Preliminary Study on Activation of the Hypothalamic-pituitary-gonadal Axis in Infancy (Minipuberty) and Its Effects on Catch-up Growth and Neurocognitive Outcome in Male Preterm Neonates Born at <32 Weeks of Gestational Age
Conditions: Infant, Newborn
Sponsor: NHS Greater Glasgow and Clyde

 
 
Version 1, 6 December 2018 
 
PRELIMINARY STUDY ON ACTIVATION OF THE HYPOTHALAMIC-
PITUITARY-GONADAL AXIS IN INFANCY (MINIPUBERTY) AND ITS 
EFFECTS ON CATCH-UP GROWTH AND NEUROCOGNITIVE 
OUTCOME IN MALE PRETERM NEONATES BORN AT <32 WEEKS OF 
GESTATIONAL AGE 
 
 
 
 
Running title:  
 
Minipuberty and its effects on preterm neonates 
Protocol Version: 
 
 
1 
Date:  
 
 
 
6 December 2018 
REC Reference Number: 
 
/ 
ISRCTN/Clinical trial.gov: 
 
/ 
Sponsor’s Protocol Number:  
GN18ME668P 
Sponsor: 
 
 
 
NHS Greater Glasgow & Clyde 
Funder: 
 
 
 
NA 
 
 
 
Amendment number 
Date 
Protocol version 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
This study will be performed according to the Research Governance Framework for Health 
and Community Care (Second edition, 2006) and WORLD MEDICAL ASSOCIATION 
DECLARATION OF HELSINKI  Ethical Principles for Medical Research Involving Human 
Subjects 1964 (as amended). 

 
 
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CONTACTS 
Chief Investigator 
 
Prof. Faisal Ahmed  
Samson Gemmell Chair of Child Health – University of Glasgow  
Work address: Office block, QUEH Campus, Ground floor, zone 1 
Work E-mail: faisal.ahmed@glasgow.ac.uk 
 
 
Co-investigator   
 
Dr Martina Rodie 
BSc, MBChB, MRCPCH, PhD 
Locum consultant Neonatologist  
Work address: Neonatal Intensive Care Unit - Royal Hospital for Children  
Work E-mail: martina.rodie@nhs.net 
Tel: 07973887549 
 
Dr Alessandra Boncompagni  
Doctor in training in Paediatrics at the University Hospital of Modena and Reggio Emilia  
Clinical fellow with the Endocrine Research group at the Royal Hospital for Children in 
Glasgow  
E-mail: ale.bonco@gmail.com  
Tel: +39 3491440692 
 
 
Dr. Andrew Brunton 
Neonatal GRID trainee 
Work address: Neonatal Intensive Care Unit – Royal Hospital for Children 
Work E-mail: a.brunton1@nhs.net  
 
 
Study Statistician 
 
Developmental Endocrinology Research Group  
Work address: Office block, QUEH Campus, Ground floor, zone 1 
 
 
 
Sponsor’s representative –  
 
Paul Dearie  
Clinical Research & Development 
NHS Greater Glasgow and Clyde 
West Glasgow Ambulatory Care Hospital 
Dalnair Street 
Glasgow G3 8SW 
Mail paul.dearie@ggc.scot.nhs.uk 
Tel 0141-232-1810 
 
Funding Body – NA 
 

 
 
Version 1, 6 December 2018 
 
STUDY SYNOPSIS 
 
Title of Study: 
Preliminary study on activation of the hypothalamic-pituitary-
gonadal axis in infancy (minipuberty) and its effects on catch-
up growth and neurocognitive outcome in male preterm 
neonates born at <32 weeks of gestational age 
Study Centre: 
Royal Hospital for Children, Glasgow  
Duration of Study: 
18 months  
Primary Objective: 
- To describe when urinary hormones (follicle stimulating 
hormone and luteinising hormone) start to raise and when 
they reach their peak among male preterm neonates born with 
a gestational age <32 weeks.  
- To investigate if there is a difference in hormone levels and 
their peak timing between extremely preterm babies (<28 
weeks of gestational age) and very preterm babies (28-32 
weeks of gestational age). 
Secondary Objective: 
- Identify a link between hormone levels and linear growth in 
the first months of life in preterm neonates. 
- Identify a link between hormone levels and the development 
of external genitalia (testicular volume and position, penile 
length).  
- Identify a link between hormone levels and short-term 
neurocognitive development. 
Rationale: 
The knowledge about the activation of the hypothalamic-
pituitary-gonadal axis (HPG) in the first months of life is 
relatively recent. It is scientifically proven that in this period, 
called "minipuberty", there is the same activation that 
occurs during puberty. This is responsible for changes in terms 
of growth, pubertal development and neurobehavioral 
patterns. Understanding all the factors involved in children's 
physical and neurocognitive development has a significant 
scientific impact and could provide useful information in case 
of future problems. As prematurity survival rates 
continue to improve, clinicians need to be familiar with the 
hormonal changes in these patients and the first months of 
life provide a ‘window of opportunity' for functional studies of 
the HPG axis prior to pubertal development 
Methodology: 
This is a preliminary prospective and crossectional study.  
Sample Size: 
20 male preterm neonates with a gestational age ≤32 weeks 
Main Inclusion Criteria: 
• 
Male preterm infants born ≤32 weeks of gestational age 
• 
Written informed consent provided  
Main Exclusion Criteria: 
• 
Female sex assignment at birth  
• 
Male preterm infants born >32 weeks of gestational age  
• 
Male infants born at term 
Statistical Analysis: 
Results shall be expressed as median (range) and analysed 
using Minitab. Some data may require log transformation. 
Pearson’s correlation shall be used for univariate analysis of 
association of and multivariate analysis shall be performed 
using backward stepwise linear regression analysis including 
factors 
significant 
from 
univariate 
analysis 
(Pearson’s 

 
 
Version 1, 6 December 2018 
 
correlation) as independent factors. The Mann Whitney U test 
shall be used for comparison of nonparametric data. Statistical 
significance shall be set at p < 0.05. 
 
 
 

 
 
Version 1, 6 December 2018 
 
Full title: PRELIMINARY STUDY ON ACTIVATION OF THE HYPOTHALAMIC-PITUITARY-
GONADAL AXIS IN INFANCY (MINIPUBERTY) AND ITS EFFECTS ON CATCH-UP GROWTH 
AND NEUROCOGNITIVE OUTCOME IN MALE PRETERM NEONATES BORN AT <32 WEEKS OF 
GESTATIONAL AGE 
 
Short title: Minipuberty and its effects on preterm neonates 
 
1. Introduction 
 
The activation of the hypothalamic-pituitary-gonadal axis (HPG) develops through three main 
moments: foetal life, first postnatal months (usually first 6 months of life) and finally during 
puberty.1 During the foetal period, there is a peak of gonadotropin secretion starting from the 
second trimester of gestation, and a subsequent suppression in the last weeks until birth, due 
to the negative feedback of placental estrogens.2 In male foetuses, LH levels exceed those of 
FSH. The foetal testis secretes testosterone and anti-müllerian hormone (AMH) from the 8th 
week of gestation and this is essential for masculinization. Formation of the active metabolite 
of testosterone, dihydrotestosterone, is required for the development of the prostate, penis 
and scrotum.3 Initial testicular development is intra-abdominal and the descent of the testes 
into the scrotum occurs in two phases. The first, transabdominal, phase is completed by 15 
weeks of gestation. The second, inguinoscrotal, phase is usually completed by the end of the 
35th week of gestation and this phase is androgen dependent. Testosterone levels are high in 
male foetuses between 10 and 20 weeks of gestational age, reaching adult values, and 
decrease thereafter towards term.4 In the same way LH and FSH levels decrease towards the 
end of gestation and are low at term in both sexes, due to the negative feedback of placental 
oestrogens.  
A second post-natal HPG activation, also called “minipuberty”, occurs at around 1 week of 
age, when placental hormones are cleared from the circulation. From the first weeks of life 
onwards there is a progressive increase in gonadotropins levels with a greater increase of 
FSH in females and of LH in males. During the “minipuberty” period gonadotropins levels 
have a peak at 3 months of life and a progressive exhaustion at around 6 months, except for 
FSH levels in females that can remain high even up to 3 or 4 years. Particularly in males, 
testosterone and androgen levels are associated with development and maturation of the 
reproductive system (penis and testis) as well as the androgynous cutaneous manifestations 
and a different neurobehavioral structure.5 Postnatal testosterone levels have been associated 
with male-type behaviour in 14-month-old infants, suggesting a role in neurobehavioral 
development.6 Recently, it has also been suggested how the increasing testosterone level 
during the first 6 months of life, as well as during puberty, translates into an increase of linear 
growth, regardless of the levels of growth hormone (GH) or insulin-like growth factor (IGF1).7 
Preterm birth does not seem to influence post-natal HPG activation, since that gonadotropin 
levels seem to raise up at the same time as term infants after the birth. The difference is that 
in preterm neonates these values last higher and longer than full-term newborns. According 
to the most recent longitudinal data, the post-natal activity of this axis declines at about the 
same in term neonates compared to premature infants with the same corrected gestational 
age, suggesting that the HPG activity is evolutionarily regulated.8,9 This higher increase of 
gonadotropin levels in premature males has been associated with a faster penile and 
testicular growth after birth compared to full-term boys. At last, in neonates small for 
gestational age (SGA) there are some evidence that post-natal FSH and testosterone levels 
are higher compared to infants with an adequate weight (AGA), although it is not known 

 
 
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whether this correlates or have any influence on the catch-up growth of this subjects.10,11 
Understanding how “minipuberty” can influence linear growth, genitalia development and 
neurocognitive processes in preterm neonates has a significant scientific impact and could 
provide us more information in understanding auxological and neurobehavioral developmental 
patterns of this population. 
 
2. Aim/Primary and Secondary Objectives 
 
This is a preliminary prospective and crosssectional study aiming to describe “minipuberty” in 
male preterm children at the Royal Hospital for Children in Glasgow. The research project has 
been discussed by the Developmental Endocrinology Research Group at the University of 
Glasgow. 
 
• 
Primary Endpoint 
- To describe when urinary hormones (follicle stimulating hormone and luteinising 
hormone) start to raise and when they reach their peak among male preterm neonates 
born with a gestational age <32 weeks.  
- To investigate if there is a difference in hormone levels and their peak timing between 
extremely preterm babies (<28 weeks of gestational age) and very preterm babies (28-
32 weeks of gestational age).  
 
• 
Secondary endpoints 
- Identify a link between hormone levels and linear growth in the first months of life in 
preterm neonates. 
- Identify a link between hormone levels and the development of external genitalia 
(testicular volume and position, penile length).  
- Identify a link between hormone levels and short-term neurocognitive development. 

 
 
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3. 
Study Design 
This is a preliminary prospective and crosssectional study on male preterm children at the 
Royal Hospital for Children in Glasgow. The study will not include any investigational medical 
product. The research project has been discussed by the Developmental Endocrinology 
Research Group at the University of Glasgow. 
 
This study will be performed according to the Research Governance Framework for Health 
and Community Care (Second edition, 2006). 
3.1 
Study Population 
We aim to enroll 20 neonates born under 32 weeks of gestational age at the Royal Hospital 
for Children in Glasgow. Neonates will be enrolled during the first 3 days of life with written 
informed consent signed by their parents.  
3.2 
Inclusion criteria 
• 
Male preterm infants born ≤32 weeks of gestational age  
• 
Written informed consent provided  
3.3 
Exclusion criteria 
• 
Female sex assignment at birth  
• 
Male preterm infants born >32 weeks of gestational age  
• 
Male infants born at term 
3.4 
Identification of participants and consent 
The family of a newborn eligible for this study will be approached in the neonatal unit after 
the birth. Information material about the study shall be provided with an initial private 
discussion and we will make the family aware that participation is voluntary and they can 
leave the study at any time without their baby’s standard care being affected. We will ask for 
contact details during this first meeting in order to contact them later checking whether they 
are interested in participating and start with the study. Consent will be taken in the Neonatal 
Intensive Care Unit in a private counselling room by the research team (Dr Martina Rodie or 
Dr Alessandra Boncompagni or Dr Andrew Brunton). Parents will be given a minimum of  24 
hours to consider participation and they could take a decision until 72 hours of newborn’s life. 
Neonates of parents who will provide written informed consent will be enrolled in this study.  
3.5 
Withdrawal of subjects 
Participants have the right to withdraw from the study at any point for any reason.   
 
 

 
 
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4 
Study Outcome Measures 
Our study will include 3 types of different evaluations on each neonate:  
- 
Multiple urine collections to evaluate urinary gonadotrophin levels 
- 
Multiple clinical evaluations of auxological parameters and external genitalia  
- 
Neurocognitive assessment at 3 months of corrected gestational age   
 
Timing of sample analysis and simultaneously clinical evaluation will be as follow:  
 
T1: 1-3 days of life 
T2: 7 days of life 
T2a – T2z: one per week until 40 weeks of post-conceptional age or until discharge from 
Neonatal Unit 
T3-TX: urinary sample analysis and clinical evaluation during the routine follow up clinics until 
12 months of corrected gestational age 
 
4.1 
Primary Outcome Measure 
Urine collection will be done with a plastic bag with a sticky strip on one end, made to fit over 
baby's genital area. In extremely preterm babies (<28 weeks of gestational age) urine 
collection will be from cotton wool in the nappy because of their sensitive skin.  
Samples will be analyzed on fresh urine for urinary gonadotropin levels (FSH, LH) as detailed 
in laboratory tests section.  
4.2. 
Secondary Outcome Measure 
 
- 
Auxological parameters for each evaluation will include: length, weight, head 
circumference. They are routinely checked both during the baby’s hospital stay and 
the routine follow-up clinics therefore we will not add any to the standard baby’s 
care.  
- 
External genitalia evaluation will include: testicular volume and position, penile 
length. This is not a part of the routine monitoring of preterm babies but it is not 
invasive and it will be done at the same time of plastic bag’s positioning for urine 
collection in order to minimize handling of the baby.  
- 
Short term neurocognitive outcome: during the hospital stay in the Neonatal Unit 
parents will be asked if they agree with video recording their baby using their mobile 
phone at home after discharge. We would like to assess the Fidgety Movements at 3 
months of corrected gestational age using the Prechtl’s method12,13. If the parents 
agree, we will give an instruction sheet before they go home briefly explaining how 
they have to record their baby and for how long. Fidgety Movements are a 
spontaneous pattern of movements which parents can record without the need for a 
healthcare professional. Parents will upload their videos onto a secure video 
messaging system (www.vcreate.tv) which already has governance approval in GGC 
and is endorsed by the NHS. Laura Lucaccioni is an Italian researcher at the 
University Hospital of Modena (Italy). She is a highly trained expert of Prechtl’s 
Method and a member of the General Movements Trust. With an expert team she will 
review all the videos giving an evaluation of the Fidgety Movements pattern. We will 

 
 
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store the videos for a maximum of 10 years and will delete them after this time. 
 
 
4.3 
Laboratory Tests 
Measurement of LH and FSH urinary levels will be made on fresh samples using electro-
chemiluminescence immunoassay (ECLIA). Sample analysis will be performed by the 
Biochemistry Department of the Royal Hospital for Children in Glasgow using Abbott Architect 
i1600 instrument.  
 
 
5. 
Assessment of Safety  
We do not expect particular problems in this study. Urine collection will be made in the same 
way of routine collection in infants. Auxological evaluations are part of the routine care of 
preterm neonates and we will add a non-invasive evaluation of external genitalia without 
particular risks for the babies. Neurobehavioural development will be evaluated using parents' 
video only if they agree with video recording their baby. This will allow parents to stay at 
home without bringing their baby to the hospital. 
 
6. Statistics and Data Analysis  
 
Results shall be expressed as median (range) and analysed using Minitab. Some data may 
require log transformation. Pearson’s correlation shall be used for univariate analysis of 
association of and multivariate analysis shall be performed using backward stepwise linear 
regression analysis including factors significant from univariate analysis (Pearson’s 
correlation) as independent factors. The Mann Whitney U test shall be used for comparison of 
nonparametric data. Statistical significance shall be set at p < 0.05. 
 
7.  STUDY CLOSURE / DEFINITION OF END OF TRIAL 
Planned start date:  February 2019  
Planned end date: August 2020 
Total duration: 18 months 
The enrolment is expected to last 6 months starting from the approval. 
The study will be finished at the last visit (12 months of corrected gestational age) of the last 
newborn enrolled.  
 
 
8. Ethical Consideration 
 
The study will be carried out in accordance with the World Medical Association Declaration of 
Helsinki (1964) and its revisions (Tokyo [1975], Venice [1983], Hong Kong [1989], South 
Africa [1996] and Edinburgh [2000]). 
Favourable ethical opinion will be sought from an appropriate REC before patients are 
entered into this clinical trial.  Patients will only be allowed to enter the study once their 

 
 
Version 1, 6 December 2018 
 
parents have provided written informed consent. The CI will be responsible for updating the 
Ethics committee of any new information related to the study. 
 
 
9. Finance and Indemnity 
 
This study is sponsored by NHS Greater Glasgow & Clyde.  The sponsor will be liable for 
negligent harm caused by the design of the trial.  NHS indemnity is provided under the 
Clinical Negligence and Other Risks Indemnity Scheme (CNORIS). 
 
 
11. Publications 
 
We may report and disseminate the results of our study through peer reviewed scientific 
journals, internal report and conference presentation.  
 
12. References 
1. Kuiri-Hänninen T, Sankilampi U, Dunkel L. Activation of the hypothalamic-pituitary-
gonadal axis in infancy: minipuberty. Horm Res Paediatr. 2014;82(2):73-80 
2. Reyes F, Winter JS, Faiman C. Studies on human sexual development. Fetal 
gonadal and adrenal sex steroids. J Clin Endocrinol Metab 1973;37:74–78. 
3. Debieve F, Beerlandt S, Hubinont C, Thomas K. Gonadotropins, prolactin, inhibin A, 
in-hibin B, and activin A in human fetal serum from midpregnancy and term 
pregnancy. J Clin Endocrinol Metab 2000;85:270–274. 
4. Takagi S, Yoshida T, Tsubata K, Ozaki H, Fujii TK, Nomura Y, Sawada M. Sex 
differences in fetal gonadotropins and androgens. J Steroid Biochem 1977;8:609–
620. 
5. Winter JS, Faiman C, Hobson WC, Prasad AV, Reyes FI. Pituitary-gonadal relations 
in infancy. I. Patterns of serum gonadotropin concentrations from birth to four 
years of age in man and chimpanzee. J Clin Endocrinol Metab 1975;40:545–551. 
6. Lamminmäki A, Hines M, Kuiri-Hänninen T,Kilpeläinen L, Dunkel L, Sankilampi U. 
Testosterone measured in infancy predicts subsequent sex-typed behavior in boys 
and in girls. Horm Behav 2012;61:611–616. 
7. Kiviranta P, Kuiri-Hänninen T, Saari A, Lamidi M-L, Dunkel L, Sankilampi U. 
Transient postnatal gonadal activation and growth velocity in infancy. Pediatrics. 
2016; 138  
8. Kuiri-Hänninen T, Seuri R, Tyrväinen E, Turpeinen U, Hämäläinen E, Stenman UH, 
Dunkel L, Sankilampi U. Increased activity of the hypothalamic-pituitary-testicular 
axis in infancy results in increased androgen action in premature boys. J Clin 
Endocrinol Metab 2011;96:98–105 
9. Kuiri-Hänninen T1, Haanpää M, Turpeinen U, Hämäläinen E, Seuri R, Tyrväinen E, 
Sankilampi U, Dunkel L.Postnatal Ovarian Activation Has Effects In Estrogen Target 
Tissues In Infant Girls. J Clin Endocrinol Metab. 2013 Dec;98(12):4709-16 

 
 
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10. Panu Kiviranta, Tanja Kuiri-Hänninen, Antti Saari, Marja-Leena Lamidi, Leo 
Dunkel, Ulla Sankilampi. Transient Postnatal Gonadal Activation and Growth 
Velocity in Infancy. Pediatrics 2016 Jul 9;138 
11. Ibáñez L, Valls C, Cols M, Ferrer A, Marcos MV, De Zegher F. Hypersecretion of FSH 
in infant boys and girls born small for gestational age. J Clin Endocrinol Metab 
2002;87:1986–1988.   
12. Einspieler C, Prechtl HFR, Ferrari F, Cioni G, Bos AF. The qualitative assessment of   
general movements in preterm, term and young infants - review of the 
methodology.  Early Hum. Dev. 50 (1997) 47-60 
13. Einspieler C, Peharz R, Marschik PB. Fidgety movements - tiny in appearance, but 
huge in impact. J. Pediatr. (Rio J) 92 (2016) S64-70