Genetic polymorphisms of CYP2D6*10 and the effectiveness of combined tamoxifen citrate and testosterone undecanoate treatment in infertile men with idiopathic oligozoospermia

Genetic polymorphisms of CYP2D6*10 and the effectiveness of combined tamoxifen citrate and testosterone undecanoate treatment in infertile men with idiopathic oligozoospermia CYP2D6*10基因多态性对他莫昔芬联合十一酸睾酮治疗特发性少精男性不育症的疗效影响 1 2 first-author 1 2 2 2 1 1 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:sfguizhou@163.comxingjpcn@gmail.com †E-mail:sfguizhou@163.comxingjpcn@gmail.com sfguizhou@163.com 2 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:sfguizhou@163.comxingjpcn@gmail.com †E-mail:sfguizhou@163.comxingjpcn@gmail.com sfguizhou@163.com Department of Urology, Affiliated Hospital of Guiyang Medical College, Guiyang 550004, China Department of Urology, Affiliated Hospital of Guiyang Medical College, Guiyang 550004, China Department of Urology, the First Affiliated Hospital of Medical College of Xi’an Jiaotong University, Xi’an 710061, China Department of Urology, the First Affiliated Hospital of Medical College of Xi’an Jiaotong University, Xi’an 710061, China Tamoxifen citrate, as the first line of treatment for infertile men with idiopathic oligozoospermia, was proposed by the World Health Organization (WHO), and testosterone undecanoate has shown benefits in semen values. Our objective was to assess the effectiveness of treatment with tamoxifen citrate and testosterone undecanoate in infertile men with idiopathic oligozoospermia, and whether the results would be affected by polymorphisms of CYP2D6*10 . A total of 230 infertile men and 147 controls were included in the study. Patients were treated with tamoxifen citrate and testosterone undecanoate. Sex hormone, sperm parameters, and incidence of spontaneous pregnancy were detected. There were no significant differences between the control and patient groups with respect to CYP2D6*10 genotype frequencies ( P > 0.05). The follicle-stimulation hormone (FSH), luteinizing hormone (LH), and testosterone (T) levels were raised, and sperm concentration and motility were increased at 3 months and became significant at 6 months, and they were higher in the wild-type allele (C/C) than in the heterozygous variant allele (C/T) or homozygous variant allele (T/T) subgroups ( P < 0.05). In addition, the percentage of normal morphology was raised at 6 months, and represented the highest percentage in the C/C subgroup ( P < 0.05). The incidence of spontaneous pregnancy in the C/C subgroup was higher than that in the C/T or T/T subgroups ( P < 0.01). This study showed that the CYP2D6*10 variant genotype demonstrated worse clinical effects in infertile men with idiopathic oligozoospermia. 目的 2 探讨细胞色素P4502D6*10（ CYP2D6*10 ）基因遗传多态性，并评估其对他莫昔芬联合十一酸睾酮治疗特发性少精男性不育症患者血清性激素、精液参数及自然妊娠率的影响。 方法 2 该病例对照研究包括230例特发性少精男性不育患者和147例正常对照。病例组服用枸橼酸他莫昔芬20mg/d和十一酸睾酮40mg/d，疗程共6个月。采用 Hph I内切酶对 CYP2D6*10 基因聚合酶链式反应（PCR）产物进行内切后，从而对其分型。分别于研究开始时、3月及6月分别检测研究对象性激素水平、精液参数及配偶自然妊娠率。 结论 2 CYP2D6*10 基因突变型特发性少精男性不育患者接受他莫昔芬联合十一酸睾酮疗效较基因野生型组差。 Infertility Cytochrome P450 Oligozoospermia Tamoxifen Testosterone 男性不育症 细胞色素P450 少精子症 他莫昔芬 性激素 Introduction 1 Tamoxifen (TMX) was used for the treatment of oligospermia for the first time by Comhaire ( 1976 1976 ) and has become one of the widely prescribed drugs for idiopathic male infertility until now (Comhaire, 1976 1976 ; Chua et al., 2013 2013 ). Tamoxifen citrate, as a first line of treatment for infertile men with idiopathic oligozoospermia, was proposed by the World Health Organization (WHO) Working Committee (Rowe et al., 2000 2000 ). Tamoxifen undergoes extensive hepatic and gut wall metabolism in humans for several primary and secondary metabolites that exhibit a range of pharmacologic activity (Squirewell et al., 2014 2014 ). Previous research demonstrated that endoxifen formation proceeds stepwise by oxidation of tamoxifen with N -desmethyltamoxifen (NDM) as the predominant intermediate (Desta et al., 2004 2004 ), and patients receiving tamoxifen are influenced by cytochrome P4502D6 ( CYP2D6 ) genetic variants (Stearns et al., 2003 2003 ; Jin et al., 2005 2005 ). According to the activity of the CYP2D6 enzyme, patients should be grouped into poor metabolizer (PM), extensive metabolizer (EM), and ultra-rapid metabolizer (UM) phenotypes. There are significant inter-ethnic differences in the frequency of the PM phenotype and PM alleles of CYP2D6 . Except for the non-functional CYP2D6 PM alleles, intermediate metabolizers (IM) have reduced enzyme activity and are common in Orientals; one of the very common IM alleles is CYP2D6*10 (Ji et al., 2002a 2002a ; Borges et al., 2006 2006 ). Among Chinese, the most common polymorphism in CYP2D6 is allelic variant *10, which generates a 188 C to T transition in exon 1, leading to a proline 34 to serine amino acid substitution and resulting in an unstable enzyme with lower catalytic activity (Johansson et al., 1994 1994 ; Garcia-Barcelo et al., 2000 2000 ; Ji et al., 2002b 2002b ). Recent studies have shown that CYP2D6*10 genotype affects the efficacy of tamoxifen treatment in Chinese women with breast cancer, although women with the CYP2D6*10 variant homozygous variant allele (T/T) genotype have a worse clinical outcome (Xu et al., 2008 2008 ). Other studies have shown that giving tamoxifen citrate alone exerts a limited effect on sperm morphology and motility; it appears that the superior functional sperm fraction response was probably related to administration of testosterone undecanoate (Adamopoulos et al., 1997 1997 ; 2003 2003 ). In this study, we assess the effectiveness of treatment with tamoxifen citrate and testosterone undecanoate in infertile men with idiopathic oligozoospermia, and whether they are affected by polymorphisms of CYP2D6*10 . Materials and methods 1 Subjects 2 This study included 230 infertile men (mean age (27±3.7) years, range 22‒40 years) with idiopathic oligospermia diagnosed at the First Affiliated Hospital of the Medical College of Xi’an Jiaotong University, China. All patients had a history of infertility for at least one year, with their spouses confirmed to have had a normal gynecological evaluation. All of the subjects had sperm concentration below the WHO ( 1999 1999 ) threshold (20×10 6 ml −1 ) on at least two semen analyses. The infertile men with known or demonstrable causes of oligozoospermia (varicocele, infections, autoimmunity, stress, chromosomal abnormalities, environmental factors, or epididymopathy) were excluded. The study also included 147 volunteers with normozoospermic semen (mean age (25±2.5) years, range 23‒35 years). Their sperm underwent the same examinations as those of the oligozoospermic men. Written informed consent was obtained from all the patients and controls included in this study. The protocol was approved by the Ethics Committee of the First Affiliated Hospital of the Medical College of Xi’an Jiaotong University, China. Protocol 2 The protocol included two phases. Initial assessment was made during the first phase. The basal follicle-stimulation hormone (FSH), luteinizing hormone (LH), and testosterone (T) were measured during this phase, and at least two semen analyses were taken after 3‒5 d of sexual abstinence in all participants. None of the participants had received any medication for at least 3 months before entry into the trial, and each of them had not participated in any recent relevant study. Additional blood samples (3 ml taken into ethylene diamine tetraacetic acid (EDTA) by venipuncture) were obtained from all participants. After collection, the whole blood was immediately stored at −80 °C until use for genomic DNA extraction. In the second phase, all the patients ( n =230) received tamoxifen citrate (20 mg/d) and testosterone undecanoate (40 mg/d), and the treatment was prescribed for 6 months. Sex hormone measurement and semen analysis were performed at the 3rd and 6th months, respectively, and the percentage of pregnancies was recorded as well. Measurements 2 The FSH, LH, and T in serum were measured by using a double-antibody recombinant immunoassay (CIS Biointernational, Paris, France, for FSH and LH; Farmos, Oulunsalo, Finland, for T). Semen samples were obtained after 3‒5 d of sexual abstinence and were analyzed within 1 h after collection. In all patients, a standard semen analysis was performed, assessing semen parameters, including sperm concentration, motility, and morphology according to the WHO ( 1999 1999 ) guidelines. DNA source, genotyping, and definition of phenotypes 2 An AxyPrep™ Genomic DNA Miniprep Kit (Axygen Biosciences, Union City, CA, USA) was used to isolate genomic DNA from blood samples. The mutant CYP2D6*10 (C188T) allele was detected by polymerase chain reaction (PCR) amplification using primers: forward, 5'-TCAACACAGCAGGTTCA-3'; reverse, 5'-CTGTGGTTTCACCCACC-3', followed by Hph I digestion (5 U, 37 °C) (Sachse et al., 1997 1997 ). PCR was performed in a 25-μl reaction buffer containing 200 μmol/L dNTPs, 1.5 mmol/L MgCl 2 , 10 pmol of each primer, approximately 200 ng of template DNA, and 2 U of thermostable Taq DNA polymerase (MBI, Vilnius, Lithuania). After a 5-min pretreatment at 95 °C, the specific conditions used for all PCR amplifications (30 cycles) were denaturation for 45 s at 94 °C, annealing for 45 s at 55 °C, and fragment extension for 45 s at 72 °C, and a final elongation step for 7 min at 72 °C. PCR products digested by restriction enzyme were electrophoresis on a 2% (0.02 g/ml) agarose gel stained with ethidium bromide to verify the correct size of the expected fragments. The mutant CYP2D6*10 (C188T) allele was detected by Hph I digestion after PCR amplification, the 433 bp amplified product was digested into 362 and 71 bp products for wild-type allele (C/C), and 262, 100, and 71 bp products for homozygous variant allele (T/T), and 362, 262, 100, and 71 bp products for heterozygous variant allele (C/T). Statistical analysis 2 The semen analyses and sex hormone results before treatment were compared with those at 3- or 6-month using the paired Student’s t -test. The comparisons of variance on semen analyses and hormone results between genotype groups and between patients taking CYP2D6*10 inhibitors and those not taking CYP2D6*10 inhibitors were performed by use of t -tests. Phenotype expression in each defined genotype group was reported as mean±standard deviation (SD). The associations between CYP2D6*10 genotype subgroups were evaluated by Chi-square test, and P < 0.05 was considered statistically significant. Results 1 At the ending of this study, 204 of 230 infertile men with idiopathic oligozoospermia and 147 controls completed the protocols. No statistically significant differences were found between the patients and controls with respect to age, smoking, or alcohol consumption. In addition, no differences were found among the subgroups with respect to the age of the female partners. Genotypes of CYP2D6*10 2 The frequencies of the CYP2D6*10 C/C, C/T, and T/T genotypes in the control and patient groups are shown in Table 1 1 . Both of these two groups did deviate from the Hardy-Weinberg equilibrium law ( P > 0.05). The frequencies of CYP2D6*10 C/C, C/T, and T/T genotypes were 19.73%, 55.78%, and 24.49%, respectively, in the control group; and 18.14%, 55.88%, and 25.98%, respectively, in the patient group. No significant differences were found between the control and patient groups with respect to the frequencies of the CYP2D6*10 genotypes ( P > 0.05). Data are expressed as number (percentage) C/C: wild-type allele; C/T: heterozygous variant allele; T/T: homozygous variant allele; HWE: Hardy-Weinberg equilibrium law; OR: odds ratio; CI: confidence interval 2 CYP2D6*10 genotype 2 Frequency * * 2 χ 2 2 P -value 2 OR (95% CI) Control Patient Total 147 204 C/C 29 (19.73%) 37 (18.14%) 0.142 0.707 0.902 (0.525‒1.547) C/T 82 (55.78%) 114 (55.88%) <0.001 0.985 1.004 (0.655‒1.539) T/T 36 (24.49%) 53 (25.98%) 0.1 0.751 1.082 (0.664‒1.765) 6 HWE ( χ 2 , P -value) 2.053, 0.152 3.165, 0.075 Association of CYP2D6*10 genotypes with sex hormone and semen parameters 2 The levels of the FSH, LH and T, sperm concentration, motility, and normal morphology in the participants are shown in Table 2 2 . No significant differences were found with the basal evaluation of FSH, LH, T, density, motility, or normal morphology between the control and patient groups, and among the subgroups in patients with respect to CYP2D6*10 genotypes ( P > 0.05). FSH: follicle-stimulation hormone; LH: luteinizing hormone; T: testosterone; C/C: wild-type allele; C/T: heterozygous variant allele; T/T: homozygous variant allele P < 0.05 vs. baseline value P < 0.05 vs. 3-month value P < 0.05 vs. C/C genotype Group No. FSH (IU/L) LH (IU/L) T (nmol/L) Sperm concentration (×10 6 ml −1 ) Motility (%) Normal morphology (%) 8 Basal evaluation Control 147 8.03±1.01 7.31±1.32 15.01±2.98 56.72±3.07 59.24±4.80 55.39±3.95 Patient 204 7.73±1.88 7.22±1.98 13.70±3.80 9.45±2.35 30.39±3.60 44.52±4.87 C/C 37 7.85±1.12 7.16±1.12 13.77±2.13 9.35±1.43 30.17±5.05 45.02±5.83 C/T 114 7.66±2.06 7.23±2.49 13.87±3.88 9.56±2.93 30.10±3.16 45.49±4.10 T/T 53 7.79±1.92 7.22±1.01 13.14±4.40 9.29±1.24 31.20±3.08 44.09±4.90 8 Three-month evaluation Patient 204 8.02±1.77 7.58±2.44 14.41±3.73 * * 9.74±1.78 31.22±4.43 * * 45.97±4.94 C/C 37 9.29±2.15 * * 8.35±1.34 * * 16.99±3.50 * * 10.23±1.56 * * 33.61±7.62 * * 46.95±6.08 C/T 114 7.72±1.10 * * 7.43±2.98 * * 13.97±3.21 * * 9.73±2.02 30.09±3.28 * * 46.31±4.24 T/T 53 7.91±2.28 * * 7.31±1.43 * * 13.55±4.18 * * 9.40±1.21 * * 31.96±2.34 * * 44.68±5.32 8 Six-month evaluation Patient 204 9.52±4.05 a a b b 8.76±3.16 a a b b 19.28±7.37 a a b b 11.35±4.60 a a b b 34.34±5.74 a a b b 47.35±3.98 a a b b C/C 37 15.69±4.30 a a b b 13.95±2.77 a a b b 31.85±6.10 a a b b 18.19±1.23 a a b b 40.76±5.21 a a b b 50.17±5.18 a a b b C/T 114 8.11±1.51 * * 7.66±2.01 * * 17.06±4.02 * * 9.98±4.13 * * 32.84±5.42 * * 47.15±2.79 * * T/T 53 8.06±3.04 * * 7.50±1.18 * * 15.27±3.46 * * 9.53±1.90 * * 33.10±3.16 * * 45.85±4.28 * * At the 3rd month, FSH, LH and T levels, motility, and the percentage of normal morphology in the C/C subgroup were all higher than those in the basal evaluation and C/T and T/T subgroups, respectively ( P =0.001, P < 0.001, P =0.002 for FSH; P =0.001, P =0.009, P =0.001 for LH; P < 0.001, P < 0.001, P < 0.001 for T; P =0.034, P =0.009, P =0.021 for motility; P =0.157, P =0.051, P =0.064 for the percentage of normal morphology). Sperm concentration in the C/C subgroup was higher than those in the basal evaluation and T/T subgroup, respectively ( P =0.015, P =0.004). At the 6th month, we found that FSH, LH, and T levels, sperm concentration, and motility in the C/C subgroup were all higher than those in the basal evaluation and 3-month evaluation, and also C/T and T/T subgroups, respectively ( P < 0.001, P < 0.001, P < 0.001, P < 0.001 for all). The percentage of normal morphology in the C/C subgroup was also higher than those in the basal evaluation, 3-month evaluation, and C/T and T/T subgroups, respectively ( P < 0.001, P =0.022, P =0.002, P < 0.001). Association of CYP2D6*10 genotypes with pregnancy incidence 2 In this study, the incidence of spontaneous pregnancy was 21.1% (43/204) in the infertile men with idiopathic oligozoospermia, after treatment with tamoxifen citrate and testosterone undecanoate (Table 3 3 ). According to the genotypes of CYP2D6*10 , the incidence of spontaneous pregnancy was 40.5% (15/37) in the C/C subgroup, 20.2% (23/114) in the C/T subgroup, and 9.4% (5/53) in the T/T subgroup. It was shown that the incidence of spontaneous pregnancy in the C/C subgroup was higher than those in the C/T and T/T subgroups, respectively ( χ 2 =6.152, P =0.013; χ 2 =12.198, P < 0.001). Data are expressed as number (percentage) Compared with the C/C genotype 2 Group 2 No. 4 Incidence of spontaneous pregnancy * * 2 χ 2 2 P -value Starting Three-month Six-month Total Patient 204 0 8 (3.2%) 35 (17.2%) 43 (21.1%) C/C 37 0 3 (8.1%) 12 (32.4%) 15 (40.5%) C/T 114 0 4 (3.5%) 19 (16.7%) 23 (20.2%) 6.152 0.013 a a T/T 53 0 1 (1.9%) 4 (7.5%) 5 (9.4%) 12.198 < 0.001 a a Discussion 1 Tamoxifen is a trans isomer of clomiphene citrate, which is a combination of two isomers that exert both anti-estrogenic and estrogenic effects simultaneously and human studies have confirmed its estrogenic activity to be minimal or negligible (Lin et al., 2010 2010 ; Lu et al., 2012 2012 ). Tamoxifen citrate enhances spermatogenesis by increasing FSH, leydig cells sensibility to LH, and testosterone levels, which lacks an intrinsic oestrogenic effect, so it may be more appropriate to use in male infertility (Buvat et al., 1983 1983 ; Kadioglu et al., 1999 1999 ). Previous studies demonstrated that tamoxifen significantly increased sperm concentration in infertile men with oligozoospermia, but does not affect other semen values, such as volume, pH, motility, morphology, or viability, because of tamoxifen’s effectiveness on the seminiferous tubules during the early stages of spermatogenesis (Vermeulen and Comhaire, 1978 1978 ; Kotoulas et al., 1994 1994 ). Other studies have shown that given tamoxifen citrate alone exerts a limited effect on sperm morphology and motility; it appears that the superior functional sperm fraction response was probably related to administration of testosterone undecanoate (Adamopoulos et al., 1997 1997 ; 2003 2003 ). In this study, the sperm concentration, motility, and percentage of normal morphology were improved in infertile men with oligozoospermia after treatment with tamoxifen citrate and testosterone undecanoate. Endoxifen is one of the most important metabolites of tamoxifen, and plasma concentrations of endoxifen appeared to be influenced by the patient’s CYP2D6 genotype. Previous studies have shown that plasma concentrations of endoxifen were statistically significantly lower in patients who were carriers of non-functional CYP2D6 allelic variants, compared with those having two functional wild-type alleles, and the CYP2D6*10 genotype is the most common polymorphism of CYP2D6 in the Chinese population (Stearns et al., 2003 2003 ; Jin et al., 2005 2005 ). The percentage of homozygous variant T/T genotype was 24.49% in the control group and 25.98% in the patient group in our present study, which was similar to previous reports (Johansson et al., 1994 1994 ; Garcia-Barcelo et al., 2000 2000 ). In vitro experiments have shown that the catalytic activity of the homozygous variant T/T genotype is 1/40 of the activity of the wild-type C/C genotype (Johansson et al., 1994 1994 ). In our present study, it was shown that the levels of the FSH, LH and T, sperm concentration, motility, percentage of normal morphology, and the incidence of spontaneous pregnancy in patients with the T/T and C/T genotypes had a lower outcome than those in patients with the C/C genotype. Conclusions 1 In summary, we found that the CYP2D6*10 mutant genotype had a worse clinical outcome in the combined treatment of tamoxifen citrate and testosterone undecanoate in infertile men with idiopathic oligozoospermia. Analyses of CYP2D6*10 genotype may be useful for patients with idiopathic oligozoospermia, and may benefit from treatment when combining tamoxifen citrate with testosterone undecanoate. Nevertheless, our study does have certain limitations. Our results should be carefully interpreted and the CYP2D6*10 genotypes should not apply in clinical practice until the data from more center studies are available. References DA Adamopoulos , S Nicopoulou , N Kapolla , 等 . 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Hormonal effects of an antiestrogen, tamoxifen, in normal and oligospermic men WHO (World Health Organization) WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interactions , 1999 . 4th Ed : London, England Cambridge University Press , 270 - 278 . . WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interactions Y Xu , Y Sun , L Yao , 等 . Association between CYP2D6*10 genotype and survival of breast cancer patients receiving tamoxifen treatment . Ann Oncol , 2008 . 19 ( 8 ): 1423 - 1429 . 18407954 DOI: 10.1093/annonc/mdn155 http://doi.org/10.1093/annonc/mdn155 . Association between CYP2D6*10 genotype and survival of breast cancer patients receiving tamoxifen treatment Our heartfelt thanks go to the research participants in this study. Compliance with ethics guidelines Kai-fa TANG, Yi-li ZHAO, Shang-shu DING, Qi-fei WU, Xing-yang WANG, Jia-qi SHI, Fa SUN, and Jun-ping XING declare that they have no conflict of interest. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008 (5). Informed consent was obtained from all patients for being included in the study. Additional informed consent was obtained from all patients for whom identifying information is included in this article. 10.1631/jzus.B1400282 Project supported by the National Natural Science Foundation of China (No. 81300541), the Technology Project of Guizhou Province (No. QKHJZ[2013]2051), and the Doctoral Fund of the Affiliated Hospital of Guiyang Medical College (No. C-2012-6), China 2014-10-21 2015-02-22 2015-01-01 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2015 2015 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 03 16