Dynamic GnRH and hCG testing: establishment of new diagnostic reference levels

in European Journal of Endocrinology
Authors:
A Kirstine BangDepartment of Growth and Reproduction, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark
International Center for Research and Research Training in Endocrine Disruption of Male Reproduction and Child Health (EDMaRC), Rigshospitalet, Denmark

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Loa NordkapDepartment of Growth and Reproduction, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark
International Center for Research and Research Training in Endocrine Disruption of Male Reproduction and Child Health (EDMaRC), Rigshospitalet, Denmark

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Kristian AlmstrupDepartment of Growth and Reproduction, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark
International Center for Research and Research Training in Endocrine Disruption of Male Reproduction and Child Health (EDMaRC), Rigshospitalet, Denmark

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Lærke PriskornDepartment of Growth and Reproduction, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark
International Center for Research and Research Training in Endocrine Disruption of Male Reproduction and Child Health (EDMaRC), Rigshospitalet, Denmark

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Jørgen Holm PetersenDepartment of Growth and Reproduction, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark
International Center for Research and Research Training in Endocrine Disruption of Male Reproduction and Child Health (EDMaRC), Rigshospitalet, Denmark
Department of Biostatistics, University of Copenhagen, Copenhagen, Denmark

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Ewa Rajpert-De MeytsDepartment of Growth and Reproduction, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark
International Center for Research and Research Training in Endocrine Disruption of Male Reproduction and Child Health (EDMaRC), Rigshospitalet, Denmark

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Anna-Maria AnderssonDepartment of Growth and Reproduction, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark
International Center for Research and Research Training in Endocrine Disruption of Male Reproduction and Child Health (EDMaRC), Rigshospitalet, Denmark

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Anders JuulDepartment of Growth and Reproduction, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark
International Center for Research and Research Training in Endocrine Disruption of Male Reproduction and Child Health (EDMaRC), Rigshospitalet, Denmark

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Niels JørgensenDepartment of Growth and Reproduction, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark
International Center for Research and Research Training in Endocrine Disruption of Male Reproduction and Child Health (EDMaRC), Rigshospitalet, Denmark

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Correspondence should be addressed to N Jørgensen; Email: niels.joergensen@regionh.dk
Free access

Objective

Gonadotropin-releasing hormone (GnRH) and human chorionic gonadotropin (hCG) stimulation tests may be used to evaluate the pituitary and testicular capacity. Our aim was to evaluate changes in follicular-stimulating hormone (FSH), luteinizing hormone (LH) and testosterone after GnRH and hCG stimulation in healthy men and assess the impact of six single nucleotide polymorphisms on the responses.

Design

GnRH and hCG stimulation tests were performed on 77 healthy men, 18–40 years (reference group) at a specialized andrology referral center at a university hospital. The potential influence of the tests was illustrated by results from 45 patients suspected of disordered hypothalamic–pituitary–gonadal axis.

Methods

Baseline, stimulated, relative and absolute changes in serum FSH and LH were determined by ultrasensitive TRIFMA, and testosterone was determined by LC–MS/MS.

Results

For the reference group, LH and FSH increased almost 400% and 40% during GnRH testing, stimulated levels varied from 4.4 to 58.8 U/L and 0.2 to 11.8 U/L and FSH decreased in nine men. Testosterone increased approximately 110% (range: 18.7–67.6 nmol/L) during hCG testing. None of the polymorphisms had any major impact on the test results. Results from GnRH and hCG tests in patients compared with the reference group showed that the stimulated level and absolute increase in LH showed superior identification of patients compared with the relative increase, and the absolute change in testosterone was superior in identifying men with Leydig cell insufficiency, compared with the relative increase.

Conclusions

We provide novel reference ranges for GnRH and hCG test in healthy men, which allows future diagnostic evaluation of hypothalamic–pituitary–gonadal disorders in men.

Abstract

Objective

Gonadotropin-releasing hormone (GnRH) and human chorionic gonadotropin (hCG) stimulation tests may be used to evaluate the pituitary and testicular capacity. Our aim was to evaluate changes in follicular-stimulating hormone (FSH), luteinizing hormone (LH) and testosterone after GnRH and hCG stimulation in healthy men and assess the impact of six single nucleotide polymorphisms on the responses.

Design

GnRH and hCG stimulation tests were performed on 77 healthy men, 18–40 years (reference group) at a specialized andrology referral center at a university hospital. The potential influence of the tests was illustrated by results from 45 patients suspected of disordered hypothalamic–pituitary–gonadal axis.

Methods

Baseline, stimulated, relative and absolute changes in serum FSH and LH were determined by ultrasensitive TRIFMA, and testosterone was determined by LC–MS/MS.

Results

For the reference group, LH and FSH increased almost 400% and 40% during GnRH testing, stimulated levels varied from 4.4 to 58.8 U/L and 0.2 to 11.8 U/L and FSH decreased in nine men. Testosterone increased approximately 110% (range: 18.7–67.6 nmol/L) during hCG testing. None of the polymorphisms had any major impact on the test results. Results from GnRH and hCG tests in patients compared with the reference group showed that the stimulated level and absolute increase in LH showed superior identification of patients compared with the relative increase, and the absolute change in testosterone was superior in identifying men with Leydig cell insufficiency, compared with the relative increase.

Conclusions

We provide novel reference ranges for GnRH and hCG test in healthy men, which allows future diagnostic evaluation of hypothalamic–pituitary–gonadal disorders in men.

Introduction

The hypothalamic–pituitary–gonadal (HPG) axis regulates the development and maintenance of the male reproductive system. Secretion of the gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), from the pituitary gland is mainly stimulated by the hypothalamic gonadotropin-releasing hormone (GnRH). Simplified, FSH and LH stimulate spermatogenesis and testosterone (T) production in the testicles respectively.

Decreased testicular function can be due to a primary testicular dysfunction or a dysfunction of the pituitary gland or hypothalamus. Dynamic testing of the HPG axis may facilitate the diagnostic process of testosterone deficiency. The capacity of the pituitary gland for gonadotropin secretion can be assessed by the GnRH test, whereas the capacity of the Leydig cells in the testicles to produce T can be assessed by the human chorionic gonadotropin (hCG) test (1, 2, 3, 4, 5, 6, 7, 8).

The single nucleotide polymorphism (SNP) FSHB -211G>T (rs10835638) in the promoter of the gene coding for the FSH B-subunit leads to a reduced FSH production (9, 10, 11). In addition, both sensitivity and transcriptional activity of the FSH receptor (FSHR) have been linked to the polymorphisms FSHR 2039A>G(rs6166) and the promoter polymorphism FSHR -29G>A (rs1394205) and all three SNPs have been associated to male reproduction (11, 12, 13). Genetic variants in the LH receptor (LHCGR) might alter the receptor function, and rs2293275 has been associated with spermatogenic damage (14) and rs7371084 and rs4953617 have been associated with testicular germ cell cancer (15). To which degree these polymorphisms affect the GnRH and hCG test responses, have, to our knowledge, not been described. To provide updated reference levels for the GnRH and hCG tests and examine the possible confounding by genetic effect of six gonadotropin-related SNPs, we examined 77 healthy Danish men. The potential usefulness of these reference levels is illustrated by results from 45 patients suspected of disordered HPG axis who had previously been tested.

Subjects and methods

Healthy participants (reference group)

Eighty healthy Danish men participating in an ongoing study of testicular function (16) were invited to take part in the hormone tests within a period of three weeks from the primary study. Inclusion criteria were no medical history of chronic diseases, testicular surgery or trauma. During the primary study, the men completed a questionnaire regarding medical history and lifestyle factors, delivered a semen sample and underwent a physical examination (body weight and height, assessment of testicular volume and ultrasound scan of the testicles). All examinations, tests and laboratory assessments were performed at Dept. of Growth and Reproduction, Rigshospitalet, Copenhagen, Denmark between 2012 and 2014. All men received 500 DKK (~68 Euro) for their participation. Seventy-seven men completed the GnRH and hCG tests. However, five did not show up for the final blood sampling for the hCG tests, leaving results for 72 men on the hCG test. The study has been approved by the ‘Ethical board of the Capital region’ (permit number H-2-2012-092).

Hormone testing

All men had a GnRH test performed directly followed by the initiation of an hCG test. All GnRH tests were started between 08:00 and 12:00 h. Prior to the tests, a baseline blood sample was drawn, which counted as the baseline blood sample for both tests.

GnRH stimulation test

An intravenous injection of 100 μg GnRH (Relefact) was given, and a blood sample was collected after 30 min.

hCG

An injection of 5000 IU hCG (Pregnyl) was given in the gluteal muscle after the final blood sample was taken for the GnRH test. The follow-up blood sample was taken 72 h later.

All men also had an adrenocorticotropic hormone (ACTH) test performed simultaneously with the GnRH test for another study. Results of this test will not be described here.

Patient group

We retrospectively identified five patient groups with a total of 45 men from our out-patient clinic. They underwent a GnRH test and/or an hCG test performed the same way as the healthy participants, as part of their clinical work-up of suspected testosterone deficiency between 1996 and 2015. Fourteen patients had been unilaterally orchiectomized due to testicular germ cell cancer and had received irradiation against germ cell neoplasia in situ (GCNIS) in the remaining testis. Eleven were suspected of hypogonadotropic hypogonadism on various backgrounds; pituitary abnormalities (five tumors and one empty sella), normal pituitary morphology (MRI validated) but absence of puberty (two with Kallmann syndrome and three with isolated hypogonadotropic hypogonadism (IHH)). Nine were suspected of medically induced testosterone deficiency due to treatment with methadone, morphine, gabapentin or baclofen. Eleven patients were obese men (BMI >30) with a low-normal baseline total testosterone where it was concluded from the clinical work-up that they did not have testosterone deficiency, and therefore, were not offered testosterone treatment.

Hormone and semen analysis

The blood samples obtained from the healthy participants were allowed to clot, thereafter centrifuged, and serum was frozen at −20°C until analysis. Hormone analyses were performed when all subjects had completed their participation. Patients had their hormone analysis done at the time of the visit in the clinic. Serum levels of FSH and LH were determined using a time-resolved immunofluorometric assay (TRIFMA) (Delfia, Wallac, Turku, Finland). Sex hormone-binding globulin (SHBG) was determined by time-resolved chemiluminescent immunoassay (Access, Beckman Coulter). Estradiol levels were determined using radioimmunoassay (Biotech-IGG, Pantex). Inter- and intra-assay coefficients of variation (CVs) for measurements of the hormones were FSH (3% and 2%), LH (2% and 3%), SHBG (5% and 4%) and estradiol (15% and 8%) respectively. Serum testosterone of the healthy volunteers was analyzed by a TurboFlow-LC–MS/MS method (17). The CVs of the low and high level quality control samples were 10% and 5.7% respectively in the detection range of 0.071–36 nmol/L. Limit of quantification (LOQ) was 0.10 nmol/L. Serum testosterone of the patients had been analyzed by radioimmunoassay/RIA (Coat-a-Count, Siemens) with a detection limit of 0.23 nmol/L, intra-assay CV was 17% and an inter-assay CV was 13%. For T, the conversion factor to LC–MS/MS from RIA was 1.24 for T (RIA) >5 nmol/L and 1.91 for T (RIA) <5 nmol/L. Patients T-values were corrected according to these factors. Free testosterone was calculated (cFT) according to Vermeulen et al. (18) assuming a fixed albumin value of 43.8 g/L.

All participants produced semen samples by masturbation, and the samples were assessed as previously described (16). Semen volume was assessed by weighing, assuming a density of 1 g/mL. For the assessment of the sperm concentration, the samples were diluted in a solution of 0.6 mol/L NaHCO3 and 0.4% (v/v) formaldehyde in distilled water, and subsequently assessed using a Bürker-Türk haemocytometer (Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany). Total sperm count was calculated as semen volume × sperm concentration.

Genotyping

Genotyping was performed as described before (19). In short, EDTA-preserved peripheral blood was used for isolation of genomic DNA using the Promega Maxwell 16 DNA purification kits (Promega Biotech AB) and quantified on a NanoDrop ND-1000 spectrophotometer (Saveen Werner, Limhamn, Sweden). KASP SNP genotyping assays (LGC Genomics, Hoddesdon, UK) were used to determine the genotypes by competitive PCR. KASP genotyping assays were designed by LGC Genomics toward the following sequences: FSHB -211G>T (rs10835638), TATCAAATTTAATTT[G/T]TACAAAATCATCAT; FSHR -29G>A (rs1394205), TCTCTGCAAATGCAG[A/G]AAGAAATCAGGTGG; FSHR2039A>G (rs6166), ATGTAAGTGGAACCA[C/T]TGGTGACTCTGGGA. Genotyping assays for the three LHCGR variants were designed by LGC Genomics toward the following sequences: rs2293275, ACAGTGTTTTRTTR[Y]TCACTKYCCTTACTGT; rs7371084, ACACTGTGGC[Y]TAGRTYKGTTAAGTA; rs4953617, TCCTGGCCAAG[Y]AGATTCTGCCCC.

A master mix containing a low concentration of the ROX fluorophore was used for internal normalization and as negative controls DNA was omitted in the reaction mixture and in all cases showed no amplification. All assays separated samples into clear genotype clusters.

All 77 healthy men were genotyped for the FSHB and the two FSHR variants but only 75 men were genotyped for the LHCGR variants.

Statistical analysis

For descriptive statistics, medians and 2.5–97.5th percentiles or frequencies were calculated for the healthy men used as reference group. Correlations between serum FSH and LH were tested by Spearman’s rank-order correlation. Responses to the hormone stimulations were calculated as absolute increase (stimulated-baseline) or relative increase ((stimulated-baseline)/baseline). Paired samples t-test was used to compare baseline and stimulated hormonal values. Multivariate linear regression analysis was used to detect the differences in the hormone levels between the different genotype groups (tested as additive, dominant and recessive models). Hormone values were transformed by natural logarithm to obtain variance stability and normalize data in the analysis. Covariates evaluated in the statistical model included factors possibly associated with levels of reproductive hormones (blood sampling hour, age, smoking status, BMI and alcohol consumption) and were included if significant. P < 0.05 was considered statistically significant. The 2.5th percentiles of the stimulated levels and increases (absolute and relative) from the healthy reference group were used as cut-off values when comparing with the results from the patients’ hormone stimulation tests. SPSS, version 22 for Windows was used for all statistical analyses.

Results

Table 1 describes the group of healthy men, including genotypes and distribution of alleles. Three men had used medicine (antihistamine; n = 2, dermally applied glucocorticoid; n = 1) at some point during the last 3 months prior to participation. Use of medicine was not significantly associated with any of the hormone levels (data not shown). Among the smokers, 14 men used cannabis or other recreational drugs; for all less than once a month and the use was not associated to any hormone levels (data not shown). Thus, adjusting for men who used the drugs or analyzing data excluding these men did not change the results.

Table 1

Basic description of 77 healthy men.

Characteristics Values
Basic description, median (min; max)
 Age (years) 27.0 (18; 40)
 Height (cm) 183 (165; 197)
 Weight (kg) 77 (58; 113)
 Body mass index (kg/m2) 22.9 (17.3; 30.9)
 Mean of testis size (mL)a 14.0 (7.7; 26.0)
 Total sperm count (million) 221 (7; 827)
 Alcohol intake (units/week)b 7.5 (0; 60)
 Smoking, % (n)
  Daily 11.7 (9)
  Occasionally 28.6 (22)
 Proven fatherhood 53.2 (41)
Genotypes, % (n)
 rs10835638 (FSHB -211G > T)
  GG 68.8 (53)
  GT 26.0 (20)
  TT 5.2 (4)
 rs1394205 (FSHR -29G > A)
  GG 55.8 (43)
  GA 36.4 (28)
  AA 7.8 (6)
 rs6166 (FSHR 2039A > G)
  AA 23.4 (18)
  AG 46.8 (36)
  GG 29.9 (23)
 rs2293275 (LHCGR S312N)
  CC 37.3 (28)
  CT 46.7 (35)
  TT 16.0 (12)
 rs7371084 (LHCGR)
  TT 64.0 (48)
  TC 36.0 (27)
  CC 0 (0)
 rs4953617 (LHCGR)
  TT 85.3 (64)
  TC 14.7 (11)
  CC 0 (0)

Measured by ultrasound and calculated as mean value of left and right testicle; b1 unit = 12 g alcohol. Alcohol intake is the sum of intake of beer, wine and strong alcohol the recent week prior to participation in study.

Of the 77 men, 41 men were proven fathers and 36 had unproven fertility status. Total sperm count ranged from 7 to 827 million, with only two men with less than 39 million (7 and 38 million). None of the participants were homozygous minor allele (TT) carriers of LHCGR variants rs7371084 and rs4953617.

GnRH test

The results of the GnRH and hCG tests in the healthy men are shown in Table 2 and colored according to baseline quartiles in Fig. 1. Tables 3, 4 and 5 show the results stratified according to the polymorphisms. The stimulated gonadotropin levels showed a large variation (Fig. 1A and B), with stimulated FSH and LH values in the ranges 0.2–11.8 IU/L and 4.4–58.8 IU/L respectively. For FSH, the stimulated level decreased in nine men. The median (min–max) testis size of these men were 14 mL (12–22 mL), and total sperm counts 202 million (45–614 million). LH increased in all 77 men. Stimulated values correlated with baseline values for both FSH (rs: 0.872, P < 0.01) and LH (rs: 0.292, P < 0.01). The relative increase of the LH level (392%) was much higher than the increase of FSH (40%), but overall, the serum levels of the two gonadotropins correlated positively (baseliners: 0.415, stimulated rs: 0.606, absolute increase rs: 0.665 and relative increase rs: 0.826, all P < 0.01) (Fig. 2). Age of the men was negatively correlated with the stimulated level (rs: −0.35, P = 0.002) and absolute increase in LH (rs: −0.31, P = 0.005), but not with the change in any other hormones. Smoking status or BMI had no influence on the hormonal increase after GnRH stimulation (P > 0.05).

Figure 1
Figure 1

(A, B, C and D) Hormone levels at baseline and after GnRH and hCG-test in the reference group of healthy men (n = 77/72). Results stratified and colored according to baseline quartiles (Blue: 1st quartile, Green: 2nd quartile, Light brown: 3rd quartile, Purple: 4th quartile). (A) LH levels at baseline and 30 min. after GnRH stimulation. (B) FSH levels at baseline and 30 min. after GnRH stimulation. (C) Testosterone levels at baseline and 72 h after hCG stimulation. (D) Calculated free testosterone levels at baseline and 72 h after hCG stimulation.

Citation: European Journal of Endocrinology 176, 4; 10.1530/EJE-16-0912

Figure 2
Figure 2

Correlation between the absolute change in FSH and LH 30 min after GnRH stimulation in the reference group of 77 healthy men.

Citation: European Journal of Endocrinology 176, 4; 10.1530/EJE-16-0912

Table 2

Serum hormone levels in the reference group of healthy men undergoing GnRH and hCG test.

Median (2.5; 97.5 percentile)
GnRH test (n = 77)
 FSH Baseline (IU/L) 2.6 (0.7; 7.2)
Stimulated (IU/L) 3.7 (0.5; 10.5)
Absolute increase (IU/L) 0.9 (−1.4; 4.7)
Relative increase (%) 40 (−52; 128)
 LH Baseline (IU/L) 3.6 (1.8; 8.4)
Stimulated (IU/L) 17.8 (7.4; 54.2)
Absolute increase (IU/L) 14.1 (3.3; 48.6)
Relative increase (%) 392 (69; 1068)
hCG test (n = 72)
 Testosterone Baseline (nmol/L) 19.1 (9.0; 34.5)
Stimulated (nmol/L) 40.9 (22.8; 67.5)
Absolute increase (nmol/L) 20.7 (7.5; 46.4)
Relative increase (%) 111 (35; 347)
 Estradiol Baseline (pmol/L) 64 (17; 121)
Stimulated (pmol/L) 166 (102; 265)
Absolute increase (pmol/L) 99 (17; 214)
Relative increase (%) 150 (14; 760)
 Free testosterone Baseline (pmol/L) 426 (239; 712)
Stimulated (pmol/L) 1079 (554; 1791)
Absolute increase (pmol/L) 606 (203; 1444)
Relative increase (%) 138 (39; 477)
Table 3

FSH and LH levels before and after GnRH stimulation stratified according to the 6 different polymorphisms in the reference group of healthy men.

P-value (ANOVA)
Median (min; max) Additive Dominant Recessive
rs10835638 (FSHB -211G > T) GG (n = 53) GT (n = 20) TT (n = 4)
 FSH Baseline (IU/L) 3.0 (0.4; 7.9) 2.3 (0.7; 5.4) 2.9 (1.1; 3.7) 0.228 0.154 0.863
Stimulated (IU/L) 4.0 (0.2; 10.4) 2.8 (1.1; 11.8) 3.4 (1.4; 5.8) 0.491 0.447 0.835
Absolute increase (IU/L) 1.0 (−1.6; 4.6) 0.6 (0.1; 6.6) 0.9 (−0.5; 2.07) 0.918 0.751 0.694
Relative increase (%) 45 (−54; 109) 36 (3; 152) 44 (−14; 57) 0.625 0.452 0.730
rs1394205 (FSHR -29G > A) GG (n = 43) GA (n = 28) AA (n = 6)
 FSH Baseline (IU/L) 2.6 (0.4; 7.9) 3.3 (1.1; 7.2) 2.2 (0.9; 6.6) 0.454 0.213 0.604
Stimulated (IU/L) 3.3 (0.2; 7.4) 4.4 (0.6; 11.8) 3.2 (1.5; 10.1) 0.164 0.066 0.933
Absolute increase (IU/L) 0.9 (−1.6; 2.7) 1.4 (−0.6; 6.6) 1.0 (0.1; 3.5) 0.027 0.010 0.626
Relative increase (%) 39 (−54; 109) 46 (−52; 152) 56 (10;80) 0.077 0.052 0.055
rs6166 (FSHR 2039A > G) AA (n = 18) AG (n = 36) GG (n = 23)
 FSH Baseline (IU/L) 2.2 (0.4; 6.6) 3.0 (0.9; 7.9) 2.6 (0.9; 6.0) 0.309 0.124 0.842
Stimulated (IU/L) 3.2 (0.2; 10.1) 4.0 (0.6; 11.8) 4.0 (1.2; 9.9) 0.238 0.200 0.493
Absolute increase (IU/L) 0.8 (−1.2; 3.5) 1.0 (−1.5; 6.6) 1.0 (−0.5; 4.6) 0.450 0.525 0.542
Relative increase (%) 46 (−54; 93) 37 (−52; 152) 54 (−14; 109) 0.569 0.784 0.373
rs2293275 (S312N) CC (n = 28) CT (n = 35) TT (n = 12)
 LH Baseline (IU/L) 3.9 (2.1; 8.9) 3.3 (1.9; 8.4) 3.6 (1.6; 5.5) 0.455 0.534 0.540
Stimulated (IU/L) 18.8 (8.2; 54.0) 17.8 (7.6; 58.8) 14.8 (4.4; 31.7) 0.276 0.498 0.249
Absolute increase (IU/L) 15.9 (3.8; 48.5) 14.1 (3.4; 50.4) 11.0 (1.8; 26.9) 0.394 0.561 0.398
Relative increase (%) 420 (60; 1122) 375 (70; 1065) 368 (71; 869) 0.392 0.414 0.569
rs7371084 TT (n = 48) TC (n = 27) CC (n = 0)
 LH Baseline (IU/L) 3.8 (1.6; 7.3) 3.2 (1.9; 8.9) 0.614
Stimulated (IU/L) 17.8 (4.4; 43.7) 18.2 (8.2; 58.9) 0.412
Absolute increase (IU/L) 13.9 (1.8; 36.7) 15.3 (3.4; 50.4) 0.358
Relative increase (%) 364 (71; 1065) 501 (60; 1122) 0.082
rs4953617 TT (n = 64) TC (n = 11) CC (n = 0)
 LH Baseline (IU/L) 3.4 (1.6; 8.9) 4.2 (2.3; 7.1) 0.458
Stimulated (IU/L) 17.7 (4.4; 58.8) 24.7 (9.2; 44.0) 0.337
Absolute increase (IU/L) 13.9 (1.8; 50.4) 19.5 (5.1; 38.1) 0.297
Relative increase (%) 400 (60; 1122) 392 (122; 869) 0.888
Table 4

Testosterone and calculated free testosterone levels before and after hCG stimulation stratified according to the 3 LHCGR polymorphisms in the reference group of healthy men.

P-value (ANOVA)
Median (min; max) Additive Dominant Recessive
rs2293275 (S312N) CC (n = 27) CT (n = 32) TT (n = 11)
 Testosterone Baseline (nmol/L) 20.3 (7.1; 33.6) 18.6 (11.2; 30.3) 15.5 (9.4; 38.9) 0.488 0.458 0.722
Stimulated (nmol/L) 42.7 (29.1; 67.5) 40.0 (18.7; 58.2) 39.0 (23.7; 67.6) 0.691 0.085 0.280
Absolute increase (nmol/L) 20.6 (7.8; 48.2) 20.2 (6.2; 35.9) 21.1 (14.3; 39.3) 0.948 0.423 0.365
Relative increase (%) 102 (37; 412) 107 (28; 297) 130 (74; 253) 0.657 0.798 0.187
 Free testosterone Baseline (pmol/L) 485 (171; 774) 416 (260; 663) 395 (258; 609) 0.338 0.435 0.445
Stimulated (pmol/L) 1135 (701; 1907) 1018 (495; 1521) 1057 (705; 1444) 0.188 0.114 0.676
Absolute increase (pmol/L) 636 (213; 1513) 586 (202; 1136) 639 (425; 1031) 0.961 0.691 0.531
Relative increase (%) 133 (39; 596) 135 (38; 437) 152 (109; 397) 0.886 0.795 0.253
rs7371084 TT (n = 46) TC (n = 24) CC (n = 0)
 Testosterone Baseline (nmol/L) 18.2 (7.1; 38.9) 20.0 (9.4; 33.6) 0.488
Stimulated (nmol/L) 39.3 (18.7; 67.6) 42.8 (23.7; 61.5) 0.285
Absolute increase (nmol/L) 20.8 (6.2; 48.2) 20.3 (7.8; 39.0) 0.824
Relative increase (%) 112 (49; 412) 108 (28; 333) 0.711
 Free testosterone Baseline (pmol/L) 415 (171; 699) 472 (302; 774) 0.385
Stimulated (pmol/L) 1026 (495; 1767) 1138 (701; 1907) 0.270
Absolute increase (pmol/L) 593 (201; 1513) 642 (213; 1351) 0.984
Relative increase (%) 141 (56; 596) 138 (38; 401) 0.851
rs4953617 TT (n = 61) TC (n = 9) CC (n = 0)
 Testosterone Baseline (nmol/L) 18.9 (7.1; 38.9) 21.3 (12.7; 30.3) 0.416
Stimulated (nmol/L) 40.9 (18.7; 67.6) 50.0 (31.4; 58.1) 0.232
Absolute increase (nmol/L) 20.6 (6.2; 48.2) 21.0 (14.8; 39.3) 0.282
Relative increase (%) 111 (28; 412) 110 (62; 253) 0.666
 Free testosterone Baseline (pmol/L) 423 (171; 774) 485 (260; 626) 0.986
Stimulated (pmol/L) 1027 (495; 1907) 1140 (872; 1457) 0.365
Absolute increase (pmol/L) 602 (201;1513) 731 (449; 1031) 0.363
Relative increase (%) 139 (38;596) 137 (83; 397) 0.376
Table 5

Testosterone and calculated free testosterone levels before and after hCG stimulation stratified according to the 3 FSH related polymorphisms, in the reference group of healthy men.

P-value (ANOVA)
Median (min; max) Additive Dominant Recessive
rs10835638 (FSHB -211G > T) GG (n = 50) GT (n = 18) TT (n = 4)
 Testosterone Baseline (nmol/L) 19.1 (7.1; 33.6) 19.4 (12.7; 38.9) 16.3 (12.5; 20.2) 0.949 0.615 0.244
Stimulated (nmol/L) 41.3 (23.7; 67.5) 40.7 (25.4; 67.6) 34.3 (18.7; 52.2) 0.162 0.499 0.026
Absolute increase (nmol/L) 20.6 (7.8; 48.2) 20.7 (9.2; 39.3) 18.0 (6.2; 31.8) 0.308 0.611 0.113
Relative increase (%) 113 (28; 412) 103 (49; 253) 106 (49; 158) 0.386 0.410 0.578
 Free testosterone Baseline (pmol/L) 432 (171; 774) 430 (258; 609) 369 (294; 423) 0.405 0.743 0.143
Stimulated (pmol/L) 1097 (701; 1907) 992 (651; 1445) 852 (495; 1189) 0.023 0.099 0.013
Absolute increase (pmol/L) 620 (212; 1513) 582 (307; 1031) 482 (201; 766) 0.097 0.283 0.037
Relative increase (%) 142 (38; 596) 138 (63; 397) 125 (56; 203) 0.267 0.363 0.316
rs1394205 (FSHR -29G > A) GG (n = 41) AG (n = 25) AA (n = 6)
 Testosterone Baseline (nmol/L) 18.3 (9.4; 38.9) 19.8 (7.1; 33.5) 22.5 (15.5; 31.0) 0.404 0.900 0.077
Stimulated (nmol/L) 40.3 (18.7; 67.6) 39.0 (24.8; 64.0) 46.5 (40.9; 58.2) 0.260 0.573 0.105
Absolute increase (nmol/L) 20.0 (6.2; 48.2) 23.0 (7.8; 35.9) 22.9 (16.7; 39.3) 0.360 0.493 0.364
Relative increase (%) 110 (28; 412) 122 (37; 322) 106 (62; 253) 0.899 0.602 0.519
Free testosterone Baseline (pmol/L) 423 (254; 773) 429 (171; 699) 467 (260; 666) 0.652 0.278 0.366
Stimulated (pmol/L) 1046 (567; 1907) 1121 (495; 1523) 1183 (938; 1319) 0.915 0.819 0.509
Absolute increase (pmol/L) 602 (203; 1513) 636 (201; 1136) 620 (485; 1031) 0.699 0.813 0.631
Relative increase (%) 137 (38; 596) 152 (39; 437) 146 (80; 397) 0.660 0.456 0.755
rs6166 (FSHR 2039A > G) AA (n = 16) AG (n = 34) GG (n = 22)
 Testosterone Baseline (nmol/L) 15.7 (7.1; 33.6) 19.8 (11.2; 38.9) 18.6 (11.7; 33.5) 0.334 0.088 0.989
Stimulated (nmol/L) 41.3 (23.7; 67.5) 40.6 (30.2; 67.6) 40.7 (18.7; 64.0) 0.064 0.146 0.115
Absolute increase (nmol/L) 23.0 (14.3; 48.2) 20.4 (11.3; 34.7) 20.4 (6.2; 39.0) 0.021 0.053 0.065
Relative increase (%) 139 (53; 412) 112 (49; 214) 105 (28; 333) 0.007 0.003 0.111
 Free testosterone Baseline (pmol/L) 382 (171; 774) 433 (280; 666) 391 (284; 644) 0.450 0.138 0.884
Stimulated (pmol/L) 1160 (764; 1906) 1055 (701; 1521) 1075 (495; 1688) 0.039 0.098 0.084
Absolute increase (pmol/L) 625 (288; 1513) 606 (336; 1027) 614 (201; 1351) 0.015 0.043 0.052
Relative increase (%) 157 (50; 596) 135 (63; 273) 135 (38; 401) 0.007 0.006 0.090

FSH levels were associated to the FSHR -29G>A genotypes; A-allele carriers had higher absolute increase in FSH: median 0.9 IU/L in the GG group, 1.4 IU/L in the GA group and 1.0 IU/L in the AA group (additive model, P = 0.027 and dominant model, P = 0.010), but no difference in baseline levels. No other SNPs were associated with the LH or FSH response (Table 3). The decrease in FSH levels after GnRH stimulation in the nine men was not associated to any genotype.

hCG test

Results of the hCG tests are shown in Table 2. Figure 1C and D illustrate the increases in T and cFT (both P < 0.01) stratified into quartiles based on baseline hormone level. The absolute increase in stimulated T and cFT ranged from 18.7 to 67.6 nmol/L and 495 to 1907 pmol/L respectively. The median relative increase in T was 111% and in cFT 138%. Estradiol also increased (P < 0.01), whereas SHBG did not change (P = 0.27) (data not shown). Stimulated levels correlated with baseline values for T (r: 0.475, P < 0.01) and cFT (r: 0.299, P = 0.011). Stimulated levels of estradiol were positively correlated with the increase in T (r: 0.524, P < 0.01) and cFT (r: 0.565, P < 0.01). Age and BMI were negatively associated with baseline levels of T (P = 0.003 and P = 0.004) and cFT (P = 0.012 and P = 0.001), but not associated with stimulated levels (P > 0.05). Smoking status was not associated to the T or cFT response (P > 0.05).

None of the 3 LHCGR polymorphisms showed any significant effect on the stimulated T levels following an hCG test (Table 4). The FSHB -211G>T and the FSHR 2039A>G genotypes were associated to the testosterone response after hCG stimulation (Table 5). The T-allele (FSHB -211G>T) carriers had lower stimulated testosterone as well as lower stimulated and absolute cFT increase (P < 0.05). For the FSHR 2039A>G genotypes, the G-allele carriers had lower absolute and relative testosterone as well as cFT increase (P < 0.05).

Patient groups

Figure 3A and B show the LH and T levels before and after GnRH and hCG stimulation in the healthy men and the 45 patients (Table 6: medians and range). The 2.5th percentiles from the healthy reference group (Table 2) were used as cut-off, and we classified the patients’ responses to the two tests on whether or not they reached the 2.5th percentile of the LH levels (stimulated: 7.4 IU/L, absolute increase: 3.3 IU/L and relative increase: 69%) and T levels (stimulated: 23 nmol/L, absolute increase: 7.5 nmol/L and relative increase: 35%).

Figure 3
Figure 3

(A and B) LH and testosterone levels before and after GnRH/hCG stimulation in the reference group of healthy men (n = 77/72), and in 5 groups of patients suspected of testosterone sufficiency. Patients with pituitary abnormalities (n = 6/4), Patients with Kallman syndrome/IHH (n = 5/5), Patients suspected of medically induced testosterone deficiency (n = 9/8), Obese patients (BMI >30) (n = 11/8), Patients who had been unilaterally orchiectomized due to testicular germ cell cancer and received irradiation against germ cell neoplasia in situ (GCNIS) in the remaining testis (n = 14). Marked line indicates 2.5th percentile for the reference group of healthy men.

Citation: European Journal of Endocrinology 176, 4; 10.1530/EJE-16-0912

Table 6

Hormon values at baseline and after hormone stimulation stratified according to 5 groups of patients suspected of testosterone sufficiency.

Patient categories
1. Pituitary abnormalities 2. Kallmann syndrome/IHH 3. On medication 4. Obese 5. GCNIS irradiated
GnRH test n = 6 n = 5 n = 9 n = 11 n = 14  FSH
 FSH Baseline (IU/L) 2.4 (1.3; 4.9) 0.8 (0.1; 1.2) 2.9 (1.5; 13.9) 3.8 (1.2; 24.4) 40.6 (23.9; 59.9)
Stimulated (IU/L) 3.3 (1.6; 7.2) 2.0 (0.4; 2.4) 4.0 (2.0; 16.3) 6.8 (1.8; 36.1) 61.1 (28.9; 92.9)
Absolute increase (IU/L) 1.0 (0.4; 2.4) 0.9 (−0.1; 1.4) 1.5 (0.3; 3.6) 2.2 (0.6; 11.7) 19.1 (−2.0; 45.7)
Relative increase (%) 43 (28; 57) 101 (−9; 465) 41 (7; 143) 48 (33; 122) 43 (−4; 100)
 LH Baseline (IU/L) 1.4 (0.8; 2.52 ) 0.5 (0.0; 0.8) 2.0 (0.4; 6.0) 2.8 (1.6; 7.5) 14.9 (7.0; 32.0)
Stimulated (IU/L) 6.5 (2.8; 12.6) 5.4 (0.1; 13.7) 11.0 (6.4; 23.4) 16.9 (9.5; 50.5) 71.2 (37.5; 123.0)
Absolute increase (IU/L) 5.3 (2.0; 11.1) 4.7 (0.0; 12.9) 9.0 (5.0; 21.1) 14.8 (7.9; 43.1) 60.4 (23.5; 108.6)
Relative increase (%) 374 (164; 757) 1097 (20; 1679) 576 (161; 1726) 496 (287; 950) 345 (96; 904)
hCG test n = 4 n = 5 n = 8 n = 8 n = 14  Testosterone
Baseline (nmol/L) 5.3 (0.4; 6.2) 2.6 (1.2; 4.4) 7.8 (0.9; 10.4) 7.4 (6.8; 11.5) 8.6 (5.8; 15.8)
Stimulated (nmol/L) 22.9 (17.9; 27.5) 6.4 (2.5; 13.5) 21.9 (18.3; 32.2) 17.9 (12.7; 53.7) 12.9 (6.5; 21.7)
Absolute increase (nmol/L) 19.6 (13.1; 21.7) 2.1 (1.0; 12.4) 12.8 (9.8; 25.9) 10.7 (5.6; 42.2) 3.6 (0.7; 8.9)
Relative increase (%) 523 (374; 6855) 128 (49; 1687) 189 (109; 4596) 122 (78; 366) 30 (11; 122)

Values are medians (min;max). Group 1: patients with pituitary abnormalities, group 2: patients with kallman syndrome/IHH, group 3: patients suspected of medically induced testosterone deficiency, group 4: obese patients (BMI >30), group 5: patients who had been unilaterally orchiectomized due to testicular germ cell cancer and received irradiation against germ cell neoplasia in situ (GCNIS) in the remaining testis.

GnRH test

All patients, except one with IHH, had a relative LH increase above the 2.5th percentile of the healthy participants. Evaluating the absolute increases showed that the two Kallmann patients and two patients with pituitary abnormalities did not reach the cut-off. However, assessing the stimulated level showed that four of the six patients with pituitary abnormalities and one in the group with suspicion of medically induced testosterone deficiency did not reach the cut-off level. All the obese patients and the GCNIS-irradiated patients had an LH response higher than the cut-off of both the stimulated and the absolute increase.

hCG test

Of the 39 patients who had hCG tests performed, only 8 men had a stimulated T level above the 2.5th percentile cut-off. For the GCNIS-irradiated patients, all had stimulated levels and absolute increases lower than the cut-off levels in accordance with their clinical symptoms (except one), whereas the relative increase in T levels only detected eight of these men as having an insufficient response. For three of the men with normal pituitary morphology (one with Kallmann syndrome and two with IHH) and four out of the eight obese men, the absolute increase in T was below the cut-off. All men suspected of medically induced testosterone deficiency and those with pituitary abnormalities had an absolute T increase higher than the cut-off.

Discussion

In this study, we examined the responses to GnRH and hCG stimulation in healthy Danish men and established normal reference ranges. For both tests, we detected large variations in the stimulated hormone levels, but polymorphisms in genes involved in gonadotropin signaling (FSHB -211G>T, FSHR -29G>A and FSHR 2039A>G and LHCGR (rs2293275, rs7371084 and rs4953617)) did not seem to contribute significantly to this variation.

When comparing the results obtained from the reference group with those of 45 patients suspected of disorders of the HPG axes, it became clear that evaluation of the GnRH test cannot rely on assessing the relative increase in LH as almost all the patients with pituitary failure had a relative increase larger than the 2.5th percentile of the reference group. Using the absolute increase alone was not always sufficient either. Thus, we recommend that a normal GnRH response should rely on a stimulated LH level ≥7.4 IU/L combined with an absolute increase of ≥3.3 IU/L. Four of the six patients with pituitary abnormalities had an impaired LH response according to the suggested criteria. The two patients with sufficient responses both had pituitary prolactinomas, and their lower baseline LH may be caused by reduced endogenous GnRH secretion rather than a failure of the gonadotrophic cells. Of the five patients with normal pituitary morphology, the two with Kallmann’s syndrome responded normally to the GnRH test, and therefore, most likely have a hypothalamic insufficiency rather than a pituitary. In the group with suspicion of medically induced testosterone deficiency, all except one had normal GnRH test responses, in accordance with the inhibitory effect of the drugs primarily on the hypothalamic function (20).

Evaluating the hCG tests of the patients, only 8 patients actually exceeded the 2.5th percentile of the stimulated T levels of the healthy participants. Although the relative increase in T level could be used to identify men with severe testosterone deficiency, a relative increase can be difficult to use in a clinical setting as very low baseline values can show a markedly relative increase without reaching normal absolute increase or stimulated level. We suggest that a normal adult response to an hCG test should be an absolute increase in T ≥ 7.5 nmol/L. This also implies that men with secondary testosterone deficiency can increase in T, but not as much as healthy men, and still be considered to have a normal Leydig cell capacity.

The group of obese men may be challenging from a diagnostic point of view. In our study, they all had normal LH responses to GnRH tests indicating that the low baseline LH level detected in this group is not due to a pituitary insufficiency. Among the obese men, four had an abnormal absolute T change after the hCG stimulation, indicating a reduced Leydig cell capacity, which is in accordance with other studies (21, 22). However, this reduced capacity does not exclude that their baseline T level is sufficient, and in our opinion, it is evident that the clinical decision on whether they need testosterone substitution should not only be based on total T but also on free T and symptoms associated with testosterone deficiency. However, if men suspected of testosterone deficiency have large testicles, good semen quality and high serum level of inhibin B, this may additionally be arguments against the presence of testosterone deficiency (23). We found a negative association between BMI and baseline total and free T, but found no association between the increase in T and BMI in the group of healthy men, probably due to the narrow range in BMI.

Our main objective for this study was to describe the hormone changes for GnRH and hCG test in healthy men and provide novel reference ranges. We included the 45 patients suspected of testosterone deficiency to illustrate the potential use of the cut-off values, not to validate them. It would have been preferable to be able to calculate the diagnostic accuracy for our suggested cut-off values, but our patient group was too small and heterogeneous. In the clinical work-up of patients, their hormone levels are sometimes in a ‘gray zone’ in contrast to other patients who show clearly abnormal or normal levels. For example, the baseline LH is often very low in patients with hypogonadotropic hypogonadism, and the diagnosis might be obvious and without need for hormone stimulation test, whereas the testosterone levels in obese men are low, and at the same time, they may have low LH. Thus, in such situations, the stimulation tests may help to distinguish between men having a hypogonadotropic hypogonadism and those having an adequately low testosterone level as an adaptation to low SHBG caused by the obesity.

We cannot provide any information of why nine men had a decrease in FSH instead of an increase after the GnRH stimulation. As mentioned in the results, the FSH levels correlated with the LH levels, and the decrease was not associated with their testis size, semen quality or genotypes. However, it is well known that the FSH response is much smaller than the LH response after GnRH stimulation (3, 8, 24).

Both tests revealed large variations in the stimulated hormone levels among the healthy men. We anticipated that especially the promoter polymorphism FSHB -211G>T could have been an important modulator of the response to GnRH stimulation due to its strong association to the FSH secretion because other studies have shown that minor allele carriers have lower FSH levels, inhibin B levels and smaller testis volumes (9, 10, 11). The T-allele carriers of our participants had a tendency for a lower baseline and a lower stimulated FSH level, however, within the range observed for non-carriers. This polymorphism has also been suggested to be adversely associated with androgen production (9, 11). In accordance, the T-allele carriers had slightly lower T and cFT levels after hCG stimulation. The promoter polymorphism FSHR -29G>A A-allele leads to a reduced FSHR gene expression and is associated to a higher baseline FSH (13, 24, 25). We also detected a slightly, but significantly, larger increase in FSH level upon GnRH stimulation in the A-allele carriers. For the FSHR 2039A>G polymorphism, which is believed to reduce the FSHR response to FSH (26), our detection of a lower T response and tendency toward higher levels of stimulated FSH in the G-allele carriers is compatible with previous publications (27, 28). Even though we detected significant associations between the FSH-related SNPs and the hormonal responses, the differences were small. None of the tested LHCGR polymorphisms modulated the hCG response. The rs2293275 is located in exon 10 near a glycosylation site, which has been hypothesized to reduce the receptor activity (14, 27, 29). Although associations between the LHCGR polymorphism and impaired spermatogenesis and under-masculinization have been detected (14, 27, 30), it has also been shown that deletion of exon 10 only affects the sensitivity to LH and not hCG, which could be an explanation for the lack of an association (31). For two of the LHCGR polymorphisms, none were homozygous carriers of the minor allele, and in the FSH-related genotype groups, some of the minor allele groups were small. Men carrying more than one minor allele might be more affected. Testing of a sufficiently large population could probably reveal significant differences in hormone responses between genotypes, and there may be other SNPs that alone or in combination could have a larger effect on the stimulation tests. The investigated SNPs are known to affect steady state levels of hormones, but the effect of genetic variants may be different when the system is stimulated. Based on our results, we recommend that interpretation of both tests can be done without accounting for the six investigated gonadotropin-related polymorphisms.

The strength of our setup was that all 77 participants were healthy without any diseases affecting the testicular function, the first such setup study found in literature. Though previous studies have published results on both GnRH and hCG tests, those studies have included administration of different doses of GnRH/hCG and often with only a few or without healthy controls (2, 5, 32, 33, 34, 35, 36). We used the LC–MS/MS method for testosterone analysis in this group of healthy men. LC–MS/MS-based tests have a higher specificity than immunoassays and are currently believed to be the golden standard in testosterone measurements.

Our study also has some limitations. The hormone levels of the patients in the retrospective part of this study were analyzed by immunoassays as LC–MS/MS was not yet available. Previous studies have shown a good agreement between the LC–MS/MS method and immunoassays, though in the low concentration range, the correlation was lower (17). However, we corrected for the difference between the two methods before comparisons. In the setup of our study, all men had an ACTH test performed simultaneously with the GnRH test for another study. In the routine clinical setting, these tests are often done simultaneously because suspicion of pituitary deficiency should lead to the validation of the other pituitary axes. The knowledge whether the tests influence each other’s response is, sparse, but studies have shown that combined pituitary testing does not lead to differences in the hormone responses compared to separate tests (37, 38, 39). Results from rodents indicate that ACTH can stimulate T production in the neonatal Leydig cells through the ACTH receptor, but apparently without any effect post-pubertally (40). Also, Leydig cells may have glucocorticoid receptors, and high levels of glucocorticoids have been shown to decrease testosterone production (41).

Even though the age range in our reference group was narrow, we detected a negative association of age on the LH response. This is in accordance with Harman et al. who showed that even though baseline gonadotropins increase slightly with age, there is a lower incremental response to GnRH test (42).

This is the first study to evaluate the dynamic changes in FSH, LH and T after GnRH and hCG stimulation respectively, in a large cohort of healthy men. Hereby, we provide new reference data, which allow future evaluation of men suspected of HPG disorders.

Declaration of interest

The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. None of the authors have any competing interests to declare.

Funding

This study was supported by the Faculty of Health and Medical Sciences, University of Copenhagen, and by the Danish Research Council grant no. DFF – 1331-00044, Nordic Research Committee grant no. R195-A16270, and the A.P. Møller Foundation for the Advancement of Medical Science grant no. 12-164.

Author contribution statement

Substantial contributions to conception and design: A K B, N J, N L and A J. Data acquisition: A K B, L N, L P and K A. Data analysis: A K B. Data interpretation: All authors. Drafting the manuscript: A K B and N J. Revising manuscript critically for important intellectual content: All authors. Final approval of the manuscript: All authors.

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  • 23

    Jørgensen N, Liu F, Andersson AM, Vierula M, Irvine DS, Auger J, Brazil CK, Drobnis EZ, Jensen TK & Jouannet P et al. Serum inhibin-b in fertile men is strongly correlated with low but not high sperm counts: a coordinated study of 1797 European and US men. Fertility and Sterility 2010 94 21282134. (doi:10.1016/j.fertnstert.2009.12.051)

    • Search Google Scholar
    • Export Citation
  • 24

    Desai SS, Achrekar SK, Pathak BR, Desai SK, Mangoli VS, Mangoli RV & Mahale SD. Follicle-stimulating hormone receptor polymorphism (G-29A) is associated with altered level of receptor expression in granulosa cells. Journal of Clinical Endocrinology and Metabolism 2011 96 28052812. (doi:10.1210/jc.2011-1064)

    • Search Google Scholar
    • Export Citation
  • 25

    Grigorova M, Punab M, Punab AM, Poolamets O, Vihljajev V, Žilaitiene B, Erenpreiss J, Matulevičius V, Laan M. Reproductive physiology in young men is cumulatively affected by FSH-action modulating genetic variants: FSHR -29G/A and c.2039 A/G, FSHB -211G/T. PLoS ONE 2014 9 110. (doi:10.1371/journal.pone.0094244)

    • Search Google Scholar
    • Export Citation
  • 26

    Casarini L, Moriondo V, Marino M, Adversi F, Capodanno F, Grisolia C, La Marca A, La Sala GB & Simoni M. FSHR polymorphism p.N680S mediates different responses to FSH in vitro. Molecular and Cellular Endocrinology 2014 393 8391. (doi:10.1016/j.mce.2014.06.013)

    • Search Google Scholar
    • Export Citation
  • 27

    Lindgren I, Bååth M, Uvebrant K, Dejmek A, Kjaer L, Henic E, Bungum M, Bungum L, Cilio C & Leijonhufvud I et al. Combined assessment of polymorphisms in the LHCGR and FSHR genes predict chance of pregnancy after in vitro fertilization. Human Reproduction 2016 31 672683. (doi:10.1093/humrep/dev342)

    • Search Google Scholar
    • Export Citation
  • 28

    Grigorova M, Punab M, Poolamets O, Sõber S, Vihljajev V, Žilaitienė B, Erenpreiss J, Matulevičius V, Tsarev I, Laan M. Study in 1790 Baltic men: FSHR Asn680Ser polymorphism affects total testes volume. Andrology 2013 1 293300. (doi:10.1111/j.2047-2927.2012.00028.x)

    • Search Google Scholar
    • Export Citation
  • 29

    Piersma D, Verhoef-Post M, Look MP, Uitterlinden AG, Pols HAP, Berns EMJJ & Themmen AP. Polymorphic variations in exon 10 of the luteinizing hormone receptor: functional consequences and associations with breast cancer. Molecular and Cellular Endocrinology 2007 276 6370. (doi:10.1016/j.mce.2007.06.007)

    • Search Google Scholar
    • Export Citation
  • 30

    Mongan NP, Hughes IA & Lim HN. Evidence that luteinising hormone receptor polymorphisms may contribute to male undermasculinisation. European Journal of Endocrinology 2002 147 103107. (doi:10.1530/eje.0.1470103)

    • Search Google Scholar
    • Export Citation
  • 31

    Müller T, Gromoll J & Simoni M. Absence of exon 10 of the human luteinizing hormone (LH) receptor impairs LH, but not human chorionic gonadotropin action. Journal of Clinical Endocrinology and Metabolism 2003 88 22422249. (doi:10.1210/jc.2002-021946)

    • Search Google Scholar
    • Export Citation
  • 32

    Schwarzstein L, Aparicio NJ, Turner D, de Turner EA, Premoli F, Rodriguez A, Schally AV. Pituitary and testicular response to hypothalamic LH-releasing hormone (LH-RH) in normal and oligospermic men. International Journal of Fertility and Sterility 1976 10 96102. (doi:10.1111/j.1439-0272.1978.tb01316.x)

    • Search Google Scholar
    • Export Citation
  • 33

    Saez JM & Forest MG. Kinetics of human chorionic gonadotropin-induced steroidogenic response of the human testis. I. Plasma testosterone: implications for human chorionic gonadotropin stimulation test. Journal of Clinical Endocrinology and Metabolism 1979 49 278283. (doi:10.1210/jcem-49-2-278)

    • Search Google Scholar
    • Export Citation
  • 34

    Saal W, Glowania HJ, Hengst W & Happ J. Pharmacodynamics and pharmacokinetics after subcutaneous and intramuscular injection of human chorionic gonadotropin. Fertility and Sterility 1991 56 225229. (doi:10.1016/S0015-0282(16)54476-8)

    • Search Google Scholar
    • Export Citation
  • 35

    Meier C, Christ-Crain M, Christoffel-Courtin C, Staub JJ & Müller B. Serum estradiol after single dose hCG administration correlates with Leydig cell reserve in hypogonadal men: reassessment of the hCG stimulation test. Clinical Laboratory 2005 51 509515.

    • Search Google Scholar
    • Export Citation
  • 36

    Gerhard I, Lenhard HK, Eggert-Kruse W & Runnebaum B. Hormone load tests in infertile male patients. Archives of Andrology 1991 27 129147. (doi:10.3109/01485019108987664)

    • Search Google Scholar
    • Export Citation
  • 37

    Cohen R, Bouquier D, Biot-Laporte S, Vermeulen E, Claustrat B, Cherpin MH, Cabrera P, Guidetti P, Ferry S & Bizollon CA et al. Pituitary stimulation by combined administration of four hypothalamic releasing hormones in normal men and patients. Journal of Clinical Endocrinology and Metabolism 1986 62 892898. (doi:10.1210/jcem-62-5-892)

    • Search Google Scholar
    • Export Citation
  • 38

    Sheldon WR, Debold CR, Evans WS, Decherney GS, Jackson RV, Island DP, Thorner MO & Orth DN. Rapid sequential intravenous administration of four hypothalamic releasing hormones as a combined anterior pituitary function test in normal subjects. Journal of Clinical Endocrinology and Metabolism 1985 60 623630. (doi:10.1210/jcem-60-4-623)

    • Search Google Scholar
    • Export Citation
  • 39

    Wehrenberg WB, Baird A, Ying SY, Rivier C, Ling N & Guillemin R. Multiple stimulation of the adenohypophysis by combinations of hypothalamic releasing factors. Endocrinology 1984 114 19952001. (doi:10.1210/endo-114-6-1995)

    • Search Google Scholar
    • Export Citation
  • 40

    O’Shaughnessy PJ, Fleming LM, Jackson G, Hochgeschwender U, Reed P & Baker PJ. Adrenocorticotropic hormone directly stimulates testosterone production by the fetal and neonatal mouse testis. Endocrinology 2003 144 32793284. (doi:10.1210/en.2003-0277)

    • Search Google Scholar
    • Export Citation
  • 41

    Whirledge S & Cidlowski JA. Glucocorticoids, stress, and fertility. Minerva Endocrinologica 2010 35 109125. (doi:10.1586/eem.10.1)

  • 42

    Harman SM, Tsitouras PD, Costa PT & Blackman MR. Reproductive hormones in aging men. II. Basal pituitary gonadotropins and gonadotropin responses to luteinizing hormone-releasing hormone. Journal of Clinical Endocrinology and Metabolism 1982 54 547551. (doi:10.1210/jcem-54-3-547)

    • Search Google Scholar
    • Export Citation

 

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    (A, B, C and D) Hormone levels at baseline and after GnRH and hCG-test in the reference group of healthy men (n = 77/72). Results stratified and colored according to baseline quartiles (Blue: 1st quartile, Green: 2nd quartile, Light brown: 3rd quartile, Purple: 4th quartile). (A) LH levels at baseline and 30 min. after GnRH stimulation. (B) FSH levels at baseline and 30 min. after GnRH stimulation. (C) Testosterone levels at baseline and 72 h after hCG stimulation. (D) Calculated free testosterone levels at baseline and 72 h after hCG stimulation.

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    Correlation between the absolute change in FSH and LH 30 min after GnRH stimulation in the reference group of 77 healthy men.

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    (A and B) LH and testosterone levels before and after GnRH/hCG stimulation in the reference group of healthy men (n = 77/72), and in 5 groups of patients suspected of testosterone sufficiency. Patients with pituitary abnormalities (n = 6/4), Patients with Kallman syndrome/IHH (n = 5/5), Patients suspected of medically induced testosterone deficiency (n = 9/8), Obese patients (BMI >30) (n = 11/8), Patients who had been unilaterally orchiectomized due to testicular germ cell cancer and received irradiation against germ cell neoplasia in situ (GCNIS) in the remaining testis (n = 14). Marked line indicates 2.5th percentile for the reference group of healthy men.

  • 1

    Forest MG. How should we perform the human chorionic gonadotrophin (hCG) stimulation test? International Journal of Andrology 1983 6 14. (doi:10.1111/j.1365-2605.1983.tb00318.x)

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  • 2

    Harman SM, Tsitouras PD, Costa PT, Loriaux DL & Sherins RJ. Evaluation of pituitary gonadotropic function in men: value of luteinizing hormone-releasing hormone response versus basal luteinizing hormone level for discrimination of diagnosis. Journal of Clinical Endocrinology and Metabolism 1982 54 196200. (doi:10.1210/jcem-54-1-196)

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  • 3

    Besser GM, McNeilly AS, Anderson DC, Marshall JC, Harsoulis P, Hall R, Ormston BJ, Alexander L & Collins WP. Hormonal responses to synthetic luteinizing hormone and follicle stimulating hormone-releasing hormone in man. British Medical Journal 1972 3 267271. (doi:10.1136/bmj.3.5821.267)

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  • 4

    Cailleux-Bounacer A, Reznik Y, Cauliez B, Menard JF, Duparc C & Kuhn JM. Evaluation of endocrine testing of Leydig cell function using extractive and recombinant human chorionic gonadotropin and different doses of recombinant human LH in normal men. European Journal of Endocrinology 2008 159 171178. (doi:10.1530/EJE-07-0876)

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  • 5

    Flanagan JN & Lehtihet M. The response to gonadotropin-releasing hormone and hCG in men with prior chronic androgen steroid abuse and clinical hypogonadism. Hormone and Metabolic Research 2015 47 668673. (doi:10.1055/s-0034-1398492)

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  • 6

    Isidori AM, Giannetta E & Lenzi A. Male hypogonadism. Pituitary 2008 11 171180. (doi:10.1007/s11102-008-0111-9)

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    Belli S, Santi D, Leoni E, Dall’Olio E, Fanelli F, Mezzullo M, Pelusi C, Roli L, Tagliavini S & Trenti T et al. Human chorionic gonadotropin stimulation gives evidence of differences in testicular steroidogenesis in Klinefelter syndrome, as assessed by liquid chromatography-tandem mass spectrometry. European Journal of Endocrinology 2016 174 801811. (doi:10.1530/EJE-15-1224)

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  • 8

    Bauman WA, La Fountaine MF, Cirnigliaro CM, Kirshblum SC & Spungen AM. Provocative stimulation of the hypothalamic–pituitary–testicular axis in men with spinal cord injury. Spinal Cord 2016 26 16. (doi:10.1038/sc.2016.50)

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  • 9

    Grigorova M, Punab M, Zilaitienė B, Erenpreiss J, Ausmees K, Matuleviĉius V, Tsarev I, Jørgensen N & Laan M. Genetically determined dosage of follicle-stimulating hormone (FSH) affects male reproductive parameters. Journal of Clinical Endocrinology and Metabolism 2011 96 15341541. (doi:10.1210/jc.2011-0632)

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  • 10

    Grigorova M, Punab M, Poolamets O, Kelgo P, Ausmees K, Korrovits P, Vihljajev V & Laan M. Increased prevalance of the -211 T allele of follicle stimulating hormone (FSH) B subunit promoter polymorphism and lower serum FSH in infertile men. Journal of Clinical Endocrinology and Metabolism 2010 95 100108. (doi:10.1210/jc.2009-1010)

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  • 11

    Tüttelmann F, Laan M, Grigorova M, Punab M, Sõber S & Gromoll J. Combined effects of the variants FSHB -211G>T and FSHR 2039A>G on male reproductive parameters. Journal of Clinical Endocrinology and Metabolism 2012 97 36393647. (doi:10.1210/jc.2012-1761)

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  • 12

    Lindgren I, Giwercman A, Axelsson J & Lundberg Giwercman Y. Association between follicle-stimulating hormone receptor polymorphisms and reproductive parameters in young men from the general population. Pharmacogenetics and Genomics 2012 22 667672. (doi:10.1097/FPC.0b013e3283566c42)

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  • 13

    Wunsch A, Ahda Y, Banaz-Yaşar F, Sonntag B, Nieschlag E, Simoni M & Gromoll J. Single-nucleotide polymorphisms in the promoter region influence the expression of the human follicle-stimulating hormone receptor. Fertility and Sterility 2005 84 446453. (doi:10.1016/j.fertnstert.2005.02.031)

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  • 14

    Simoni M, Tüttelmann F, Michel C, Böckenfeld Y, Nieschlag E & Gromoll J. Polymorphisms of the luteinizing hormone/chorionic gonadotropin receptor gene: association with maldescended testes and male infertility. Pharmacogenet and Genomics 2008 18 193200. (doi:10.1097/FPC.0b013e3282f4e98c)

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  • 15

    Brokken LJS, Lundberg-Giwercman Y, Rajpert De-Meyts E, Eberhard J, Ståhl O, Cohn-Cedermark G, Daugaard G, Arver S & Giwercman A. Association of polymorphisms in genes encoding hormone receptors ESR1, ESR2 and LHCGR with the risk and clinical features of testicular germ cell cancer. Molecular and Cellular Endocrinology 2012 351 279285. (doi:10.1016/j.mce.2011.12.018)

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  • 16

    Jørgensen N, Joensen UN, Jensen TK, Jensen MB, Almstrup K, Olesen IA, Juul A, Andersson AM, Carlsen E & Petersen JH et al. Human semen quality in the new millennium: a prospective cross-sectional population-based study of 4867 men. BMJ Open 2012 2 11971198. (doi:10.1136/bmjopen-2012-000990)

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  • 17

    Søeborg T, Frederiksen H, Fruekilde P, Johannsen TH, Juul A & Andersson AM. Serum concentrations of DHEA, DHEAS, 17α-hydroxyprogesterone, δ4-androstenedione and testosterone in children determined by TurboFlow-LC-MS/MS. Clinica Chimica Acta 2013 419 95101. (doi:10.1016/j.cca.2013.01.019)

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  • 18

    Vermeulen A, Verdonck L & Kaufman JM. A critical evaluation of simple methods for the estimation of free testosterone in serum. Journal of Clinical Endocrinology and Metabolism 1999 84 36663672. (doi:10.1210/jcem.84.10.6079)

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  • 19

    Henriksen LS, Hagen CP, Assens M, Busch AS, Skakkebæk NE, Almstrup K & Main KM. Genetic variations in FSH action affect sex hormone levels and breast tissue size in infant girls: a pilot study. Journal of Clinical Endocrinology and Metabolism 2016 101 31913198. (doi:10.1210/jc.2016-1672)

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  • 20

    Gudin JA, Laitman A & Nalamachu S. Opioid related endocrinopathy. Pain Medicine 2015 16 S9S15. (doi:10.1111/pme.12926)

  • 21

    Isidori AM, Caprio M, Strollo F, Moretti C, Frajese G, Isidori A & Fabbri A. Leptin and androgens in male obesity: evidence for leptin contribution to reduced androgen levels. Journal of Clinical Endocrinology and Metabolism 1999 84 36733680. (doi:10.1210/jc.84.10.3673)

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  • 22

    Roumaud P & Martin LJ. Roles of leptin, adiponectin and resistin in the transcriptional regulation of steroidogenic genes contributing to decreased Leydig cells function in obesity. Hormone Molecular Biology and Clinical Investigation 2015 24 2545. (doi:10.1515/hmbci-2015-0046)

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    • Export Citation
  • 23

    Jørgensen N, Liu F, Andersson AM, Vierula M, Irvine DS, Auger J, Brazil CK, Drobnis EZ, Jensen TK & Jouannet P et al. Serum inhibin-b in fertile men is strongly correlated with low but not high sperm counts: a coordinated study of 1797 European and US men. Fertility and Sterility 2010 94 21282134. (doi:10.1016/j.fertnstert.2009.12.051)

    • Search Google Scholar
    • Export Citation
  • 24

    Desai SS, Achrekar SK, Pathak BR, Desai SK, Mangoli VS, Mangoli RV & Mahale SD. Follicle-stimulating hormone receptor polymorphism (G-29A) is associated with altered level of receptor expression in granulosa cells. Journal of Clinical Endocrinology and Metabolism 2011 96 28052812. (doi:10.1210/jc.2011-1064)

    • Search Google Scholar
    • Export Citation
  • 25

    Grigorova M, Punab M, Punab AM, Poolamets O, Vihljajev V, Žilaitiene B, Erenpreiss J, Matulevičius V, Laan M. Reproductive physiology in young men is cumulatively affected by FSH-action modulating genetic variants: FSHR -29G/A and c.2039 A/G, FSHB -211G/T. PLoS ONE 2014 9 110. (doi:10.1371/journal.pone.0094244)

    • Search Google Scholar
    • Export Citation
  • 26

    Casarini L, Moriondo V, Marino M, Adversi F, Capodanno F, Grisolia C, La Marca A, La Sala GB & Simoni M. FSHR polymorphism p.N680S mediates different responses to FSH in vitro. Molecular and Cellular Endocrinology 2014 393 8391. (doi:10.1016/j.mce.2014.06.013)

    • Search Google Scholar
    • Export Citation
  • 27

    Lindgren I, Bååth M, Uvebrant K, Dejmek A, Kjaer L, Henic E, Bungum M, Bungum L, Cilio C & Leijonhufvud I et al. Combined assessment of polymorphisms in the LHCGR and FSHR genes predict chance of pregnancy after in vitro fertilization. Human Reproduction 2016 31 672683. (doi:10.1093/humrep/dev342)

    • Search Google Scholar
    • Export Citation
  • 28

    Grigorova M, Punab M, Poolamets O, Sõber S, Vihljajev V, Žilaitienė B, Erenpreiss J, Matulevičius V, Tsarev I, Laan M. Study in 1790 Baltic men: FSHR Asn680Ser polymorphism affects total testes volume. Andrology 2013 1 293300. (doi:10.1111/j.2047-2927.2012.00028.x)

    • Search Google Scholar
    • Export Citation
  • 29

    Piersma D, Verhoef-Post M, Look MP, Uitterlinden AG, Pols HAP, Berns EMJJ & Themmen AP. Polymorphic variations in exon 10 of the luteinizing hormone receptor: functional consequences and associations with breast cancer. Molecular and Cellular Endocrinology 2007 276 6370. (doi:10.1016/j.mce.2007.06.007)

    • Search Google Scholar
    • Export Citation
  • 30

    Mongan NP, Hughes IA & Lim HN. Evidence that luteinising hormone receptor polymorphisms may contribute to male undermasculinisation. European Journal of Endocrinology 2002 147 103107. (doi:10.1530/eje.0.1470103)

    • Search Google Scholar
    • Export Citation
  • 31

    Müller T, Gromoll J & Simoni M. Absence of exon 10 of the human luteinizing hormone (LH) receptor impairs LH, but not human chorionic gonadotropin action. Journal of Clinical Endocrinology and Metabolism 2003 88 22422249. (doi:10.1210/jc.2002-021946)

    • Search Google Scholar
    • Export Citation
  • 32

    Schwarzstein L, Aparicio NJ, Turner D, de Turner EA, Premoli F, Rodriguez A, Schally AV. Pituitary and testicular response to hypothalamic LH-releasing hormone (LH-RH) in normal and oligospermic men. International Journal of Fertility and Sterility 1976 10 96102. (doi:10.1111/j.1439-0272.1978.tb01316.x)

    • Search Google Scholar
    • Export Citation
  • 33

    Saez JM & Forest MG. Kinetics of human chorionic gonadotropin-induced steroidogenic response of the human testis. I. Plasma testosterone: implications for human chorionic gonadotropin stimulation test. Journal of Clinical Endocrinology and Metabolism 1979 49 278283. (doi:10.1210/jcem-49-2-278)

    • Search Google Scholar
    • Export Citation
  • 34

    Saal W, Glowania HJ, Hengst W & Happ J. Pharmacodynamics and pharmacokinetics after subcutaneous and intramuscular injection of human chorionic gonadotropin. Fertility and Sterility 1991 56 225229. (doi:10.1016/S0015-0282(16)54476-8)

    • Search Google Scholar
    • Export Citation
  • 35

    Meier C, Christ-Crain M, Christoffel-Courtin C, Staub JJ & Müller B. Serum estradiol after single dose hCG administration correlates with Leydig cell reserve in hypogonadal men: reassessment of the hCG stimulation test. Clinical Laboratory 2005 51 509515.

    • Search Google Scholar
    • Export Citation
  • 36

    Gerhard I, Lenhard HK, Eggert-Kruse W & Runnebaum B. Hormone load tests in infertile male patients. Archives of Andrology 1991 27 129147. (doi:10.3109/01485019108987664)

    • Search Google Scholar
    • Export Citation
  • 37

    Cohen R, Bouquier D, Biot-Laporte S, Vermeulen E, Claustrat B, Cherpin MH, Cabrera P, Guidetti P, Ferry S & Bizollon CA et al. Pituitary stimulation by combined administration of four hypothalamic releasing hormones in normal men and patients. Journal of Clinical Endocrinology and Metabolism 1986 62 892898. (doi:10.1210/jcem-62-5-892)

    • Search Google Scholar
    • Export Citation
  • 38

    Sheldon WR, Debold CR, Evans WS, Decherney GS, Jackson RV, Island DP, Thorner MO & Orth DN. Rapid sequential intravenous administration of four hypothalamic releasing hormones as a combined anterior pituitary function test in normal subjects. Journal of Clinical Endocrinology and Metabolism 1985 60 623630. (doi:10.1210/jcem-60-4-623)

    • Search Google Scholar
    • Export Citation
  • 39

    Wehrenberg WB, Baird A, Ying SY, Rivier C, Ling N & Guillemin R. Multiple stimulation of the adenohypophysis by combinations of hypothalamic releasing factors. Endocrinology 1984 114 19952001. (doi:10.1210/endo-114-6-1995)

    • Search Google Scholar
    • Export Citation
  • 40

    O’Shaughnessy PJ, Fleming LM, Jackson G, Hochgeschwender U, Reed P & Baker PJ. Adrenocorticotropic hormone directly stimulates testosterone production by the fetal and neonatal mouse testis. Endocrinology 2003 144 32793284. (doi:10.1210/en.2003-0277)

    • Search Google Scholar
    • Export Citation
  • 41

    Whirledge S & Cidlowski JA. Glucocorticoids, stress, and fertility. Minerva Endocrinologica 2010 35 109125. (doi:10.1586/eem.10.1)

  • 42

    Harman SM, Tsitouras PD, Costa PT & Blackman MR. Reproductive hormones in aging men. II. Basal pituitary gonadotropins and gonadotropin responses to luteinizing hormone-releasing hormone. Journal of Clinical Endocrinology and Metabolism 1982 54 547551. (doi:10.1210/jcem-54-3-547)

    • Search Google Scholar
    • Export Citation