COPENHAGEN Minipuberty Study (CPHMINIPUB)

Minipuberty is a term used to describe the transient activation of the pituitary-gonadal axis 2-3 months after birth in both boys and girls. It is, however, not known why infants reach adult levels of reproductive hormones in early life, nor is the exact timing of the peak known. Furthermore, what determines the timing of peaks and suppressions of reproductive hormones from infancy throughout childhood and into adolescence remains to be elucidated.

The study aims to described and evaluate dynamic changes in the hypothalamic-pituitary- gonadal axis in early postnatal life.

Study Overview

• Status: Unknown
• Conditions: Disorders of Sex Development; Child Development

Detailed Description

Minipuberty is a term used to describe the transient activation of the pituitary-gonadal axis 2-3 months after birth in both boys and girls. It is, however, not known why infants reach adult levels of reproductive hormones in early life, nor is the exact timing of the peak known. Furthermore, what determines the timing of peaks and suppressions of reproductive hormones from infancy throughout childhood and into adolescence remains to be elucidated.

Few studies have investigated minipuberty and one, for example, found that it is affected in premature infants (before gestation week 37). However, no studies on normative data throughout minipuberty in infants exist.

Furthermore, using minipuberty as a window for diagnosis of endocrine disorders and future reproductive function has been suggested. Defining minipuberty, both in terms of circulating hormone levels and urinary metabolites, in healthy infants is therefore essential in order to utilize this window. Studies using patients with Disorders of Sex Development during minipuberty have been carried out, but they are hampered by small sample sizes and lack of control groups.

In addition, little is known about the genetic and epigenetic factors that drive the onset, progression and termination of minipuberty as well as the actual puberty, i.e. the factors responsible for the quiescence of the HPG axis during childhood and the dis-inhibition responsible for pubertal onset. Therefore, much attention was drawn on the study performing whole exome sequencing in patients and relatives with central precocious puberty (CPP). For the first time, MKRN3 was suggested as the primary factor responsible for HPG inhibition during mid-childhood. A number of studies support that MKRN3 mutations cause CPP, and genetic variation of MKRN3 affect pubertal timing in healthy girls. Our findings of declining serum levels of MKRN3 prior to pubertal onset in healthy girls support MKRN3 as a regulator of pubertal onset. The exact mechanism through which MRKN3 exceeds its effect remains to be elucidated; however, its zink-finger structure indicates regulation of superior cellular processes such as epigenetic regulation of DNA transcription.

Twin studies suggest that 60% of the inter-individual variation is caused by genetic factors. However, genome wide association (GWA) studies only explain a fraction of the variation in age at puberty. Recently, our research group has revealed the largest effect of a single SNP on age at pubertal onset in girls. The location of the SNPs in genes regulating FSH action emphasizes the need of a wide focus including downstream processes in the HPG axis when evaluating factors regulating puberty.

In general, the abovementioned studies have led to a spark in the interest in epigenetic studies, i.e. studies of genetic changes that are not caused by changes in the DNA sequences themselves, but rather regulatory mechanisms of DNA expression. Generally, this is thought to include DNA methylation, histone modifications and small RNAs. Epi-mutations (improper epigenetic regulation) possibly account for more of the variation in puberty than genetic factors. Previously, both gene-specific and genome-wide DNA methylation patterns have been studied. Genome-wide hypomethylation seen in peripheral leukocytes has been shown to be linked with an array of cancers, including colorectal cancers. As multiple histone modifications exist and analysis requires special sample treatment procedures, DNA methylation is the most appropriate epigenetic marker to analyze. A study of rats found that specific gene hypomethylation was accountable for lack of pubertal onset, but the link between epigenetics and mini- and pubertal timing and progression has, however, only scarcely been studied. Understanding this link would greatly add to our knowledge of reproductive function and normal sex development.

Disorders of Sex Development (DSD) is an umbrella term covering conditions with congenital disordered development of chromosomal, gonadal or anatomical sex. Genital abnormalities may include as many as up to 4-6 in 1000 births, although individual disorders are much rarer, e.g. 45,X/46,XY mosaicism is seen in about 1 in 15000 live births. Previously DSD diagnoses were labeled with different and often imprecise terms such as 'intersex', 'sex reversal' and 'hermaphroditism' etc. In 2006, DSD nomenclature was renamed and grouped according to genetic sex into sex chromosome DSD, 46,XY DSD and 46,XX DSD.

DSD patients are diagnosed at different periods in life depending on their diagnosis, phenotype and primary and secondary sexual development. Patients with sex chromosome DSD can be diagnosed at prenatal screenings, patients with affected external genitalia at birth, some during childhood due to growth abnormalities, some during adolescence due to abnormal pubertal progression and lastly, some in adulthood due to infertility.

Understanding normal sex development is therefore the key to identifying and optimizing diagnosis and treatment of patients with DSD. A project, as the present, that seeks to investigate normal minipuberty while comparing to minipuberty in patients with DSD is therefore of great importance. Furthermore, knowledge of the genetic and epigenetic control mechanisms of minipuberty will aid the understanding of reproductive physiology and in particular DSD pathology.

• Study Type: Observational
• Enrollment (Anticipated): 280

Contacts and Locations

Study Locations

• Denmark
  • Copenhagen, Denmark, 2100
    • Department of Growth and Reproduction, Rigshospitalet

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: No older than 1 year (CHILD)
• Accepts Healthy Volunteers: No
• Genders Eligible for Study: All

• Sampling Method: Non-Probability Sample

Study Population

Pregnant women, meeting the inclusion criteria, and the fathers-to-be followed at the Department of Obstetrics, Rigshospitalet. Further, parents and infants with disordered sexual development (DSD). These patients will be recruited via The Department of Growth and Reproduction, Rigshospitalet. Parents whose fetuses have been diagnosed prenatally with any DSD diagnosis or during the first 6 months of life will be invited to participate.

Three groups of participants in this study:

1. A group of healthy infants
2. All infant patients diagnosed with or under evaluation for DSD
3. The parents of the healthy infants and DSD patients

Number (approximately) of participants:

1. 200 healthy infants (100 boys and 100 girls)
2. unknown number of DSD infants that will be referred within a year to the Department of Growth and Reproduction; estimation: 15 DSD infants.
3. 400 parents of healthy infants (200 fathers and 200 mothers) - unknown number of parents of DSD patients

Description

Inclusion Criteria:

• Singleton pregnancy
• Maternal and paternal Caucasian origin
• Maternal pre-pregnancy BMI between 18 and 35 kg/m2
• No serious maternal illness, including no pre-existing maternal diabetes nor thyroid gland diseases
• Term pregnancy (week 37+0 to 41+7)
• No gestational diabetes
• No fetal malformations or chromosomal disorders
• Birth weight of child between 3rd and 97th percentile

Only healthy infants born at term will be included in the study which all prospective participants will be informed of.

Exclusion Criteria:

-

Study Plan

How is the study designed?

Number of groups / cohorts

2

Cohorts and Interventions

Group / Cohort
1 year follow-up group; 1 year follow-up group including 6 measurements
40 days diaper study subgroup; Subgroup of the "1 year follow-up group" including 15 girls undergoing daily measurement of urinary hormone excretion

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Serum and urinary metabolites of reproductive hormones (e.g. steroid hormone metabolites and gonadotropins) (newborn)	change/course serum and urinary metabolites	3-7d, and 1,3,5,7,12m or 2,4,6,8,12m after birth plus 40 days daily measurement (urine, female 40 days diaper study subgroup)
Urinary metabolites of endocrine disrupting chemicals (e.g. phthalates, phenols, perfluorinated compounds and parabens) (newborn)	change/course urinary metabolites	3-7d, & 1,3,5,7,12m or 2,4,6,8,12m after birth plus 40 days daily measurement (urine, female 40 days diaper study subgroup)
Basic clinical examination (newborn) (size and proportions)	change/course: measurements of length, weight, skin folds and hip-waist ratio	3-7d, and 1,3,5,7,12m or 2,4,6,8,12m after birth
Basic clinical examination (newborn) (pubertal staging)	change/course: pubertal staging using Tanners classification (including testicular size in boys assessed by Prader's orchidometer and ultrasound	3-7d, and 1,3,5,7,12m or 2,4,6,8,12m after birth
Basic clinical examination (newborn) (genitalia)	change/course: classification of external genitalia (classification of genital tubercle, location of gonads, position of urethra, labia/scrotal fusion)	3-7d, and 1,3,5,7,12m or 2,4,6,8,12m after birth
Basic clinical examination (newborn) (penile measurement)	change/course: penile measurement with a ruler (in boys)	3-7d, and 1,3,5,7,12m or 2,4,6,8,12m after birth
Basic clinical examination (newborn) (AGD)	change/course: ano-genital distance (AGD) measured with a ruler	3-7d, and 1,3,5,7,12m or 2,4,6,8,12m after birth
Genetic profiling	Genotyping of different genetic loci (genetic variation of loci regulating hormone signalling, e.g. FSHB, etc.)	single determination or 3-7d, and 1,3,5,7,12m or 2,4,6,8,12m after birth
Epigenetic profiling	change/course: epigenetic variation of loci regulating hormone signalling	single determination or 3-7d, and 1,3,5,7,12m or 2,4,6,8,12m after birth

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Basic clinical examination (parents) (height)	Height	postpartal (within first 3 months)
Basic clinical examination (parents) (weight)	self-reported pre-pregnancy weight for the mother and postpartal weight of the father	postpartal (within first 3 months)
Basic clinical examination (parents)	Skinfolds measured above the biceps, triceps, at the flank, and below the scapula	postpartal (within first 3 months)
Pregnancy and perinatal outcome (newborn and mother)	Perinatal outcome including birth weight, -length, partus mode, adverse events/complications, pre- and perinatal drug intake, pregnancy outcomes including gestational age, pregnancy complications, IVF Treatment etc.	before birth and perinatal phase
Medical history and exposure (parents) (basic)	Basic medical history (parents) (questionaire / journal)	postpartal (within first year)
Medical history and exposure (parents) (obstetrical)	Obstetrical history including outcomes of previous pregnancies and births (mother), smoking and drug intake during pregnancy (mother) (questionaire / journal)	postpartal (within first year)
Medical history and exposure (parents) (puberty)	Pubertal history (parents) including age at menarche, pubertal timing with regard to peers, age at menopause of the mother of the parents etc. (questionaire)	postpartal (within first year)
Breastfeeding and food intake (newborn)	change/course: breastfeeding and food intake of the newborn during the course of the first year (questionaire)	first year of life

Collaborators and Investigators

• Sponsor: Rigshospitalet, Denmark
• Investigators: Study Chair: Anders Juul, Prof., Rigshospitalet, Denmark; Principal Investigator: Alexander S Busch, MD, Rigshospitalet, Denmark

Publications and helpful links

General Publications

• Ljubicic ML, Busch AS, Upners EN, Fischer MB, Petersen JH, Raket LL, Frederiksen H, Johannsen TH, Juul A, Hagen CP. A Biphasic Pattern of Reproductive Hormones in Healthy Female Infants: The COPENHAGEN Minipuberty Study. J Clin Endocrinol Metab. 2022 Aug 18;107(9):2598-2605. doi: 10.1210/clinem/dgac363.
• Ljubicic ML, Busch AS, Upners EN, Fischer MB, Main KM, Andersson AM, Johannsen TH, Hagen CP, Juul A. Dynamic changes in LH/FSH ratios in infants with normal sex development. Eur J Endocrinol. 2022 Jun 1;187(1):135-142. doi: 10.1530/EJE-21-0999.

Study record dates

Study Major Dates

• Study Start: August 1, 2016
• Primary Completion (ANTICIPATED): December 31, 2019
• Study Completion (ANTICIPATED): December 31, 2019

Study Registration Dates

• First Submitted: May 17, 2016
• First Submitted That Met QC Criteria: May 23, 2016
• First Posted (ESTIMATE): May 27, 2016

Study Record Updates

• Last Update Posted (ACTUAL): February 15, 2019
• Last Update Submitted That Met QC Criteria: February 13, 2019
• Last Verified: February 1, 2019

More Information

Terms related to this study

• Keywords: Hypothalamic-pituitary-gonadal axis
• Additional Relevant MeSH Terms: Endocrine System Diseases; Gonadal Disorders; Urogenital Abnormalities; Congenital Abnormalities; Disorders of Sex Development
• Other Study ID Numbers: RH-H-15014876; RH-2015-210-04146 (OTHER: Danish Data Protection Agency)

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: NO