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DERMATOLOGICA SINICA xxx (2016) 1e4 Contents lists available at ScienceDirect Dermatologica Sinica journal homepage: http://www.derm-sinica.com ORIGINAL ARTICLE Effects of acitretin on semen quality and reproductive hormone levels in patients with psoriasis vulgaris Hui Liu*, Jie Li, Li Yu Department of Dermatology, 210 Hospital of People's Liberation Army, Dalian 116021, Liaoning, China a r t i c l e i n f o a b s t r a c t Article history: Received: Aug 5, 2016 Revised: Sep 21, 2016 Accepted: Oct 31, 2016 Background: The aim of this study was to investigate the effect of different doses of acitretin (ACI) on the main parameters of semen, sperm morphology, and reproductive hormone levels in patients with psoriasis vulgaris Methods: The main parameters of semen and the sperm morphology of 31 psoriatic patients were analyzed before and after treatment with different doses of ACI, and the changes in reproductive hormone levels were measured and compared with those of 14 healthy control individuals Results: At treatment doses of 20 mg/d (n ¼ 15) and 30 mg/d (n ¼ 16) of ACI, the semen volume, sperm motility, viability, concentration, and normal appearance percentage, and the serum reproductive hormone levels in the psoriatic patients showed no significant difference from those in healthy control individuals (n ¼ 14) after month and months of treatment, as well as after withdrawing the treatment for months (p > 0.05) Conclusion: Different doses of ACI did not significantly affect semen quality in psoriatic patients at different treatment stages Copyright © 2016, Taiwanese Dermatological Association Published by Elsevier Taiwan LLC This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/) Keywords: acitretin erythrocyte innate immune psoriasis semen quality sperm Introduction Retinoids are a group of compounds that have similar structure and biological activity as vitamin A Their bioactive isomers include alltrans retinoid and 9-cis retinoid, which regulate cell proliferation and differentiation, thus exhibiting bioactivities such as antikeratosis, proliferation inhibition, antisebum synthesis, antiinflammation, and immunomodulation.1 Their molecular structure is composed of one cyclic group, one polyene side chain, and one terminal polar group The change in any of the three parts could result in compounds with different bioactivities Among the three generations of retinoids,2 acitretin (ACI) is the second-generation derivative, and is suitable for treatment of all types of psoriasis and keratosis Previously, the treatments of psoriasis with retinoids were mostly focused on severe erythrodermic psoriasis and pustular psoriasis,3,4 which are types of psoriasis with significant side Conflicts of interest: The authors declare that they have no financial or non-financial conflicts of interest related to the subject matter or materials discussed in this article * Corresponding author Department of Dermatology, 210 Hospital of People's Liberation Army, Dalian 116021, Liaoning, China E-mail address: huiliudoc@126.com (H Liu) effects Their indications kept on expanding, and low dose longterm treatment protocols were more widely used for psoriasis vulgaris, with satisfactory results and minimal side effects.5 Currently, it has been established that the major side effects of retinoids mainly manifest in muscle and bone, the central nervous system, liver, blood lipids, skin, and mucous membranes They have particularly potent teratogenic effects in women and female animals, and women of childbearing age must use contraception during retinoid use and for at least years after the withdrawal of retinoids.6 The specific effects of retinoids occur mainly in the cardiovascular system, central nervous system, and auditory system, and the occurrence rate could be as high as approximately 30% The embryotoxicity manifests as a high proportion of stillbirths and miscarriages, and the teratogenicity is still high a long time after withdrawal.6 Thus, a consensus has been formed by today’s medical community that women of childbearing age must strictly use contraception during retinoid use The combination of sperm and egg, formation of a fertilized egg, and fetal development are all carried out in utero, so the effect of maternal factors on fetal development is self-evident Sperm cells are one of the most active and damage-susceptible cells in humans The father’s sperm quality, especially the sperm concentration, mobility, and morphology, affects the formation and development of the fertilized egg and the http://dx.doi.org/10.1016/j.dsi.2016.10.005 1027-8117/Copyright © 2016, Taiwanese Dermatological Association Published by Elsevier Taiwan LLC This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/) Please cite this article in press as: Liu H, et al., Effects of acitretin on semen quality and reproductive hormone levels in patients with psoriasis vulgaris, Dermatologica Sinica (2016), http://dx.doi.org/10.1016/j.dsi.2016.10.005 H Liu et al / Dermatologica Sinica xxx (2016) 1e4 fetus It was reported that infertility caused by male factors accounted for about half of the total number of cases of infertility.7 Thus, we must determine whether the increasingly widespread usage of retinoids influences the reproductive system of male patients This has not received enough attention for a long time, and the effect of retinoids on the semen quality and genital safety in male psoriatic patients of childbearing age in China and abroad is rarely reported Reproductive safety-targeted research was firstly done by Parsch et al8 in 1990, who used a manual method to detect the semen quality changes in five patients after one retinoid treatment (50 mg/d, 3e4 months) and concluded that retinoids had no impact on sperm quality However, it is concerning that the majority of the literature on the effects of retinoids on male genital safety thereafter cited this report In recent years, foreign scholars have begun to focus on the issue of retinoids’ application toward male genital safety Because of ethical reasons, direct intervention studies of ACI on the human male reproductive system in psoriatic patients are very difficult Choosing animal models to obtain teratogenicity data and referring to the results of oral administration of vitamin A and its major metabolites in the volunteers are the new methods being used by certain research institutes to solve this problem; however, the conclusions were different.9,10 Using in vitro tests, Pilkington and Brogden11 found that high concentrations of ACI could inhibit the fructose decomposition inside sperm, significantly decreasing the contents of lactic acid and CO2 Sigg et al12 conducted a small study on the effect of ACI on eight male patients, and reported no change in sperm concentration Although it is still unknown whether ACI has residual effects in the semen of male patients taking ACI during or after the treatment, it would not constitute a risk to the fetus The maximum concentration of ACI observed in human semen after taking ACI or etretinate was 12.5 ng/mL, namely about 125 ng/10 mL in semen However, there is a warning that women of childbearing age and their spouses must use contraception for at least months before or after taking isotretinoin in its package inserts Therefore, the aim of this study was to investigate the changes of semen quality in 31 psoriatic patients of childbearing age before and after treatment with different doses of ACI Parameters analyzed included sperm concentration, motility, viability, and morphology, as well as levels of three serum reproductive hormones: luteinizing hormone (LH), follicle stimulating hormone (FSH), and serum testosterone (T) The aim was to preliminarily explore the reproductive safety issues in male patients using this kind of drug In this study, the detection of physical indicators and parameters was done using a manual method, and the sperm concentration and motility were analyzed using a computerized automatic semen analysis system Methods General information Thirty-one psoriatic patients were recruited from the in-patient department of our hospital All were: young soldiers, aged 18e24 years, without any history of contact with radar or nuclear radiation; unmarried with no children; with their primary disease as psoriasis without orchitis, prostatitis, or varicocele; without infectious diseases (such as hepatitis B, hepatitis C, or human immunodeficiency virus); without endocrine or genetic diseases; without familial high incidence of cancer; with a disease duration of 3e48 months; and with a Psoriasis Area and Severity Index (PASI) score of over eight points All of the patients were in the progressive or stationary phase when semen examination was performed The 31 patients were randomly divided into two groups according to the doses of ACI The major parameters of semen [sperm morphology and serum reproductive hormone levels (T, FSH, and LH)] were observed before the treatment, month after the treatment, months after the treatment, and months after withdrawal Group A: 15 cases, orally administered 20 mg once a day; Group B: 16 cases, orally administered 30 mg once a day The PASI score, age, disease duration, and body weight of the two groups showed no statistically significant difference (p > 0.05) All of the patients were prohibited from undergoing any treatment during the withdrawal, except for external ointment therapy such as tazarotene or mometasone The 14 healthy volunteers in the control group were young soldiers recruited from the surrounding area All were unmarried, without children, healthy, and aged 20e25 years All of the patients and volunteers were familiar with the study contents and gave informed consent Semen tests All participants were asked to abstain from sexual activity for 2e7 days before the semen collection During the sampling, benzalkonium bromide solution and saline were used to swab the urethra and surrounding skin Then, the semen was obtained in a sterile wide-mouth glass bottle by masturbation so as to avoid contamination and leakage The semen was liquefied, and the sperm count, motility, and morphological analyses were measured according to the standards described in the Laboratory Manual of WHO human semen and spermdcervical mucus interactions (fifth edition).13 The semen volume was measured, and the color, sticky degree, or liquefaction were observed The pH was determined using pH test strips after the semen was liquefied A sperm quality automatic analyzer (SCA 5.2; Microptic, Barcelona, Spain) was used to measure the sperm concentration, viability, and motility.14 Sperm morphology observations The results of sperm morphology observations were manually counted using the Diff-Quik method.15 Detection of sperm nuclear DNA fragments Sperm nuclear DNA fragments were detected using flow cytometry (Accuri C6; Becton Dickinson Immunocytometry Systems, San Jose, CA, USA).16 Detection of reproductive hormones by radioimmunoassay The levels of T, FSH, and LH were detected using the BHP9507 chemiluminescence immunoassay analyzer and kit (Yamasa Shoyu Co., Ltd., Chiba, Japan),17 with the intrabatch error < 5% Statistical analysis The comparison between different ACI dose groups, different treatment period groups, and the control group was performed using the t test The measurement data were expressed as x±SD, and all the data were processed using SPSS13.0 statistical package (SPSS Company, Chicago, Illinois, USA), with a ¼ 0.05 set as the test level Ethical approval This study was conducted in accordance with the declaration of Helsinki This study was conducted with approval from the Ethics Committee of 210 Hospital of People's Liberation Army Written informed consent was obtained from all participants Please cite this article in press as: Liu H, et al., Effects of acitretin on semen quality and reproductive hormone levels in patients with psoriasis vulgaris, Dermatologica Sinica (2016), http://dx.doi.org/10.1016/j.dsi.2016.10.005 H Liu et al / Dermatologica Sinica xxx (2016) 1e4 Results Table The variety of DNA fragments determination of sperm nuclear for men between psoriatic patients and normal controls in different periods ðx±sÞ Comparison of routine semen tests Group Course (mo) Red fluorescence intensity (FL1-H) The concentration, motility, viability, and morphology of sperm, the semen volume, and the pH in Groups A and B showed no significant difference compared with those in the control group before the treatment or month and months after the treatment (p > 0.05, Table 1) Red fluorescence intensity/total fluorescence intensity (at) A (20 mg/d, n ¼ 15) 3 13.92 ± 1.03 13.77 ± 0.90 14.18 ± 1.16 14.47 ± 1.40 13.92 ± 1.07 13.82 ± 1.01 14.13 ± 1.48 9.92 ± 3.05 10.26 ± 3.90 9.90 ± 2.37 10.77 ± 4.01 9.89 ± 2.64 9.90 ± 6.31 10.10 ± 5.61 B (30 mg/d, n ¼ 16) Comparison of sperm nuclear DNA fragments Control (n ¼ 14) The red fluorescence intensity (FL1-H) and red fluorescence intensity/total fluorescence intensity (at) in Groups A and B showed no significant difference compared with those in the control group before the treatment or month and months after the treatment (p > 0.05, Table 2) Table The variety of the genital hormone for men between psoriatic patients and normal controls in different treatment stage ðx±sÞ Group Course (mo) T (ng/mL) FSH (ng/mL) LH (ng/mL) A (20 mg/d, n ¼ 15) 3 744 ± 189 746 ± 240 739 ± 171 751 ± 201 737 ± 223 742 ± 193 740 ± 235 7.2 ± 1.4 7.3 ± 1.5 7.3 ± 0.9 7.3 ± 1.0 7.3 ± 0.9 7.4 ± 1.3 7.3 ± 1.6 2.7 ± 1.1 2.8 ± 1.7 2.7 ± 1.6 2.8 ± 1.3 2.7 ± 1.4 2.9 ± 1.9 2.8 ± 1.8 Comparison of reproductive hormone levels The levels of T, FSH, and LH in Groups A and B showed no significant difference compared with those in the control group before the treatment or month and months after the treatment (all p > 0.05, Table 3) B (30 mg/d, n ¼ 16) Control (n ¼ 14) FSH ¼ follicle stimulating hormone; LH ¼ luteinizing hormone; T ¼ testosterone Comparison of semen quality and reproductive hormone levels The semen quality indexes and levels of T, FSH, and LH in Groups A and B showed no significant difference compared with those in the control Group months after withdrawal (p > 0.05, Table 4) Table Comparison of semen quality and genital hormone between psoriatic patients and normal controls after months of withdrawal ðx±sÞ Discussion It has been confirmed that ACI has strong teratogenic effects in humans and animals Thus, women of childbearing age must use contraception for at least years after the withdrawal of treatment.6 There is a large number of studies on the teratogenicity and embryotoxicity of retinoids after their oral and topical administration in animals, but retinoid risk assessments in humans mainly include indirect data of pharmacokinetic analysis in animals and humans after systematic administration experiments and topical retinoid application in humans, which also referred to the results of in vitro teratogenic tests in animals and humans.18e20 The most direct determinants of teratogenic effects depend on the concentration-time relationship of drugs in target tissues, the drugs’ physicochemical and structure-activity properties, and their concentration and affinity in the terminal target organs.21 Although pharmacological and pharmacokinetic preventive measures have been proposed, they are still widely used clinically because of their efficacy.22 Currently, there are few studies on the effects of retinoids Index A (20 mg/d, n ¼ 14) B (30 mg/d, n ¼ 13) Control (n ¼ 14) Semen volume (mL) pH Total mobility (PR ỵ NP) (%) Ratio of live sperm (%) Sperm concentration ( 106/L) Ratio of sperm with normal morphology (%) T (ng/mL) FSH (ng/mL) LH (ng/mL) Red fluorescence intensity Red fluorescence intensity/total fluorescence intensity 3.3 ± 0.4 7.4 ± 0.2 63.7 ± 2.7 81.1 ± 11.3 48.6 ± 9.9 3.8 ± 0.3 3.4 ± 0.2 7.3 ± 0.3 62.8 ± 3.1 80.9 ± 12.9 47.1 ± 10.2 3.7 ± 0.6 3.4 ± 0.3 7.5 ± 0.2 63.8 ± 3.6 80.4 ± 12.2 47.6 ± 10.9 3.8 ± 0.5 737 ± 179 7.3 ± 1.3 2.7 ± 1.6 14.60 ± 1.45 9.94 ± 5.61 743 ± 201 7.4 ± 0.6 2.8 ± 1.3 13.93 ± 1.36 10.30 ± 3.44 740 ± 235 7.3 ± 1.6 2.8 ± 1.8 14.13 ± 1.48 10.10 ± 5.61 FSH ¼ follicle stimulating hormone; LH ¼ luteinizing hormone; NP ¼ nonprogressive sperm; PR ¼ progressive sperm; T ¼ testosterone on the male reproductive system These studies are based on tests and literature, and most of them consider retinoid treatment to be safe from the viewpoint of andrology Given the wide clinical usage of retinoids, clarification of whether retinoids affect the reproductive system in male patients of childbearing age is urgently needed Table Comparison of semen quality between psoriatic patients and normal controls in different periods ðx±sÞ Group Course (mo) Semen volume (mL) pH Total motility (PRỵNP) (%) Ratio of live sperm (%) Sperm concentration (106/mL) Percentage of sperm with normal morphology (%) A (20 mg/d, n ¼ 15) 3 3.4 ± 0.4 3.3 ± 0.5 3.4 ± 0.2 3.3 ± 0.6 3.4 ± 0.1 3.4 ± 0.2 3.4 ± 0.3 7.5 ± 0.1 7.4 ± 0.3 7.5 ± 0.1 7.4 ± 0.2 7.6 ± 0.1 7.5 ± 0.3 7.5 ± 0.2 63.1 ± 3.8 64.1 ± 4.0 63.9 ± 2.7 64.0 ± 2.9 63.7 ± 3.1 63.1 ± 4.1 63.8 ± 3.6 79.8 ± 13.6 81.1 ± 14.1 79.6 ± 12.7 80.1 ± 11.7 81.4 ± 12.9 79.2 ± 14.0 80.4 ± 12.2 46.9 ± 10.2 48.1 ± 11.0 47.5 ± 9.8 48.0 ± 9.9 46.9 ± 8.9 47.1 ± 11.3 47.6 ± 10.9 3.9 ± 0.7 3.8 ± 0.3 3.8 ± 0.2 3.8 ± 0.4 3.7 ± 0.6 3.9 ± 0.5 3.8 ± 0.5 B (30 mg/d, n ¼ 16) Control (n ¼ 14) NP ¼ nonprogressive sperm; PR ¼ progressive sperm Please cite this article in press as: Liu H, et al., Effects of acitretin on semen quality and reproductive hormone levels in patients with psoriasis vulgaris, Dermatologica Sinica (2016), http://dx.doi.org/10.1016/j.dsi.2016.10.005 H Liu et al / Dermatologica Sinica xxx (2016) 1e4 As one of the most active and damage-susceptible cells in vivo, the development and maturation of sperm cells are complex These processes mainly occur in testes and seminiferous tubes, and consist of three phases that together last 2e3 months: the proliferation and division of spermatogonia, reduction division of spermatocytes, and spermatogenesis.7 Routine tests and analysis of semen and sperm are a direct and objective experimental method in clinical practice The comprehensive assessment of semen volume and liquefaction time, as well as sperm concentration, motility, viability, morphology, and other indexes could be used as an important basis for understanding male fertility The male reproductive hormone levels depend on the hypothalamic-pituitarygonadal axis, and changes to this axis are directly related to changes in sperm concentration, motility, and morphology seen in semen analysis To understand the effects of different doses of ACI on sperm, we compared the semen volume, motility, survival ratio, sperm concentration, percentage of sperm with normal morphology, damaged DNA fragment index, and certain genital hormones among 15 psoriatic patients administered 20 mg of ACI/d, 16 psoriatic patients administered 30 mg of ACI/d, and 14 healthy volunteers before the treatment and month and months after treatment The results showed that ACI can improve the clinical symptoms of psoriatic patients, but different doses of ACI did not cause significant changes in sperm concentration, sperm morphology, total sperm count, sperm motility, or reproductive hormone levels month or months after treatment Three months after withdrawal of treatment (one period of spermatogenesis), we rechecked the semen quality and reproductive hormone levels of 27 patients (3 patients retired from active military service, and patient quit) The results did not reveal any abnormalities, indicating that long-term oral treatment with ACI did not affect spermatogenesis and that ACI did not affect sperm concentration, reduce sperm motility, or affect sperm morphology ACI had no significant effect on the male hypothalamic-pituitary-gonadal axis or its regulated reproductive hormone levels during or after treatment Compared with the previous experiments,8 the patients in this study were younger and the volunteers were young soldiers without psoriasis; therefore, the study samples were easily managed and followed up, and their compliance was better The sample size was larger than in previous studies, the treatment protocol used conventional doses (no more than 30 mg/d), and the conclusions obtained were consistent Therefore, the study confirmed that long-term treatment with therapeutic doses of retinoids has no adverse effects on reproductive function in psoriatic male patients ACI has confirmed efficacy in the treatment of psoriasis, and despite its relatively many side effects, it shows no adverse effects in the reproductive system of male patients of childbearing age during treatment Many issues regarding the use of ACI have not been completely understood, such as post-treatment long-term follow-up and reproductive safety issues of other drugs used concomitantly with ACI (such as isotretinoin) in male patients of childbearing age Confirming whether ACI affects the reproductive system of male psoriatic patients of childbearing age is an important public health problem that requires urgent solutions Answering this question would provide a strong theoretical basis for clinical and scientific application and safety assessment of retinoids References Alsenaid A, Eder I, Ruzicka T, Braun-Falco M, Wolf R Successful treatment of nail lichen planus with alitretinoin: report of cases and review of the literature Dermatology 2014;229:293e6 Kong R, Cui Y, Fisher GJ, et al A comparative study of the effects of retinol and retinoic acid on histological, molecular, and clinical properties of human skin J Cosmet Dermatol 2016;15:49e57 Tang MM, Spanou Z, Tang H, Schibler F, Pelivani N, Yawalkar N Rapid downregulation of innate immune cells, 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Novotny J, Aziz N, Rybar R, et al Relationship between reactive oxygen species production in human semen and sperm DNA damage assessed by Sperm Chromatin Structure Assay Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2013;157:383e6 17 Safarinejad MR Clinical significance of reproductive hormone test J Urol 2009;182:237e48 18 Nya-Ngatchou JJ, Arnold SL, Walsh TJ, et al Intratesticular 13-cis retinoic acid is lower in men with abnormal semen analyses: a pilot study Andrology 2013;1: 325e31 19 Hogarth CA, Arnold S, Kent T, Mitchell D, Isoherranen N, Griswold MD Processive pulses of retinoic acid propel asynchronous and continuous murine sperm production Biol Reprod 2015;92:1e11 20 Busada JT, Chappell VA, Niedenberger BA, et al Retinoic acid regulates Kit translation during spermatogonial differentiation in the mouse Dev Biol 2015;397:140e9 21 Zheng L, Xu T, Li D, Zhou J A representative retinoid X receptor antagonist UVI3003 induced teratogenesis in zebrafish embryos J Appl Toxicol 2015;35: 280e6 22 Monfrecola G, Baldo A Retinoids and phototherapy for psoriasis J Rheumatol Suppl 2009;83:71e2 Please cite this article in press as: Liu H, et al., Effects of acitretin on semen quality and reproductive hormone levels in patients with psoriasis vulgaris, Dermatologica Sinica (2016), http://dx.doi.org/10.1016/j.dsi.2016.10.005 ... 16) Control (n ¼ 14) FSH ¼ follicle stimulating hormone; LH ¼ luteinizing hormone; T ¼ testosterone Comparison of semen quality and reproductive hormone levels The semen quality indexes and levels. .. Please cite this article in press as: Liu H, et al., Effects of acitretin on semen quality and reproductive hormone levels in patients with psoriasis vulgaris, Dermatologica Sinica (2016), http://dx.doi.org/10.1016/j.dsi.2016.10.005... Comparison of reproductive hormone levels The levels of T, FSH, and LH in Groups A and B showed no significant difference compared with those in the control group before the treatment or month and months