Original Article pISSN: 2287-4208 / eISSN: 2287-4690 World J Mens Health 2020 Apr 38(2): 198-207 https://doi.org/10.5534/wjmh.180099 New Insights on the Mechanisms Affecting Fertility in Men with Non-Seminoma Testicular Cancer before Cancer Therapy Tania R. Dias1,2,3 , Ashok Agarwal1 , Peter N. Pushparaj4 , Gulfam Ahmad5 , Rakesh Sharma1 1American Center for Reproductive Medicine, Cleveland Clinic, Cleveland, OH, USA, 2Universidade da Beira Interior, Covilhã, Portugal, 3Department of Microscopy, Laboratory of Cell Biology, Institute of Biomedical Sciences Abel Salazar and Unit for Multidisciplinary Research in Biomedicine, University of Porto, Porto, Portugal, 4Center of Excellence in Genomic Medicine Research, Faculty of Applied Medical Sciences, Jeddah, Saudi Arabia, 5Division of Pathology, School of Medical Sciences, Sydney University, Sydney, Australia Purpose: Patients with non-seminoma testicular cancer (NSTC) cancer can be subfertile or infertile, and present reduced sperm quality, but the underlying mechanisms are unknown. The aim of this study was to compare the sperm proteome of patients with NSTC, who cryopreserved their sperm before starting cancer treatment, with that from healthy fertile men. Materials and Methods: Semen volume, sperm motility and sperm concentration were evaluated before the cryopreservation of samples from patients with NSTC (n=15) and the control group (n=15). Sperm proteomic analysis was performed by liquid chromatography-tandem mass spectrometry and the differentially expressed proteins (DEPs) between the two groups were identified using bioinformatic tools. Results: A total of 189 DEPs was identified in the dataset, from which five DEPs related to sperm function and fertilization were selected for validation by Western blot. We were able to validate the underexpression of the mitochondrial complex subunits NADH:Ubiquinone Oxidoreductase Core Subunit S1 (NDUFS1) and ubiquinol-cytochrome C reductase core pro- tein 2 (UQCRC2), as well as the underexpression of the testis-specific sodium/potassium-transporting ATPase subunit alpha-4 (ATP1A4) in the NSTC group. Conclusions: Our results indicate that sperm mitochondrial dysfunction may explain the observed decrease in sperm concen- tration, total sperm count and total motile count in NSTC patients. The identified DEPs may serve as potential biomarkers for the pathophysiology of subfertility/infertility in patients with NSTC. Our study also associates the reduced fertilizing ability of NSTC patients with the dysregulation of important sperm molecular mechanisms. Keywords: Cancer therapy; Male fertility; Non-seminoma; Sperm quality; Testicular cancer This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. INTRODUCTION representing only 1% to 2% of all tumors [1]. The USA National Cancer Institute reported 8,850 new cases in Testicular cancer (TC) is a rare tumor among men, 2017 [2] and this number is estimated to increase to Received: Oct 23, 2018 Revised: Nov 22, 2018 Accepted: Nov 25, 2018 Published online Dec 21, 2018 Correspondence to: Ashok Agarwal https://orcid.org/0000-0003-0585-1026 Andrology Center, American Center for Reproductive Medicine, Cleveland Clinic, Mail Code X-11, 10681 Carnegie Avenue, Cleveland, OH 44195, USA. Tel: +82-1-216-444-9485, Fax: +1-216-445-6049, E-mail: [email protected] Copyright © 2020 Korean Society for Sexual Medicine and Andrology Tania R. Dias, et al: Sperm Molecular Dysregulation in Testicular Non-Seminoma 9,310 new cases in 2018 [3]. The etiology of TC is not tain disease conditions in its early stage. In this study, fully understood but several risk factors were identi- we aimed to analyze the sperm proteome of patients fied, including genetic background, age, race/ethnicity, with NSTC who had cryopreserved semen samples undescended testicle, human immunodeficiency virus before initiating cancer therapy and identify potential infection and/or AIDS [4,5]. One of the main types of sperm protein biomarkers responsible for the altered TC is non-seminoma testicular cancer (NSTC). It rep- reproductive function in these patients. resents about half of the cases of germ cell tumors [6], which account up to 95% of all TC cases [7]. NSTC is MATERIALS AND METHODS commonly diagnosed in young men (25–35 years) [8,9], thus constituting a major threat for couples during 1. Study population and ethics statement reproductive years. Although the etiology of NSTC This study was ethically approved by the Institution- is largely unknown, its treatment is highly effective, al Review Board (IRB) of Cleveland Clinic. The control showing a 5-year survival rate of 99% in early stages, group included 15 healthy volunteers with proven and 48% to 91% in more advanced and metastatic stag- fertility, i.e., men who fathered a child in the last two es [6]. Surgery, radiotherapy or chemotherapy are fre- years before entering the study, whereas the NSTC quently used to treat this type of cancer [10]. However, group comprised 15 NSTC patients who cryopreserved these therapies are associated with secondary effects semen samples before starting cancer therapy. All the on male fertility, especially due to gonadal damage participants signed an informed written consent at [11]. After the treatment, many patients show reduced the time of sample collection at the Andrology Center, sperm quality and impaired spermatogenesis, and often Cleveland Clinic. result in permanent infertility [12,13]. A 30% decrease in the probability to conceive by natural conception af- 2. Semen analysis and cryopreservation ter cancer therapy has been reported [14]. Thus, in most Semen samples were collected after 2 to 3 days of of NSTC cases the clinicians advise the patients to abstinence and placed at 37°C for 20 to 30 minutes to undergo sperm banking before initiating any kind of allow liquefaction. Volume, sperm motility, and sperm cancer therapy [15,16]. NSTC tends to grow rapidly and concentration were evaluated according to World spread to other tissues [17], highlighting the urgency to Health Organization (WHO) 2010 guidelines [29]. Total initiate the treatment. sperm count and total motile count were also calculated Most of the cases of NSTC are found when couples and results were expressed as mean±standard error of are not able to have children and seek for medical as- mean (SEM). Semen samples were then cryopreserved sistance. In fact, the development of NSTC is facilitat- in TEST-yolk buffer (TYB; Irvine Scientific, Santa ed by the presence of abnormal semen parameters [18] Ana, CA, USA) in a ratio 1:1 as previously described [30] and subfertility/infertility [19-21]. There are many cases and finally stored in liquid nitrogen at -196°C. of men presenting poor semen quality and reduced fer- tilizing ability prior to NSTC diagnosis [12,22,23]. Part 3. Sperm protein extraction and quantification of the problem starts with the impairment of sper- Cryopreserved samples were thawed and centrifuged matogenesis, as the tumor affects the normal hormonal at 4,000×g for 10 minutes to isolate spermatozoa. The regulation [24]. It is of extreme importance for the field pellet was washed four times with consecutive resus- of male reproduction to investigate the mechanisms pensions in phosphate buffered saline (PBS; Irvine Sci- by which NSTC affects male fertility. In this context, entific) and centrifugations at 4,000×g for 10 minutes, proteomics has emerged as a valuable tool to explore at 4°C. Radio-immunoprecipitation assay (RIPA; Sigma- alterations in sperm proteome triggered by certain Aldrich, St. Louis, MO, USA) buffer supplemented health conditions [25-27]. Due to their post-translational with Protease Inhibitor Cocktail, cOmpleteTM ULTRA modifications, spermatozoa are highly susceptible to Tablets, ethylene diamine tetraacetic acid-free (Roche, proteome alterations during maturation throughout Mannheim, Germany) was added to each sperm pellet the male reproductive tract [28]. These changes are (100 µL RIPA/106 sperm) and left overnight at 4°C for highly dependent on the individual health status and cell lysis. Then, samples were centrifuged at 10,000×g may be a useful source of biomarkers to identify cer- for 30 minutes, at 4°C and the supernatant containing www.wjmh.org 199 https://doi.org/10.5534/wjmh.180099 the protein fraction was transferred to a new centri- 6. Validation of selected differentially fuge tube. Protein quantification was performed using expressed proteins the Pierce BCA Protein Assay kit (Thermo Fisher Sci- Validation of key DEPs was performed by WB using entific, Waltham, MA, USA) according to the manufac- six individual samples per group. Sperm proteins (25 turer’s instructions. µg/sample) were mixed with 4× Laemmli sample buf- fer in a ratio 1:3 in a total volume of 25 µL in PBS. The 4. Liquid chromatography-tandem mass prepared samples were immediately boiled at 95˚C for spectrometry 10 minutes for protein denaturation and ran into a 4% The present study was conducted in compliance to 15% sodium dodecyl sulfate-polyacrylamide gel elec- with the Minimum Information about a Proteomics trophoresis gel at constant voltage (90 V) for 2 hours. Experiment (MIAPE) guidelines of the Human Pro- A molecular weight marker (Precision Plus ProteinTM teome Organization’s Proteomics Standards Initiative Dual Xtra Standards) was loaded in the first well of (HUPO-PSI) for reporting proteomics studies [31]. Three each gel. Transfer of proteins from the gels to metha- samples from control or NSTC groups (n=3/group) nol-activated polyvinylidene difluoride membranes was were pooled using the same amount of protein from performed with constant voltage (18 V) for 30 minutes. each sample and analyzed by liquid chromatography- Membranes were blocked for 90 minutes at room tem- tandem mass spectrometry (LC-MS/MS). The samples perature, with a 5% non-fat milk solution prepared in in each pool were randomly selected within the experi- tris-buffered saline tween-20.