Possible role of androgen receptor gene in therapeutic response of infertile men with hypogonadotropic hypogonadism

ABSTRACT

Hypogonadotropic hypogonadism (HH) is defined as a dysfunction of hypothalamic–pituitary–gonadal axis, which causes impairments in gametogenesis, pubertal maturation, and/or secretion of the gonadal sex hormones. Human chronic gonadotropin (hCG) stimulates the Leydig cells of the testis to secrete testosterone, which is essential for spermatogenesis. Testosterone replacement therapy is one of the possible options to manage HH treatment. Given the fact that testosterone functions are mediated via androgen receptor (AR), the aim of the present study was to evaluate whether the CAG/GGN triple repeat expansion in AR gene can modulate the response to hCG and testosterone treatment in HH men. Sixty-two men who diagnosed with HH and treated with testosterone and hCG were assessed after treatment. They were classified into two groups, 31 subjects with a positive and 31 subjects with a negative response to replacement therapy within 12–18 months. Androgen receptor CAG and GGN repeat numbers were measured in both groups by hot start polymerase chain reaction (PCR)-sequencing technique. Subjects who reached complete spermatogenesis showed the 20 and 23 as the median numbers of AR CAG/GGN repeats, respectively. In individuals who did not respond to treatment the median length for both CAG/GGN repeats were 23. The average of CAG repeats was statistically lower in patients who had the positive response in comparison to patients who did not respond to hormone therapy (p < 0.05), but the length of GGN repeats were not statistically different between these groups of patients (p > 0.05). The number of CAG repeats are negatively and significantly associated with better hormone therapy response. Our results suggest that the length of CAG repeat polymorphism in AR gene might affect the response to treatment in men suffering from HH, whereas no relationship was found between AR gene GGN repeat polymorphism and testosterone and hCG replacement therapy response.

Abbreviations: AR: androgen receptor; FSH: follicle stimulating hormone; Gn: gonadotropins; GnRH: gonadotropin-releasing hormone; hCG: human chronic gonadotropin; HH: hypogonadotropic hypogonadism; LH: luteinizing hormone; PCR: polymerase chain reaction

KEYWORDS:

• Androgen receptor
• CAG repeats
• GGN repeat
• polymorphism
• Human chorionic gonadotropin (hCG)
• hypogonadotropic hypogonadism

Introduction

Hypogonadotropic hypogonadism (HH) or secondary hypogonadism is a disorder which is characterized by hypogonadism due to an impaired secretion of gonadotropins. As the main harm involves the hypothalamus and the pituitary gland, normal levels of the luteinizing hormone (LH) and the follicle stimulating hormone (FSH) are reduced and result in low circulating testosterone levels. Several treatments are proposed based on the defect type (Bosdou et al. 2016). In men with HH, puberty and spermatogenesis both can be induced by testosterone, gonadotropins (Gn), or the gonadotropin-releasing hormone (GnRH) therapy. In hypothalamic disorders such as idiopathic hypogonadotropic hypogonadism pulsatile GnRH or Gn administration can be used alternatively, while male patients with primary pituitary disorders can only be treated with Gn (Alen et al. 1999; Giagulli et al. 2012). In the complete form of isolated hypogonadotropic hypogonadism, treatment with pulsatile GnRH for the first 2 years does not accelerate or boost testicular growth, amplify the initiation of sperm production, or increase sperm output significantly in comparison to hCG/hMG. Liu, L., et al. (1988) showed that consuming hCG/hMG is more beneficial than GnRH therapy in the term of testosterone production. Testosterone has its effect via the androgen receptor on target organs for the development of male primary sexual characteristics. Testosterone also seems to play a fundamental role in the evolution of spermatocytes to round spermatids during the spermatogenesis (Liu et al. 1988; Beitel et al. 2013; Fraietta et al. 2013). Previous studies reported that androgens influence many genes’ expression by the help of activated AR (Sun et al. 2014). Accordingly, the AR exerts a key role by regulating the transcription of androgen-dependent proteins from embryogenesis to adolescence. In the first exon of the AR gene, there are two regions consisting of a varying number of CAG and GGN triplets, which encode the polyglutamine and polyglycine stretch, respectively. AR CAG polymorphism seems to influence androgen action (Chamberlain et al. 1994; Khan et al. 2018). In healthy individuals, the CAG normal ranges are from 9 to 36 repeats (Borjian Boroujeni et al. 2018). Depending on ethnic origin, the reported CAG repeats length for fertile populations differs and ranges from 8 to 30 among American whites, 8–39 among Europeans, and 11–31 among Asians, whereas Europeans of Caucasian origin have a bigger range in their AR-CAG repeat lengths (Rajpert-De Meyts et al. 2002). The ligand-induced transactivation activity of the AR is inversely associated with the length of the CAG repeat chain. Longer CAG repeats are associated with a decreased transcriptional activity of the receptor, which results in deficient effects on target tissues (Chamberlain et al. 1994; Mostafa et al. 2012; Tirabassi et al. 2014c). The possible underlying mechanisms for this phenomenon can be attributed to differential affinity of coactivator proteins for the encoded polyglutamine stretch of the AR protein (Lapauw et al. 2007). Moreover, in vitro studies indicated a possible effect of the GGN repeat polymorphism on the AR gene transcription (Bogaert et al. 2009). The clinical observations suggest that GGN repeat might modulate the androgen action whereas this effect appears to be small. The GGN repeat can form a hairpin structure in which the increase of repeat length can cause free energy reduction and increased hairpin stability. This suggests that hairpin stability may interfere with translation, which illustrates the inverse effect of GGN repeat length on AR protein yields (Ding et al. 2005). It has been shown that the GGN longer allele contributes to contradictory androgen receptor function, associated with spermatogenic failure (Castro‐Nallar et al. 2010). Accordingly, in this study, we aimed to evaluate the role of the CAG and GGN repeat polymorphisms in the conditioning of the spermatogenesis recovery in HH males undergoing testosterone and combined hCG and FSH therapy.

Results

In the positive response group, the median numbers of repeats were 20.61 and 22.06 repeats for CAG and GGN, respectively. The range of the repeats for CAG was 16 to 25 in which CAG20 and CAG21 showed the highest frequency in the population (19%). After 20 and 21 repeats of CAG, 19 had the highest frequency (16%). The range of GGN repeat in this group was 17 to 26 which the repeat in 23 showed the highest frequency in the population (32%). After GGN23, GGN24 had the highest frequency.

In the Negative response group, the median numbers of repeats were 22.45 and 22.35 repeats for CAG and GGN, respectively. The range of the repeats for CAG was 17 to 26 in which the repeat CAG24 showed the highest frequency in the population (19%). After 24 repeats of CAG, CAG22 and 25 had the highest frequency (16%). The range of GGN repeat in this group was 17 to 25 and the repeat of GGN23 showed the highest frequency in the population (38%). After GGN23, GGN24 had the highest frequency (Table1.)

Table 1. Frequency of CAG and GGN repeats in each of the studied groups.

	No. of CAG repeats (n = 31)				No. of GGN repeats (n = 31)
	Positive respondersto the treatment	n	Negative responders to the treatment	n	Positive responders to the treatment	n	Negative responders to the treatment	n
	16	1	17	4	17	1	17	1
	17	1	20	3	18	1	19	1
	18	3	21	2	19	3	20	5
	19	5	22	5	20	3	21	1
	20	6	23	3	21	2	22	3
	21	6	24	6	22	3	23	12
	22	3	25	5	23	10	24	6
	23	2	26	3	24	7	25	2
	24	1			26	1
	25	3
Mean ± SD	20.61 ± 2.29		22.45 ± 2.75		22.06 ± 2.13		22.35 ± 1.87
P-value	0.006^a				0.617^b

^aUsing t-test

^bUsing Mann–Whitney U test

Student’s paired T-test results indicated that the group who reached complete spermatogenesis after hormone therapy had a significantly lower number of CAG repeats (p = 0.006*) compared to the group who did not obtain a complete spermatogenesis. Statistical analysis did not illustrate any significant difference between the two groups regarding the GGN number (p > 0.05). (Figure1.)

Figure 1. Percent distribution of the number of CAG and GGN repeats in hypogonadotropic hypogonadism patients with the positive response and negative response. Besides the significant shorter median number of CAG repeats (P = 0.006) in positive responders than that of in negative one, also a shift toward shorter repeat length of CAG repeats among positive responders was observed which was not seen in GGN repeats.

Discussion

One of the important functions of the hypothalamic–pituitary–gonadal axis is to regulate gonadotropin and testosterone releasing. Production of GnRH in the hypothalamus stimulates the pituitary gland to secrete LH and FSH. Both of which ultimately control gonadal function. In men, FSH initiates, and in conjunction with high intratesticular testosterone, maintains spermatogenesis, whereas LH controls androgens (T and DHT) synthesis by testicular Leydig cells stimulation (Achermann and Jameson 1999). Male hypogonadism is a disorder characterized by the absence of pubertal development, or cessation or regression of the maturation of secondary sex characteristics, and infertility. This disorder is divided into two groups; I) primary hypogonadism due to testis failure (hypergonadotropic hypogonadism) and II) hypogonadotropic hypogonadism due to insufficient secretion of gonadotropins (LH, FSH). HH disorder is treated with testosterone replacement therapy (TRT) and/or gonadotropins replacement therapy (GRT) and hCG/hMG (Sato et al. 2015). Testosterone therapy for men with symptomatic testosterone deficiency can induce and maintain secondary sex characteristics and correct symptoms of hypogonadism (Bhasin et al. 2018). Testosterone has an effect on target organs via the androgen receptor (AR). Several studies represented the expansions of CAG repeats length in AR gene associated with an increased risk of male infertility (Mosaad et al. 2011). This emphasizes the AR important role in spermatogenesis regulation in man. Moreover, the AR CAG repeat polymorphism seems to play a critical role in the pharmacogenetics of T treatment (Francomano et al. 2013). Previously, it was shown that the length of the repeat tract is inversely associated with the transcriptional and physiological activity induced by T in vitro (Mhatre et al. 1993), both in mouse (Albertelli et al. 2006) and in human models (Zeegers et al. 2004).

In this work we evaluated the impact of AR gene CAG and GGN polymorphism length on therapeutic effects of TRT, focusing on male hypogonadotropic hypogonadism. The significantly lower CAG repeats in patients who had positive response to hCG and Testosterone, can be justified by the hypothesis that CAG trinucleotide repeats coding glutamine amino acid repeats in N-terminal ligand-independent AF-1 domain of AR protein. AF1 considers interacting with transcription factors. Poly-glutamine repeat expansions have an effect on protein folding and reduce AF1 interaction with transcription factors which finally diminished transcription of androgen target genes. This finding indicates that shorter CAG repeats in the AR gene might be associated with a more effective response to testosterone and hCG therapy to reach complete spermatogenesis. This result is in agreement with the study of Giagulli, V., et al. which demonstrated that the length of AR CAG repeat in HH subjects who reached complete spermatogenesis within 12 months, was the same as control group, while the CAG repeat number of HH patients who obtained mature sperms in their ejaculate beyond a year to within 30 months, was significantly higher. Accordingly, they suggested that the length of AR CAG repeat polymorphism might affect the response to GnTh in men suffering from HH (Giagulli et al. 2012) which supports our results as well.

In the other studies, men affected by post-surgical hypogonadotropic hypogonadism who underwent several replacement hormone therapies, were evaluated before and after TRT and the influence of AR triplet CAG repeat on different parameters such as sexual characteristic recovery, central body fat and bone metabolism were investigated (Tirabassi et al. 2013, 2014a, 2014b, 2014c). Tirabassi, G., et al. (2014c) showed that there was an association between shorter AR-CAG repeats with the better and earlier recovery of sexual function after TRT which can also confirm our result. Tirabassi, et al. (2014a) also demonstrated that shorter length of AR CAG repeat tract is independently associated with a more remarkable decrease of abdominal fat after TRT. In other research Tirabassi et al. (2013) suggested that in postsurgical HH male, shorter AR gene CAG repeats length yield greater metabolic improvement after TRT. They also demonstrated that, in these men, shorter AR CAG tract is independently associated with greater TRT-induced improvement of bone mineral density (Tirabassi et al. 2014c). Francomano, D., et al. (2013) tried to introduce the AR polymorphism investigation as a future option to tailor testosterone therapy.

In our research, no significant difference between GGN tract length and its impact on treatment effectiveness was observed. There is no other study in this regard which we can compare our results. Based on the absence of a simple relationship between AR‐GGN repeat length, transcriptional activation, sperm count, and male infertility (Rajender et al. 2006) its impact on TRT is significantly less than CAG repeats.

It should be mentioned that our study is based on the genetic alterations which are polymorphisms and most polymorphisms are ethnic-related and this may prove limiting. Accordingly, the decisive results should be drawn by a larger number of cases. In conclusion, our study suggests that, in men affected by hypogonadotropic hypogonadism, in contrast to GGN, the shorter length of CAG tract in AR gene may be a predictor of response to TRT. Accordingly, pharmacogenomic investigations of the AR polymorphism may in the future provide one option to tailor testosterone titration individually and also to better identify subjects as potentially more or less responsive to treatments.

Materials and methods

Subjects

Considering the principle of Respect for Persons, the investigation was performed with the Helsinki declaration on research with human participants. It was approved by the Ethical Committee of Royan reproductive and biomedicine research center (reference number EC/91,000,539). Written informed consent was provided for every patient. The predominant ethnic background of all groups was Iranian (>95%). All samples were collected during a 3-year period (2015–2017). In this study, 62 hypogonadotropic hypogonadism subjects (age range 23 to 44 years) who were treated with testosterone and combined hCG and FSH were enrolled. They were considered as two groups based on their response to therapy within 6–18 months. Thirty-one men positively responded to T and hCG therapy and 31 men did not. All subjects were selected with a normal karyotype and without any AZF micro deletions. None were referred with the problem of sexual ambiguity. Clinical history and physical examination were directed to evaluate specific signs and symptoms consistent with hypogonadism. Testis size (TS) was measured by the Prader orchidometer. Secondary hypogonadism was diagnosed according to the Endocrine Society criteria (Bhasin et al. 2010): age greater than 18 years, symptoms and signs of hypogonadism, serum testosterone level below 150 ng/dl in two different tests and low to low normal gonadotrophin levels. In all individuals under examination, serum glucose levels were below 100 mg/dl and kidney and liver functions were also normal. Exclusion criteria were: neoplastic and endocrine disorders, alcohol or drug dependence, male-gender-specific disorders (e.g., benign hypertrophy of prostate, chronic prostatitis, urinary incontinence), and intellectual disabilities.

Study design

After at least 90 days of testosterone treatment withdrawal, basal seminal and hormonal parameters were determined twice, at a 15-day interval. To achieve complete spermatogenesis, all hypogonodal men were given GnTh starting with both β HCG (5000 IU/week im) (Choriomon, IBSA, Lugano, Switzerland) and human recombinant FSH (rhFSH) (75 IU/day im) (Gonal F, Serono, Italy) for 18 months at the most (Giagulli and Carbone 2006). Serum and seminal parameters were assessed after 6, 12, and 18 months of GnTh. T, FSH, and LH hormone levels for each man were recorded and sperm analysis performed after 3–5 days of abstinence before and after hormonal replacement therapy to select patients responding to treatment. Sperm analysis was performed according to the World Health Organization criteria (Cao et al. 2011).

PCR-sequencing

For AR gene CAG and GGN repeat genotyping, genomic DNA was isolated from peripheral blood by salting out method. After designing the specific primers (Table 2) with Perlprimer v 1.1.14 software, a PCR-sequencing strategy was applied for genotyping of samples. Extracted DNA (150 ng) was then amplified in a polymerase chain reaction. Thermal cycling (Bio-Rad) included: 94°C for 5 min, 35 cycles of 94°C for 1 h, 60°C and 61°C to annealing two pairs of primers to amplifying CAG and GGN regions, respectively, for 1 h, and 72°C to extension for 1 h, then 72°C for 5 min to final extension. CAG and GGN triplets’ repeats number for each group determined by sequencing method. The products of gene amplification were sent for sequencing to Fazabiotech Company. FinchTV software v.1.4.0 and BLASTN at the NCBI Blast server were used to analyze and recognize the probable variations in comparison with the human AR gene sequence at the NCBI-Gene database (NC_000023.11 as the reference sequence). The possible similarities and the differences were evaluated and the number of triplet repeats of CAG and GGN of each group was counted and compared between two groups.

Table 2. Oligonucleotide primer sequences for CAG & GGN evaluation.

Analysis Type	Primer sequences	Product Size	Primer Tm
CAG evaluation	F: 5-TCCAGAATCTGTTCCAGAGCGTGC-3 R: 5ʹ-GCTGTGAAGGTTGCTGTTCCTCAT-3’	286	60°C
GGN evaluation	F: 5ʹ-CGGTTCTGGGTCACCCTCA-3ʹ R: 5ʹ-TCACCATGCCGCCAGGGTA-3’	263	61°C

Statistical analysis

Statistical analysis was carried out with SPSS version 20.0 (SPSS Inc, Chicago, IL, USA). Continuous variables were expressed as mean ± SD and categorical variables as number (percentage). Normality of the variables was checked with the Kolmogorov–Smirnov Test. T-test was used to evaluate the difference between groups in normal variables and Mann–Whitney U test was used to evaluate difference in non-normal variables. P-value ≤0.05 was considered statistically significant.

Acknowledgments

This study was funded by a grant from the Royan Institute, Tehran, Iran. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. We are thankful to the participants of the study.

Authors’ contributions

Prepared the samples, performed the experiment, and wrote the manuscript draft: PBB, VF; Collected and prepared the samples, and performed biochemical tests of the patients: ZR; Prepared the sample size and analysed the data statistically: MM; Performed the clinical consultation for patients to enter the study and she was also the scientific consultant: MASG; Conceived and designed the experiment, controlled the samples based on the right criteria, analyzed the data, interpreted the data, contributed in writing the manuscript, and revised and edited the paper: MS., AMM. All authors approved revisions and the final paper.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This study was funded by a grant from Royan Institute, Tehran, Iran.