4. Statistical Analyses

Descriptive statistics were performed for all variables and expressed as means with standard deviations. The relationship between sLHCGR in serum and semen and outcome variables (hormones, pubertal onset, testicular size, age, and BMI) was performed using regression analysis. The Gaussian distribution of the numerical variables was assessed by residual pp plots to ensure the validity of the regression models. As a result, sLHCGR in serum and urine was used as the dependent variable in all cohorts, sLHCGR in the semen of young healthy men was used as the dependent variable, and sLHCGR in semen/serum was log-transformed as the dependent variable in the combined analysis. Measurements of hormones and sLHCGR below the detection limit were set to half of the detection limit. In the large cohort, sLHCGR was further divided into quartiles and Kruskal-Wallis was performed using the Dunn-Bonferroni post hoc test to adjust for multiple comparisons. Longitudinal measures of puberty, outcomes of clinical interventions, and comparisons of sLHCGR in different body fluids of the same male were analyzed using the Wilcoxon paired test. When multiple paired tests were performed, the significance levels of multiple tests were corrected using the Bonferroni method. The following ratios were calculated and analyzed in independent regression models: inhibin B/FSH, testosterone/LH, and testosterone/estradiol. In all prepubertal boys, analyses of testosterone and estradiol (including ratios) were ignored because all but two measurements were below the detection limit. For all analyses, p <0.05 was considered a statistically significant difference. All presented semen quality analyses were adjusted for the duration of ejaculatory abstinence.

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Results

1. Several Isoforms of LHCGR Is Expressed in Testis and Detected in Serum

Several isoforms of LHCGR were identified in reproductive tissues using primers for exons 2-4. Two strong bands of approximately 100 and 200 base pairs were detected in human testis samples and ovarian RNA and confirmed as LHCGR by direct sequencing. exon 11 was also expressed in the testis and ovary. LHCGR was detected in human testicular tissue and two serum samples; high sLHCGR male serum samples and low sLHCGR male serum samples by ELISA using the western blotting (WB) technique with antibodies against OASG04237.WB showed two bands of 50 and 75 kDa in the testis, smaller bands of similar size in both serum samples and serum samples A circular band of approximately 75 kDa was predominant in the serum sample. Two bands of 50 and 75 kDa were also detected using the LHR029 antibody bound to the extracellular portion of LHCGR, although the intensity was lower in both serum samples compared to the testis, and a 75 kDa band was again detected in the serum samples. The SC-26341 antibody detected an unknown portion of the extracellular structural domain of LHCGR, showing in all samples approximately 50 and 75 kDa bands, and the 75 kDa band was much stronger in testis than in serum samples. Notably, control β-2 microglobulin levels in serum were significantly lower than expected compared to testicular tissue, which hinders the quantitative use and comparison of LHCGR levels in tissue and serum. The study shows densitometric readings of strips and uncropped blots. Immunohistochemistry of testicular tissue from an adult male with in situ germ cell tumors and mild Leydig cell hyperplasia using the same antibody (LHR029) as the ELISA assay showed membrane binding and mild cytoplasmic expression of LHCGR in most Leydig cells.

2. sLHCGR Is Detectable in Body Fluids but Undetectable in Serum from Xenograft Mice

No sLHCGR was detected in the sera of nude mice with or without TCam-2 xenograft tumors treated in vivo with vector, LH, or hCG.

However, cultured TCam-2 cell medium showed low but detectable levels of sLHCGR, and the combination of the two demonstrated that the human ELISA did not cross-react with any of the mouse proteins in the serum. sLHCGR was also not detected in the unconditioned medium containing fetal bovine serum.

sLHCGR was detectable in the urine of healthy and hypogonadal men at comparable levels (mean 0.024 pmol/mL vs. 0.021 pmol/mL). Urine levels were on average approximately more than 100 times lower than serum (mean 2.21 pmol/mL, hypogonadism mean 1.80 pmol/mL), suggesting that renal elimination is not associated with hypogonadism and is not affected by age differences over 10 years. Furthermore, the low concentration in urine suggests that sLHCGR, like other proteins, is not filtered or excreted in amounts that exceed the renal reabsorption capacity. Notably, the mean serum concentration of sLHCGR in the eight men who had both testes removed was comparable to that of young men (mean 2.04 pmol/mL vs. 2.21 pmol/mL), and hypogonadal men (mean 2.04 pmol/mL vs. mean 1.80 pmol/mL). In addition, sLHCGR levels were detected in both human fetal adrenal and human fetal kidney tissues (mean 0.15 pmol/mL and 0.14 pmol/mL, respectively) (Figure 1E), which were higher than the sLHCGR levels released from TCam-2 cells in vitro (mean 0.03 pmol/mL). Total sLHCGR was measured in the serum of three men and three postmenopausal women from the arterial and venous phases of the liver, kidney, and lower extremities. in all samples, one man and two women had levels above the limit of detection, while the rest had undetectable levels. We did not detect any significant difference in sLHCGR from the arterial side to the venous side in any organ and therefore excluded these organs as the site of origin (t-test: p-value all > 0.31)

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3. LC-MS/MS on Human Serum

Sera from normal men (n:2), patients with testicular germ cell carcinoma (1 seminoma, 1 nonseminoma), and pregnant women (n:2) were used for protein gel electrophoresis after albumin and globulin depletion. Four bands of 75, 60, 50, and 35 kDa were extracted from the pregnancy sera and analyzed by low-sensitivity LC-MS/MS because the signal was strongest here. none of the LC-MS/MS detected tryptic peptides of LHCGR. After removing IgG and albumin, the peptides with the highest protein content were albumin, complement C3, α -1-antitrypsin, and vitamin D binding protein, and all detected proteins were plasma proteins.

4. Serum sLHCGR. Diurnal Variation, Stability, Reproducibility, Freeze, and Thaw

We used three different batches of LHCGR kits from the NBCL ELISA assay with CVs of 16 and 19% and concentrations of 0.4 and 1.7 pmol/mL, respectively. season of the year or time of day of blood sampling had no effect on sLHCGR levels (either semen levels or serum levels). 18 adult Klinefelter syndrome Male patients with sLHCGR were measured continuously for 4-40 months, with low intraindividual variability. The sLHCGR levels were stable in all but two men. In addition, serial measurements over 16-30 months in 23 men who had their testes removed showed that serum sLHCGR levels were significantly elevated in only one man during this period, while there was no overall difference in serum sLHCGR levels in the remaining 22 men. Multiple freeze-thaws reduced sLHCGR concentrations by ~40%.

5. Serum and Urine LHCGR Are Influenced by Treatment with Human Chorion Gonadotropin

Injection of 5000 IU hCG reduced serum sLHCGR after 8 hours (Wilcoxon paired test p = 0.031), and the reduction was further enhanced after 24 hours (Wilcoxon paired test p = 0.016, Bonferroni corrected, p <0.025). Only two men with the highest baseline sLHCGR values were detected in serum 24 hours after injection. The rapid effect of hCG on circulating sLHCGR levels was also evident in urine, where urinary sLHCGR levels fell below the lower limit of detection in all men after 24 hours (Wilcoxon paired test p = 0.0078). Baseline serum sLHCGR before hCG injection did not correlate with baseline urine-sLHCGR, serum testosterone, or serum LH levels, or with δ serum testosterone or δ serum LH levels. Urinary sLHCGR did not correlate with either age or serum reproductive hormones. hCG induced an increase in serum testosterone from 19.6 nmol/L (3.3 SD) to 28.2 nmol/L (4.4 SD) after 24 hours (Wilcoxon paired test: p= 0.001) and to 43.9 nmol/L (8.6 SD) after 72 hours, while 24 LH significantly decreased after 24 hours (Wilcoxon paired test: p= 0.43 at 8 hours, p= 0.001 at 24 hours, p= 0.001 at 72 hours).

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6. Longitudinal Measurement in Children Show a Decline in sLHCGR during Puberty Only in Healthy Boys

To determine how pubertal episodes of increased gonadotropin secretion affect sLHCGR, 36 normal boys were followed longitudinally, and pubertal episodes were assessed using Tanner staging.

Individual post-pubertal sLHCGR levels were significantly lower than pre-pubertal levels (pre-pubertal vs. post-pubertal Wilcoxon paired test: mean (1.36 (2.2 SD) pmol/mL vs. 0.60 (0.89 SD) pmol/mL p = 0.0001). This supports that sLHCGR may decrease with increasing serum gonadotropins, but reproductive hormone levels, BMI, age, or testicular size were not associated with sLHCGR at any time point during pubertal development. Thus, we found no association between sLHCGR and urinary LH or FSH around puberty. As a comparison, 7 KS boys were followed longitudinally without testosterone-induced puberty.

Boys in the KS group did not show a similar decline in sLHCGR during puberty as boys without chromosomal aberrations. All KS boys (except one) experienced peak sLHCGR concentrations at the onset of puberty but did not reach statistical significance. Klinefelter boys also experienced an increase in gonadotropins, but for unknown reasons, they did not experience the same decrease in sLHCGR, suggesting that high sLHCGR may be associated with impaired gonadal function. Serum sLHCGR levels were slightly lower in young KS men compared to young healthy men (0.92 pmol/mL vs. 2.21 pmol/mL, p = 0.031, Tables 1 and 3). The four KS men with the highest serum sLHCGR levels did not differ phenotypically from the remaining KS men.

7. Serum sLHCGR Is Associated with the Gonadal Marker Inhibin/FSH Ratio and Estradiol in Healthy Men

Baseline characteristics of 148 men in the general population. Serum sLHCGR was negatively correlated with the gonadal marker inhibin B/FSH ratio (β-0.004, p = 0.027, and removal of the two extreme right outliers did not alter the correlation:β-0.003, p = 0.018). In the subsequent slicer quartile stratification, a significant downward trend was found, with men in the lowest sLHCGR quartile having a significantly higher ratio of inhibin B/FSH compared to men in the highest sLHCGR quartile after adjustment for multiple comparisons. However, serum sLHCGR was not associated with any other hormone or semen quality, age, BMI, or testicular size. For comparison, 297 infertile men without severe comorbidities underwent cross-sectional examination and had a mean serum sLHCGR of 1.05 pmol/mL (SD 3.15), with 117 of the 297 men having even undetectable sLHCGR levels. In this cohort, we did not find an association between sLHCGR and the inhibin B/FSH ratio (β-0.0010, p = 0.28) or any measured hormone, testicular size, BMI, or age. There was no difference in the inhibin B/FSH ratio when men were divided into sLHCGR quartiles (Kruskal-Wallis p = 0.56). We pooled all men with normal sex chromosomes and pubertal onset from the cohort used. This increased the total number of cases with available serum sLHCGR (n = 488), increasing the age span (11.7-58.0 years) and BMI span (16.3-56.3 years). Serum sLHCGR was negatively correlated with estradiol (β-0.009, p = 0.020), age (β-0.060, p = 2.11 × 10-7), and BMI (β-0.072, p = 0.004), but not with other hormones independently. The same negative correlation was found for the inhibin B/FSH ratio (β-0.003,p = 0.045) when only healthy men from all the above cohorts were analyzed.

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Conclusions

In conclusion, this study shows that sLHCGR is released into serum and some body fluids. sLHCGR serum levels are associated with pubertal development and gonadal function and are temporarily suppressed by high levels of serum hCG. Serum sLHCGR may have prognostic value as a marker of gonadal function. sLHCGR is released into serum via the testes and other organs, which may suggest an extragonadal role for LH or hCG in boys and men.

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Li Juel Mortensen ¹, Mette Lorenzen ¹, Anne Jørgensen ², Jakob Albrethsen ², Niels Jørgensen ²,Søren Møller ^3,4, Anna-Maria Andersson ², Anders Juul ^2,4 and Martin Blomberg Jensen ^1,5

1. Group of Skeletal, Mineral, and Gonadal Endocrinology, University Department of Growth and Reproduction, Rigshospitalet, 2100 Copenhagen, Denmark; li.juel.mortensen@regionh.dk (L.J.M.); mette.lorenzen.01@regionh.dk (M.L.)

2. Department of Growth and Reproduction and International Center for Research and Research Training in Endocrine Disruption of Male Reproduction and Child Health (EDMaRC), Rigshospitalet, University of Copenhagen, Blegdamsvej 9, 2100 Copenhagen, Denmark; Anne.Joergensen.02@regionh.dk (A.J.); Jakob.Christian.Albrethsen@regionh.dk (J.A.); Niels.Joergensen@regionh.dk (N.J.); Anna-Maria.Andersson@regionh.dk (A.-M.A.); Anders.Juul@regionh.dk (A.J.)

3. Center for Functional and Diagnostic Imaging and Research, Department of Clinical Physiology and Nuclear Medicine 260, Hvidovre Hospital, 2650 Copenhagen, Denmark; Soeren.Moeller@regionh.dk

4. Department of Clinical Medicine, Faculty of Health Sciences, Copenhagen University, 2200 Copenhagen, Denmark

5. Division of Bone and Mineral Research, Harvard School of Dental Medicine/Harvard Medical School, Boston, MA 02115, USA