (Oestrogel) 

 
A suitable vendor will be identified to carry out the randomisation procedure to protect 
against any accidental breach of randomisation codes and maintain allocation concealment, 
which will minimise selection bias. So far, we have identified two possible vendors (Altus 
Solutions and ClinData Solutions Sdn Bhd)  who will assist in the trial participant 
randomization (a) Miss Lim Jie Ying – jylim@altussolutions.com.my (03-40418655); b) Jau 
Shya – jaushya@clinidata.com.my (contact no: 0122865193)) 
 
 
4.14 Allocation Concealment and Masking  
 
The vendor will conduct central randomisation and the randomisation sequence will 
be uploaded to the electronic data capture software, REDCAP. After confirming the study 
eligibility of the study participants, the recruiter will fill in the study participants’ clinical 
details in the REDCAP and the vendor will perform the randomisation and the treatment 
allocation will only be notified REDCAP to the recruiter to maintain allocation concealment. 
The recruiter will then inform the pharmacist dispensing the Oral Estradiol Valerate and 
Transdermal 17β Estradiol. The pharmacist will subsequently notify the interventionists 
(clinicians in charge of the trial participants, exclusive of the trial investigators), who will first 
demonstrate to the trial participants the appropriate ways to use the interventions. There will 
be two pharmacists who will be involved in the storing and labelling of the interventional 
agents (including placebo) at the central trial locations (Hospital Canselor Tuanku Muhriz 
(HCTM) and Hospital Pakar Kanak-Kanak (HPKK UKM; also known as Hospital Tuanku Ampuan 
Rohani, UKM).  
 
The study participants investigators, data handlers, healthcare givers (including 
physicians/specialists in charge, nurses and other support staff), outcome assessors and the 
trial statistician will not be masked to the identities of the assigned intervention. 
 
 
4.15. Clinical Protocol  
 
4.15.1 Trial Procedures 
 
For all eligible Turner Syndrome patients that fulfil the trial eligibility criteria, they 
will be randomised into two groups: 
 
Group A: Oral Estradiol Valerate (Progynova) group  
Group B: Transdermal 17β Estradiol gel (Oestrogel) group  
 
They will receive the treatment according to the protocol below (Table 5). 
 
 
 
Table 5: The estrogen regimes based on the allotted HRT types 
 
Duration 
            Oral          
        Transdermal  

         Estradiol  
          valerate  
           Group 
        17β Estradiol     
            Group 
Twice a week for a 
month 
1.0 mg 
0.75mg 
(1/2 ruler) 
Once daily for 6 
months 
1.0 mg 
0.75mg 
(1/2 ruler) 
Once daily for 6 
months 
2.0 mg 
1.5mg         
(1 ruler) 
Once daily for 6 
months until total 2 
years 
4.0 mg 
3.0mg         
(2 rulers) 
 
The detailed clinical procedural steps for this trial are given in Figure 3 below: 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 3: The overall process for the conduct of the trial 
i) Consent received from concurrent trial participants. Identity 
of treatment allocation obtained from the REDCAP system 
ii) Unique subject ID number allocated to each concurrent trial 
participant 
Perform baseline  measurements of clinical, laboratory and 
imaging parameters  
(refer table 6 below) 
Initiate either Progynova or Oestrogel for each trial 
participant in the concurrent group  
(based on the allotted intervention) 
1st year of intervention 
       i) Trial endpoint measurements (months 1 and 7 after intervention initiation) 
          ii) Compliance Monitoring (every 3 months) 
         iii) Finalize all data entry for year 1 of the intervention and lock the database 
               for 1st-year measurements 
Tailor the Progynova / Oestrogel dose according to the 
protocol for 2nd year of intervention 
2nd year of intervention 
i) Trial endpoint measurements (months 13 and 19) 
ii) Compliance Monitoring (every 3 months) 
   iii) Finalize all data entry for this phase  of intervention   
        and database  locked (3 months after  data entry for   
        the last  patient’s final visit)) 
Final data verification and statistical analyses 

Table 6. Planned Measurements of Clinical, Laboratory, Imaging and Quality of Life Parameters for 
each participant for the entire trial duration (the allowable observation window is ± 7 days from the 
actual measurement time points). 
 
Variables 
Timepoints 
 
 
Pre-intervention 
(Baseline) 
Month 1 
Month 7 and 
Month 13  
Month 19 
Karyotype 
 
X 
 
 
 
Sociodemographic 
Profile 
 
X 
 
 
 
a) Clinical 
Parameters 
 
 
 
 
 
Height (cm) 
 
X 
X 
X 
X 
Weight (kg) 
 
X 
X 
X 
X 
BMI (kg/m2) 
 
X 
X 
X 
X 
Height increment  
rate (cm/year) 
 
X 
 
 
X 
Vital signs (SBP, 
DBP, HR, RR, 
Temperature) 
 
X 
X 
X 
X 
Tanner Breast 
Stage  
 
X 
 
X 
X 
Pubic hair 
development 
X 
 
 
 
Withdrawal 
Bleeding 
 
 
 
X 
X 
Compliance 
Monitoring 
 
 
X 
X 
X 
Safety Monitoring 
 
 
X 
X 
X 
Concomitant 
Medication 
 
 
X 
X 
X 
b) Laboratory 
Parameters 
 
 
 
 
Serum FSH, LH, 
prolactin 
 
X 
 
 
 
Serum Estrone 
X 
 
X 
X 

 
Serum Estrone / 
Estradiol ratio 
 
X 
 
X 
X 
Serum SHBG 
 
X 
 
X 
X 
Free T4 and TSH 
 
X 
 
 
X 
Liver Function 
Test (LFT) 
 
X 
 
 
X 
Fasting Blood 
Glucose  
 
X 
 
X 
X 
HbA1c 
 
X 
 
 
X 
Fasting Lipid 
Profile (Total 
Cholestrol, LDL, 
HDL and 
Triglycerides) 
 
X 
 
X 
X 
Imaging 
 
 
 
 
 
Pelvic Ultrasound 
(Uterine Length, 
AP Diameter, 
Endometrial  
lining presence 
and endometrial 
thickness (ET) 
X 
 
X 
X 
 
For trial endpoints that may result in poor inter-rater agreement (e.g., Tanner Staging for 
breast development, USS Uterine images that are difficult to classify), training sessions will be 
provided to the raters (O&G registrars with at least 5 years of experience working in the O&G 
setting) to standardize rating procedures. This will be followed by a trial endpoint realignment 
meeting to discuss difficult cases and assessments of inter-rater agreement for all trial 
endpoints with poor inter-rater agreement. Any rater that exhibits poor inter-rater 
agreement will be excluded as a clinical assessor for the trial endpoints. Statistical procedures 
that will be employed to evaluate inter-rater agreement are elaborated in detail in Section 
4.20.4. To assess the patient’s withdrawal bleeding status and time to the first withdrawal 
bleeding, a monitoring log (diary) will be provided to each trial participant (Appendix IV). The 
severity of withdrawal bleeding will be graded by the number of pads/tampons used per HRT 
cycle, as advocated by previous research (81-83). The operational definition of spotting by 
Zhang et al will be utilised for this research (84). 
 
All laboratory tests will be conducted in laboratories with Good Laboratory Practice (GLP) 
certification or at least are accredited with the MS ISO 15189:2014 and MS ISO/IEC 17025 
(testing and calibration laboratories) credentials by Standards Malaysia. 

 
4.15.2: Study Instruments (captured REDCAP) 
 
The REDCAP system (eCRF) will capture the following information for each trial participant 
 
1. Socio-demographic profile 
 
2. Baseline blood investigations: FSH, LH, Estradiol, and DNA Karyotyping. 
 
3. Ultrasound measurement of uterine size and endometrial thickening: before and 
after treatment commenced at 6-monthly intervals until attainment of first 
withdrawal bleed (according to regime protocol). Pelvic ultrasound is used to 
measure the uterus (length, width, depth, volume) and endometrial thickening (in 
the sagittal plane). Two readings will be obtained for each, and the average will be 
taken. 
 
4. Tanner staging: 
 
a) breast development: before and after treatment commenced, at intervals of 6 
months.  
 
b) pubic hair development: before treatment only (for baseline information only). 
 
Breast and pubic hair (for baseline information only) development will be assessed 
using Tanner Staging (Appendix 1), also known as Sexual Maturity Rating (SMR), 
developed by Marshall and Tanner. This is an objective classification system that 
is used to document and track the development and sequence of secondary sex 
characteristics of children during puberty. The descriptions for each Tanner stage 
for breast and pubic hair development are given below: 
 
Breast development: 
Stage 1: No glandular breast tissue palpable  
Stage 2: Breast bud palpable under the areola (1st pubertal sign in females) 
Stage 3: Breast tissue palpable outside areola; no areolar development 
Stage 4: Areola elevated above the contour of the breast, forming a “double 
scoop” appearance 
Stage 5: Areolar mound recedes into a single breast contour with areolar 
hyperpigmentation, papillae development, and nipple protrusion 
Pubic Hair development: 
Stage 1: No hair 

Stage 2: Downy hair 
Stage 3: Scant terminal hair 
Stage 4: Terminal hair that fills the entire triangle overlying the pubic region 
Stage 5: Terminal hair that extends beyond the inguinal crease onto the thigh 
5. The date of first withdrawal menses and the time duration from treatment 
commencement, and the details of this withdrawal bleed: duration, menstrual flow, and 
number of pads used per day. 
 
 
4.15.3 Labelling, Packaging, Storage, Accountability and Return of Clinical Supplies and 
Interventional Agents 
 
4.15.3.1 Investigational Medicinal Products (IMPs) 
 
It is the responsibility of the investigators at each trial site to ensure satisfactory and adequate 
handling, supply, storage, distribution and usage of IMPs according to the trial protocol and 
all relevant rules and regulations. The clinical supplies provided by the sponsor (UKM) are 
detailed below (Table 7). 
 
Table 7. The descriptions of clinical supplies. 
 
Product Name 
Dosage Form 
Manufacturer 
Oral 
estradiol 
valerate 
(Progynova®) 
Tablet, 1 and 2mg  
Bayer 
(Leverkusen, 
Germany) 
Transdermal 17β estradiol 
(Oestrogel®) 
Non-greasy, 
non-staining 
clear 
and 
colourless 
transdermal 
gel. 
It 
is 
provided in an 80 g bottle 
with a metered dose pump. 
Each pump pack delivers 64  
metered doses. 
Each 1.25 grams of estradiol 
gel contains 0.75 mg (as 
hemihydrate) 
Besins Healthcare (Monaco, 
France) 
 
 

 
4.15.3.2 Packaging and Labeling of the Clinical Supplies 
 
The conditions for IMP storage (temperature, humidity) will be provided on the trial 
package. Packaging will be performed by Besins Healthcare for both the transdermal 17β-
estradiol (Oestrogel) gel. Oral estradiol valerate (Progynova) will be purchased from Bayer 
(Leverkusen, Germany). All packaging and labelling processes will adhere to the Good 
Manufacturing Practices (GMP) and the Good Clinical Practice (GCP) guidelines, as well as all 
Malaysian legal requirements. All IMP packages will have a tear-off part that needs to be 
affixed to the study documents upon IMP dispensation by the investigators/pharmacists to 
the study participants at each trial site. 
 
4.15.3.3 Storage and Handling Requirements 
 
All clinical supplies (IMPs) shall be stored at a secured and locked facility at each trial 
centre and under the conditions stated on the labels (storage temperature: 1) Oral Estradiol 
valerate (Progynova): below 30oC; 2) transdermal 17β estradiol (Oestrogel): between 20o to 
25oC, temperature excursion (fluctuation) is permitted to 15o to 30o,  with access restricted 
to the principal investigators/local pharmacists/authorized personnel in charge of storing and 
distributing 
the 
clinical 
supplies 
at 
the 
specific 
trial 
site. 
The 
principal 
investigators/pharmacists should maintain an updated temperature log to provide evidence 
of satisfactory storage of clinical supplies (IMPs) for the entire duration of the trial. 
 
The receipt and dispensing of clinical supplies (IMPs) shall be compulsorily 
documented by the principal investigator/local pharmacists/authorized personnel at each 
trial site. The principal investigator/local pharmacists/authorized personnel should sign the 
receipt forms for clinical supplies (IMPs). The details and records of the delivery of clinical 
supplies (IMPs) to the trial site, the cataloguing, listing and counting of IMPs, inventory 
maintenance at the trial site and the return of unused IMPs to the Sponsor shall also be 
documented by the principal investigator/pharmacist/authorized personnel at each trial site. 
The details shall include batch numbers, dates, quantities and the unique numbers allotted 
to the IMPs and study participants. Clinical supplies must be used by the investigators for trial 
purposes and administered only to enrolled trial participants. 
 
4.15.3.4 Discard, Destruction, Returns and Reconciliation  
 
It is the investigator’s responsibility to maintain and safeguard the accuracy of the 
records for clinical supplies provided by the Sponsor at each trial site. The investigators at 
each trial site should also document the number of IMPs allocated to each study participant 
and the number of IMPs returned by each subject at the end of the trial. 
 
The Sponsor shall provide the investigators with documentation forms (including 
format) that should be filled in to assess the drug accountability or return (or discard and 
destruction). Any unused IMPs must be returned to the Sponsor for accountability and 
returned upon trial conclusion. 
 
4.15.4 Patient Compliance 

 
Study participants will be required to bring used and unused oral estradiol valerate 
(Progynova) blister packs and transdermal 17β-estradiol (Oestrogel) tubes during each 
follow-up visit. Participant compliance will be evaluated by counting the remaining tablets or 
measuring the length of the tubes containing transdermal 17β-estradiol (Oestrogel). The 
compliance rate at each visit should not be below 80% to be considered as adequate 
compliance. Apart from that, they will also be required to supply the photographs (in a 
suitable format) of their blister packs and gel tubes as objective evidence of their compliance. 
Reminders will also be sent periodically about the schedule for their next follow-up visits to 
maximise attendance at their monitoring visits. 
 
 
4.15.5 Protocol Deviation  
 
Protocol deviations are defined as 
 
1. Not meeting/fulfilling any of the eligibility criteria (inclusion and exclusion criteria) 
during subsequent reviews after the patients have been enrolled into the trial. 
2. Consumption or use of concomitant medications that are forbidden during the clinical 
trial 
3. No show during the visit dates or visits are performed outside the acceptable visit 
windows (± 1 week) 
4. Non-adherence to the study protocol 
5. Medication Storage / Administrative Issues 
 
For condition no 3, visit windows should be calculated by assuming 1 month is equivalent to 
30 days and 1 week is equivalent to 7 days.  
 
4.15.6 Participant Withdrawal and Loss to Follow-Up 
 
Trial participants who withdraw from the study will not be replaced with new 
participants. A participation withdrawal is operationally defined as a scenario in which the 
participant does not return for a trial final visit as scheduled in the trial protocol. The statistical 
treatment of participants who withdraw from the study is outlined in Section 4.19.1: 
Statistical Analysis Plan for Efficacy Endpoints. 
 
A participant may withdraw from the trial or rescind their consent to trial participation 
at any time, and for any reason, without prejudicing the clinical management and treatment 
they may receive in the future. Participants will be asked to complete all pending research 
procedures prior to trial withdrawal. 
 
If the trial participant wishes to withdraw due to intervention-associated Adverse 
Events (AEs), the participant will be requested to undergo follow-up until the resolution of 
AEs before the investigator completes the end-of-study electronic Clinical Record Form (eCRF) 
on REDCap. All information associated with participant withdrawal will be documented on 
the eCRF, including whether the decision to withdraw from the trial was made by the 

participant or the investigator at the trial site. One of the following will be documented as the 
reason for trial withdrawal: 
 
i) AEs (state the actual AEs) 
 
ii) Serious AEs (state the actual SAEs) 
 
iii) Death 
 
iv) Lost to follow-up 
 
v) Major Protocol Deviation  
 
vi)  Trial termination by Sponsor 
 
vii) Investigator / Clinician-In-Charge Decision 
 
viii) Consent withdrawal by the trial participant (state reason) 
 
ix) Others 
 
Participants who withdraw from the trial may request that their collected but 
untested samples be destroyed. In this case, the investigator must document this. All the 
results obtained before such a request is made should be discarded. All samples collected 
must thus be destroyed. If such a request is not made, the investigator is allowed to retain 
and use the collected data before consent is withdrawn. 
 
Participants are deemed lost to follow-up if they repeatedly fail to attend their trial 
visits without explicitly requesting to withdraw from the trial, or if they cannot be contacted 
by their respective trial sites. In this case, the following steps should be taken to resolve the 
problem: 
 
i) Multiple attempts should be made to contact the participants and missed visits should be 
rescheduled. Counselling should be given to the participants on the importance of attending 
the follow-up visits and the investigator should also explore whether the participants are still 
interested in being part of the trial. 
 
ii) Prior to concluding that the participants are lost to follow-up, at least three phone calls/ 
text messages should be made and a registered letter should be sent to the patient’s last 
known home address to re-establish communication with the participants. These attempts to 
re-establish communication with the participants should be documented, signed and dated 
in the patient’s eCRF and medical records. 
 
iii) After the final attempt to contact the participant is unsuccessful, the participant is 
considered to have been lost to follow-up and withdrawn from the trial. 
 
4.16 Interventional Safety Assessment  

4.16.1 Adverse Events (AEs) / Serious Adverse Events (SAEs): Definitions 
 
For this study, the study investigators define adverse events as “an abnormal sign, 
symptom, laboratory test, syndromic combination of such abnormalities, untoward or 
unplanned occurrence (e.g., an accident), or any unexpected deterioration of concurrent 
illness” (85). For serious adverse events (SAEs), they are defined according to the USA Code 
of Federal Regulations Title 21, Section 312.32, which states that “adverse events result in the 
following outcomes: 
 
1) Death;  
 
2) Life-threatening AEs;  
 
3) Inpatient hospitalisation or prolongation of existing hospitalisation;  
 
4) A persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions, or a congenital anomaly/birth defect” (86).  
 
Other significant medical events are also considered SAEs if the events compromise 
study participants and require medical or surgical intervention to prevent one of the 
outcomes above. As an example, allergic bronchospasm requiring intensive treatment in an 
emergency setting to prevent the outcomes above. 
 
4.16.2: The likelihood of AEs / SAEs: Classification of causality 
 
The likelihood of AEs and SAEs is classified based on the modified Naranjo et al 
recommendations as follows (87): 
 
a) Unrelated: The AEs / SAEs are unlikely to be related to oral estradiol valerate (Progynova) 
or transdermal 17β estradiol  (Oestrogel) application. 
 
b) Possible: The connection between AEs / SAEs and oral estradiol valerate (Progynova) or 
transdermal 17β estradiol  (Oestrogel) cannot be excluded with absolute certainty: 
 
i) There is a temporal relationship between oral estradiol valerate (Progynova) or transdermal 
17β estradiol  (Oestrogel) application and the occurrence of AEs / SAEs. 
 
ii) However, there is an alternative factor (e.g., characteristic of the patient’s disease / clinical 
state) that can possibly or likely explain such a relationship, or a significant uncertainty exists 
over the cause of AEs / SAEs 
 
c) Probable: The connection between AEs / SAEs and oral estradiol valerate (Progynova) or 
transdermal 17β estradiol (Oestrogel) has a high degree of certainty 
 
i) There is a temporal relationship between oral estradiol valerate (Progynova) or transdermal 
17β estradiol (Oestrogel) application and the occurrence of AEs / SAEs 
 

ii) AEs / SAEs disappear or decrease upon the withdrawal of oral estradiol valerate 
(Progynova) or transdermal 17β estradiol  (Oestrogel) application, but do not reappear upon 
subsequent oral estradiol valerate (Progynova) or  transdermal 17β estradiol  (Oestrogel) 
application 
 
iii) Alternative causes cannot reasonably explain the relationship between oral estradiol 
valerate (Progynova) or transdermal 17β estradiol  (Oestrogel) application and the 
occurrence of AEs / SAEs. 
 
e) Definite: The AEs / SAEs are obviously linked to oral estradiol valerate (Progynova) or 
transdermal 17β estradiol  (Oestrogel) application with the occurrence of AEs / SAEs 
 
i) There is a temporal relationship between oral estradiol valerate (Progynova) or transdermal 
17β estradiol  (Oestrogel) application and the occurrence of AEs / SAEs 
 
ii) AEs / SAEs disappear or decrease in severity upon the withdrawal of oral estradiol valerate 
(Progynova) or transdermal 17β estradiol  (Oestrogel) application and reappear on 
subsequent oral estradiol valerate (Progynova) or transdermal 17β estradiol  (Oestrogel) 
application. 
 
iii) No other alternative causes. 
 
4.16.3 Methods for assessing and recording AEs / SAEs 
 
The study protocol will be halted at any moment of the study period if the study 
participants develop any sudden (expected or unexpected) severe complications / AEs. The 
withdrawal of a study participant from the trial will be documented on the adverse event 
page of the CRF and the participant will be further followed up for the study outcomes and 
included in the analysis as per the original randomisation group (intention-to-treat analysis). 
All AEs / SAEs will be recorded and graded according to the Common Terminology Criteria for 
Adverse Events (CTCAE) version 5 and the US FDA’s Toxicity Grading Scale for Healthy Adults 
and Adolescents Volunteers Enrolled in Preventive Vaccine Clinical Trials criteria (88-89). The 
statistical analysis plan for interventional safety assessment is given in section 4.20. 
 
4.16.4 Reporting of AEs / SAEs to the Institution Review Board (IRB) and Malaysian National 
Pharmaceutical Regulatory Agency (Malaysian NPRA) 
 
The safety reporting window will start from the first administration of the intervention 
(Progynova/Oestrogel) randomly allotted to the first trial participant at baseline (first patient 
first visit (FPFV)) until 30 days after the last administration of the randomly allotted 
intervention to the last trial participant. All AEs will be recorded in the eCRFs in the REDCap 
system. AEs of grade 3 and above will be reported to the UKM Human Ethics Committee (IRB) 
and the Data Monitoring Committee (DMC, a subcommittee of the IRB) within 5 business days 
or in accordance with the IRB guidelines for other trial centres. All SAEs (including Serious 
Unexpected Suspected Adverse Events (SUSARs)) will be reported within 24 hours of 
occurrence (expedited reporting) to the UKM Human Ethics Committee. The reporting 
procedures for SAEs should adhere to the following steps: 

 
i) The SAEs initial report form should be filled in by the assessors (O&G registrars) and 
reviewed and signed by the clinical PI (Associate Professor Ani Amelia Dato Zainuddin) or the 
co-investigator (Professor Nur Azurah Abdul Ghani) at the trial centres. 
 
ii) The SAEs initial report should then be submitted to the UKM Ethics Committee (expedited). 
 
iii) SAEs should be reviewed and followed to resolution by the O&G registrars / PI/co-
investigator in charge of the study participants. 
 
iv) A subsequent follow-up report will be submitted that documents any additional important 
information, including the resolution of SAEs. 
 
 
The UKM Ethics Committee will then be responsible for relaying all the information on 
the SAEs experienced by the individual participants (recorded in the Individual Case Study 
Report (ICSR)) to the Malaysian NPRA. Besides, the aggregate reporting of the SAEs will be 
analysed and prepared by an independent statistician (see Section 4.20.1) and submitted to 
the Data Monitoring Committee for further adjudication and submission to the Malaysian 
NPRA. 
 
 
4.16.5 AEs / SAEs follow-up at the end of the study period 
 
If AEs/SAEs occur or are still ongoing at the end of the study, trial participants will 
continue to be followed up and monitored until AEs/SAEs resolve, unless the PI deems no 
further follow-up is necessary. The follow-up may take the form of 1) additional subject visits 
to the trial centre/hospital, 2) telephone calls to the subjects, and 3) additional reporting in 
the form of letters from the treating physicians. 
 
4.16.6 Study Halting Criteria 
 
Participant enrolment and allocation and institution of interventions will be stopped 
if one of the following occurs: 
 
a) Death related to the intervention (oral estradiol valerate (Progynova) or transdermal 17β 
estradiol (Oestrogel)) application 
 
b) Any participant experiences bronchospasm, laryngospasm, or anaphylaxis within 24 hours 
post-intervention (oral estradiol valerate (Progynova) or transdermal 17β estradiol  
(Oestrogel)) usage or application 
 
c) Any SAE related to interventions (oral estradiol valerate (Progynova) or transdermal 17β 
estradiol (Oestrogel)) 
 
d) Any AE of grade 3 and above or any SAE that cannot be obviously implicated by other 
causes 
 

e) Any study participant who develops abscess/ulceration/erosion at the site(s) of 
transdermal 17β estradiol  Oestrogel application 
 
 
4.16.7 Management of AEs linked to oral Estradiol Valerate (Progynova) and transdermal 
17β estradiol (Oestrogel) 
 
Oral estradiol valerate (Progynova) administration or transdermal 17β estradiol 
(Oestrogel) should be promptly halted if the following AEs occur during the oral estradiol 
valerate (Progynova) or transdermal 17β estradiol (Oestrogel) use: 
 
i) Pregnancy or lactation 
 
ii) Undiagnosed vaginal bleeding, defined as excessive vaginal blood loss that warrants 
tampon, pad, or pantyliner use 
 
iii) Known or suspected breast cancer 
 
iv) Known or suspected premalignant conditions or malignancies, if they are influenced by sex 
steroids  
 
v) Presence or history of hepatic tumours (e.g. hepatic adenoma) 
 
vi) Severe liver disease resulting in cholestatic jaundice or deterioration of liver function 
 
vii) Acute arterial thromboembolic events (myocardial infarction or stroke) or a recent history 
of myocardial infarction or stroke 
 
viii) Active deep vein thrombosis, thromboembolic disorders, thrombophlebitis, or a recent 
history of deep vein thrombosis, thromboembolic disorders, or thrombophlebitis 
 
ix) New onset of migraine-type headache or unusually severe headache that occurs for the 
first time 
 
x) Severe hypertriglyceridemia that leads to pancreatitis 
 
xi) Any hypersensitivity (anaphylaxis, angioedema) to the oral estradiol valerate (Progynova) 
or  transdermal 17β estradiol  (Oestrogel) components 
 
For other AEs, withhold Progynova or Oestrogel until the AEs have fully disappeared 
if the severity is Grade 3 and above based on the CTCAE Version 5 classification. Reintroduce 
oral estradiol valerate (Progynova) or transdermal 17β estradiol  (Oestrogel), and if AEs recur, 
permanently stop oral estradiol valerate (Progynova) or transdermal 17β estradiol  
(Oestrogel), and the trial participant will be withdrawn. 
 
4.16.8 Dermatological Toxicity 
 

A) Grade 1 CTC-AE maculopapular rash/desquamation 
 
Definition  
 
Macules / Papules covering 10%< Body Surface Area (BSA) with or without symptoms 
(pruritus, burning, tightness). 
 
Management  
 
Oestrogel / Progynova will be continued and the study participant will be treated with 
hydroxyzine 100mg/day for 8 days. 
 
B) Grade 2 CTC-AE maculopapular rash/desquamation  
 
Definition  
 
Macules/papules covering 10 - 30% BSA with or without symptoms (e.g., pruritus, 
burning, tightness); limiting instrumental ADL; rash covering > 30% BSA with or without mild 
symptoms. 
 
Management  
 
1) The participant will be clinically managed with hydroxyzine 100 mg/day for 8 days and with 
prednisone for 8 days (1 mg/kg for the first 2 days, 0.5 mg/kg for the next 2 days, then 20 
mg/day for the next 2 days, and  10 mg/day for the last 2 days).  
 
2) If grade 2 maculopapular rash/desquamation remains despite symptomatic treatment, 
Progynova / Oestrogel will be stopped. 
 
B) Grade 3-4 CTC-AE of any dermatological toxicity 
 
Definitions 
 
1) Macules/papules covering >30% BSA with moderate or severe symptoms; limiting self-care 
ADL 
 
2) Macules/papules covering >30% BSA with moderate or severe symptoms; limiting self 
care activity of daily living (ADL) 
 
3) Skin sloughing covering 10 - 30% BSA with associated signs (e.g., erythema, purpura, 
epidermal detachment and mucous membrane detachment) 
 
Management 
 
Consult a dermatologist for diagnosis and immediately stop the interventions. 
 
4.16.9 Liver toxicity 

 
A)  Baseline Liver toxicity grade 1 (Baseline Serum Alanine Aminotransferase (ALT) & 
Aspartate Aminotransferase (AST) ≤ 3 Upper Limit of Normal (ULN) or Gamma GT ≤ 2.5 ULN 
or bilirubin ≤ 1.5 ULN) 
 
Management 
 
1) In case of grade 2 liver enzymes increased, maintain Progynova / Oestrogel 
 
2) In case of grade 3 CTC-AE liver enzymes increase, interrupt Progynova/Oestrogel until 
return to grade 1 or to baseline level, then resume. 
 
3) In case of grade 4 liver enzymes increased, halt Progynova / Oestrogel. 
 
B) Baseline liver toxicity grade 2 (Baseline Serum Alanine Aminotransferase (ALT) & 
Aspartate Aminotransferase (AST) transaminases > 3 and ≤5 ULN, gamma GT > 
2.5 and ≤ 5 ULN, bilirubin > 1.5 and ≤3 ULN) 
 
Management 
 
In case of liver enzymes increased ≥ 20% from baseline, halt Progynova / Oestrogel until 
return to baseline level, then resume. 
 
C) Any grade 4 liver toxicity 
 
Management:  
 
Stop Progynova / Oestrogel indefinitely. 
 
4.16.10 Cardiac-related toxicity 
 
Management 
 
At each follow-up visit, cardiac symptoms need to be carefully elicited during history taking 
and clinical examination. 
 
If a cardiac event is suspected based on these symptoms; dyspnoea, thoracic chest pain, 
increased blood pressure, the following steps should be undertaken by the clinicians in charge 
of the study participants: 
 
1) Perform an ECG: If there is any change compared to the previous ECG(s) readings, a 
cardiologist’s advice should be sought. 
 
2) Obtain serum troponin level: If the result exceeds the Upper Limit of Normal (ULN) (more 
than the 99th percentile of  upper reference limit (local-assay dependent)), a cardiologist’s 
advice must be sought. 
 

3) For blood pressure increase (hypertension): Start or adapt existing antihypertensive 
medications. If hypertension persists, a cardiologist must be consulted and the cessation of 
study intervention must be considered based on the risk-benefit ratio for the study 
participant. 
 
4.16.11 Endocrinological toxicity 
 
A) Hypertriglyceridemia Grade 1 and 2 (Grade 1: 1.71 – 3.42 mmol/L; Grade 2: >3.42 – 5.7 
mmol/L) 
 
Management: 
 
1) If grade 1, maintain the same dose or stop the assigned estrogen replacement therapy until 
serum triglyceride level returns to normal (at physician’s discretion). 
 
2) If grade 2, stop the assigned estrogen replacement therapy until serum triglyceride level 
returns to grade 1 or less. Consider starting fenofibrate (267 mg daily) if serum triglyceride is 
above 5 mmol/L - 10 mmol/L if  the 10-year CVD risk is above 8% based on the SCORE2-ASIA 
risk prediction model. 
 
B) Hypertriglyceridemia Grade 3 and 4 (Grade 3: >5.7 – 11.4 mmol/L; Grade 4: >11.4 
mmol/L) 
 
Management: 
 
1) Discontinue the assigned estrogen therapy and start fenofibrate 267 mg daily immediately 
if serum triglyceride level is above 10 mmol/L.  
 
2)For those with serum TG between >5.7 mmol/L and 10 mmol/L, start fenofibrate 267 mg 
daily if  the 10-year risk of CVD event is above 8% based on the SCORE2-ASIA risk prediction 
model (90). 
 
3) Reintroduce the assigned estrogen therapy at one lower dose level after 
hypertriglyceridemia returns to grade 1 and below. Permanently halt the assigned 
intervention if hypertriglyceridemia grade 3 and above reoccurs. 
 
 
4.16.12 Haematological toxicity 
 
A) Thromboembolic events (TEE) 
 
I) Grade 1 (Superficial venous Thrombosis) or Grade 2 (uncomplicated DVT) 
 
Management 
 
1) No medical intervention is required for Grade 1 TEE. Continue assigned intervention. 
 

2) Perform serum D-Dimer and venous ultrasonography to confirm DVT. Medical intervention 
using appropriate anticoagulants is required for grade 2 TEE (No INR monitoring for 
rivaroxaban, apixaban; regular serum platelet count is required for heparin use due to 
heparin-induced thrombocytopenia). Temporary discontinuation of the IMP until TEE reduces 
to grade 1 is required. Rechallenge with 25% reduction in the originally assigned intervention 
dose. Permanently halt the assigned intervention if grade 2 TEE recurs. 
 
3)Consult a haematologist to further refine the patient management.  
 
II) Grade 3  (uncomplicated pulmonary embolism (PE), non-embolic cardiac thrombus) or 
Grade 4 (Life threatening TEE – complicated PE, cerebrovascular event, arterial 
insufficiency, with or without hemodynamic or neurological instability) 
 
Management 
 
1)Confirm PE with CT Pulmonary Angiography (CTPA) or V/Q Scan or CT Brain for 
cerebrovascular event. Medical intervention using appropriate anticoagulants is required for 
Grade 3 TEE. Rechallenge with 50% reduction in the originally assigned intervention dose if 
Grade 3 reduces to grade 1. Permanently stop the assigned intervention if Grade 3 TEE recurs. 
 
2) URGENT medical intervention (fibrinolytic therapy with tissue plasminogen activator (tPA)) 
is warranted for Grade 4 TEE. Refer cardiothoracic surgeons for urgent catheter-guided 
pharmacomechanical  therapy (physical thrombus destruction or dissolution plus catheter-
guided low-dose thrombolysis) in the presence of complicated massive PE. ICU admission is 
warranted (consult intensivist). Stop the assigned interventions permanently. 
 
 
4.17 Operational Definitions of Study Variables and Data Standards 
 
The operational definitions for important trial variables are given below (Table 8). 
 
 
Table 8: The Operational Definitions of important trial variables 
 
Variables 
Definitions 
Demographic Profiles 
  

Age 
  
Age of the Turner Syndrome patients at trial enrolment. This 
will be recorded in years and months. Continuous variable. 
  
Gender 
Gender of the participant. Since the participants are all 
females, this will be recorded as a single-group categorical 
variable. 
  
Ethnicity 
  
The racial identity of the participants. This will be recorded as 
one of the following multinomial categories: Malay, Chinese, 
Indian, Other Ethnicities. 
  
Education level 
  
The highest education level of the participant at the time of 
enrolment. This will be recorded as one of the following ordinal 
categories: 
Primary, 
Secondary, 
Pre-college 
(Matriculation/STPM/A-Level/IB), 
Undergraduate 
Degree, 
Post-Graduate Degree 
  
Socioeconomic Status 
  
The socioeconomic status of the family of the trial participants. 
This will be ordinally recorded as low (B40), intermediate 
(M40) and high (T20). The income range for each category will 
be based on the Household Income and Basic Amenities Survey 
2019 by the Department of Statistics Malaysia. 
  
Employment 
The employment status of the participants at the time of trial 
enrolment. If the participants are employed during the course 
of the trial, the participant’s employment status will not be 
changed since it represents the participant’s baseline 
employment status. This will be recorded as a binary variable 
(yes, no) should include if the participant is a student as well 

Clinical  Parameters 
  
Weight 
 The weight of the participants in kilograms measured at 
baseline, months 3, 6, 12, 15, 18, 21 and 24 This will be 
recorded as a continuous variable. 
Height 
 The height of the participants in centimetres measured at 
baseline, months 3, 6, 12, 15, 18, 21 and 24. This will be 
recorded as a continuous variable. 
BMI 
 The Body Mass Index (BMI) of the participants in kg/m2  
measured at baseline, months 3, 6, 12, 15, 18, 21 and 24. This 
will be initially recorded as a continuous variable and 
subsequently categorized according to the WHO classification. 
Systolic Blood Pressure 
(SBP) 
 The SBP of the participants in mmHg measured at baseline, 
months 3, 6, 12, 15, 18, 21 and 24. This will be recorded as a 
continuous variable. 
Diastolic Blood Pressure 
(DBP) 
 The DBP of the participants in mmHg measured at baseline, 
months 3, 6, 12, 15, 18, 21 and 24. This will be recorded as a 
continuous variable. 
Heart Rate (HR) 
 The HR of the participants in beats/minutes measured at 
baseline, months 3, 6, 12, 15, 18, 21 and 24. This will be 
recorded as a continuous variable. 
Respiratory Rate (RR) 
 The RR of the participants in breaths/minutes measured at 
baseline, months 3, 6, 12, 15, 18, 21 and 24. This will be 
recorded as a continuous variable. 
Temperature 
  
 The temperature of the participants in Celsius measured at 
baseline, months 3, 6, 12, 15, 18, 21 and 24. This will be 
recorded as a continuous variable. 

Trial Endpoints 
 Tanner Staging (Breast 
development)  
 See Section 4.15.2 for classification. It is measured as a 5-
category ordinal variable at baseline, months 6, 12, 18 and 24. 
 Uterine Length 
 Self- explanatory.  Measured from the fundus of the uterus  to 
the  lower margin of the cervix. It is measured in cm at baseline, 
month 6, 12, 18 and 24. It is treated as a continuous variable. 
 Uterine 
Antero-
posterior (AP) Diameter 
 The  width of the uterus from the anterior (front) to posterior 
(back) uterine wall at the largest diameter. It is measured in cm 
at baseline, months 6, 12, 18 and 24. It is treated as a 
continuous variable. 
Uterine 
Withdrawal 
bleeding 
Uterine bleeding occurring during the treatment with either 
estrogen.  It is treated as a binary categorical variable (yes / 
no). 
Time 
to 
uterine 
withdrawal bleeding 
The time from the initiation of oral estradiol valerate 
(Progynova) or transdermal 17β estradiol  (Oestrogel)  until the 
first uterine withdrawal bleeding occurs. It is measured in days 
and is treated as a continuous variable. 
 
4.18 Data Collection and Study Monitoring  
 
4.18.1 Data Collection 
 
The trial investigator/coordinator at each trial site must record the data collected in 
the electronic record form (eCRF) (REDCAP) developed, provided and maintained by the 
Sponsor (UKM). Details on each trial endpoint must also be accurately recorded in the 
participant’s medical records since the information documented in the records will be 
regularly cross-checked with information in pCRF and eCRF for data consistency. Besides, 
double data entry procedure (i.e. the same patient information will be entered twice in 
another duplicated eCRF) will be carried out to minimize errors in data entry. 
 
4.18.2 Study Monitoring (Audits) 
 
The Sponsor will perform a six-monthly internal audit to ensure all research activities 
at each trial centre comply with the trial protocol and the GCP principles. The eCRFs that have 
been thoroughly verified and monitored at the final visit for each trial participant will be 

locked and transferred to the Sponsor’s Data Management Team. At the conclusion of the 
trial, a copy of each completely-endorsed eCRF will be shared with each respective trial site.  
 
In addition, the Malaysian National Pharmaceutical Regulatory Agency (NPRA) might 
also want to inspect each or one particular trial site. In this case, the site investigator must 
promptly notify the Sponsor (UKM) of such requests. The investigator must therefore furnish 
the NPRA with relevant documents and access to the trial site. Besides, the IMP provider, 
Besins Healthcare, may also conduct an independent periodic external audit for the purposes 
mentioned above. 
 
 
4.18.3 Data Management and Standards  
 
Each trial dataset will be stored in triplicate in separate folders within the protected 
Amazon Cloud Drive. This will ensure there are two backup files for every original trial dataset. 
The information entered in the eCRF will be regularly and systematically evaluated in three 
ways: 
 
1. Online with programmable system-based automatic check 
2. Offline by the Data Management Personnel 
3. Based on error messages obtained via additional validation programs or database 
listings. 
 
Each error message in data entry (e.g. incorrectly entered BMI values of 398 kg/m2) will 
be captured by Data Clarification Forms and the investigators will be promptly notified to 
rectify the issues. The investigators are then required to sign the final eCRF electronically.  As 
part of Quality Assurance, the Data Manager will then check the accuracy of the data for each 
efficacy and safety endpoint before the database is locked. Any subsequent changes to the 
database at this stage will require the explicit written approval of the Principal Investigator 
(UKM) and the principal statistician (UKM). 
 
Concomitant medications should be coded according to the WHO Drug Reference List 
based on the Anatomical Chemical Classification System. The MedDRA classification system 
will be used to code all other comorbidities. 
  
The trial data acquisition and handling will conform to the Clinical Data Interchange 
Standards Consortium (CDISC) guidelines. The types and fields for the data entered in the 
REDCAP database will be specified according to the Clinical Data Acquisition Standards 
Harmonization (CDASH) standards. The conversion of trial data to standard data tables will 
follow the Study Data Tabulation Model (SDTM). On the other hand, the primary efficacy 
variables will be derived based on the Analysis Data Model (ADAM) standards. 
 
4.19 Ethical issues  
 
4.19.1 Ethical Approval and Trial Registration 
 

The trial will be conducted in accordance with the Declaration of Helsinki (64th World 
Medical Association General Assembly, 2013) and International Conference on 
Harmonization Good Clinical Practice (ICH-GCP) guidelines. Upon the finalisation of the 
protocol version, ethical approval will be sought and obtained from the UKM Research Ethics 
Committee (UKM-REC) and the participating institutions’ IRBs. The trial protocol will be 
subsequently registered with the ClinicalTrials.gov and the Malaysian National Medical 
Research Register NMRR) registries and the document containing the full trial protocol will 
be uploaded to both registries.  
 
4.19.2 Participant Consent  
 
Signed and dated informed consent will be obtained from parents or legal guardians 
for under-18 participants (and participant’s assent), along with the dated signature of the 
person conducting the informed consent process. A copy of the signed informed consent will 
be given to each participant before trial commencement. Any new information regarding the 
IMPs (e.g. new information on the safety and efficacy of the IMPs) that may affect the 
participant’s willingness to continue participating in the trial will be conveyed to the 
participants, parents or legal guardians in a timely fashion. The disclosure of such information 
will be conveyed and documented in a revised patient information sheet (PIS) and consent 
form that should be signed and dated by the parents or legal guardians for all participants. 
The revised PIS and consent form should receive approval from the UKM-REC and respective 
IRBs before use. Hence, the general format of the consent form will comply with the general 
requirements of each IRBs, the National Pharmaceutical Regulatory Agency (NPRA) laws and 
regulations and the Sponsor (UKM). 
 
4.19.3 Data Protection and Participant Confidentiality 
 
All participant data will be made accessible to the primary investigators and 
collaborators of the trial only. The participants are only identifiable by their unique trial 
identification number. All information that may identify individual participants, including the 
participant’s hospital registration number (RN) and National Identity Card (NIC), will be stored 
in a separate password-protected database system that can only be accessed by the principal 
investigator and collaborators of this trial. However, for safety monitoring, the participant’s 
identifying information can only be divulged by cross-referencing the participant’s unique 
randomisation number with the subject identification number. This can only be performed 
upon obtaining written approval and at the discretion of the trial’s principal investigator 
(UKM). 
  
The participants’ trial data will only be published in summary group measures, and 
therefore, it is unfeasible to identify the individual Turner Syndrome patients. If individual 
participants’ trial data are needed as part of the publication requirements, the participant’s 
identifying information will be removed. 
 
To ensure the integrity of record keeping and prevent further accidental/non-
accidental breaches of patient information, a data entry and access log will be created on the 
data management system (REDCAP). Each trial personnel (participant recruiters, assessors, 
interventionists, trial principal investigators and collaborators, trial coordinators) will be given 

a unique ID and password for REDCAP access that can only be used by that trial personnel. 
This information should not be shared with other trial personnel and upon discovery of any 
access sharing with other trial personnel or outside individuals, the access to the REDCAP 
system for such trial personnel will be promptly rescinded. The person will then be disbarred 
from the trial due to trial misconduct. 
 
4.19.4 Indemnity 
 
Clinical trial insurance provided by an appropriate insurer/underwriter (e.g. no-fault 
compensation insurance coverage provided by the Great Eastern General Insurance 
(Malaysia) Incorporation or Chubb Insurance (Malaysia) Incorporation)   will be purchased to 
protect against any foreseen/unforeseen liabilities related to research activities at the trial 
site. Additional research funding will be secured from the Faculty of Medicine, UKM to cover 
the cost of the trial insurance premium. The projected premium costs will be based on 
participant numbers and study duration, and the justification will be aligned with the 
institutional risk‑management policies.  
Upon approval, these funds will be ring‑fenced in the trial budget and disbursed 
directly to the insurer before the first participant visit, thereby ensuring uninterrupted 
coverage from study initiation until the end of the follow‑up period. 
 
 
4.20 Statistical Analysis Plan (SAP) 
 
4.20.1 Personnel 
 
There will be three statisticians involved in the data analyses and safety data 
monitoring: the principal trial (study) statistician, the data monitoring committee (DMC) 
statistician and an independent statistician (contractually hired throughout the trial 
duration). The study statistician (Dr Muhammad Irfan) will be responsible for the overall 
design, conduct, analyses and reporting of final trial data. The DMC statistician (Prof Shamsul 
Azhar, an experienced triallist and a clinician) and the other members of DMC in the UKM 
ethics committee will be in charge of assessing the interim reports produced by the 
independent statistician so that recommendations on the trial conduct can be made (e.g. 
whether the trial can be halted prematurely or should be modified due to obvious efficacy or 
serious safety concern). The independent statistician, who will be hired later, will be tasked 
with producing interim reports on safety and efficacy parameters that the DMC members will 
appraise for further evaluation. The rationale for our hierarchical organisation of statistical 
personnel is based on the recommendations by Pocock in 2014 that guarantee the 
impartiality of trial conduct, design, data analysis and interpretations, and reporting of results 
(91). 
 
4.20.2  Main Statistical Paradigm: Bayesian Paradigm 
 
Since this trial includes both data from historical patients who have already received 
Progynova/Oestrogel and concurrent trial participants who will be randomly assigned to 
Progynova or Oestrogel, synthetic control methods (92) will be used to analyse the data using 
a Bayesian paradigm. The recency of data from historical participants and the data obtained 

from trial participants make the synthetic method based on the Bayesian paradigm the best 
way to combine the information from both data sources to obtain a better posterior summary 
of each trial endpoint (92). 
 
To address potential imbalance in confounder profiles due to the lack of randomization in the 
historical cohort, propensity score matching will be used to match Progynova/Oestrogel users 
in the historical cohort with concurrent trial participants. However, due to the potential high-
dimensional nature of the inclusion of many confounders,  machine learning methods such 
as Variational Autoencoders (VAE) based on the Variational Bayes (VB) and Generative 
Adversarial Network (GAN) will be utilized to create the propensity scores (93-94). To account 
for the effect of patient drift due to unmeasured confounders and  possible changes of the 
measured confounders across time – a known limitation of synthetic control methods - 
simulated values will be generated from different statistical distributions with varying 
parameters for categorical and continuous unmeasured confounders. These unmeasured 
confounders will be included in the model and their impacts on the biasedness of the 
estimates will be analyzed using the method proposed by Jiang et al. (95) or based on Power 
/ Commensurate Prior Distribution within the Bayesian paradigm (96-97) to account for 
heterogeneity in data sources (i.e historical data is given lower weight (discounted) compared 
to internal trial information). 
 
To combine information from historical Progynova/Oestrogel users and concurrent trial 
participants, the Bayesian Hierarchical Model (BHM) will be used. As an illustration, the data 
generation process for this trial is represented in Figure 4 below (adapted from (92)): 
 
 
Figure 4. Sources of data and proposed synthetic control methods to combine the external 
data generated by historical Progynova / Oestrogel users with concurrent trial participants  
 

The proposed BHM is represented by the schematics below (Figure 5) (adapted from (98)): 
 
 
  
Figure 5. Schematic of the BHM to combine the information from historical 
Progynova/Oestrogel users with the concurrent trial participants who will be randomly 
assigned to Progynova/Oestrogel. 
 
Appropriate prior distributions for each trial endpoint will be elicited and constructed 
to represent the findings from the previous trials (prior information). The prior information 
will be combined with the information in our trial data (likelihood) to obtain posterior updates 
for each trial endpoint parameter. To obtain the full conditional distribution (FCD) for each 
trial endpoint parameter, Markov Chain Monte Carlo (MCMC) techniques (Gibbs Sampling or 
Metropolis-Hastings algorithm) will be utilised. The procedures will be implemented in R 
Version 4.3.1 (R Core Team, 2023) using the Rjags version 4.14 (Plummer 2022) package. 
Hamiltonian Monte Carlo (HMC) algorithm will also be carried out using RStan or Integrated 
Laplace Approximation (INLA) method using R-INLA. Bayes factor will be computed to test 
statistical hypotheses and for statistical model selection. Posterior predictive checks will also 
be employed as part of model-checking procedures under the Bayesian paradigm. If the 
results obtained using the Bayesian paradigm conform with those obtained under the 
frequentist analyses, the veracity of such findings will be made more strengthened. 
 
4.20.3 Main Statistical Analysis Steps for Efficacy Endpoints  

 
Data will be cleaned for inconsistencies, wrong data entry and missing observations. 
Multiple imputation will be performed to impute the missing observations under the Missing 
At Random (MAR) assumption. The analysis will adhere to the intention-to-treat (ITT) 
principle as the major analytical principle, but the modified ITT (i.e. the analysis will adhere 
to the ITT principles, but the participants who prematurely withdraw from the trials due to 
non-treatment related reasons such as withdrawal of consent not related treatment efficacy 
or safety, non-intervention related deaths will be excluded from the analyses) and per-
protocol analyses will also be performed as part of sensitivity analyses.  No interim analyses 
will be performed. 
 
Descriptive analyses will then be carried out to explore the data. Normally-distributed 
continuous variables (age, BMI, hormonal levels, uterine length and volume) will be 
summarised in mean (SD) or median (IQR) if the variables do not follow normal distributions. 
Categorical variables (Tanner Stage, HRT-associated adverse events) will be described in 
frequency and percentage. The normality of the continuous variables will be graphically 
assessed using histograms with overlying Gaussian (normal) distribution curves and 
subjectively using the Shapiro-Wilks test and Fisher’s skewness and kurtosis coefficients 
(threshold ± 1.96).  
 
For univariable analysis, the association between different types of HRTs and 
proportions of subjects achieving Tanner Stage B3 and above  will be investigated using the 
exact version (Mehta & Patel algorithm) of chi-square. The differences in the mean (median) 
uterine length and volume between those receiving different HRT types will be examined 
using either the independent or Mann-Whitney test. All statistical test assumptions 
(homogeneity of variance, normality of the variables) will also be evaluated and reported. The 
Kaplan-Meier method will be employed to obtain the median times to Tanner stage B3 and 
above in both the oral estradiol valerate (Progynova) and Transdermal 17β estradiol 
(Oestrogel) groups. The equality of median times to Tanner stage B3 and above in both groups 
will be evaluated using the log-rank test. 
 
For multivariable analysis, multiple logistic regression will be performed to evaluate 
the association between HRT types and Tanner Stage B3 or higher. On the other hand, the 
association between different HRT types and uterine length and volume will be investigated 
using multiple linear regression. Sequential model-building strategies will be employed to 
assess the relevance of the predictors in the model based on the sequential inclusion and 
exclusion of variables with p<0.25 at the simple logistic/linear regression level and clinically 
significant predictors. Multicollinearity will be assessed using inter-variable correlations 
(threshold: ≥ 0.75), Variance Inflation Factor (VIF) (threshold VIF ≥ 4), Tolerance (≤0.25) and 
Collinearity diagnostics (Condition Index VIF ≥ 30). The presence of statistical interaction will 
be investigated by assessing the significance of clinically plausible multiplicative interaction 
terms. 
 
The residual versus predictor value scatter plot and Durbin-Watson statistics will be 
utilised to assess the multiple linear regression assumptions (normality, heteroscedasticity, 
independence and linearity of the residuals). For the multiple logistic regression model, the 
Hosmer-Lemeshow test will be carried out to assess the model's overall fit, and the 

percentage of correct classification will be obtained to assess the model’s accuracy. The area 
under the receiver operating curve (ROC) for the sensitivity versus 1-specificity plot will be 
obtained to assess the discriminatory properties of the model. The deviance (threshold ± 4) 
and dfBeta (threshold ± 1) will be used to evaluate the presence of influential observations. 
If present, they will be removed if the values are clinically implausible (i.e. incorrectly 
measured values) and retained if they are correctly measured observations. 
 
No subgroup analyses will be performed. For these frequentist analyses, two-tailed 
statistical tests will be employed and the significance threshold will be fixed at 0.05. The 
results will be reported in accordance with the 2010 and 2022 CONSORT outcome reporting 
guidelines. 
 
 
4.20.2 Statistical Analysis for Safety Parameters 
 
A separate dataset will be created for safety parameters. The Medical Dictionary for 
Regulatory Activities (MedDRA) coding scheme will be used for coding the AEs. Results will be 
presented for each intervention group and the whole patient population. Separate results will 
be presented for those randomised but who do not receive the allotted interventions. All 
subjects who receive the allotted interventions at Day 1 and have follow-up data will be 
included in the analysis for safety parameters. 
 
The following descriptive analytical measures will be carried out and presented for each 
time point (Day 1, Months 6, 12, 18, 24): 
 
1. Frequency (percentage) of study participants with at least one AE 
 
2. Frequency (percentage) of study participants with at least one SAE (death, non-fatal 
SAEs) 
 
3. Frequency (percentage) of study participants with at least one grade 3 or 4 SAE  
 
4. Frequency (percentage) of study participants with at least 1 AE that results in trial 
discontinuation 
 
In addition, the incidence rate for each AE/SAE (number of study participants with the 
relevant AE or SAE at the end of each relevant timepoint/total number of study participants 
at each relevant timepoint * 100) and its associated 95% confidence interval will be calculated 
and presented. 
 
4.20.3 Statistical Analysis for Compliance Monitoring 
 
The medication adherence (compliance) of study participants will be descriptively 
summarised as the number (percentage) of participants who achieved at least 80% 
compliance at the end of each time period, including the baseline (Day 1). 
 
4.20.4 Statistical Analysis for Assessing Inter-rater Agreement 

 
Fleiss Kappa will be used to assess the inter-rater agreement for categorical trial endpoints 
(e.g., Tanner Staging for breast development). For continuous trial endpoints, the intraclass 
correlation coefficient will be computed. Any rater that causes the inter-rater agreement to 
fall below 0.60 for Fleiss Kappa and/or ICC will be excluded from being the assessor for the 
clinical trial endpoints (99-100). 
 
4.23 Plans for Dissemination of Results 
 
Upon trial completion, the trial results will be uploaded to the clinicaltrials.gov registry within 
6 months of the last patient visit to facilitate early dissemination of trial results. The final and 
cleaned-up version of the trial dataset containing individual clinical-trial participant data (IPD) 
for all study variables will be deposited in a public repository such as the Harvard Dataverse 
data repository (available from: https://dataverse.harvard.edu/) to ensure transparency in 
trial reporting and compliance with the clinicaltrials.gov data sharing policies. The trial results 
will then be published as presentations at scientific conferences and a complete manuscript 
publication, even if the trial has to be prematurely halted. All authors will be provided with 
the results for all primary and secondary endpoints before publication. Upon the acceptance 
of the manuscript, the principal investigators and research collaborators from all trial sites 
allow the publication of key sections of this protocol (trial objectives, the eligibility criteria of 
the subjects, the design of the trial (including revisions), the statistical analysis plan (including 
amendments) and the originally planned measurements of efficacy and safety parameters.  
 
Authorship eligibility will depend on fulfilling ALL the criteria below: 
 
1. 
Significant contributions to the conception and design of the trial, trial 
implementation, data acquisition, statistical analysis and data interpretation; 
 
2. 
Significant contributions to the drafting of the manuscript 
 
3. 
Final endorsement for the publication of the final manuscript version 
 
The final decision on the order of the authors’ names will be subject to actual participation 
and contributions to the trial and manuscript or conference proceeding write-ups. The 
primary (first) author shall be the person who contributes most significantly to all three 
criteria above. The primary (first) author shall be responsible for safeguarding and preserving 
the trial data’s integrity, the soundness of the trial methodology, data analysis and the 
manuscript’s overall scientific content.  
 
The corresponding author will be responsible for all communications during manuscript 
submission, the peer-review process and after publication. The responsibilities include 
responding to queries by the journal editors and peer reviewers and managing 
communications related to ethical oversight, data sharing requests and any post-publication 
clarifications. Besides, the corresponding author shall also be the principal point of contact 
for any future queries related to trial results and design presented in the accepted manuscript 
for future readers.  
 

SECTION 5 
 
EXPECTED RESULTS 
 
 
4.1 Flow diagram for participant recruitment (modified CONSORT diagram for hybrid RWD-
concurrent group trial) 
 
Figure 4: The modifiedCONSORT diagram depicting the procedural flow of trial participants  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
4.2 Baseline Characteristics of Study Participants 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

4.3 Comparisons of trial efficacy endpoints for binary outcome (dummy table for one efficacy endpoint) 
 
 
 
 

4.4 Comparisons of trial efficacy endpoints for continuous outcome (dummy table for one efficacy endpoint) 
 
 
 
  

4.5 Comparisons of time to withdrawal bleeding between intervention groups 
 
 
 
 
4.6 Comparisons of safety endpoints between Intervention groups 
 
 
 
 
 
 
 

 
 
REFERENCES 
 
1. 
Gravholt CH, Viuff MH, Brun S, Stochholm K, Andersen NH. Turner syndrome: 
mechanisms and management. Nat Rev Endocrinol. 2019 Oct;15(10):601-614. doi: 
10.1038/s41574-019-0224-4. Epub 2019 Jun 18. PMID: 31213699. 
2. 
Klein KO, Rosenfield RL, Santen RJ, Gawlik AM, Backeljauw PF, Gravholt CH, et al. 
Estrogen Replacement in Turner Syndrome: Literature Review and Practical Considerations. J 
Clin Endocrinol Metab. 2018;103(5):1790-803. 
3. 
Webber L, Anderson RA, Davies M, Janse F, Vermeulen N. HRT for women with 
premature ovarian insufficiency: a comprehensive review. Hum Reprod Open. 
2017;2017(2):hox007. 
4. 
Torres-Santiago L, Mericq V, Taboada M, Unanue N, Klein KO, Singh R, et al. Metabolic 
effects of oral versus transdermal 17beta-estradiol (E(2)): a randomized clinical trial in girls 
with Turner syndrome. J Clin Endocrinol Metab. 2013;98(7):2716-24. 
5. 
Chetkowski RJ, Meldrum DR, Steingold KA, Randle D, Lu JK, Eggena P, et al. Biologic 
effects of transdermal estradiol. N Engl J Med. 1986;314(25):1615-20. 
6. 
Goodman MP. Are all estrogens created equal? A review of oral vs. transdermal 
therapy. J Womens Health (Larchmt). 2012;21(2):161-9. 
7. 
Stevenson JC, Crook D, Godsland IF, Lees B, Whitehead MI. Oral versus transdermal 
hormone replacement therapy. Int J Fertil Menopausal Stud. 1993;38 Suppl 1:30-5. 
8. 
 Backeljauw P, Klein K. Sex hormone replacement therapy for individuals with Turner 
syndrome. Am J Med Genet C Semin Med Genet. 2019 Mar;181(1):13-17. doi: 
10.1002/ajmg.c.31685. 
9.  
Zaiem F, Alahdab F, Al Nofal A, Murad MH, Javed A. Oral Versus Transdermal Estrogen 
In Turner Syndrome: A Systematic Review And Meta-Analysis. Endocr Pract. 2017 Apr 
2;23(4):408-421. doi: 10.4158/EP161622.OR. 
10.  
Wakatsuki A, Okatani Y, Ikenoue N, Fukaya T. Different effects of oral conjugated 
equine estrogen and transdermal estrogen replacement therapy on size and oxidative 
susceptibility of low-density lipoprotein particles in postmenopausal women. Circulation. 
2002 Oct 1;106(14):1771-6. doi: 10.1161/01.cir.0000032261.12632.d7 
11.  
Ho JY, Chen MJ, Sheu WH, Yi YC, Tsai AC, Guu HF, Ho ES. Differential effects of oral 
conjugated equine estrogen and transdermal estrogen on atherosclerotic vascular disease 
risk markers and endothelial function in healthy postmenopausal women. Hum Reprod. 2006 
Oct;21(10):2715-20. doi: 10.1093/humrep/del245. 
12.  
Shah S, Forghani N, Durham E, Neely EK. A randomized trial of transdermal and oral 
estrogen therapy in adolescent girls with hypogonadism. Int J Pediatr Endocrinol. 
2014;2014(1):12. doi: 10.1186/1687-9856-2014-12. 
13. 
Popat VB, Calis KA, Kalantaridou SN, Vanderhoof VH, Koziol D, Troendle JF, et al. Bone 
mineral density in young women with primary ovarian insufficiency: results of a three-year 
randomized controlled trial of physiological transdermal estradiol and testosterone 
replacement. J Clin Endocrinol Metab. 2014;99(9):3418-26. 
14. Taboada M, Santen R, Lima J, Hossain J, Singh R, Klein KO, Mauras N. Pharmacokinetics 
and pharmacodynamics of oral and transdermal 17β estradiol in girls with Turner syndrome. 
J Clin Endocrinol Metab. 2011 Nov;96(11):3502-10. doi: 10.1210/jc.2011-1449. 
15. 
Stanczyk FZ, Hapgood JP, Winer S, Mishell DR, Jr. Progestogens used in 
postmenopausal hormone therapy: differences in their pharmacological properties, 
intracellular actions, and clinical effects. Endocr Rev. 2013;34(2):171-208. 

 
 
16. 
Nabhan ZM, Dimeglio LA, Qi R, Perkins SM, Eugster EA. Conjugated oral versus 
transdermal estrogen replacement in girls with Turner syndrome: a pilot comparative study. 
J Clin Endocrinol Metab. 2009;94(6):2009-14. 
17. 
Kraus J, Unanue N, Espinoza A, Iniguez G, Torres-Santiago L, Singh R, et al. Is There a 
Difference between Ultrasonographic (US) Uterine Changes of Oral Versus Transdermal (TD) 
17beta Estradiol (17beta E2) in Girls with Turner Syndrome (TS)? Own Experience and 
Literature Review. Pediatr Endocrinol Rev. 2018;16(1):178-85. 
18. 
Hillard TC, Whitcroft SJ, Marsh MS, Ellerington MC, Lees B, Whitehead MI, et al. Long-
term effects of transdermal and oral hormone replacement therapy on postmenopausal bone 
loss. Osteoporos Int. 1994;4(6):341-8. 
19. 
Hautamaki H, Haapalahti P, Piirila P, Tuomikoski P, Sovijarvi A, Ylikorkala O, et al. Effect 
of hot flushes on cardiovascular autonomic responsiveness: a randomized controlled trial on 
hormone therapy. Maturitas. 2012;72(3):243-8. 
20.  
Derzko C, Sergerie M, Siliman G, Alberton M, Thorlund K. Comparative efficacy and 
safety of estradiol transdermal preparations for the treatment of vasomotor symptoms in 
postmenopausal women: an indirect comparison meta-analysis. Menopause. 2016;23(3):294-
303. 
21.  
Canonico M, Plu-Bureau G, Lowe GD, Scarabin PY. Hormone replacement therapy and 
risk of venous thromboembolism in postmenopausal women: systematic review and meta-
analysis. BMJ. 2008 May 31;336(7655):1227-31. doi: 10.1136/bmj.39555.441944. 
22.  
Renoux C, Dell'Aniello S, Suissa S. Hormone replacement therapy and the risk of 
venous thromboembolism: a population-based study. J Thromb Haemost. 2010 
May;8(5):979-86. doi: 10.1111/j.1538-7836.2010.03839.x. 
23.  
Mohammed K, Abu Dabrh AM, Benkhadra K, Al Nofal A, Carranza Leon BG, Prokop LJ, 
Montori VM, Faubion SS, Murad MH. Oral vs Transdermal Estrogen Therapy and Vascular 
Events: A Systematic Review and Meta-Analysis. J Clin Endocrinol Metab. 2015 
Nov;100(11):4012-20. doi: 10.1210/jc.2015-2237. PMID: 26544651. 
24.  
Rossouw JE, Prentice RL, Manson JE, Wu L, Barad D, Barnabei VM, Ko M, LaCroix AZ, 
Margolis KL, Stefanick ML. Postmenopausal hormone therapy and risk of cardiovascular 
disease by age and years since menopause. JAMA. 2007 Apr 4;297(13):1465-77. doi: 
10.1001/jama.297.13.1465. Erratum in: JAMA. 2008 Mar 26;299(12):1426. PMID: 17405972. 
25. 
 Hsia J, Langer RD, Manson JE, Kuller L, Johnson KC, Hendrix SL, Pettinger M, Heckbert 
SR, Greep N, Crawford S, Eaton CB, Kostis JB, Caralis P, Prentice R; Women's Health Initiative 
Investigators. Conjugated equine estrogens and coronary heart disease: the Women's Health 
Initiative. Arch Intern Med. 2006 Feb 13;166(3):357-65. doi: 10.1001/archinte.166.3.357. 
Erratum in: Arch Intern Med. 2006 Apr 10;166(7):759. PMID: 16476878. 
26.  
Hulley S, Grady D, Bush T, Furberg C, Herrington D, Riggs B, Vittinghoff E. Randomized 
trial of estrogen plus progestin for secondary prevention of coronary heart disease in 
postmenopausal women. Heart and Estrogen/progestin Replacement Study (HERS) Research 
Group. JAMA. 1998 Aug 19;280(7):605-13. doi: 10.1001/jama.280.7.605. PMID: 9718051. 
27.  
Grady D, Herrington D, Bittner V, Blumenthal R, Davidson M, Hlatky M, Hsia J, Hulley 
S, Herd A, Khan S, Newby LK, Waters D, Vittinghoff E, Wenger N; HERS Research Group. 
Cardiovascular disease outcomes during 6.8 years of hormone therapy: Heart and 
Estrogen/progestin Replacement Study follow-up (HERS II). JAMA. 2002 Jul 3;288(1):49-57. 
doi: 10.1001/jama.288.1.49. Erratum in: JAMA 2002 Sep 4;288(9):1064. PMID: 12090862. 
28. 
 Curb JD, Prentice RL, Bray PF, Langer RD, Van Horn L, Barnabei VM, Bloch MJ, Cyr MG, 
Gass M, Lepine L, Rodabough RJ, Sidney S, Uwaifo GI, Rosendaal FR. Venous thrombosis and 

 
 
conjugated equine estrogen in women without a uterus. Arch Intern Med. 2006 Apr 
10;166(7):772-80. doi: 10.1001/archinte.166.7.772. PMID: 16606815. 
29.  
Cushman M, Kuller LH, Prentice R, Rodabough RJ, Psaty BM, Stafford RS, Sidney S, 
Rosendaal FR; Women's Health Initiative Investigators. Estrogen plus progestin and risk of 
venous thrombosis. JAMA. 2004 Oct 6;292(13):1573-80. doi: 10.1001/jama.292.13.1573. 
PMID: 15467059. 
30.  
Abdullah WZ, Kumaraguru S, Ghazali S, Yusoff NM. Factor V Leiden and prothrombin 
G20210A mutations among healthy Indians in Malaysia. Laboratory Medicine. 
2010;41(5):284-7. 
31. 
 Yusoff NM, Abdullah WZ, Ghazali S, Othman MS, Baba AA, Abdullah N, Isa MN, Chong 
CL. The absence of factor V Leiden mutation in Malays with recurrent spontaneous abortions. 
Aust N Z J Obstet Gynaecol. 2002 May;42(2):164-6. 
32.  
Ayadurai T, Muniandy S, Omar SZ. Thrombophilia investigation in Malaysian women 
with recurrent pregnancy loss. J Obstet Gynaecol Res. 2009 Dec;35(6):1061-8. doi: 
10.1111/j.1447-0756.2009.01067.x. PMID: 20144172. 
33.  
Grady D, Gebretsadik T, Kerlikowske K, Ernster V, Petitti D. Hormone replacement 
therapy and endometrial cancer risk: a meta-analysis. Obstet Gynecol. 1995 Feb;85(2):304-
13. doi: 10.1016/0029-7844(94)00383-O. PMID: 7824251. 
34.  
Beral V, Bull D, Reeves G; Million Women Study Collaborators. Endometrial cancer and 
hormone-replacement therapy in the Million Women Study. Lancet. 2005 Apr 30-May 
6;365(9470):1543-51. doi: 10.1016/S0140-6736(05)66455-0. 
35.  
Sjögren LL, Mørch LS, Løkkegaard E. Hormone replacement therapy and the risk of 
endometrial 
cancer: 
A 
systematic 
review. 
Maturitas. 
2016 
Sep;91:25-35. 
doi: 
10.1016/j.maturitas.2016.05.013. Epub 2016 Jun 1. PMID: 27451318. 
36. Stefanick ML, et al. Effects of Conjugated Equine Estrogens on Breast Cancer and 
Mammography Screening in Postmenopausal Women With Hysterectomy. JAMA. 
2006;295:1647-1657. 
37.  
Chlebowski RT, et al. Influence of Estrogen Plus Progestin on Breast Cancer and 
Mammography in Healthy Postmenopausal Women. JAMA. 2003;289:3234-3253. 
38.  
Lee AW, Ness RB, Roman LD, Terry KL, Schildkraut JM, Chang-Claude J, Doherty JA, 
Menon U, Cramer DW, Gayther SA, Risch H, Gentry-Maharaj A, Goodman MT, Modugno F, 
Eilber U, Moysich KB, Berchuck A, Rossing MA, Jensen A, Wicklund KG, Cushing-Haugen KL, 
Hogdall E, Rudolph A, Thompson PJ, Wilkens LR, Kjaer SK, Carney ME, Stram DO, Ramus SJ, 
Wu AH, Pike MC, Pearce CL; Ovarian Cancer Association Consortium. Association Between 
Menopausal Estrogen-Only Therapy and Ovarian Carcinoma Risk. Obstet Gynecol. 2016 
May;127(5):828-836. doi: 10.1097/AOG.0000000000001387. 
39. 
Shumaker SA, Legault C, Kuller L, Rapp SR, Thal L, Lane DS, Fillit H, Stefanick ML, 
Hendrix SL, Lewis CE, Masaki K, Coker LH; Women's Health Initiative Memory Study. 
Conjugated equine estrogens and incidence of probable dementia and mild cognitive 
impairment in postmenopausal women: Women's Health Initiative Memory Study. JAMA. 
2004 Jun 23;291(24):2947-58. doi: 10.1001/jama.291.24.2947. PMID: 15213206. 
40. 
Espeland MA, Rapp SR, Shumaker SA, Brunner R, Manson JE, Sherwin BB, Hsia J, 
Margolis KL, Hogan PE, Wallace R, Dailey M, Freeman R, Hays J; Women's Health Initiative 
Memory Study. Conjugated equine estrogens and global cognitive function in 
postmenopausal women: Women's Health Initiative Memory Study. JAMA. 2004 Jun 
23;291(24):2959-68. doi: 10.1001/jama.291.24.2959. PMID: 15213207. 

 
 
41. 
Pourhadi N, Mørch LS, Holm EA, Torp-Pedersen C, Meaidi A. Menopausal hormone 
therapy and dementia: nationwide, nested case-control study. BMJ. 2023 Jun 
28;381:e072770. doi: 10.1136/bmj-2022-072770. Erratum in: BMJ. 2023 Jun 29;381:p1499. 
PMID: 37380194; PMCID: PMC10302215. 
42. 
 Cirillo DJ, Wallace RB, Rodabough RJ, Greenland P, LaCroix AZ, Limacher MC, Larson 
JC. Effect of estrogen therapy on gallbladder disease. JAMA. 2005 Jan 19;293(3):330-9. doi: 
10.1001/jama.293.3.330. PMID: 15657326. 
43. Beckett VL. Hypercalcemia associated with estrogen administration in patients with breast 
carcinoma. Cancer. 1969 Sep;24(3):610-6. doi: 10.1002/1097-0142(196909)24:3<610::aid-
cncr2820240328>3.0.co;2-4. 
44. Coelingh Bennink HJ, Verhoeven C, Dutman AE, Thijssen J. The use of high-dose estrogens 
for 
the 
treatment 
of 
breast 
cancer. 
Maturitas. 
2017 
Jan;95:11-23. 
doi: 
10.1016/j.maturitas.2016.10.010. 
45.  
Cahill M, O’Toole L, Acheson RW. Hormone replacement therapy and retinal vein 
occlusion. Eye (Lond). 1999;13(Pt 6):798-800. doi: 10.1038/eye.1999.236 
46. 
Girolami A, Vettore S, Tezza F, Girolami B. Retinal central artery occlusion in a young 
woman after ten days of a drospirenone-containing oral contraceptive (Yasmin). Thromb 
Haemost. 2007;98(2):473-474. 
47. 
Song D, Nadelmann J, Yu Y, VanderBeek BL. Association of Retinal Vascular Occlusion 
With Women Filling a Prescription for Female Hormone Therapy. JAMA Ophthalmol. 2021 Jan 
1;139(1):42-48. doi: 10.1001/jamaophthalmol.2020.4884. 
48. Smith DC, Prentice R, Thompson DJ, Herrmann WL. Association of exogenous estrogen 
and endometrial carcinoma. New England Journal of Medicine. 1975 Dec 4;293(23):1164-7. 
doi: 10.1056/ NEJM197512042932302. PMID: 1186789. 
49. Ziel HK, Finkle WD. Increased risk of endometrial carcinoma among users of conjugated 
estrogens. New England Journal of Medicine. 1975 Dec 4;293(23):1167-70. doi: 
10.1056/NEJM197512042932303. PMID: 171569. 
50. Weiderpass E, Adami HO, Baron JA, Magnusson C, Bergström R, Lindgren A, Correia N, 
Persson I. Risk of endometrial cancer following estrogen replacement with and without 
progestins. J Natl Cancer Inst. 1999 Jul 7;91(13):1131-7. doi: 10.1093/jnci/91.13.1131. PMID: 
10393721. 
51. Doherty JA, Cushing-Haugen KL, Saltzman BS, Voigt LF, Hill DA, Beresford SA, Chen C, Weiss 
NS. Long-term use of postmenopausal estrogen and progestin hormone therapies and the risk 
of endometrial cancer. American Journal Obstetrics & Gynecology. 2007 Aug;197(2):139.e1-
7. doi: 10.1016/j.ajog.2007.01.019. PMID: 17689625. 
52. Schairer C, Lubin J, Troisi R, Sturgeon S, Brinton L, Hoover R. Menopausal estrogen and 
estrogen-progestin replacement therapy and breast cancer risk. JAMA. 2000 Jan 
26;283(4):485-91. doi: 10.1001/jama.283.4.485. Erratum in: JAMA 2000 Nov 22-
29;284(20):2597. PMID: 10659874. 
53.  
Kalenga CZ, Hay JL, Boreskie KF, Duhamel TA, MacRae JM, Metcalfe A, Nerenberg KA, 
Robert M, Ahmed SB. The Association Between Route of Post-menopausal Estrogen 
Administration and Blood Pressure and Arterial Stiffness in Community-Dwelling Women. 
Front Cardiovasc Med. 2022 Jun 10;9:913609. doi: 10.3389/fcvm.2022.913609. 
54. 
Kalenga CZ, Metcalfe A, Robert M, Nerenberg KA, MacRae JM, Ahmed SB. Association 
Between the Route of Administration and Formulation of Estrogen Therapy and Hypertension 
Risk in Postmenopausal Women: A Prospective Population-Based Study. Hypertension. 2023 
Jul;80(7):1463-1473. doi: 10.1161/HYPERTENSIONAHA.122.19938. 

 
 
55. 
 Aljenedil S, Hegele RA, Genest J, Awan Z. Estrogen-associated severe 
hypertriglyceridemia with pancreatitis. J Clin Lipidol. 2017 Jan-Feb;11(1):297-300. doi: 
10.1016/j.jacl.2016.12.006. Epub 2016 Dec 23. PMID: 28391900. 
56. 
Glass SJ, Edmonson HA, Soll SN. Sex hormone changes associated with liver disease. 
Endocrinology. 1940 Nov 1;27(5):749-52. 
57. 
Arafah BM. Increased need for thyroxine in women with hypothyroidism during 
estrogen 
therapy. 
N 
Engl 
J 
Med. 
2001 
Jun 
7;344(23):1743-9. 
doi: 
10.1056/NEJM200106073442302. PMID: 11396440. 
58. 
Stachenfeld NS, Taylor HS. Effects of estrogen and progesterone administration on 
extracellular fluid. Journal of Applied Physiology. 2004 Mar;96(3):1011-8. 
59. 
Burckhardt P, Ruedi B, Felber JP. Estrogen-induced tetany in idiopathic 
hypoparathyroidism. Hormone Research in Paediatrics. 1975 Nov 21;6(5-6):321-8. 
60. 
Phillips MR, Kaiser P, Thabane L, Bhandari M, Chaudhary V; Retina Evidence Trials 
InterNational Alliance (R.E.T.I.N.A.) Study Group. Risk of bias: why measure it, and how? Eye 
(Lond). 2022 Feb;36(2):346-348. doi: 10.1038/s41433-021-01759-9. 
61.  
Berry DA. Introduction to Bayesian methods III: use and interpretation of Bayesian 
tools in design and analysis. Clin Trials. 2005;2(4):295-300; discussion 301-4, 364-78. doi: 
10.1191/1740774505cn100oa. PMID: 16281428. 
62.  
Pocock SJ, Ariti CA, Collier TJ, Wang D. The win ratio: a new approach to the analysis 
of composite endpoints in clinical trials based on clinical priorities. Eur Heart J. 2012 
Jan;33(2):176-82. doi: 10.1093/eurheartj/ehr352. Epub 2011 Sep 6. PMID: 21900289. 
63. 
Herman D, Lor KY, Qadree A, Horn D, D'Souza R. Composite adverse outcomes in 
obstetric studies: a systematic review. BMC Pregnancy Childbirth. 2021 Feb 5;21(1):107. doi: 
10.1186/s12884-021-03588-w. PMID: 33546638; PMCID: PMC7863533. 
64. 
Montori VM, Permanyer-Miralda G, Ferreira-González I, Busse JW, Pacheco-Huergo V, 
Bryant D, Alonso J, Akl EA, Domingo-Salvany A, Mills E, Wu P, Schünemann HJ, Jaeschke R, 
Guyatt GH. Validity of composite end points in clinical trials. BMJ. 2005 Mar 
12;330(7491):594-6. doi: 10.1136/bmj.330.7491.594. PMID: 15761002; PMCID: PMC554039. 
65. 
Palileo-Villanueva LM, Dans AL. Composite endpoints. J Clin Epidemiol. 2020 
Dec;128:157-158. doi: 10.1016/j.jclinepi.2020.07.017. Epub 2020 Jul 30. PMID: 32739473. 
66.    Ministry of Health Malaysia. Clinical practice guidelines: management of obesity. 2nd 
ed. Putrajaya (Malaysia): Malaysia Health Technology Assessment Section (MaHTAS), Ministry 
of 
Health 
Malaysia; 
2023. 
ISBN 
978-967-2887-53-9. 
Available 
from: 
https://www.moh.gov.my/moh/resources/Penerbitan/CPG/Endocrine/CPG_Management_
of_Obesity_(Second_Edition)_2023.pdf 
67.  
Archer DF, Maheux R, DelConte A, O'Brien FB. A new low-dose monophasic 
combination oral contraceptive (Alesse) with levonorgestrel 100 micrograms and ethinyl 
estradiol 20 micrograms. North American Levonorgestrel Study Group (NALSG). 
Contraception. 1997 Mar;55(3):139-44. doi: 10.1016/s0010-7824(97)00024-3. 
68. 
Bannemerschult R, Hanker JP, Wünsch C, Fox P, Albring M, Brill K. A multicenter, 
uncontrolled clinical investigation of the contraceptive efficacy, cycle control, and safety of a 
new low dose oral contraceptive containing 20 micrograms ethinyl estradiol and 100 
micrograms levonorgestrel over six treatment cycles. Contraception. 1997 Nov;56(5):285-90. 
doi: 10.1016/s0010-7824(97)00157-1. 
69. 
 Schonhoff P, Körner A, Kratzsch J, Pfäffle R, Kiess W. Long term clinical management of girls 
with Turner syndrome at a center of pediatric endocrinology. Exp Clin Endocrinol Diabetes. 
2011;119(6):327-33. doi: 10.1055/s-0031-1277140. Epub 2011 May 6. PMID: 21553360. 

 
 
70. 
 Labarta JI, Moreno ML, López-Siguero JP, Luzuriaga C, Rica I, Sánchez-del Pozo J, Gracia-
Bouthelier R; Spanish Turner working group. Individualised vs fixed dose of oral 17β-oestradiol for 
induction of puberty in girls with Turner syndrome: an open-randomised parallel trial. Eur J Endocrinol. 
2012;167(4):523-9. doi: 10.1530/EJE-12-0444. Epub 2012 Jul 17. PMID: 22807477. 
71. 
 Kim NY, Lee DY, Kim MJ, Yoon BK, Choi D. Estrogen requirements in girls with Turner 
syndrome; how low is enough for initiating puberty and uterine development? Gynecol Endocrinol. 
2012;28(2):130-3. doi: 10.3109/09513590.2011.588752. Epub 2011 Jul 27. PMID: 21793702. 
72. 
Shah S, Forghani N, Durham E, Neely EK. A randomized trial of transdermal and oral estrogen 
therapy in adolescent girls with hypogonadism. Int J Pediatr Endocrinol. 2014;2014(1):12. doi: 
10.1186/1687-9856-2014-12. Epub 2014 Jun 20. PMID: 24982681; PMCID: PMC4074834. 
73. 
Schoenfeld DA, Richter JR. Nomograms for calculating the number of patients needed for a 
clinical trial with survival as an endpoint. Biometrics. 1982 Mar;38(1):163-70. 
74. 
Cohen J. Statistical Power Analysis for the Behavioral Sciences.  New York (USA): Routledge 
Academic; 1988 
75. 
 Thompson B. Effect sizes, confidence intervals, and confidence intervals for effect sizes. 
Psychology in Schools. 2007;44(5): 423–432. doi: 10.1002/pits.20234 
76. 
Kunzmann, K., M. J. Grayling, K. M. Lee, D. S. Robertson, K. Rufibach, and J. M. S. Wason (2021). 
A review of Bayesian perspectives on sample size derivation for confirmatory trials. The American 
Statistician 75, 424–432.  
77. 
 Lindley, D. V. (1997). The choice of sample size. The Statistician 46, 129–138.  
78.  
 Wilson KJ, Williamson SF, Allen AJ, Williams CJ, Hellyer TP, Lendrem BC. Bayesian sample size 
determination for diagnostic accuracy studies. Stat Med. 2022 Jul 10;41(15):2908-2922. doi: 
10.1002/sim.9393. 
79.  
  Barnett V. Comparative Statistical Inference. Chichester: Wiley; 1999. 
80.  
  Stallard N. Optimal sample sizes for phase II clinical trials and pilot studies. Stat Med. 2012 
May 20;31(11-12):1031-42. doi: 10.1002/sim.4357.  
81. 
 Wise LA, Mikkelsen EM, Rothman KJ, Riis AH, Sørensen HT, Huybrechts KF, Hatch EE. A 
prospective cohort study of menstrual characteristics and time to pregnancy. Am J Epidemiol. 
2011;174(6):701-9. doi: 10.1093/aje/kwr130. 
82. 
Mejia M, McNicholas C, Madden T, Peipert JF. Association of baseline bleeding pattern on 
amenorrhea with levonorgestrel intrauterine system use. Contraception. 2016;94(5):556-560. doi: 
10.1016/j.contraception.2016.06.013. 
83. 
Wesselink AK, Wise LA, Hatch EE, Rothman KJ, Mikkelsen EM, Stanford JB, McKinnon CJ, 
Mahalingaiah S. Menstrual cycle characteristics and fecundability in a North American preconception 
cohort. Ann Epidemiol. 2016;26(7):482-487.e1. doi: 10.1016/j.annepidem.2016.05.006. 
84.  
Zhang J, Zhu C, Yan L, Wang Y, Zhu Q, He C, He X, Ji S, Tian Y, Xie L, Liang Y, Xia W, Mol BW, 
Huirne JAF. Comparing levonorgestrel intrauterine system with hysteroscopic niche resection in 
women with postmenstrual spotting related to a niche in the uterine cesarean scar: a randomized, 
open-label, 
controlled 
trial. 
Am 
J 
Obstet 
Gynecol. 
2023;228(6):712.e1-712.e16. 
doi: 
10.1016/j.ajog.2023.03.020. 
85. 
Aronson JK. Medication errors: what they are, how they happen, and how to avoid them. QJM. 
2009;102(8):513-21. doi: 10.1093/qjmed/hcp052. 
86. 
Code of Federal Regulation, Title 21, Section 312.32 (21CFR312.32). (2020). Accessible from: 
https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/cfrsearch.cfm?fr=312.32. Date of 
access: 31st / 08 / 2021 
87.  
Naranjo CA, Busto U, Sellers EM, Sandor P, Ruiz I, Roberts EA, Janecek E, Domecq C, Greenblatt 
DJ. A method for estimating the probability of adverse drug reactions. Clin Pharmacol Ther. 
1981;30(2):239-45. doi: 10.1038/clpt.1981.154.  
88. 
 US Department of Health and Human Services. Common Terminlogy Criteria for Adverse 

 
 
Events 
(CTCAE) 
version 
5. 
(2017) 
Accessible 
from: 
https://ctep.cancer.gov/protocoldevelopment/electronic_applications/docs/CTCAE_v5_Quick_Refer
ence_5x7.pdf. Date of access: 31st March 2023 
89. 
Center for Biologics Evaluation and Research, US Food and Drug Administration (US FDA). 
Guidance for Industry: Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers Enrolled in 
Preventive 
Clinical 
Trials. 
(Sept 
2007). 
Accessible 
from: 
https://www.fda.gov/media/73679/download. Date of access: 31st March 2023. 
90. 
Hageman S, Huang Z, Pennells L, Kaptoge S, Venkataraman K, Kaneko H et al. SCORE2-ASIA 
risk prediction algorithms: revised models to estimate 10- year risk of cardiovascular disease in the 
Asia 
Pacific 
Region. 
European 
Society 
of 
Cardiology 
(ESC) 
Congress 
2023. 
https://esc365.escardio.org/presentation/272307 
91. 
Pocock SJ. A major trial needs three statisticians: why, how and who? Stat Med. 2004 May 
30;23(10):1535-9. doi: 10.1002/sim.1791. PMID: 15122734. 
92.  
Thorlund K, Dron L, Park JJH, Mills EJ. Synthetic and External Controls in Clinical Trials - A 
Primer for Researchers. Clin Epidemiol. 2020 May 8;12:457-467. doi: 10.2147/CLEP.S242097.  
93.  Kingma DP, Welling M. Auto-encoding variational bayes. arXiv preprint arXiv:1312.6114. 2013 Dec 
20. 
94. 
 Goodfellow I, Pouget-Abadie J, Mirza M, Xu B, Warde-Farley D, Ozair S, Courville A, Bengio Y. 
Generative adversarial networks. Communications of the ACM. 2020 Oct 22;63(11):139-44. 
95.  
Jiang L, Thall PF, Yan F, Kopetz S, Yuan Y. BASIC: A Bayesian adaptive synthetic-control design 
for phase II clinical trials. Clin Trials. 2023 Oct;20(5):486-496. doi: 10.1177/17407745231176445. 
96. Ibrahim JG, Chen MH, Gwon Y, Chen F. The power prior: theory and applications. Stat Med. 2015 
Dec 10;34(28):3724-49. doi: 10.1002/sim.6728. 
97.  
Hobbs BP, Carlin BP, Mandrekar SJ, Sargent DJ. Hierarchical commensurate and power prior 
models for adaptive incorporation of historical information in clinical trials. Biometrics. 2011 
Sep;67(3):1047-56. doi: 10.1111/j.1541-0420.2011.01564.x. 
98.  
Johnson AA, Ott MQ, Mine Dogucu. Bayes rules! : an introduction to Bayesian modeling with 
R. Boca Raton: Chapman & Hall/Crc; 2022. 
99.  
 Landis JR, Koch GG. The measurement of observer agreement for categorical data. 
Biometrics. 1977;33(1):159–174. 
100. 
 McHugh ML. Interrater reliability: the kappa statistic. Biochemia Medica. 2012;22(3):276–
282. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
BUDGET (ONLY FOR PROGYNOVA SUBCOMPONENT OF THE GUP PROJECT) 
 
Vot Number 
Details 
 
Total 
27000 
Progynova (tablets) for 12 
patients 
assigned 
to 
the 
Progynova arm based on the 
planned intervention schedule 
(cost per unit: RM 21.90 per 
Progynova box containing 28 
Progynova tablets) 
Hence, per patient, 38.75 
Progynova boxes are required. 
 
38.75 boxes x RM 21.90 X 12 
patients  = RM 9997.50 
Grand Total 
 
RM 9997.50 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
APPENDIX A: Pictorial details on the Tanner Staging for Breast and Pubic Hair Development 
 
 
 
 
 

 
 
APPENDIX I 
 
 
 
PATIENT INFORMATION SHEET 
 
Research Title:  
 
 
Comparison of Hormone Replacement Therapy Regimes for Pubertal Induction in Adolescents 
and Young Women with Turner Syndrome Introduction 
 
Introduction:  
You are invited to participate in a research study. Before participating in this study, it is crucial 
that you thoroughly read and understand the information provided in this sheet. However, 
before you take part or agree to continue in this research study, the study will be verbally 
explained to you, and you will be allowed to ask questions. After you are adequately satisfied 
that you understand this study and you wish to take part or continue to participate in this study, 
you must sign this informed consent form. You will be given a copy of these patient 
information sheet and consent forms to take home with you. 
 
Purpose of Study: 
Pubertal delay or absence is a common finding in Turner Syndrome patients. Therefore, 
pubertal induction using hormone replacement therapy (HRT) is required in individuals with 
Turner Syndrome. However, the best HRT regime for the induction of puberty in Turner 
Sydrome patients is still unknown for certain. Hence, this study aims to determine whether oral 
estradiol valerate (Progynova) or Transdermal formulation of 17β Estradiol (Oestrogel) is the 
better HRT regime for inducing puberty in Turner Syndrome patients. 
 
What will the study involve? 
You / Your child will undergo a pubertal induction treatment. You / Your child will be 
randomly allocated to either Progynova (and a dummy gel) or Oestrogel (and a dummy tablet) 
intervention arm based on the intervention sequences that we have created. You / Your child 
will be then followed up for every three monthly for the span of two years (24 months). During 
the follow-up visits, your compliance with  the allotted interventions will be evaluated and a 
series of clinical examinations and radiological assessments to evaluate breast development, 
withdrawal bleeding status, time to first withdrawal uterine bleeding from the first day of 
intervention and bone mineral profile using DEXA scan will be performed. 
 
For this study, we will be evaluating whether Progynova or Oestrogel will have a better pubertal 
induction efficacy as evidenced from speedier breast development, withdrawal bleeding 
occurrence, shorter time to the first withdrawal bleeding occurrence and better bone mineral 
profile that will be assessed at several periodic follow-up time points. 
 
Risks:  
The interventions (Progynova or Oestrogel) pose several risks to the study participants. Risks 
of oestrogen replacement therapy with Progynova or Oestrogel may involve, but are not 
restricted to heart attacks, clot development in the blood vessels, stroke, gallstone formation, 
liver disease, breast cancer, womb cancer and fibroid tumours (non-cancerous growth). Among 
the side effects associated with Progynova or Oestrogel are increased body fat, bloating, water 
retention (excess fluid) in the body, bleeding from the womb, low mood, headaches, worsening 
of migraine and impaired blood sugar level. However, these risks are small (around 1-10% in 

 
 
post-menopausal women undergoing hormone replacement therapy) and can be much smaller 
in young Turner Syndrome patients (none so far in our experience).  
 
Should you / your child experience one of the side effects above, we will promptly give 
appropriate treatments to mitigate such side effects. If the side effects are serious, the 
interventions will be promptly and completely halted. You/your child will be then withdrawn 
from the trial. 
 
Benefits:   
You / Your child may experience normal pubertal development. You / Your child may also 
experience other HRT-associated benefits such as a general sense of well-being, increased 
energy, reduced hot flushes and/or night sweats, reduced bone loss, enhanced memory and 
cognitive ability, reduction in sleep problems (insomnia) and possible reductions in future risks 
of dementia and heart diseases. 
 
Do you have to take part? 
Participation in this study is absolutely voluntary. Your / your child's medical care is not 
affected if you / your child decide(s) not to participate in this study. You / Your child will have 
the usual standard of care according to the day-care protocol.  
 
If you agree to participate, you will be asked to sign the "Informed Consent Form". You will 
be given a copy of the informed consent form and this Patient Information Sheet. Should you 
decide to participate, you are still free to withdraw from the study at any time without giving a 
reason or penalty. If you decide to cease participating in this study, you must inform your study 
investigator and no new data will be collected from your child. The researcher may also remove 
your child from the study for various reasons. In this event, your child will not lose his/her 
rights as a patient and will still receive the usual standard of care. 
 
Data & Confidentiality: 
Participant’s confidentiality will be maintained throughout the investigation. The personal data 
will be anonymized. Hence your identity will be kept confidential. Data collected and entered 
into the Case Report Form and the Electronic Data Capture System remain the property of 
UKM. In the event of any publication regarding this study, your identity will remain 
confidential. 
 
By signing the Informed Consent form attached, you (or your legally acceptable representative, 
if relevant) are authorising such access to your study records. 
 
Payment and compensation: 
You do not have to pay, nor will you be paid to participate in this study. You do have to pay 
for the usual hospital charges.   
 
Whom can I ask about the study? 
If you have any questions about this study or your rights, please contact 
 
Principal Investigators:   
Profesor Madya Dr Ani Amelia Dato' Zainuddin 
Jabatan Obstetrik dan Ginekologi 
Pusat Perubatan Universiti Kebangsaan Malaysia 
No. Telefon : 03-9145 5950 

 
 
Co-investigator  
Profesor Dr Nur Azurah Abdul Ghani 
Jabatan Obstetrik dan Ginekologi 
Pusat Perubatan Universiti Kebangsaan Malaysia 
No. Telefon : 03-9145 6485 
 
 
Signatures 
 
To be entered into this study, you or a legal representative must sign and date the signature 
page [APPENDIX II]        
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
APPENDIX II 
 
Patient/Subject Information and Consent Form 
(Signature Page) 
 
Research Title: Bayesian Pragmatic Trial for Pubertal Induction in Turner Syndrome: Transformation 
Initiative For Efficient Clinical TriAl Design Advancement in RarE Diseases (TRIFECTA-DARED 
framework) 
 
Researcher's Name: Professor Dr Ani Amelia Dato Zainuddin / Prof. Dr Nur Azurah Abdul Ghani  
 
To become a part of this study, you or your legal representative must sign this page. By signing this page, I am 
confirming the following: 
• 
I have read all of the information in this Patient Information and Consent Form, including any information 
regarding the risk in this study and I, have had time to think about it. 
• 
All of my questions have been answered to my satisfaction. 
• 
I voluntarily agree to be part of this research study, to follow the study procedures, and to provide necessary 
information to the doctor, nurses, or other staff members, as requested. 
• 
I may freely choose to withdraw from this study at any time without reason or without repercussion. 
• 
I understand that my anonymity will be ensured during research write-up 
• 
I have received a copy of this Patient Information and Consent Form to keep for myself. 
 
 
 
Patient Name (Print or type) 
 
 
 
 
 
Patient Initials and Number 
 
 
 
Patient I.C No. (New) 
 
 
 
 
 
 
 
 
 
 
 
Signature of Patient or Legal Representative  
 
 
 
Date (dd/MM/yy) 
(Add time if applicable) 
 
 
 
Name of Individual  
Conducting Consent Discussion (Print or Type) 
 
 
 
 
Signature of Individual  
 
 
 
 
 
              Date (dd/MM/yy) 
Conducting Consent Discussion 
 
 
 
 
 
Name & Signature of Witness 
 
 
           
 
 
Date (dd/MM/yy) 
 
 
Note:  
i) 
All subject/patients who are involved in this study will not be covered by insuranc

 
 
APPENDIX III 
 
 
20, AVENUE APPIA – CH-1211 GENEVA 27 – SWITZERLAND – HTTP://WWW.WHO.INT/ETHICS/REVIEW-COMMITTEE 
 
 
 
 
 
 
 
 
 
 
Informed Assent Form Template for Children/Minors 
Research Ethics Committee UKM  
(RECUKM) 

 
 
 
An Informed Assent Form does not replace a consent form signed by parents or guardians. The 
assent is in addition to the consent and signals the child's willing cooperation in the study.  
 
 
Informed Assent Form for Turner Syndrome Children Aged Between 11-17 years old 
who attend Pediatric and Adolescent Gynaecology Clinic and who we are inviting to 
participate in research “Bayesian Pragmatic Trial for Pubertal Induction in Turner 
Syndrome: TRansformation Initiative For Efficient Clinical TriAl Design Advancement 
in RarE Diseases (TRIFECTA-DARED framework”) 
 
 
 
 
 
 
Principal Investigator: Professor Dr Ani Amelia Zainuddin 
Location: Universiti Kebangsaan Malaysia 
Sponsor: Universiti Kebangsaan Malaysia 
Project Title: Bayesian Pragmatic Trial for Pubertal Induction in Turner Syndrome: 
TRansformation Initiative For Efficient Clinical TriAl Design Advancement in RarE 
Diseases (TRIFECTA-DARED framework) 
 
 
This Informed Assent Form has two parts: 
• Information Sheet (gives you information about the study) 
• Certificate of Assent (this is where you sign if you agree to participate) 
 
You will be given a copy of the full Informed Assent Form 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
Part I: Information Sheet 
Introduction  
 
My name is ____and my job is to research and test two types of hormone replacement therapy 
(Oestrogel and Progynova) to see which works best to stimulate normal development of breast 
and womb in children with Turner Syndrome and we think this research could help tell us that.  
 
 I am going to give you information and invite you to be part of a research study. You can 
choose whether or not you want to participate. We have discussed this research with your 
parent(s)/guardian and they know that we are also asking you for your agreement. If you are 
going to participate in the research, your parent(s)/guardian also have to agree. But if you do 
not wish to take part in the research, you do not have to, even if your parents have agreed.  
 
You may discuss anything in this form with your parents or friends, or anyone else you feel 
comfortable talking to. You can decide whether to participate or not after you have talked it 
over. You do not have to decide immediately. 
 
There may be some words you don't understand or things that you want me to explain more 
about because you are interested or concerned. Please ask me to stop at any time, and I will 
take the time to explain. 
 
Purpose: Why are you doing this research? 
We want to find better ways to improve the pubertal development in Turner Syndrome 
children. We have two types of medications for the delivery of the hormone estrogen. We thus 
want to know which one is better in inducing normal development of the breast and womb in 
children with Turner Syndrome. We need to test it to determine if it is better. 
 
Choice of participants: Why are you asking me? 
 
We are testing these two types of types of medications for hormone replacement on participants 
who are at your age - between 12 and 17 years old - who have Turner Syndrome. We want to 
know which medication is better in terms of its efficacy for stimulating puberty in children 
with Turner Syndrome.  
 
Participation is voluntary: Do I have to do this?  
You don't have to be in this research if you don't want to be. It’s up to you. If you decide not 
to be in the research, it is okay and nothing changes. This is still your clinic, everything stays 
the same as before.  Even if you say "yes" now, you can change your mind later and it is still 
okay.  
 
 If anything changes and we want you to stay in the research study even if you want to stop, 
we will talk to you first. 
 
 
I have checked with the child, and they understand that participation is voluntary 
__(initial) 
 
 
 
 

 
 
 
 
Information on the Trial Drug [Name of Drug]: What is this drug and what do you know 
about it? 
 
The types of medications that we are testing in this research are Progynova (in pill form) and 
Oestrogel (in gel form). Both medications have been in pre-menopausal adults to alleviate pre-
menopausal signs and symptoms. However, we now want to test the medications on teenagers 
and young women with Turner Syndrome to induce normal development of the breast and 
uterus.  This research is called a "phase 2" trial.  
 
Both medications have several side effects. Possible side effects include changes in mood, 
headaches, feeling bloated, gaining weight, swelling in the legs or hands, soreness in the 
breasts, and some bleeding from the womb. In rare cases, hormone treatment can cause more 
serious side effects such as blood clots, stroke, heart problems, liver problems, or breast and 
womb cancer. These risks are much more common in older women who use hormone therapy 
for many years. In young patients with Turner Syndrome, these serious side effects are 
extremely rare, and none have been reported in our experience so far. 
 
Procedures: What is going to happen to me? 
 
We are going to test the medications by giving some of the children in the research Progynova 
and others are going to receive Oestrogel.  You will be randomly allocated to receive either 
Progynova or Oestrogel.  By doing the research like this, we can compare which of the types 
of hormone replacement therapy is better without being influenced by what we think or hope 
the research will show.  
 
If you decide that you want to do this, several things will happen:  
 
1. You will come to the clinic with your parents and your eligibility (your suitability) for trial 
participation will be evaluated against certain criteria. 
  
2. If you are eligible to take part in this study, the doctor will ask about your history, will take 
blood samples, examine your breasts and will perform an ultrasound scan to measure your 
uterus.  
 
3. You will then receive either Progynova or Oestrogel and you will have to take them for two 
years (24 months) 
 
4. You also need to come to the clinic every three months to check if you are taking the 
medicine correctly, check your breast development and ultrasound scan to measure your uterus. 
And to check if you have side effects, so we can help.   
 
Altogether, you will come to the clinic 7 times over 2 years (24 months). At the end of 2 years, 
the research will be completed.   
 
 
I have checked with the child and they understand the procedures ________(initial) 
 
Risks: Is this bad or dangerous for me?  

 
 
 
Both Progynova and Oestrogel are considered safe. They have already been tested on pre-
menopausal adults. There has been nothing that has worried us at all. If anything unusual 
happens to you, however, we need to know and you should feel free to call us anytime with 
your concerns or questions. Another way for us to know how you are is by having you come 
to the clinic every 3 months for a check-up. If you get sick or have concerns or questions in-
between the scheduled visits to the clinic, you should inform me or the staff nurse. You don't 
have to wait for a scheduled visit. 
 
 
Discomforts: Will it hurt? 
 
There are a few other things I would like you to know. 
 
I do not anticipate any significant discomfort when taking the study medications. This is 
because the medications being used — Progynova, which is a tablet, and Oestrogel, which is 
applied as a gel to the skin — are both given in ways that are generally easy and painless. You 
will not be getting any injections as part of this treatment. 
 
However, as with any medication, there is a chance that you might experience some mild side 
effects. If anything feels unusual, uncomfortable, or if you’re worried about how you feel after 
using the medicine, please tell your parents or let me know as soon as possible. 
 
We don’t expect that taking part in the study will interfere much with your daily life. You may 
have to come to the clinic from time to time, but we will try to make those visits as short and 
easy as possible. If you ever feel unsure, upset, or uncomfortable about anything, it’s okay to 
speak up. 
 
I have checked with the child and they understand the risks and discomforts ____(initial) 
 
Benefits: Is there anything good that happens to me? 
Yes, there may be some good things that happen if you take part in this study. By being in this 
study, you (or your child) may start puberty in a way that is more like how it happens naturally. 
This means the body may begin to develop in ways that include breast development, the start 
of periods, and the growth of the womb. These are important parts of growing up and becoming 
more like other girls your age. 
 
The hormone treatment may also help you feel better in other ways. It might help with: 
• 
Having more energy 
• 
Sleeping better 
• 
Thinking more clearly 
• 
Feeling happier 
• 
Building stronger bones 
Starting puberty at the right time may also reduce the chances of having health problems 
later in life, such as heart disease or dementia when you're older. 
Even if you don’t get all of these benefits, the information we learn from this study 
could help doctors take better care of other girls with Turner Syndrome in the future. 
 
I have checked with the child and they understand the benefits_____ (initial) 
 

 
 
You will not have to pay to take part in this study, and you will not be paid for participating. Whilst 
you are in the trial, the initial blood tests, the ultrasound scans, visits to the doctor and the medicine 
(hormones) will be given to you free of charge. 
However, you will still need to cover the usual hospital charges that are not related to the research. 
These are the same fees you would normally pay if you were not in the study. 
 
Confidentiality: Is everybody going to know about this? 
 
We will not tell other people that you are in this research and we won't share information about 
you with anyone who does not work in the research study. After the research is over, you and 
your parents will be told which of the two injections you received and the results. 
 
Information about you that will be collected from the research will be kept away and no one 
but the researchers will be able to see it. Any information about you will have a number on it 
instead of your name. Only the researchers will know what your number is and we will lock 
that information up with a lock and key. It will not be shared with or given to anyone except 
[name who will have access to the information, such as research sponsors and your doctor. 
 
Compensation: What happens if I get hurt? 
 
If you become sick during the research, we will look after you. We have given your parents 
information about what to do if you are hurt or get sick during the research. 
 
Sharing the Findings: Will you tell me the results? 
 
When we are finished the research, I will sit down with you and your parent and I will tell you 
about what we learnt. Afterwards, we will be telling more people, scientists and others, about 
the research and what we found. We will do this by writing and sharing reports and by going 
to meetings with people who are interested in the work we do.  
 
Right to Refuse or Withdraw: Can I choose not to be in the research? Can I change my 
mind? 
 
You do not have to be in this research. No one will be mad or disappointed with you if you say 
no. It is your choice. You can think about it and tell us later if you want. You can say "yes” 
now and change your mind later and it will still be okay. 
 
Who to Contact: Who can I talk to or ask questions to? 
 
You can ask me questions now or later. I have written a number and address where you can 
reach us, or, if you are nearby, you can come and see us. If you want to talk to someone else 
that you know, like your personal doctor or relatives, that's okay too. 
 
If you choose to be part of this research, I will also give you a copy of this paper to keep 
for yourself. You can ask your parents to look after it if you want.  
You can ask me any more questions about any part of the research study, if you wish to. Do 
you have any questions?   
  

 
 
PART 2: Certificate of Assent 
 
I understand that this research is about helping girls with Turner Syndrome start puberty 
using special hormone treatments. In this study, I will get one of two types of medicine for 
estrogen replacement — either a pill called Progynova or a skin gel called Oestrogel. I won’t 
choose which one I get; it will be decided by chance (like flipping a coin). The treatment will 
continue for two years, and I will come to the hospital every three months for check-ups. 
 
At each visit, the doctor will check how I’m doing, whether I’m taking the medicine correctly, 
and how my body is developing — like whether my breasts are growing or if my periods have 
started. 
 
I have read this information (or had the information read to me), I have had my questions 
answered, and I know that I can ask questions later if I have them.  
 
I agree to take part in the research. 
 
OR 
 
I do not wish to take part in the research and I have not signed the assent 
below.___________(initialled by child/minor) 
 
Only if child assents: 
Print name of child ___________________ 
Signature of child: ____________________ 
Date:________________ 
           day/month/year    
 
 
If illiterate: 
A  literate witness must sign (if possible, this person should be selected by the participant, not be a 
parent, and should have no connection to the research team). Participants who are illiterate should 
include their thumb print as well.   
 
I have witnessed the accurate reading of the assent form to the child, and the individual 
has had the opportunity to ask questions. I confirm that the individual has given consent 
freely.  
 
Print name of witness (not a parent)_________________  AND    Thumb print of participant 
Signature of witness ______________________ 
Date ________________________ 
                Day/month/year 
 
 
 
 
 
 
 

 
 
 
I have accurately read or witnessed the accurate reading of the assent form to the 
potential participant, and the individual has had the opportunity to ask questions. I 
confirm that the individual has given assent freely.  
 
Print name of researcher_________________ 
Signature of researcher___________________  
Date__________________ 
             Day/month/year 
 
Statement by the researcher/person taking consent 
I have accurately read out the information sheet to the potential participant, and to 
the best of my ability, made sure that the child understands that the following will be 
done: 
1. 
2. 
3. 
I confirm that the child was given an opportunity to ask questions about the study, and 
all the questions asked by him/her have been answered correctly and to the best of my 
ability. I confirm that the individual has not been coerced into giving consent, and the 
consent has been given freely and voluntarily.  
   
 A copy of this assent form has been provided to the participant. 
 
Print Name of Researcher/person taking the assent________________________ 
 
 
 
 
Signature of Researcher /person taking the assent __________________________ 
Date ___________________________ 
 
 
 
                 Day/month/year 
 
 
 
 
 
 
Copy provided to the participant ________(initialed by researcher/assistant)  
 
Parent/Guardian has signed an informed consent ___Yes   ___No  _____(initialed by 
researcher/assistant) 
 
 
 
 
 
 

 
 
 
 
 
 
 
 
 
 
 
 
APPENDIX IV 
CASE REPORT FORM 
 
Research title: Bayesian Pragmatic Trial for Pubertal Induction in Turner Syndrome: 
Transformation Initiative For Efficient Clinical TriAl Design Advancement in RarE Diseases 
(TRIFECTA-DARED framework) 
 
Research Investigators: Dr Muhammad Irfan Abdul Jalal, Associate Prof Dr Ani Amelia 
Zainuddin, Professor Dato’ Dr A Rahman A Jamal, Professor Dr Nur Azurah Abdul Ghani, Dr 
Esther Loh Sweet Yi. 
 
 
 
Randomisation ID: 
 
 
A. SUBJECT’S DETAIL 
 
 
Subject’s name: 
 
 
 
 
Age 
: 
         years 
          months 
 
Registration No 
(RN)  : 
 
 
Telephone no 
: 
Handphone: 
 
 
 
 
Home         : 
 
 
 
 
 
Date of birth 
: 
 
/  
/ 
 
 
 
 
 
 
 
 
 
 
 
 
 
Date of consent: 
 
/  
/  
 
 
 
 
 
 
 
 
Hospital Sticker 
Subject ID 
 
 
 
 
 
 
Date: 
 
 
/  
 
/  
 
 
 
 
Day 
 
Month 
 
Year 
 

 
 
Ethnicity 
: 
 
Malay 
 
Chinese 
 
Indian 
 
 
 
 
 
 
 
 
 
Others (please specify): 
 
 
 
B. FAMILY HISTORY OF ILLNESS 
 
Has any of the family members having any significant disease in their history of illness. 
 
First degree 
 
Yes 
No 
 
 
 
 
 
 
 
 
Father 
 
 
 
 
 
 
 
 
 
Mother 
 
 
 
 
 
 
 
 
 
 
Siblings 
 
 
 
 
 
 
 
 
 
 
 
Others: 
 
 
 
 
 
 
 
If Yes, please specify the types of diseases 
and age at diagnosis: 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
C. REVIEW OF MEDICAL HISTORY / MEDICATIONS CHECKLIST 
 
Relevant medical history (Y/N) 
 
Diabetes  
 
: 
 
 
 
Hypertension 
 
: 
 
 
 
Hyperlipidemia 
 
: 
 
 
 
Hypothyroidism  
: 
 
 
 
 Autoimmune Disease 
: 
 
 
 
Cancer 
 
 
:           
 
 
 
Osteoporosis 
 
:           
 
 
Others 
 
   : 
 
 
 
Medications (please specify the name, indications, dosage and administration 
frequency) 
 
Name 
Reasons 
Doses 
Frequency of 
administration 
Duration been 
on this 
medication 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Has subject ever experienced any allergy or adverse event from any medications? 
 
 
Yes *  
 
 
No 
 
 
 
 
 
 
 
 
 
 

 
 
 
*If yes, please specify the 
type of medication: 
 
 
D. CLINICAL DATA 
 
Date of first physical examination:  
 
i) Anthropometric measurement 
 
1. Weight at recruitment : 
kg 
 
 
2. Height at recruitment : 
cm 
 
3. Body mass index (BMI) at recruitment : 
kg/𝑚2 
 
4. Systolic Blood Pressure (SBP): 
 
mmHg 
 
5. Diastolic Blood Pressure (DBP):  
mmHg 
 
ii) Turner Syndrome’s profile 
 
1. Karyotype  
:    
 
2.  Breakpoint locations :  
 
3.  Cell line ratios (for mosaicism) :  
 
4.  Confirmed with the genetic report 
:  
 
5.  Type of genetic test  
 
: 
 
6.  Date of diagnosis (genetic test) 
: 
 
7. Age at diagnosis 
 
 
: 
 
7. Height at diagnosis 
:      
cm 
 
8. Weight at diagnosis :     
kg 
 
9.   Father’s height 
:     
cm 
 
10. Father’s weight 
: 
kg 
 
11. Mother’s height 
: 
cm 
 
12. Mother’s weight 
: 
kg 
 

 
 
 
13. Menstrual status  (Y/N)* (if yes, give date of 1st menstruation) 
: 
 
14. Puberty status (Y/N) : 
 
15. Has gonadectomy been done? Y/N 
If have been performed, please answer questions no 16 & 17 
 
16. Left gonad histopathology 
(describe)  
: 
 
17. Right gonad histopathology (describe) 
: 
 
18. Inspection of External genitalia (describe) 
: 
 
19. Genital surgery (Y/N) (if yes, what was the indication and what surgery was 
performed and when - year) 
: 
 
20. Tanner Stage (Breast)  
: 
 
21. Tanner Stage (Pubic Hair) 
: 
 
22. Presence of axillary hair: Y / N 
 
 
iii) USS Uterine profile (baseline) date of baseline ultrasound scan :  
 
Ultrasonographer’s name:  
 
i) Uterine length :  
cm 2 readings each 
 
ii) Uterine AP diameter:   cm 2 readings each 
 
 
 
 

 
 
APPENDIX V 
ADVERSE EVENTS TRACKING LOG 
 
No. 
Date 
reported 
Adverse event 
description 
Start 
date 
End 
date 
Ongoing 
(Yes or No) 
Outcome1 
Severity / 
grade2 
Serious 
(Yes or No) 
AE 
treatment3 
Expected 
(Yes or No) 
Intervention 
Attribution / 
Relatedness4 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Scales: 
Outcome1 
Severity / grade2 
AE treatment3 
Intervention Attribution / 
Relatedness4 
0- Fatal 
1- Not recovered / Not resolved 
2- Recovered w/sequelae 
3- Recovered w/o sequelae 
4- Recovering / Resolving 
0- Mild 
1- Moderate 
2- Severe 
3- Life-threatening 
4- Death / Fatal 
0- None 
1- Medication(s) 
2- Medication TX 
0- Definite 
1- Probable 
2- Possible 
3- Unrelated 
4- Not applicable 
(did not receive intervention) 
 
Verified by (Prior to data entry): 
Signature: 
Name: 
Date 

 
 
APPENDIX VI 
BASELINE AND FOLLOW UP MEASUREMENTS 
Visit No 
1 
2 
3 
4 
5 
Scheduled Time 
Day 1  
Month 1 
Month 7 
Month 13 
Month 19 
Visit Windows 
  
Baseline 
(Consent) Visit 
1 month after 
Visit 1 ± 1 week 
6 Months after Visit 2 ± 1 
week 
6 Months after Visit 3 
± 1 week 
6 Months After Visit 4 ± 1 week 
Clinical Parameters 
 
 
 
 
 
Systolic Blood Pressure 
(SBP) (mmHg) 
 
 
 
 
 
Diastolic Blood Pressure 
(DBP) (mmHg) 
 
 
 
 
 

 
 
Pubertal Development 
 
 
 
 
 
Tanner Stage (Breast 
Development) 
 
 
 
 
 
Withdrawal bleeding 
status (Y/N) if yes, put in 
date of forst withdrawal 
bleeding  
 
 
 
 
 
Time to the 1st 
withdrawal bleeding (only 
fill in when withdrawal 
bleeding 1st occurs) 
 
 
 
 
 
Laboratory Parameters 
 
 
 
 
 
Serum FSH Level (U/L) 
 
 
 
 
 

 
 
Need only 2 readings at 
the most at baseline 
Serum LH Level (U/L) 
Need only 1 reading at 
baseline 
 
 
 
 
 
Serum Prolactin Level 
(U/L) 
Need only 1 reading at 
baseline 
 
 
 
 
 
Serum Estradiol Level 
(pg/mL) 
Need only 1 reading at 
baseline 
 
 
 
 
 
USS Uterine  
  
 
  
 
  

 
 
Uterine length (cm) 
 
 
 
 
 
Uterine AP Diameter (cm) 
 
 
 
 
 
Endometrial thickening 
(hyperplasia) (Yes/No) if 
yes, ET size (mm) 
 
 
 
 
 
Quality of Life 
Psychometrics 
(Questionnaires) 
 
 
 
 
 
WHOQOL-BREF (General 
QOL) 
 
 
 
 
 
Breast-Q (breast 
satisfaction score) 
 
 
 
 
 

 
 
Safety Profile (based on 
adverse event tracking 
log) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
APPENDIX VI 
 
WITHDRAWAL BLEEDING MONITORING LOG (adapted from Zhang et al. 2023) 
 
Subject ID No 
 
Month / Year 
 
Days 
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 
Bleeding 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
No of pads 
/ tampons 
used 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Grading 
(for 
withdrawal 
bleeding) 
 
The participant has to mark either X = red vaginal bleeding; o = brown discharge. For withdrawal bleeding grading (Wise et al. 2011; Mejia et al. 
2016; Wesselink et al. 2016) (33-35): 1 = no bleeding; 2 = spotting, 3 = light bleeding (≤10 pads/tampons per menses); 4 = moderate bleeding (11-
20 pads/tampons per menses); 5 = heavy bleeding (20-30 pads/tampons per menses); 6 = very heavy bleeding (>30 pads/tampons per menses). 
Spotting is defined as bloody discharge that requires not more than a panty liner (Zhang et al. 2023) (83).