A Biphasic Pattern of Reproductive Hormones in Healthy Female Infants: The COPENHAGEN Minipuberty Study (2024)

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Abstract

The early postnatal and transient activation of the hypothalamic-pituitary gonadal (HPG) hormone axis is termed minipuberty. It has been suggested to peak at around 2 to 3 months of age, after which the HPG axis becomes somewhat quiescent until puberty (1, 2). Although the exact function remains largely unknown, minipuberty has been suggested to play a role as an essential imprinting period for genital development (3-5), anthropometry (6), and cognitive function (7, 8).

In girls, follicular growth and increasing levels of reproductive hormones during infancy have been described in both serum (4, 9, 10) and urine samples (3, 4, 11, 12). With limited data from prospective, longitudinal cohorts of healthy, infant girls, the dynamics of the activation and silencing of the HPG axis in the individual girl have not been fully elucidated. Only a few studies have followed healthy girls with more than a single serum sample throughout the first year of life (4, 13), and a thorough evaluation based on longitudinal serum samples of reproductive hormones quantified by highly sensitive methods of quantification is needed to describe the dynamics of minipuberty in detail.

Sex- and age-adjusted reference ranges for the serum concentrations of the most clinically relevant reproductive hormones are scarce, often based on a narrow timeframe (2-5 months of age) and less sensitive assays (9, 14). In fact, most pediatric references are age-specific, tabulated intervals rather than continuous reference ranges, which limits the interpretation of patient samples (15). These reference intervals are often based on data from laboratory information systems and thereby from patient cohorts not designed for the creation of reference intervals (15, 16). Despite the importance of continuous reference ranges rather than intervals by age groups (17, 18), their lack is largely from the challenge in recruiting healthy children for venous blood sampling (15, 16).

Thus, in this study we aimed to (1) establish age-specific reference ranges for LH, FSH, inhibin B, anti-Müllerian hormone (AMH), estrone (E1), estradiol (E2), and SHBG from birth throughout the first year of life in healthy girls; (2) evaluate whether an infant girl maintains her relative hormone levels over time; and (3) evaluate the dynamics of the female HPG hormone axis from birth to 1 year of age.

Materials and Methods

The COPENHAGEN Minipuberty Study

Healthy, pregnant women were recruited as part of The COPENHAGEN Minipuberty Study at the Department of Growth and Reproduction, Rigshospitalet, Copenhagen, between 2016 and 2018. The study has been registered in Clinical Trials.gov (ID: NCT02784184). A detailed description of study design and participants including flow charts of inclusion/exclusion, demographics, and methods has recently been presented (19). In brief, a total of 233 infants (119 boys and 114 girls) born to mothers from affluent and educated backgrounds (eg, high proportion of academic backgrounds, only 1% smokers) were followed from birth throughout the first year of life with 1201 examinations. In total, 98 girls began follow-up, whereas 89 completed the sixth examination. All infants followed the same examination schedule with 6 visits, each including a clinical examination, a urine sample, and a blood sample. Each clinical examination included anthropometric outcomes including body length and weight, anogenital distance (20), presence of breast tissue, and scoring of external genital phenotype (19).

In the present study, a total of 98 infant girls in whom blood sampling was successful at least once were included. In total, 266 blood samples drawn between 5 days and 14.2 months of age were available for data analysis. The median number of blood samples per infant girl was 3 (interquartile range: 1-4). The variation in available samples per child was due to a limit of 2 attempts at blood sampling per visit for ethical reasons; sampling was only attempted if the infant was calm and not crying; and specific parental wishes. For further details on study design including participants with missing data for each hormone, please refer to Busch et al (19).

Hormone Assays

Serum sampling at each visit has been described in detail previously (19). Preanalytical processing took place within 8 hours of sample collection. Identical preanalytical sample handling including thawing, mixing, and internal quality control protocols took place. Serum samples were stored at -20°C up to 1 year before analysis. Based on internal quality control data, it has been concluded that storage up to 10 years does not affect analyte stability for the analytes included in this study (data not published). Samples were analyzed in several batches, and each batch included samples from different age groups, which abated systematic bias. Serum concentrations of LH and FSH were analyzed by time-resolved fluoroimmunometric assays (AutoDELFIA, Perkin Elmer, Turku, Finland; RRID: AB_2783737, https://antibodyregistry.org/search.php?q=AB_2783737 for LH and RRID: AB_2783738, https://antibodyregistry.org/search.php?q=AB_2783738 for FSH). For both assays, the limits of detection (LOD) were 0.05 IU/L, and the inter-assay coefficients of variation (CVs) were < 6%, whereas intra-assay CVs were ≤ 3%. Inhibin B was determined using a double antibody enzyme-immunometric assay (Inhibin B GenII ELISA, Beckman-Coulter, Brea, CA, USA; RRID:AB_2827405, https://antibodyregistry.org/search.php?q=AB_2827405) with an LOD of 3 pg/mL and inter- and intra-assay CVs of < 11% and < 7%, respectively. AMH and SHBG were measured by a chemiluminescent immunoassay (Access 2 Immunoassay System, Beckman-Coulter; RRID: AB_2892998, https://antibodyregistry.org/search?q=AB_2892998 for AMH and RRID: AB_2893035, https://antibodyregistry.org/search?q=AB_2893035 for SHBG) with LODs of 0.14 pmol/L and 0.33 nmol/L and inter-assay CVs of < 6% and ≤ 10% and intra-assay CVs of < 3% and < 5%, respectively. E1 and E2 were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as previously described (10). Limits of quantification (LOQs) were 2.9 pmol/L and 4.0 pmol/L, respectively, with inter-assay CVs < 7% and intra-assay CVs < 20%. Values below LOD or LOQs were reported as LOD/2 or LOQ/2 for all hormones.

All analyses were accredited by The Danish Accreditation Fund for medical examination according to a European and international standard approved in Denmark (the standard DS/EN ISO 15189).

Statistical Methods

Age-specific reference ranges for all analytes were created using the Generalized Additive Model for Location, Scale and Shape (GAMLSS) based on a Box-Cox distribution with age-varying coefficients. The data were summarized by 3 smoothed, age-dependent curves: L (age-dependent skewness), M (age-dependent median), and S (age-dependent coefficient of variation). Age-related SD scores were calculated based on the GAMLSS model using the following equation: SD score = ((X/M)^L-1)/(L × S), where X is the measurement and L ≠ 0. The GAMLSS model estimates a cross-sectional reference range and thus assumes that the data are cross-sectional. Because the population studied was assumed to be representative of the healthy background population and the follow-up measurements were decided in advance, this assumption was deemed appropriate. The GAMLSS model also helped determine the needed study size of approximately 90 to 100 infants to allow for generating the reference ranges while also being practically feasible.

Dispersions were calculated to describe the within-child variation in concentrations of consecutive samples of the reproductive hormones. Calculations were performed using IBM Statistics SPSS, version 25.

To explore the dynamics of the HPG axis in detail while making use of the longitudinal study design, we fitted random-coefficient spline models to each hormone using maximum-likelihood estimation procedures for linear mixed models. This approach enabled modeling and prediction of the individual hormone level trajectories as well as estimation of population-level local peak times of hormones. Specifically, each mean hormone trajectory was modeled using a natural spline basis and systematic subject deviation from the mean was modeled using random spline coefficients in the same natural spline basis, where the random coefficients were modeled as 0-mean Gaussian with a free covariance structure. Furthermore, the model included independent Gaussian measurement noise. The degrees of freedom (between 3 and 12) of the spline basis were chosen by the Akaike Information Criterion.

Ethical Considerations

Parents gave written consent for their children to be enrolled in the study. The COPENHAGEN Minipuberty Study received the necessary approvals by the regional Ethics Committees (H-15014876) and the Danish Data Protection Agency (RH-2015-210, I-Suite: 04146).

Results

Serum concentrations of LH, FSH, inhibin B, AMH, E1, E2, and SHBG as well as the corresponding age-specific reference lines (-2 SD, -1 SD, median, +1 SD, and +2 SD corresponding to the following percentiles: 2.5, 16, 50, 84, and 97.5) are shown in Fig. 1. Supplemental Table 1A-G provides the datapoints for the reference lines as well as L, M, and S for each hormone (available in a digital research material repository (21)). Moreover, Suppl. Fig. 1 provides an overview of the individual longitudinal serum concentrations according to age for all girls (available in a digital research material repository (22)). Notably, the vast majority of samples revealed detectable concentrations of ovarian hormones from 0.2 to 0.6 years of age (number of undetectable samples: inhibin B: 1, AMH: 0, E2: 2) (Suppl. Fig. 1 (22)). After 1 year of age, LH and E1 were undetectable, whereas almost all FSH, inhibin B, AMH, E2, and SHBG concentrations were detectable at all time points (Fig. 1, Suppl. Fig 1 (22)).

To assess whether individual girls maintained their relative hormone levels over time (ie, maintained stable SD scores despite significant alterations in the absolute concentrations, also termed tracking), the within-child variation in SD scores was evaluated (Fig. 2). Specifically, the SD scores of the respective hormones measured in the consecutive samples for each girl and the dispersions (SD) of these SD scores were plotted (Fig. 2). For the girls individually, dispersions varied from 0 to +2 SD scores for all hormones, with E1 presenting with the largest dispersions (Fig. 2). The average within-child variation (the overall mean dispersion for each hormone multiplied by 2) was ±1.0 SD scores for AMH, inhibin B, and SHBG, ±1.2 SD scores for LH, FSH, and E2, whereas it was ±1.4 for E1. The degree of dispersion for each hormone did not depend on the absolute concentration level (data not shown).

To explore the dynamics and physiology of the reproductive hormones in infancy, the longitudinal hormone trajectories were modelled using random-coefficient spline models on the entire dataset from 0 to 1 year of age (Fig. 3). Here, 2 peaks were visible for all hormones. The first peak for all hormones was timed around days 15 to 27 (0.04-0.07 years), whereas the second peaks appeared to occur around days 107 to 125 (0.29-0.34 years) for inhibin B, AMH, E1, E2, and SHBG. For LH and FSH, the second peaks occurred slightly later (around days 164-165 for both [0.45 years]). The initial peaks for LH, FSH, and E1 were relatively higher than the second peaks, which was unlike inhibin B, AMH, and E2, in which both peaks reached similar levels. For all hormones, the nadirs between the 2 peaks were observed between days 58 and 92 (0.15-0.25 years).

Discussion

This prospective, longitudinal study of healthy, infant girls allowed for a detailed and novel description of the transient activation of the HPG hormone axis revealing a biphasic pattern in infant girls. Moreover, age-specific reference ranges from birth throughout the first year of life, all based on highly sensitive methods of quantification, were produced.

We present age-specific, continuous reference ranges of the most relevant reproductive hormones throughout the first year of life. Each girl maintained relatively stable concentrations when expressed as SD scores despite great fluctuations in absolute concentrations. Importantly, the within-child variation for all hormones was small (±1 SD) and smaller than the between-child variation, which, by definition, is ±2 SD. This is imperative for the clinical applicability of the reference ranges, particularly whenever consecutive sampling may be relevant (monitoring). Previously, reference ranges and intervals have included serum samples from infants at 2 to 5 months of age only (9, 14), urine samples only (11), or 3 certain time points within the first 5 months of life (23). The need for continuous, age-specific reference ranges of the reproductive hormones during the entire first year of life, and particularly during minipuberty, is apparent when diagnosing and managing patients with suspected hypogonadism such as differences of sex development (9, 24). The unique reference ranges presented here can be directly implemented in other centers if applied with caution. Depending on local methods for hormone analyzes, recalibration or factorization may be needed.

The longitudinal setup of our study, with serum samples available from shortly after birth until 1 year of age, revealed that minipuberty in girls is biphasic, illustrated by 1 initial, early peak at around days 15 to 27 and a second, later peak around days 107 to 125 for the ovarian hormones and days 164 to 165 for the gonadotropins. Notably, the nadir between the 2 peaks was simultaneously timed for all hormones. To our knowledge, a female minipuberty consisting of 2 adjacent, but separate, activations of the HPG axis has not previously been described in a single dataset. Rather, a single, postnatal activation has been outlined (25). However, in a review of the existing literature on minipuberty, two peaks were observed in combined metadata of females for FSH, inhibin B, and testosterone (26).

An older, longitudinal study of 73 females in whom blood samples were drawn because of clinical indications up to 6 times during the first month of life also noted an early peak of reproductive hormones (27). When including umbilical cord sera, the data even suggested 2 peaks during the first month of life that could likely be an artefact from the small number of samples available at the investigated timepoints. Nevertheless, their data seem to confirm the initial peak observed at 1 month of age in our present data (27). In another study, urinary FSH concentrations similarly showed an apparent peak around 1 month of age (4), but the cohort did not include samples past 5 months (150 days), which could likely explain why monthly urinary LH and FSH concentrations from the same cohort did not allude to 2 peaks (28). In general, studies including consecutive serum samples taken during the entire first year of life from healthy infants have not previously been carried out, and thus, our findings are best confirmed by the metadata discussed (26).

A female 2-peak minipuberty could theoretically be attributed to the immediate, postnatal release of the fetal pituitary suppression exerted by the placental steroidogenesis. This fits with rapidly increasing gonadotropin concentrations observed in boys just hours after birth (29) and during the first month of life (30) and would explain the similar rise in ovarian products that we observed. Of note, the timings of the second peaks were not synchronous for the ovarian and pituitary hormone, (ie, inhibin B, AMH, and E2 peaked around days 107-125-127, whereas the gonadotropins [LH and FSH] peaked around days 164-165). Gonadotropic control of the gonadal hormone production does therefore not appear to be as definite in girls as observed in boys (31). Interestingly, in our cohort, very low LH concentrations seemed sufficient for E2 production, contradicting a previous study which observed that increases in urinary E2 levels followed the gonadotropin surge (3). The discrepancy in timings of peaks may be explained by a more complex and cyclical nature of the female HPG axis or by limitations in our study design and the associated mathematical modeling. However, this interpretation of our results is limited by several factors and stresses the need for further studies to ascertain the exact mechanisms underlying the female minipuberty.

Our study clearly challenges the general impression of minipuberty being a single and short-lived activation of the HPG axis in infancy, which has repercussions for the clinicians aiming to use the window of diagnostic opportunity that minipuberty presents. The timing of the 2 peaks observed in our study suggest that sampling at 3 months of age may not be the optimal timing. Moreover, the high degree of detectability of the ovarian hormones at both 6 months and 1 year of age indicates that the period of HPG axis activation appears to be longer than previously believed and compared with what has been observed in male infants (2). In line with this, data on females with Turner syndrome indicated that even sampling beyond 1 year of age may produce an insight into gonadal function (32). We therefore suggest that a single sample around 3 months of age is not necessarily the only or best way to elucidate the HPG axis in a newborn girl. In fact, consecutive samples may be desirable to fully investigate the axis and ovarian activation.

The strengths of this study include: (1) the longitudinal, prospective design of the study with consecutive blood samples in a large number of healthy, infant girls; (2) the study spans from birth until 12 months of age, thus including all of minipuberty; (3) it is a single-center study; (4) all blood samples were measured by highly sensitive analytical methods in the same laboratory including gold standard LC-MS/MS estradiol quantification; and (5) preanalytical processes mimicked those in our outpatient clinic. Limitations of this study include that: (1) all girls were Caucasian and conclusions may not directly apply to non-Caucasians; (2) the cohort was made of up of infants from singleton pregnancies and born to mothers from affluent and educated backgrounds and may not be representative of other demographic groups; (3) the reference ranges of the included reproductive hormones were restricted to the specific analytical method used and should be interpreted with caution when applied to other laboratories; (4) the use of immunoassays risks the possibility of interference, which has specifically been reported for the Beckman-Coulter AMH assay; (5) a more sensitive assay than the one used is in this study is available for AMH quantification (Ansh Labs); (6) because of the limit of 2 attempts at blood sampling per infant, missing samples were not rare. Theoretically this may have skewed the data; however, we consider this risk minimal because no association between factors compromising blood sampling and serum levels of hormones was suspected; (7) serum sampling was performed randomly between 8 am and 4 pm and consequently, diurnal variations of hormones are not accounted for in this setup; (8) factors that may affect inter-child variation have not been investigated in this study (eg, the possible influence of maternal age, gestational age, birth weight, delivery method, and maternal hormone concentrations); and (9) serum data on adrenal and ovarian androgen production were not available in this study because of limited volumes of the serum samples.

In conclusion, in this longitudinal study of minipuberty in healthy, infant girls, we present reference ranges of reproductive hormones throughout the first year of life. Interestingly, we observed a biphasic and prolonged female minipuberty. Two infant peaks within the first 6 months of life for both the gonadotropins and ovarian hormones represent hitherto undescribed HPG axis physiology.

Abbreviations

Abbreviations

• AMH

  anti-Müllerian hormone

• CV

  coefficients of variation

• E1

  estrone

• E2

  estradiol

• GAMLSS

  Generalized Additive Model for Location, Scale and Shape

• HPG

  hypothalamic-pituitary-gonadal axis

• LC-MS/MS

  liquid chromatography tandem mass spectrometry

• LOD

  limits of detection

• LOQ

  limits of quantification

Acknowledgments

The authors thank all participants and their parents for their contribution to this study.

Funding

The COPENHAGEN Minipuberty Study received funding from: (1) The Absalon Foundation, no. F-23653-01 (M.L.L.); (2) Aase og Ejnar Danielsens Fond: no. 10-001874 (A.J.U.); (3) Candy Foundation, nos. 2017-224, 2020-344 (E.N.U.); (4) EDMaRC: no. 1500321/1604357 (A.S.B.); (5) The Danish Environmental Protection Agency (Miljøstyrelsen: MST-621-00012 Center on Endocrine Disrupters) (HF); (6) The European Union’s Horizon 2020 research and innovation program under grant agreement No 733032 HBM4EU (H.F.); (7) The Research Council of Capital Region of Denmark no. E-22717-11 (A.J.U.); and (8) The Research Council of Rigshospitalet (A.J.U., A.S.B., M.L.L.) (nos. E-22717-12, E-22717-07, E-22717-08).

Disclosures

L.L.R. is a full-time employee of Novo Nordisk A/S. A.J.U. has received speaker’s fees from Novo Nordisk A/S, Ferring, and IPSEN. The other authors have no other conflicts of interest to declare.

Data Availability

Restrictions apply to the availability of some or all data generated or analyzed during this study to preserve patient confidentiality or because they were used under license. The corresponding author will on request detail the restrictions and any conditions under which access to some data may be provided.

References

© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

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