Abstract
The ratio between luteinizing hormone (LH) and follicle-stimulating hormone (FSH) has previously been described as an excellent marker of sex in healthy infants. However, LH/FSH remains not fully described in patients with differences of sex development (DSD). The aim was therefore to describe LH/FSH in infants with DSD. This was a retrospective study of DSD patients, all aged 0–1.2 years. In total, 87 infants with DSD and at least one serum sample per infant were included. Longitudinal samples from single patients were included whenever possible. Serum LH/FSH ratios in these patients were plotted against recently published age-related and sex-dimorphic cutoffs. Overall, LH/FSH sometimes corresponded to assigned sex without any obvious pattern in terms of diagnoses. LH/FSH corresponded to the biological sex in all patients with Turner or Klinefelter syndrome. In patients with 46,XX or 46,XY DSD (except congenital adrenal hyperplasia (CAH)), the ratios did not correspond to the assigned sex in all cases and were interchangeably within the male and female range. In patients with CAH, the ratio corresponded to biological sex (based on sex chromosomes) in some cases but also ranged across the cutoffs. In the 15 patients with 45,X/46,XY mosaicism, the LH/FSH ratios corresponded to the assigned sex in all cases (12 were raised as males, 3 as females) and at all time points in cases with multiple sampling. While this study describes LH/FSH in infants with DSD, the exact clinical role of the ratio in the management of these patients remains to be further elucidated.
Introduction
Luteinizing hormone (LH) and follicle-stimulating hormone (FSH), both produced in the gonadotropic cells of the anterior pituitary gland, have recently been described as an excellent marker of sex when combined in the LH/FSH ratio based on their serum concentrations (1, 2, 3). Interestingly, the marker only appears to be sex-dimorphic during a limited time period which covers at least the first year of life (1, 3); however, by the age of 5 years, the marker is no longer a viable discriminator due to a large overlap between the sexes (2). How the same cells in the anterior pituitary gland can produce two different products whose ratio varies from birth into adulthood is a biological conundrum and of physiological interest.
Yet, the LH/FSH ratio and its sex-dimorphisms remain not fully described in patients. With newly published cutoff values for the LH/FSH ratio for the entire first year of life (3), it is now more feasible for a study to explore the marker clinically. Particularly, in patients with differences of sex development (DSD), a heterogeneous group of conditions in which the anatomical, gonadal, or chromosomal sex is affected (4), the ratio would be of potential interest. In some newborns with DSD and ambiguous genitalia, sex assignment can be clinically and ethically difficult. International trends are moving toward limiting early sex-adjusting surgical interventions until the child can actively partake in the decision (5, 6, 7). In spite of this, legal sex must be assigned following birth in most countries.
Currently, multiple factors are thoroughly examined at birth or at diagnosis in infants with DSD and atypical genitalia and include quantification of reproductive hormones, evaluation of surgical possibilities and future fertility potential as well as estimation of the potential risk of gonadal malignancy (6, 8). Cultural factors and parental wishes also play an important role in decision-making. In addition, the presence of specific genetic mutations may provide important knowledge on the natural course of the conditions and their development in some cases. It is also important to consider the possible genital response to hormone therapy (6).
Despite this panel of factors considered, assigned sex does not always align with gender identity later in life, and the addition of other factors may further guide clinicians and families in appropriate sex assignment. Here, the LH/FSH ratio has been suggested as a supplementary marker in some cases (1). Additionally, the ratio may potentially add diagnostic information in other cases (1), which is of particular value in cases within the DSD spectrum as the exact diagnosis in many patients with DSD remains unknown. In line with this, many patients with DSD are not diagnosed during the narrow time interval of minipuberty previously studied (1). Moreover, while minipuberty is a period of pituitary–gonadal activation, reproductive hormones are still quantifiable, particularly in girls, at 1 year of age (9, 10). Thus, the LH/FSH ratio in a larger cohort of patients with DSD during the entire first year of life remains to be examined.
The aim of this study was therefore to explore and describe the LH/FSH ratio in infants with DSD aged 0–1 year by applying the recently published cutoffs for the ratio in these patients.
Methods
Patients with differences of sex development
In total, 87 infants with a diagnosis within the spectrum of DSD as defined by the 2006 Consensus Statement (4) were included in this study. The patients were identified via a database of patients with DSD followed at the Department of Growth and Reproduction, Copenhagen University Hospital, between January 1, 1996 and October 30, 2019. Inclusion criteria were: (i) a DSD diagnosis and (ii) available serum LH and FSH concentrations analyzed as part of routine evaluation during the first year of life. The diagnoses of the 87 patients were as follows (listed according to n): Klinefelter syndrome including variants (n = 29), congenital adrenal hyperplasia (CAH, classical n = 14; non-classical n = 2), 45,X/46,XY mosaicism (n = 15), idiopathic 46,XY DSD (n = 8), Turner syndrome including mosaic variants (SRY negative) (n = 8), complete androgen insensitivity syndrome (CAIS, n = 3), persistent Müllerian duct syndrome (n = 3), 46,XX DSD (n = 2), nuclear receptor subfamily 5 group A member 1 mutation (n = 1), a 46,Xvar(Y)/46,XY-karyotype (n = 1), and a 48,XXYY-karyotype (n = 1). Patients with CAH were subgrouped into classical and non-classical according to genotype; the two patients with non-classical CAH were diagnosed in infancy due to affected, older siblings.
Serum LH/FSH ratios in 27 of the 87 included patients with DSD (Klinefelter syndrome: n = 13; 45,X/46,XY mosaicism: n = 8; Turner syndrome: n = 4; and CAIS: n = 2; all aged 2–5 months) have previously been published (1), and other hormonal data have been published on several of the other patients as well (11, 12, 13, 14, 15). However, in this study, we present LH/FSH ratios and serial measurements whenever possible, which have not previously been published.
The retrospectively collected data on patients with DSD were extracted from medical records and included information on assigned sex, external genital phenotype at birth, and possible treatment. All infants with classical CAH received relevant gluco- and mineralocorticoid treatment at the time of blood sampling, while all other patients in this study were untreated at the time of sampling. Based on medical records, the external genitalia in patients with 45,X/46,XY mosaicism were retrospectively scored (prior to any surgical intervention) according to the external genitalia score (EGS) with scores ranging from 0 (female) to 12 (male) (16).
Hormone assays
Serum samples were taken as part of routine follow-up, and all preanalytical handling was identical and carried out the same day as sampling. The serum samples were centrifuged and allocated into aliquots, which were stored at −20°C for a maximum of 5 weeks before analysis.
Serum LH and FSH were quantified using time-resolved fluoroimmunometric assays (AutoDELFIA, Perkin Elmer). For both assays, the limits of detection (LODs) were 0.05 IU/L and the inter-assay coefficients of variations (CVs) were ≤5%. The analyses have been accredited by The Danish Accreditation Fund for medical examination (the standard DS/EN ISO 15189). The LH/FSH ratios were simply obtained by dividing the LH concentrations (nominator) by the FSH concentrations (denominator). For concentrations below LOD, the ratios were calculated using the LOD/2 for the corresponding hormone.
In a single patient, we report testosterone concentrations measured by in-house liquid chromatography–mass spectrometry with an LOD of 0.012 nmol/L and an inter-assay CV of 7%.
Statistical methods
The Mann–Whitney U test was used to compare EGSs. P-values below 0.05 were considered statistically significant. Statistical analyses were carried out using IBM SPSS Statistics version 22.
Data from the included patients in this study were plotted against age-related LH/FSH ratio cutoffs previously published by our group based on healthy infants from The COPENHAGEN Minipuberty Study (3, 17). For details pertaining to the receiver-operating analysis-derived cutoffs, please refer to (3).
Ethical considerations
For all included patients, the Danish Patient Safety Authority approved retrieval of information from medical records (3-3013-1376/1/) and the Danish Data Protection Agency approved the data handling (RH-2015-235, I-Suite 04204).
Results
The LH/FSH ratio corresponded to the biological sex in all patients with Turner or Klinefelter syndrome, except for a single infant with Klinefelter syndrome in whom the LH/FSH ratio of 0.53 was below the age-relevant LH/FSH cutoff value of 0.54 (Fig. 1A and B). All patients with Turner syndrome (and no Y-chromosome material) had a female phenotype and all patients with Klinefelter syndrome had a male phenotype. In patients with 46,XX or 46,XY DSD due to different underlying conditions (except CAH), the ratio did not correspond to the assigned sex in all cases (Fig. 2A and B). In fact, the LH/FSH ratios were interchangeably within the male and female range in several patients with 46,XX and 46,XY DSD.
The ratio of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), i.e. the LH/FSH ratio in serum, according to age in patients with Turner syndrome and variants (A) or Klinefelter syndrome and variants (B). Patient data are plotted against a previously published cutoff (solid black line (3)).
Citation: Endocrine Connections 12, 3; 10.1530/EC-22-0275
The ratio of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), i.e. the LH/FSH ratio in serum, according to age in patients with 46,XX DSD (A) or 46,XY DSD (B). Patient data are plotted against a previously published cutoff (solid black line (3)). CAIS, complete androgen insensitivity syndrome; NR5A1, nuclear receptor subfamily 5 group A member 1 mutation; PMDS, persistent Müllerian duct syndrome; SRY, sex-determining region of the Y chromosome.
Citation: Endocrine Connections 12, 3; 10.1530/EC-22-0275
In patients with CAH, the ratio corresponded to biological sex (based on sex chromosomes) in some cases, but not in all (Fig. 3A). In some patients with multiple samples, the LH/FSH ratio ranged across the cutoffs. In one female with classical CAH and a high and a low LH/FSH ratio, corresponding testosterone concentrations were available. These revealed an elevated serum testosterone concentration of 8.5 nmol/L and an LH/FSH ratio of 1.0 (‘male’ range) at the initial sampling and subsequently, a serum testosterone concentration of 0.38 nmol/L and an LH/FSH ratio of 0.18 (‘female’ range) after the onset of glucocorticoid treatment.
The ratio of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), i.e. the LH/FSH ratio in serum, according to age in patients with congenital adrenal hyperplasia (CAH) (A) or 45,X/,46,XY mosaicism (B). Patient data are plotted against a previously published cutoff (solid black line (3)). NC, non-classical; SW, salt-wasting.
Citation: Endocrine Connections 12, 3; 10.1530/EC-22-0275
In the 15 patients with 45,X/46,XY mosaicism, the LH/FSH ratios corresponded to assigned sex in all cases (12 were raised as males, 3 as females, Table 1) and at all time points in cases with multiple sampling (Fig. 3B). There was a significant difference in EGSs at birth between males (median = 12, range: 7–12) and females (median = 5, range: 2–8) (P = 0.004), despite the overlap. In the patients diagnosed due to external genital phenotype (i.e. atypical genitalia), there was no significant difference in EGSs between the sexes (range: 2–10).
Descriptive characteristics of 15 infants with 45,X/46,XY mosaicism.
| No | Age at serum sampling (years) | Assigned sex at birth | LH/FSH | External Genitalia Score |
|---|---|---|---|---|
| 1 | 0.24 | M | 1.69 (M) | 12 |
| 2 | 0.25 | M | 1.41 (M) | 12 |
| 3 | 0.25 | M | 1.47 (M) | 12 |
| 4 | 0.25 | M | 1.59 (M) | 12 |
| 5 | 0.26 | M | 1.71 (M) | 12 |
| 6 | 0.26 | M | 1.71 (M) | 12 |
| 7 | 0.28 | M | 1.50 (M) | 12 |
| 8 | 0.25 | M | 0.97 (M) | 10 |
| 9 | 0.24 | M | 2.06 (M) | 9 |
| 10 | 0.24 | M | 1.85 (M) | 9 |
| 11 | 0.11 | M | 1.15 (M) | 7 |
| 12 | 0.68 | M | 0.26 (M) | 7 |
| 13 | 0.11 | F | 0.30 (F) | 7 |
| 14 | 0.05 | F | 0.34 (F) | 4.5 |
| 15 | 0.14 | F | 0.37 (F) | 1.5 |
Age at serum sampling, first serum sampling if several were available for a single patient; (F), female range according to reference material; F, female; FSH, follicle-stimulating hormone; LH, luteinizing hormone; (M), male range according to reference material; M, male; No, patient number (patients listed in order of descending External Genitalia Score (4).
Discussion
This is the largest study to date to describe the serum LH/FSH ratio as a marker of sex in infants with DSD aged 0–1 year. In this exploratory study of 87 infants with DSD, the LH/FSH ratio did not appear to add individual diagnostic or treatment value to most DSD diagnoses. Still, it may be considered as an addition to the existing panel of parameters evaluated during diagnostic work-up of infants with DSD and atypical genitalia. However, larger studies are needed to ascertain the potential clinical role of the LH/FSH ratio.
The LH/FSH ratio was examined in 87 patients with 12 different DSD diagnoses and while the overall number of patients with DSD included was large, the small number of patients per diagnosis and the retrospective design did not allow for firm conclusions to be drawn. With this in mind, this exploratory study alluded to the potential clinical relevance of the ratio in aiding sex assignment alongside the existing panel of markers in patients with 45,X/46,XY mosaicism and atypical external genitalia. Specifically, the ratio corresponded to the assigned sex in all these patients, while the EGSs varied and overlapped between the sexes in patients with atypical external genitalia. For example, an EGS of 7 was observed in both female and male sex of rearing highlighting that the EGS alone cannot predict sex assignment. This group of patients often pose clinicians with the ethical and clinical dilemma of sex assignment as the phenotypes in cases of atypical genitalia, by definition, are neither clearly female nor male. The LH/FSH ratio may thus serve as additional information to help clinical decision-making, although further studies are needed to verify this potential. In particular, larger studies including more patients with diagnoses in which gonadal and genital sex may vary between patients are required to study the potential use of the LH/FSH ratio.
While the LH/FSH ratio in the other DSD diagnoses may not have a distinct clinical relevance per se, the ratio likely reflects the underlying biology, perhaps intrauterine androgen exposure in some cases, as well as any postnatal hormone treatment. The latter was likely the case in the female with classical CAH whose LH/FSH ratio was in the male range (and with high serum testosterone) prior to the onset of glucocorticoid treatment and an LH/FSH ratio in the female range after the onset of treatment. Still, we did not observe any pattern or link with the ratio that could aid diagnostics or treatment.
In a previous study by our group which included data on adult patients with DSD, a similar lack of pattern for diagnoses was observed (2). However, interestingly, many of the adult male patients with 45,X/46,XY mosaicism had very low LH/FSH ratios, some even within the female range, which is in contrast to the findings in this current study. This highlights that the first year of life provides information on the hypothalamic–pituitary–gonadal axis not available later in life (2). While the gonadotropins separately have been widely described in most DSD diagnoses (such as 11, 12, 13, 18, 19)), the conundrum remains of how the same cells produce the two gonadotropins with a varying interrelationship throughout life (20).
The strengths of this study included: (i) the inclusion of 87 patients with DSDs covering 12 different diagnoses including both biochemical and clinical data; (ii) the inclusion of some serial measurements; (iii) all patients were followed at a single center; and (iv) the quantification of LH and FSH by highly sensitive analytical methods. However, the following limitations to our study should also be considered: (i) although the total number of patients with DSD is relatively large, the high variety of diagnoses and the fact that several of the DSD diagnoses were represented by only a single or few heterogeneous patients do not allow for firm conclusions; (ii) blood sampling took place during normal working hours, consequently diurnal variations may have skewed the data; (iii) different ethnic backgrounds unaccounted for the in the analyses of the data; and (iv) as 27 of the patients have previously been included in studies on DSD from our department, a risk of skewing published data exists.
In conclusion, this study explored and described the LH/FSH ratio in infants with DSD, and while the ratio may provide useful clinical information on some patients with certain forms of DSD and atypical genitalia during the first year of life, the overall clinical relevance of the ratio remains unknown.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review.
Funding
The COPENHAGEN Minipuberty Study received financial support from: (1) The Candy Foundation, 2017-224 and 2020-344 (E N U); (2) The Absalon Foundation, F-23653-01 (M L L); (3) The European Union’s Horizon 2020 research and innovation program, No 733032 HBM4EU (A M A); (4) The Danish Environmental Protection Agency (Miljøstyrelsen): MST-621-00012 Center on Endocrine Disrupters (A J); (5) The Research council of Capital Region of Denmark: E-22717-11 (A J); (6) Research council of Rigshospitalet: Nos. E-22717-12, E-22717-07, E-22717-08 (A J, A S B, M L L); (7) Aase og Ejnar Danielsens Fond: 10-001874 (A J); and (8) International Research and Research Training Centre for Male Reproduction and Child Health (EDMaRC): 1500321/1604357 (A S B).
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