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Is methotrexate safe for men with an immune-mediated inflammatory disease and an active desire to become a father? Results of a prospective cohort study (iFAME-MTX)

Is methotrexate safe for men with an immune-mediated inflammatory disease and an active desire to... Treatment TRANSLATIONAL SCIENCE Is methotrexate safe for men with an immune- mediated inflammatory disease and an active desire to become a father? Results of a prospective cohort study (iFAME- MTX) 1 1 1,2 Luis Fernando Perez- Garcia , Esther Röder , Bouwe P Krijthe, 1,3 1 4 Laura JC Kranenburg- van Koppen, Roxanne van Adrichem, Els Zirkzee, 5 6 7 7 8 Pieter H Griffioen , Kris Peeters, Marry Lin, Eduard A Struys, Gerrit Jansen, 9 7 10 1 Martijn BA van Doorn, Robert de Jonge, Gert R Dohle, Radboud JEM Dolhain Handling editor Josef S ABSTRACT WHAT IS ALREADY KNOWN ON THIS TOPIC Smolen Introduction Current scientific evidence guiding ⇒ Although methotrexate (MTX) is one of the the decision whether men with an active desire to ► Additional supplemental most frequently prescribed immunosuppressive become a father should be treated with methotrexate material is published online medication for several immune- mediated only. To view, please visit (MTX) remains controversial. We aimed to prospectively the journal online (http:// dx. inflammatory diseases (IMIDs), the evidence evaluate the testicular toxicity profile of MTX focusing doi. org/ 10. 1136/ ard- 2023- regarding the testicular toxicity profile of on several markers of male fertility, including semen 224032). MTX is scarce. Ultimately, this has resulted in parameters and sperm DNA fragmentation index (sDFI). conflicting recommendations respecting the For numbered affiliations see As a secondary outcome, we aimed to evaluate whether end of article. safety of MTX in men with a wish to have MTX- polyglutamates can be detected in spermatozoa children. and seminal plasma and to evaluate the enzymatic Correspondence to activity in spermatozoa of folylpolyglutamate synthetase Dr Luis Fernando Perez- Garcia, WHAT THIS STUDY ADDS (FPGS). Rheumatology, Erasmus Medical Methods In a prospective cohort study, men ≥18 years ⇒ This is the first prospective study that evaluates Center, Rotterdam, Netherlands; l. perez@ erasmusmc. nl who started therapy with MTX were invited to participate the testicular toxicity profile of MTX in men (MTX- starters). Participants were instructed to produce diagnosed with IMIDs. We evaluated the effect Received 15 February 2023 two semen samples (a pre- exposure and a post- exposure of MTX on multiple markers of testicular toxicity Accepted 12 May 2023 sample after 13 weeks). Healthy men ≥18 years were and demonstrated that the semen parameters, invited to participate as controls. Conventional semen the male reproductive axis and the sperm DNA analyses, male reproductive endocrine axis and sDFI were fragmentation index were comparable between compared between groups. FPGS enzymatic activity and healthy controls and patients exposed to MTX. MTX- PG1- 5 concentrations were determined by mass Furthermore, our study also demonstrates spectrometry analytical methods. that MTX can be detected in spermatozoa and Results In total, 20 MTX- starters and 25 controls seminal fluid, but that the concentration of were included. The pre- exposure and postexposure MTX- polyglutamates, especially in spermatozoa, semen parameters of MTX-starters were not statistically is very low. Finally, we revealed the mechanistic significant different. Compared with healthy controls, the basis for these latter findings, demonstrating conventional semen parameters and the sDFI of MTX- that in spermatozoa, the catalytic activity of the starters were not statistically significant different. These enzyme responsible for the polyglutamylation data were corroborated by the marginal accumulation of of MTX, folylpolyglutamate synthetase, is very MTX- PGs in spermatozoa, consistent with the very low low. FPGS enzymatic activity associated with the expression of an alternative FPGS splice- variant. HOW THIS STUDY MIGHT AFFECT RESEARCH, Discussion Treatment with MTX is not associated PRACTICE OR POLICY with testicular toxicity, consistent with the very low ⇒ Altogether, our data suggest that MTX is not concentration of intracellular MTX- PG. Therefore, therapy associated with testicular toxicity. Therefore, with MTX can be safely started or continued in men and therapy with MTX can be safely started or with a wish to become a father. continued in men diagnosed with an IMID and © Author(s) (or their employer(s)) 2023. Re- use with an active wish to become a father. permitted under CC BY. Published by BMJ. INTRODUCTION To cite: Perez- Garcia LF, Methotrexate (MTX) is one of the most frequently Röder E, Krijthe BP, et al. for men with an active desire to become a father, the prescribed immunosuppressive drugs for the treat- Ann Rheum Dis Epub ahead decision of whether they should stop or continue of print: [please include Day ment of several immune- mediated inflammatory Month Year]. doi:10.1136/ therapy with MTX before conception remains diseases (IMIDs) such as rheumatoid arthritis (RA), ard-2023-224032 psoriatic arthritis (PsA) and psoriasis. Remarkably, controversial. Perez- Garcia LF, et al. Ann Rheum Dis 2023;0:1–8. doi:10.1136/ard-2023-224032 1 Treatment In this regard, the American College of Rheumatology were proven fertile (history of a partner’s positive pregnancy recently updated its recommendations on paternal immuno- test). Three groups of men were included in the study. First, suppressive exposure before conception. Concerning MTX, men diagnosed with an IMID (RA, Spondyloarthritis (SpA), PsA, they recommend that MTX can be ‘conditionally continued’ in psoriasis) based on the expert opinion of their rheumatologist or men planning to father a child. Furthermore, the British Society dermatologist, who were not exposed to MTX in the last year for Rheumatology guideline on prescribing drugs in pregnancy and were going to start therapy with MTX, were included in the considers paternal exposure to low- dose MTX (≤25 mg/week) ‘MTX- starters’ group. Second, to evaluate the effect of chronic as compatible with pregnancy. Conversely, other federal agen- MTX exposure, men who were exposed to MTX (≥15 mg/week cies and medical associations recommend that MTX should be for≥1 year) were included in the ‘MTX- chronic’ group. Third, 3–5 stopped at least 3–6 months before conception. healthy men were included as ‘healthy controls’. A fundamental reason for these contradictory recommen- Men who were not proven fertile, who were exposed to drugs dations is the scarcity of solid scientific data on the testicular with known testicular toxic side effects (ie, oligospermia) were toxicity profile of MTX. In its guideline to evaluate testicular excluded. Importantly, concomitant therapy with prednisone toxicity, the Food and Drug Administration (FDA) considers (≤7.5 mg/day), hydroxychloroquine or tumour necrosis factor semen analysis parameters as the main outcomes of interest. alpha (TNFa) inhibitors was allowed (see online supplemental Furthermore, in studies evaluating testicular toxicity, the FDA file 1), table 1. Exclusion criteria and table 2 Exclusion criteria, recommends evaluating at least one baseline and one follow-up drug exposure). semen sample (at the end of the first 13 weeks). In addition to spermatogenesis, the production of hormones is Study visits another important function of the testicles. Therefore, the eval- A study visit consisted of three parts. First, the demographic uation of the male reproductive endocrine axis (ie, testosterone, and medical history were obtained from an interview, followed luteinising hormone (LH), follicle stimulating hormone (FSH)) by a concise physical evaluation. Second, a blood sample was should also be considered an important outcome. obtained. Finally, participants provided a semen sample. Two Another novel outcome of interest that reflects the integrity study visits were required from participants from the MTX- or damage of the sperm DNA and that can be evaluated is the starters group, one before exposure to MTX (pre- exposure) and sperm DNA fragmentation index (sDFI). Sperm DNA integrity another one at least 13 weeks after their initial MTX exposure is indispensable for the birth of healthy offspring and sperm (postexposure). Participants from the MTX-chronic and healthy DNA damage has been strongly associated with male infertility control groups were just required to complete one study visit. and a higher risk of miscarriages. Sperm DNA damage can be induced by either the pharmacological exposure itself or the Demographic characteristics, medical history and physical oxidative stress and inflammation states associated with a diag- evaluation nosis of IMID. Participants answered a questionnaire that included questions Finally, it is known that the pharmacological efficacy of MTX concerning their demographic characteristics, medical history critically depends on the intracellular bioactivation of MTX to (including reproductive history) and their current and previous MTX- polyglutamates (MTX- PG). This process is catalysed by medication exposure. the enzyme folylpolyglutamate synthetase (FPGS). Lower FPGS A physical evaluation was performed to determine height, activity and subsequent inefficient polyglutamylation is a well- weight and testicular volume (using an orchidometer). Disease known phenomenon associated with a rapid efflux of MTX from activity was calculated using validated scores (DAS28 for RA, cells. MTX-PG have been detected in several human cells such BASDAI for SpA, DAPSA for PsA and PASI for psoriasis). as erythrocytes or peripheral blood mononuclear cells (PBMCs) and low levels of MTX- PG have been associated with increased 12 13 Blood sample resistance to therapy. In this regards, recent molecular Blood from all participants was obtained between 09:00 and studies have shown that a significant reduction of FPGS activity 11:00 hours by venipuncture. The concentration of testosterone, is associated with aberrant pre- mRNA splicing of this enzyme, LH, FSH, inhibin B, sex hormone binding globulin (SHBG) and including partial retention of intron 8 (8PR) and a higher ratio C reactive protein were evaluated. Erythrocytes and PBMCs of FPGS 8PR over FPGS wild type (WT). Whether intracel- 12 15 were isolated using the protocol previously described. lular MTX-PG can be detected in the spermatozoa and whether the spermatozoa have the enzymatic capabilities of forming and Semen sample collection retaining intracellular MTX- PG has never been studied before. Semen samples were delivered by masturbation. To ensure a To establish whether MTX can be safely used by men diag- timely analysis of the samples, participants provided the sample nosed with an IMID and a wish to conceive, we aimed to prospec- in the hospital. All samples were evaluated within 30 min of tively evaluate the testicular toxicity profile of MTX focusing on production. The fresh semen sample was analysed for semen several markers of male fertility such as semen parameters, the volume, sperm concentration and motility. Slides were prepared male reproductive endocrine axis and the sDFI. Furthermore, as for the morphology analysis and sent to a specialised centre a secondary outcome, we aimed to evaluate whether MTX-PG (Radboud UMC, Nijmegen The Netherlands). can be detected in spermatozoa and seminal plasma and to eval- Thereafter, the semen samples were individually processed to uate the FPGS enzymatic activity in spermatozoa. prepare pellets for further analysis. First, samples were processed for future sDFI measurement following the steps previously METHODS published. Second, the semen samples were centrifuged and Study design and patient selection washed out three times with PBS and spermatozoa were isolated The iFAME-MTX study was a prospective cohort study for future MTX- PG concentration, FPGS catalytic activity and conducted in the Erasmus University Medical Center, Rotterdam, mRNA expression measurements. All samples were stored at The Netherlands. All participants were aged 18–55 years and −80°C. 2Perez- Garcia LF, et al. Ann Rheum Dis 2023;0:1–8. doi:10.1136/ard-2023-224032 Treatment Table 1 Demographic characteristics MTX- naïveMTX- naïve Pre- exposure (n=20)Post- exposure (n=18) Healthy controls (n=25) MTX chronic§ (n=5) P value General information Age years, mean (95% CI) 35.2 (31.4 to 39.1) 34.7 (32.9 to 36.7) 36.6 (32.1 to 41.1) NS Smoking, n (%) 4 (20) 4 (20) 6 (24) 1 (20) NS BMI %, mean (95% CI) 27.1 (24.8 to 29.2) 26.8 (24.5 to 29.1) 25.5 (24.2 to 26.8) 25.5 (21.4 to 29.6) NS Testicular volume, mean (95% CI) 22.9 (21.4 to 24.3) 22.6 (21.3 to 23.8) 22.6 (21.3 to 23.8) NS Inflammatory arthritis Diagnosis: RA, n (%) 7 (35.0) 6 (33.3) - 2 (40.0) PsA, n (%) 8 (40.0) 7 (38.9) - 1 (20.0) SpA, n (%) 1 (5.0) 1 (5.6) - 2 (40.0) Psoriasis, n (%) 4 (20.0) 4 (22.2) - 0 (0.0) Age at diagnosis, mean (SD) 27.4 (22.1–32.6) 28.5 (24.5–34.5) – 30.3 (18.5–42.1) Disease duration, mean (SD) 6.9 (2.1–11.8)5.6 (- 5.5–16.8) MTX dose (mg/week), mean (95% CI) – 16.0 (13.6 to 18.4) – 18.3 (15.6 to 21.1) Prednisone exposure, n (%) 2 (10.0) 6 (33.3) -- 1 (20) NS TNFa inhibitor exposure, n (%) 3 (15.0) 5 (27.8) -- 2 (40) NS C reactive protein mg/dL, median (IQR) 2.1 (0.6–5.0) 1.4 (1.0–3.2) 0 (0.0–0.9) 1.1 (0–1.8) * p=0.011 † p=0.008 Disease activity scores VAS general health mm, 42 (19 to 67) 20 (12 to 36) 17.7 (10.5 to 25- 5) 20.20 (0.3 to 40.1) * p=<0.001 mean (95% CI) ‡ p=0.008 VAS pain mm, mean (IQR) 42 (5.5–75) 14 (4–36) -- 11 (4–34) VAS activity mm, mean (IQR) 68 (51–78) 27 (16–49) -- 10 (4–52) RA: DAS28, mean (IQR) 2.7 (2.4–2.9) 2.55 (1.34–3.40) -- 2.3 (1.4–2.7) PsA: DAPSA, mean (IQR) 24.1 (20.5–33.3) 17.8 (11.2–22.7) -- 1.1 (1.1–1.1) Psoriasis: PASI, mean (IQR) 1.9 (1.3–5.1) 1.1 (0.7–3.1) -- 0.8 (0.3–1.2) *Statistically significant difference between pre- exposure and healthy controls. †Statistically significant difference between post- exposure and healthy controls. ‡Statistically significant difference between pre- exposure and post- exposure. §Presented only for descriptive purposes, no statistical analyses were conducted. BMI, body mass index; DAPSA, disease activity index for psoriatic arthritis; DAS28, disease activity score 28; MTX, methotrexate; PsA, psoriatic arthritis; RA, rheumatoid arthritis; SpA, spondyloarthritis; TNFa, tumour necrosis factor alpha; VAS, visual analogue scale. Sperm DNA fragmentation index for 45 min. After washing, the pellets were incubated in fresh Assessment of sDFI was performed using the terminal deosy- permeabilisation solution (0.1% sodium citrate, 0.1% triton nucleotidyl transferase dUTP nick end labeling (TUNEL) assay X–100, both Sigma-Aldrich, Belgium) for 5 min at 4°C. The described by Mitchell et al. In short, the spermatozoa pellets positive control samples were treated with 5 µl of DNase I were, after thawing, washed in phosphate buffered saline and (Qiagen, Germany) 1500 Kunitz Units for 30 min. The assay was incubated in 2 mM dithiothreitol (Sigma- Aldrich, Belgium) performed using the fluorescein In Situ Cell Death Detection Table 2 Conventional semen parameters and sperm morphology MTX- naïveMTX- naïve Pre- exposure (n=20)Post- exposure (n=18) Healthy controls (n=25) MTX chronic* (n=5) P value Conventional semen parameters Sperm concentration x10 /mL, median (IQR) 57.0 (35.0–90.5) 54.0 (41.0–82.0) 60.0 (37.0–111.0) 37.0 (32.0–59.9) NS Progressive motility* %, 63.2 (55.4 to 70.9) 60.1 (49.5 to 70.6) 56.9 (51.1 to 62.8) 50.4 (34.8 to 65.9) NS mean (95% CI) Semen volume mL, median (IQR) 2.4 (1.6–3.2) 3.0 (1.5–3.2) 3.0 (2.0–4.0) 2.0 (1.6–2.4) NS Sperm morphology evaluation Normal morphology %, mean (95% CI) 6.4 (4.5 to 8.3) 7.1 (5.6 to 8.4) 6.3 (4.7 to 7.9) 5.9 (2.6 to 9.1) NS Teratozoospermia index, mean (95% CI) 1.2 (1.2 to 1.3) 1.3 (1.2 to 1.4) 1.2 (1.2 to 1.3) 1.2 (1.1 to 1.4) NS Excess residual cytoplasm, median (IQR) 2.0 (0.7–4.3) 2.0 (1.0–4.5) 2.0 (1.0–4.0) 2.0 (1.0–4.0) NS Abnormalities in head (%), mean (95% CI) 92.8 (90.8 to 94.7) 93.0 (91.2 to 94.6) 92.7 (91.0 to 94.3) 92.3 (88.0 to 96.5) NS Abnormalities in middle piece (%), mean (95% CI) 19.2 (14.2 to 24.1) 24.5 (18.6 to 30.2) 19.9 (15.3 to 24.5) 22.9 (5.7 to 40.0) NS Abnormalities in tail (%), mean (95% CI) 7.1 (3.8 to 10.5) 7.3 (3.7 to 11.1) 6.6 (3.9 to 9.3) 4.6 (1.4 to 7.7) NS *Presented only for descriptive purposes, no statistical analyses were conducted. MTX, methotrexate. Perez- Garcia LF, et al. Ann Rheum Dis 2023;0:1–8. doi:10.1136/ard-2023-224032 3 Treatment Kit (Roche Diagnostics, Mannheim, Germany) with an Accuri polyglutamylation in acute lymphoblastic leukaemia and RA C6 flow cytometer (BD Sciences, Erembodegem, Belgium). For patients. To evaluate if alterations in FPGS pre-mRNA splicing each sample, 5000–10 000 events were recorded at a flow rate levels in spermatozoa could explain our findings, an additional of 35 µL/min. experiment was performed. Shortly, RNA was isolated from PBMCs and spermatozoa according to the manufacturer’s protocol (BD Biosciences). RNA (250 ug) was reverse transcribed MTX polyglutamates quantification in erythrocytes, to cDNA using Moloney Murine Leukaemia Virus (M- MLV; spermatozoa and seminal plasma Thermo Fisher Scientific, Waltham, Massachusetts) in a reaction Procedure EDTA erythrocyte cell pellets buffer containing random hexamer primers (Roche, Basel, Swit- Considering that MTX-PG have never been measured in sper - zerland), Deoxynucleotide triphosphates (dNTPs) (Roche) and a matozoa, we opted to measure MTX-PG in erythrocytes as a ribonuclease inhibitor RNasin (Promega, Madison, Wisconsin). control measure for MTX adherence. The erythrocyte cell pellets Primer sequences (see online supplemental figure 1) and methods are measured accordingly to our previously described validated used to quantify the levels of FPGS 8PR, FPGS WT are described LC- MS/MS method using custom- made stable isotopes of MTX- 12 15 elsewhere. pg1- 7 as internal standards. This method with minor alter- ations was also used to measure MTX-PG in spermatozoa and Statistical analysis seminal fluid. Comparisons between the pre- exposure and postexposure MTX- starters groups and healthy controls were tested. Because of the Procedure spermatozoa cell pellets low number of participants included in the MTX-chronic group, Spermatozoa cell pellets were kept at −80°C until analysis. we present their data in this article only for descriptive purposes. To avoid polyglutamate deconjugation activity by γ-glutamyl Categorical variables were presented as number (percentage), hydrolase (GGH), semen cells were placed on ice and denatured and continuous variables are reported as mean±SD, or medi- immediately. A volume of 320 µL perchloric acid (16%) was an±IQR, as appropriate. Continuous variables were compared added to the pellets and mixed immediately to denature. After using a one-way analysis of variance, Tukey post hoc test, paired denaturation samples were supplemented with 200 µL saline t- test, Mann- Whitney test and Wilcoxon signed- rank. Categor- and 200 µL internal standards mixture (pg1–pg7). Supernatants ical variables were compared using χ tests and Fisher’s exact were transferred two times into clean tubes after centrifugation tests. For linear correlation analysis, we used the Pearson correla- (Hettich micro 220R, 10 min, 21 250×g, 5°C) and used for tion coefficient. The level of significance was set as a two-tailed measurements of MTX-PG. p≤0.05, and statistical analyses were completed using Stata V.15 (StataCorp- LP). Procedure for seminal fluid Seminal fluid samples were kept at −80°C until analysis. To Ethics avoid GGH activity, seminal fluids were shortly thawed on ice This study was approved by the ethic review board of the before denaturation. A sample volume of 200 µL of previously Erasmus University Medical Center in compliance with the isolated seminal fluid was used. Because of its high viscosity, Declaration of Helsinki (NL64218·078·18). All participants samples were denatured with 320 µL acidic methanol (16% gave their informed consent. perchloric acid in methanol) and mixed immediately to dena- ture the seminal plasma. After denaturation, 200 µL of internal Patient and public involvement standard mixture (pg1–pg7) was added. Supernatants are trans- Three male patients diagnosed with inflammatory arthritis and ferred two times into clean tubes after centrifugation (Hettich who are active members of the research advisory board from the micro 220R, 10 min, 21 250×g, +5°C.) Samples were dried Department of Rheumatology of the Erasmus University Medical under nitrogen flow (Evaporex EVX-192,+50°C) and dissolved Center were involved in the design of the questionnaire and the in 720 µL purified water (Millipore) and mixed (IKA MTS4, invitation letter. Together, we carefully assessed the burden on 10 min, 600 rpm, room temperature (RT)). Samples were filtered participating patients. We intend to share the results to partic- (Whatmann mini-uniprep, UN203NPUPP) before being used for ipating patients and will appropriately disseminate the results. MTX- PG measurements. RESULTS FPGS activity in PBMCs and spermatozoa Between February 2019 and January 2022, a total of 118 (46 FPGS catalytic activity in PBMCs (as positive control) and sper- MTX- starters, 49 healthy controls and 23 MTX- chronic) men matozoa of healthy controls and MTX-starters were analysed in were invited to participate in the study. In total, 50 men agreed 10 µg protein extracts and assay mixtures containing 250 mmol/L to participate (20 MTX- starters, 25 healthy control and 5 MTX- MTX, and 4 mmol/L N- labelled L-glutamic acid as subtract chronic). Most men who did not participate in the study provided concentrations as described by Muller et al. FPGS activity their reasons not to do so (no time for study visits (n=23), is reported as pmol MTX- PG - N formed/hour/mg protein. unwilling to provide semen samples (n=16), COVID- 19 lock- In addition, cell extracts of CCRF-CEM and CEM/R30dm down (n=12), no interest in the topic (n=8), Erasmus University leukaemia cells were used a positive and negative controls for Medical Center being too far away (n=2), reason not provided FPGS activity, respectively. (n=7). The demographic and clinical characteristics of these men are presented in table 1. mRNA expression profiles of folate genes in spermatozoa An important mechanism of loss of FPGS activity and subse- quent inefficient polyglutamylation can occur due to aber- Conventional semen parameters and sperm morphology rant pre- mRNA splicing of FPGS. We recently identified a There were no statistically significant differences in the median partial retention of FPGS intron 8 (8PR) as a prominent splice sperm concentration, semen volume, sperm motility and sperm variant conferring FPGS dysfunction and decreased MTX morphology parameters between MTX- starters and healthy 4Perez- Garcia LF, et al. Ann Rheum Dis 2023;0:1–8. doi:10.1136/ard-2023-224032 Treatment Table 3 Sperm DNA fragmentation index MTX- naïveMTX- naïve Pre- exposure (n=20)Post- exposure (n=18) Healthy controls (n=25) MTX chronic* (n=5) P value Sperm DNA fragmentation index %, median (IQR) 24.3 (7.1–30.7) 13.1 (9.5–19.9) 13.5 (8.7–20.2) 13.5 (13.3–26.1) NS *Presented only for descriptive purposes, no statistical analyses were conducted. MTX, methotrexate. controls. Only one case of oligospermia (<15 million sperma- MTX- PG2- N formed/hour/mg protein) was statistically signif- tozoa/mL) was observed in an MTX- starter (pre- exposure and icant higher in PBMCs (183 pmol MTX- PG2- N formed/hour/ postexposure samples, table 2). mg protein) compared with spermatozoa from healthy controls (15 pmol MTX- PG2- N formed/hour/mg protein) and sper- matozoa from MTX- starters (5 pmol MTX- PG2- N formed/h/ Sperm DNA fragmentation index mg protein) (figure 1D). Remarkably, FPGS activity in sperma- The median sDFI was higher in the pre- exposure samples from tozoa is even lower than a control cell line (CEM/R30dm) with the MTX- starters (22.0% (IQR 10.7–30.7), but this was not acquired resistance to MTX due to loss of FPGS activity and statistically significant different when compared with the post- impairment of MTX-PG ( figure 1D). exposure sample (13.1% (IQR 9.5–16.3), p=0·247) and to the healthy controls (13.5% (IQR 8.7–20.2), p=0.257). See table 3. mRNA expression profiles of folate genes in spermatozoa Male reproductive endocrine axis To explore whether molecular alterations underlie the extremely The median serum concentrations of testosterone, SHBG, LH low FPGS activity in spermatozoa, mRNA expression profiles and FSH were not statistically significant different between the of FPGS and other folate genes were evaluated in spermatozoa groups. The median serum concentration of inhibin B was statis- from four healthy controls and six MTX-starters (see online tically significant lower in the pre-exposure (132.5 ng/L (IQR supplemental figure 1). Of note, a relatively high FPGS/8PR and 101.5–179.5)) and postexposure samples of the MTX-starters FPGS 8PR/WT ratio was observed in spermatozoa compared group (123.0 ng/L (IQR 116.0–179.0)) compared with the with PBMCs (results are not shown). healthy controls (189.0 ng/L (170.0–236.0)). See table 4. DISCUSSION MTX polyglutamate quantification The iFAME-MTX study is the largest study to date that prospec- MTXpg1–5 were detected in erythrocytes from all partic- tively evaluated the potential impact of MTX on many important ipants (figure 1A), consistent with MTX- pg accumulation markers of testicular toxicity and to evaluate the potential under- profiles in erythrocytes of patients under MTX therapy and lying mechanisms explaining why MTX does not impair sperm thus confirming their intake of MTX. In seminal fluid, quality. It shows that MTX is not associated with conventional mainly MTX- pg1 was detected with a median concentration semen analysis abnormalities, disturbances in the male repro- of 184 nmol/L (IQR 39–265), whereas MTXpg2,3 were barely ductive endocrine axis or with increased sperm DNA damage. noticeable (figure 1B). In spermatozoa, mainly MTXpg1 was Furthermore, to the best of our knowledge, our study reports detected (median 0.26 fmol/10 spermatozoa, IQR 0.16–0.69), for the first time that the enzyme responsible for intracellular whereas MTXpg2,3 levels were at the lower limit of detection polyglutamylation and hence the bioactivation of MTX, that is, (figure 1C). Total MTXpg levels in spermatozoa were approxi- FPGS, has an extremely low activity in spermatozoa. Ultimately mately 18- fold lower than in erythrocytes (0.32 fmol/10 sper- resulting in very low concentrations of intracellular MTX-PG in matozoa vs 5.8 fmol/10 erythrocytes, respectively, figure 1A/C). spermatozoa. Our results provide long-waited answers to important clin- FPGS activity in PBMCs and spermatozoa ical questions. First, studies that evaluated the effect of MTX To determine whether the marginal MTX- pg accumulation on semen parameters and/or the male reproductive endo- is related to FPGS catalytic activity, this enzyme activity was crine axis have resulted in conflicting results. Most of these measured in PBMCs and spermatozoa from four healthy controls studies included a small number of patients, lacked prospec- and 10 MTX-starters (postexposure). FPGS activity (pmol tively collected samples, did not have a control group or did Table 4 Male reproductive endocrine axis MTX- naïveMTX- naïve Pre- exposure (n=20)Post- exposure (n=18) Healthy controls (n=25) MTX chronic‡ (n=5) P value Testosterone (nmol/L) median (IQR) 14.6 (11.3–16.2) 13.4 (12.0–15.6) 14.1 (12.8–16.7) 16.3 (16.3–17.1) NS SHBG (nmol/L) median (IQR) 26.6 (22.6–34.6) 28.8 (22.5–34.6) 32.6 (25.7–41.9) 35.4 (34.1–38.7) NS LH (U/L) median (IQR) 3.1 (2.3–3.9) 2.7 (2.2–3.2) 2.9 (2.2–3.4) 4.10 (4.0–4.1) NS FSH (U/L) median (IQR) 4.6 (3.5–5.3) 4.2 (3.2–5.0) 3.7 (3.0–4.5) 4.1 (4.0–4.1) NS Inhibin B (ng/L) median (IQR) 132.5 (101.5–179.5) 123.0 (116.0–179.0) 189.0 (170.0–236.0) 92.2 (87.0–203.0) * p=<0.001 p=<0.001 *Statistically significant difference between pre- exposure and healthy controls. †Statistically significant difference between post- exposure and healthy controls. ‡Presented only for descriptive purposes, no statistical analyses were conducted. FSH, follicle stimulating hormone; LH, luteinising hormone; MTX, methotrexate; SHBG, sex hormone binding globulin. Perez- Garcia LF, et al. Ann Rheum Dis 2023;0:1–8. doi:10.1136/ard-2023-224032 5 Treatment Figure 1 MTX- polyglutamate (PG) accumulation in erythrocytes, seminal fluid and spermatozoa of RA patients and FPGS activity in spermatozoa. Please note that the y axis varies between figures. (A) Individual and total MTX- PG accumulation in erythrocytes of male RA patients (n=17) on MTX therapy for >12 weeks. Data are depicted as fmol MTX- PG/106 erythrocytes and presented in box plots with the sample median and IQR. (B) MTX-PG concentrations (in nmol/L) in seminal fluid of RA patients (n=17) on MTX therapy. (C) Individual and total MTX- PG accumulation in spermatozoa of RA patients (n=17) on MTX therapy. Data are depicted as fmol MTX-PG/106 spermatozoa and presented in box plots with the sample median and IQR . (D) FPGS catalytic activity in spermatozoa of healthy donors (n=4) and RA patients (n=10) for>12 weeks on MTX therapy, and for comparison in PBMCs of healthy individuals (n=4). For comparative and analytical controls, FPGS data are shown for proliferative CCRF-CEM leukaemia cells (n=24) and CEM/R30dm cells (n=15), a subline of CCRF- CEM with 1% residual FPGS activity and with acquired resistance to MTX due to impairment of MTX- PG formation. Data for FPGS catalytic activity are depicted as pmol MTX-PG2- 15N formed/hr/mg protein and presented in box plots with the sample median and IQR. FPGS, folylpolyglutamate synthetase; MTX, methotrexate; MTX-PG , methotrexate polyglutamate; RA, rheumatoid arthritis. not correct for relevant confounders (ie, disease activity or high FPGS gene (8PR) over the WT transcript. Regarding seminal dose glucocorticoids). Recently, Kazutaka et al evaluated the fluid, similar to the recent findings of Grosen et al, we detected testicular toxicity profile of MTX in a cross-sectional study that predominantly MTX- pg1. included 14 patients mainly diagnosed with Crohn’s disease. The iFAME-MTX study provides a strong scientific basis to Although this was not a prospective study, their findings are consider that MTX is safe for men with an active wish to become similar to ours, as they report that MTX therapy is not associ- a father. Our study showed that exposure to MTX did not result ated with abnormalities in semen parameters or the male repro- in abnormalities in semen parameters and other male fertility ductive endocrine axis. outcomes. Furthermore, although this study was not designed to Second, sperm DNA integrity is essential for producing evaluate the potential teratogenic effect of paternal MTX, three normal spermatozoa and DNA damage has been associated with pregnancies that were exposed to paternal MTX were reported male infertility. Based on the known effects of MTX on DNA by MTX- starters (conception within 1 year after their first study synthesis, we were concerned that MTX could result in sperm visit). No negative pregnancy outcomes or congenital malforma- DNA damage. Reassuringly, we did not find a negative impact of tions were reported. This goes in line with our data that shows MTX on sDFI. Noteworthy, the median pre-exposure sDFI of that MTX is not associated with sperm DNA damage and that 24.3% in MTX- starters may be a reflection of a negative impact polyglutamylation is inefficient in spermatozoa is reassuring. of disease activity on spermatogenesis. Although not statisti- In this regard, the risk of birth defects associated to paternal cally significant, after exposure to MTX, the sDFI decreased to MTX has been evaluated before in more than 250 men and it 13.5%. This may be caused by a reduction of disease activity. was concluded that MTX was not associated with an increased 21 24 Third, another concern of patients and healthcare profes- risk of birth defects. sionals is that it was not known whether MTX could be detected Other secondary findings from our study warrant further in spermatozoa. Therefore, we aimed to measure the concentra- discussion. Inhibin B is secreted by the Sertoli cells and is consid- tion of MTXpg, the active forms of MTX in spermatozoa and in ered a marker of Sertoli cell function and spermatogenesis. seminal fluid. Reassuringly, we detected only MTXpg1 in very Sertoli cells are one of the most important cells necessary for low concentrations in spermatozoa and barely detected longer sperm production in men. Comprehensive evaluation of the retained MTXpg2,3. Furthermore, the findings of our comple- reproductive axis revealed statistically significant lower serum mentary experiments are reassuring, as we report a very low concentrations of inhibin B in the MTX-starters before expo- activity of FPGS in spermatozoa, indicating that MTX polyglu- sure to MTX. Lower serum concentrations of inhibin B have tamylation in spermatozoa is limited. Mechanistically, the low also been reported in men diagnosed with ankylosing spondy- 26 27 FPGS activity in spermatozoa may be associated with a higher litis and systemic lupus erythematosus. These findings further ratio of mRNA expression of an alternatively spliced form of the support the evidence that autoimmunity and inflammation can 6Perez- Garcia LF, et al. Ann Rheum Dis 2023;0:1–8. doi:10.1136/ard-2023-224032 Treatment 22 28 29 Consejo Nacional de Ciencia y Tecnologia (CONACYT) (project number 601574). All result in Sertoli cell dysfunction. Further research is needed are non- profit organisations. to corroborate these findings. Contributors All authors met the authorship criteria, they had a substantial Furthermore, both findings (higher sDFI and lower inhibin contribution to the conception or design of the work (LFPG, ER, BPK, GRD, RJEMD) B before exposure to MTX) go in line with the conclusion of or the acquisition (LFPG, LJCKvK, RvA, MBAvD, RJEMD), analysis (LFPG, RJEMD, our recent study where we reported that inflammatory arthritis ML, GJ, EAS, PHG, RdJ) or interpretation of data for the work (all authors) and were might impair male fertility. Inflammation, especially via mech- involved in revising a draft of this work, gave final approval of this version to be anisms associated with oxidative stress, was considered as a published and are accountable for all aspects of the work in ensuring accuracy and integrity. LFPG and RJEMD accept full responsability for the work and/or conduct of potential contributor to these findings. Whether inflammation the study, had access to the data, and controlled the decision to publish. secondary to IMIDs such as IA results in an increased oxidative Funding The funder had no role in study design, data collection, data analysis, or stress state in the testicles (or elsewhere) with the potential to data interpretation; writing the report; or the decision to submit this manuscript for disrupt the required homeostasis for normal spermatogenesis publication. remains unknown and warrants further research. Altogether, Competing interests RJEMD has received fees from Galagapos and UCB this may imply that in men with a wish to conceive, treating unrelated to this work and research support from UCB. LFPG has received fees the disease with immunosuppressive drugs (without known from Galapagos unrelated to this work. All the other authors declare no competing testicular toxicity profiles) while aiming at lower disease activity interest related to this work. states, may improve their chances of a successful pregnancy. Patient and public involvement Patients and/or the public were involved in the Our study has several strengths. It is the first prospective study design, or conduct, or reporting, or dissemination plans of this research. Refer to the Methods section for further details. that included cases and healthy controls and that was specifically designed to evaluate the impact of MTX on several markers of Patient consent for publication Consent obtained directly from patient(s) testicular toxicity. Our low loss to follow-up rate maximises the Ethics approval This study was approved by the ethics review board of the validity of our data. Furthermore, our results were corroborated Erasmus University Medical Center in compliance with the Declaration of Helsinki. All patients gave their informed consent (Erasmus MC - Ethics Committee: by the results of our complementary experiments that reported NL64218.078.18). Participants gave informed consent to participate in the study for the first time that polyglutamylation of MTX is very limited before taking part. in spermatozoa, leading to very low concentrations of the active Provenance and peer review Not commissioned; externally peer reviewed. forms of MTX in seminal fluid and spermatozoa. Our study has Data availability statement Data are available upon reasonable request. important limitations. First, our results are significant and repre- sentative for the MTX- starters group and not necessarily of the Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have MTX- chronic group. Second, we only have one semen sample been peer-reviewed. Any opinions or recommendations discussed are solely those per study visit and not the ideal two (with an average value of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and reported). Thus, the wide known variability of sperm concentra- responsibility arising from any reliance placed on the content. Where the content tion might have influenced our results. includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, In conclusion, treatment with MTX is not associated with terminology, drug names and drug dosages), and is not responsible for any error testicular toxicity in men diagnosed with an IMID. It can also and/or omissions arising from translation and adaptation or otherwise. be concluded that the concentration of intracellular MTX-PG in Open access This is an open access article distributed in accordance with the seminal fluid and spermatozoa is very low. Therefore, therapy Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits with MTX can be safely started or continued in men diagnosed others to copy, redistribute, remix, transform and build upon this work for any with an IMID and with an active wish to become a father. purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. See: https://creativecommons.org/ licenses/by/4.0/. Author affiliations Department of Rheumatology, Erasmus Medical Center, Rotterdam, Netherlands 2 ORCID iDs Department of Rheumatology, Sint Franciscus Vlietland Group, Rotterdam, Luis Fernando Perez- Garcia http://orcid.org/0000-0002-8958-9493 Netherlands Esther Röder http://orcid.org/0000-0003-2139-3838 Department of Rheumatology, IJsselland Hospital, Capelle aan den IJssel, Pieter H Griffioen http://orcid.org/0000-0002-1299-2866 Netherlands Department of Rheumatology, Maasstad Ziekenhuis, Rotterdam, Netherlands Department of Clinical Chemistry, Erasmus Medical Center, Rotterdam, Netherlands REFERENCES Centre for Reproductive Medicine, University of Antwerp, Antwerpen, Belgium 1 Sammaritano LR, Bermas BL, Chakravarty EE, et al. American college of Department of Laboratory Medicine, Amsterdam University Medical Center, rheumatology guideline for the management of reproductive health in rheumatic and Amsterdam, Netherlands musculoskeletal diseases. Arthritis Care Res (Hoboken) 2020;72:461–88. Department of Rheumatology and Clinical Immunology, Amsterdam University 2 Russell MD, Dey M, Flint J, et al. British Society for rheumatology guideline on Medical Center, location VUmc, Amsterdam, Netherlands prescribing drugs in pregnancy and breastfeeding: immunomodulatory anti- rheumatic Department of Dermatology, Erasmus Medical Center, Rotterdam, Netherlands drugs and corticosteroids. Rheumatology 2023;62:e48–88. Department of Urology, Erasmus Medical Center, Rotterdam, Netherlands 3 Menter A, Gelfand JM, Connor C, et al. Joint American Academy of Dermatology– National psoriasis Foundation guidelines of care for the management of psoriasis with Twitter Luis Fernando Perez- Garcia @DrReumatologo systemic nonbiologic therapies. Journal of the American Academy of Dermatology Acknowledgements We extend our gratitude to Ron Buijs, data manager of 2020;82:1445–86. the department of Rheumatology, Erasmus MC for his invaluable help with regards 4 FDA. Methotrexate label. Available: https://www.accessdata.fda.gov/drugsatfda_docs/ to technical support, data collection and data management. We also extend our label/2020/040054s015,s016,s017.pdf gratitude to our colleagues from the Reproductive Medicine department of the 5 Agency EM. Mtx label. n.d. Available: https://www.ema.europa.eu/en/documents/ Erasmus MC for their support especially to K. van Veen- Buurman , who introduced product-information/jylamvo-epar-product-information_en.pdf me to the lab and patiently taught me how to perform a semen analysis and who 6 U.S. Department of Health and Human Services Food and Drug Administration Center sadly did not get a chance to see the final results of the study (Special thanks for 488 Drug Evaluation and Research. Testicular toxicity: evaluation during drug also to Dr. E.B. Baart, M. Chan, M. Rijken, V. Gobardhan- Jagesser, A.J.A.M. Dons, development guidance for industry, draft guidance. 2015. Available: http://www. Y. van Doorn, S.G.C. Waterval, K. Karremans), our colleagues from the Andrology fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ department of the Radboud UMC and our colleagues from the Department of UCM455102.pdf [Accessed 20 2016]. Clinical Chemistry of the Erasmus University Medical Center and Amsterdam UMC. 7 Ramaswamy S, Weinbauer GF. Endocrine control of Spermatogenesis: role of FSH and This work was supported by research grants from the Dutch Arthritis Foundation LH/ testosterone. Spermatogenesis 2014;4:e996025. (ReumaNederland) (project number: 16–3- 402), The Netherlands Organization for 8 Esteves SC, Zini A, Coward RM, et al. Sperm DNA fragmentation testing: summary Health Research and Development (ZonMw) (project number 849200009) and evidence and clinical practice recommendations. Andrologia 2021;53:e13874. Perez- Garcia LF, et al. Ann Rheum Dis 2023;0:1–8. doi:10.1136/ard-2023-224032 7 Treatment 9 Sakkas D, Alvarez JG. Sperm DNA fragmentation: mechanisms of origin, impact on 20 Stark M, Wichman C, Avivi I, et al. Aberrant splicing of Folylpolyglutamate reproductive outcome, and analysis. Fertil Steril 2010;93:1027–36. synthetase as a novel mechanism of antifolate resistance in leukemia. Blood 10 Vaughan DA, Tirado E, Garcia D, et al. DNA fragmentation of Sperm: a radical 2009;113:4362–9. examination of the contribution of oxidative stress and age in 16 945 Semen samples. 21 Perez- Garcia LF, Dolhain RJEM, Vorstenbosch S, et al. The effect of Paternal exposure Hum Reprod 2020;35:2188–96. to immunosuppressive drugs on sexual function, reproductive hormones, fertility, 11 Rots MG, Pieters R, Peters GJ, et al. Role of folylpolyglutamate synthetase and pregnancy and offspring outcomes: a systematic review. Hum Reprod Update folylpolyglutamate hydrolase in methotrexate accumulation and polyglutamylation in 2020;26:961–1001. childhood leukemia. Blood 1999;93:1677–83. 22 Kazutaka S, Winnall WR, Muir JA, et al. Regulation of Sertoli cell Activin A and 12 Hebing RC, Lin M, Bulatovic Calasan M, et al. Pharmacokinetics of oral and Inhibin B by tumour necrosis factor Α and interleukin 1Α: interaction with Follicle- subcutaneous methotrexate in red and white blood cells in patients with stimulating hormone/adenosine 3’,5’-Cyclic phosphate signalling. Mol Cell Endocrinol early rheumatoid arthritis: the methotrexate monitoring trial. Ann Rheum Dis 2011;335:195–203. 2022:ard- 2022- 223398. 23 Gunes S, Al-Sadaan M, Agarwal A. DNA damage and DNA 13 van de Meeberg MM, Hebing RCF, Nurmohamed MT, et al. A meta-analysis of repair mechanisms in male infertility. Reprod Biomed Online methotrexate Polyglutamates in relation to efficacy and toxicity of methotrexate in 2015;31:S1472- 6483(15)00304- 1:309–19.:. inflammatory arthritis, colitis and dermatitis. Br J Clin Pharmacol 2023;89:61–79. 24 Mouyis M, Flint JD, Giles IP. Safety of anti- rheumatic drugs in men trying to conceive: 14 Muller IB, Lin M, Lems WF, et al. Association of altered Folylpolyglutamate synthetase A systematic review and analysis of published evidence. Semin Arthritis Rheum pre- mRNA splicing with methotrexate Unresponsiveness in early rheumatoid arthritis. 2019;48:S0049- 0172(18)30194- X:911–20.:. Rheumatology (Oxford) 2021;60:1273–81. 25 Kumanov P, Nandipati K, Tomova A, et al. Inhibin B is a better marker of 15 den Boer E, Meesters RJW, van Zelst BD, et al. Measuring methotrexate Spermatogenesis than other hormones in the evaluation of male factor infertility. polyglutamates in red blood cells: a new LC- MS/MS- based method. Anal Bioanal Fertil Steril 2006;86:332–8. Chem 2013;405:1673–81. 26 Almeida BP, Saad CGS, Souza FHC, et al. Testicular Sertoli cell function in Ankylosing 16 Ribeiro S, Sharma R, Gupta S, et al. Inter- and intra- laboratory standardization Spondylitis. Clin Rheumatol 2013;32:1075–9. of TUNEL assay for assessment of Sperm DNA fragmentation. Andrology 27 Suehiro RM, Borba EF, Bonfa E, et al. Testicular Sertoli cell function in male systemic 2017;5:477–85. lupus erythematosus. Rheumatology (Oxford) 2008;47:1692–7. 17 Mitchell LA, De Iuliis GN, Aitken RJ. The TUNEL assay consistently Underestimates 28 Hasan H, Bhushan S, Fijak M, et al. Mechanism of inflammatory associated DNA damage in human Spermatozoa and is influenced by DNA Compaction and cell impairment of Sperm function, Spermatogenesis and Steroidogenesis. Front vitality: development of an improved methodology. Int J Androl 2011;34:2–13. Endocrinol (Lausanne) 2022;13:897029:897029.:. 18 Muller IB, Lin M, Struys EA, et al. Development and validation of a sensitive UHPLC- 29 Hedger MP, Winnall WR. Regulation of Activin and Inhibin in the adult Testis and the MS/MS- based method for the analysis of Folylpolyglutamate synthetase enzymatic evidence for functional roles in Spermatogenesis and Immunoregulation. Mol Cell activity in peripheral blood mononuclear cells: application in rheumatoid arthritis and Endocrinol 2012;359:30–42. leukemia patients. Ther Drug Monit 2019;41:598–606. 19 McCloskey DE, McGuire JJ, Russell CA, et al. Decreased folylpolyglutamate synthetase 30 Perez- Garcia LF, Röder E, Goekoop RJ, et al. Impaired fertility in men diagnosed with activity as a mechanism of methotrexate resistance in CCRF- CEM human leukemia inflammatory arthritis: results of a large Multicentre study (iFAME- fertility). Ann sublines. J Biol Chem 1991;266:6181–7. Rheum Dis 2021;80:1545–52. 8Perez- Garcia LF, et al. Ann Rheum Dis 2023;0:1–8. doi:10.1136/ard-2023-224032 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of the Rheumatic Diseases British Medical Journal

Is methotrexate safe for men with an immune-mediated inflammatory disease and an active desire to become a father? Results of a prospective cohort study (iFAME-MTX)

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British Medical Journal
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© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY. Published by BMJ.
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0003-4967
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1468-2060
DOI
10.1136/ard-2023-224032
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Abstract

Treatment TRANSLATIONAL SCIENCE Is methotrexate safe for men with an immune- mediated inflammatory disease and an active desire to become a father? Results of a prospective cohort study (iFAME- MTX) 1 1 1,2 Luis Fernando Perez- Garcia , Esther Röder , Bouwe P Krijthe, 1,3 1 4 Laura JC Kranenburg- van Koppen, Roxanne van Adrichem, Els Zirkzee, 5 6 7 7 8 Pieter H Griffioen , Kris Peeters, Marry Lin, Eduard A Struys, Gerrit Jansen, 9 7 10 1 Martijn BA van Doorn, Robert de Jonge, Gert R Dohle, Radboud JEM Dolhain Handling editor Josef S ABSTRACT WHAT IS ALREADY KNOWN ON THIS TOPIC Smolen Introduction Current scientific evidence guiding ⇒ Although methotrexate (MTX) is one of the the decision whether men with an active desire to ► Additional supplemental most frequently prescribed immunosuppressive become a father should be treated with methotrexate material is published online medication for several immune- mediated only. To view, please visit (MTX) remains controversial. We aimed to prospectively the journal online (http:// dx. inflammatory diseases (IMIDs), the evidence evaluate the testicular toxicity profile of MTX focusing doi. org/ 10. 1136/ ard- 2023- regarding the testicular toxicity profile of on several markers of male fertility, including semen 224032). MTX is scarce. Ultimately, this has resulted in parameters and sperm DNA fragmentation index (sDFI). conflicting recommendations respecting the For numbered affiliations see As a secondary outcome, we aimed to evaluate whether end of article. safety of MTX in men with a wish to have MTX- polyglutamates can be detected in spermatozoa children. and seminal plasma and to evaluate the enzymatic Correspondence to activity in spermatozoa of folylpolyglutamate synthetase Dr Luis Fernando Perez- Garcia, WHAT THIS STUDY ADDS (FPGS). Rheumatology, Erasmus Medical Methods In a prospective cohort study, men ≥18 years ⇒ This is the first prospective study that evaluates Center, Rotterdam, Netherlands; l. perez@ erasmusmc. nl who started therapy with MTX were invited to participate the testicular toxicity profile of MTX in men (MTX- starters). Participants were instructed to produce diagnosed with IMIDs. We evaluated the effect Received 15 February 2023 two semen samples (a pre- exposure and a post- exposure of MTX on multiple markers of testicular toxicity Accepted 12 May 2023 sample after 13 weeks). Healthy men ≥18 years were and demonstrated that the semen parameters, invited to participate as controls. Conventional semen the male reproductive axis and the sperm DNA analyses, male reproductive endocrine axis and sDFI were fragmentation index were comparable between compared between groups. FPGS enzymatic activity and healthy controls and patients exposed to MTX. MTX- PG1- 5 concentrations were determined by mass Furthermore, our study also demonstrates spectrometry analytical methods. that MTX can be detected in spermatozoa and Results In total, 20 MTX- starters and 25 controls seminal fluid, but that the concentration of were included. The pre- exposure and postexposure MTX- polyglutamates, especially in spermatozoa, semen parameters of MTX-starters were not statistically is very low. Finally, we revealed the mechanistic significant different. Compared with healthy controls, the basis for these latter findings, demonstrating conventional semen parameters and the sDFI of MTX- that in spermatozoa, the catalytic activity of the starters were not statistically significant different. These enzyme responsible for the polyglutamylation data were corroborated by the marginal accumulation of of MTX, folylpolyglutamate synthetase, is very MTX- PGs in spermatozoa, consistent with the very low low. FPGS enzymatic activity associated with the expression of an alternative FPGS splice- variant. HOW THIS STUDY MIGHT AFFECT RESEARCH, Discussion Treatment with MTX is not associated PRACTICE OR POLICY with testicular toxicity, consistent with the very low ⇒ Altogether, our data suggest that MTX is not concentration of intracellular MTX- PG. Therefore, therapy associated with testicular toxicity. Therefore, with MTX can be safely started or continued in men and therapy with MTX can be safely started or with a wish to become a father. continued in men diagnosed with an IMID and © Author(s) (or their employer(s)) 2023. Re- use with an active wish to become a father. permitted under CC BY. Published by BMJ. INTRODUCTION To cite: Perez- Garcia LF, Methotrexate (MTX) is one of the most frequently Röder E, Krijthe BP, et al. for men with an active desire to become a father, the prescribed immunosuppressive drugs for the treat- Ann Rheum Dis Epub ahead decision of whether they should stop or continue of print: [please include Day ment of several immune- mediated inflammatory Month Year]. doi:10.1136/ therapy with MTX before conception remains diseases (IMIDs) such as rheumatoid arthritis (RA), ard-2023-224032 psoriatic arthritis (PsA) and psoriasis. Remarkably, controversial. Perez- Garcia LF, et al. Ann Rheum Dis 2023;0:1–8. doi:10.1136/ard-2023-224032 1 Treatment In this regard, the American College of Rheumatology were proven fertile (history of a partner’s positive pregnancy recently updated its recommendations on paternal immuno- test). Three groups of men were included in the study. First, suppressive exposure before conception. Concerning MTX, men diagnosed with an IMID (RA, Spondyloarthritis (SpA), PsA, they recommend that MTX can be ‘conditionally continued’ in psoriasis) based on the expert opinion of their rheumatologist or men planning to father a child. Furthermore, the British Society dermatologist, who were not exposed to MTX in the last year for Rheumatology guideline on prescribing drugs in pregnancy and were going to start therapy with MTX, were included in the considers paternal exposure to low- dose MTX (≤25 mg/week) ‘MTX- starters’ group. Second, to evaluate the effect of chronic as compatible with pregnancy. Conversely, other federal agen- MTX exposure, men who were exposed to MTX (≥15 mg/week cies and medical associations recommend that MTX should be for≥1 year) were included in the ‘MTX- chronic’ group. Third, 3–5 stopped at least 3–6 months before conception. healthy men were included as ‘healthy controls’. A fundamental reason for these contradictory recommen- Men who were not proven fertile, who were exposed to drugs dations is the scarcity of solid scientific data on the testicular with known testicular toxic side effects (ie, oligospermia) were toxicity profile of MTX. In its guideline to evaluate testicular excluded. Importantly, concomitant therapy with prednisone toxicity, the Food and Drug Administration (FDA) considers (≤7.5 mg/day), hydroxychloroquine or tumour necrosis factor semen analysis parameters as the main outcomes of interest. alpha (TNFa) inhibitors was allowed (see online supplemental Furthermore, in studies evaluating testicular toxicity, the FDA file 1), table 1. Exclusion criteria and table 2 Exclusion criteria, recommends evaluating at least one baseline and one follow-up drug exposure). semen sample (at the end of the first 13 weeks). In addition to spermatogenesis, the production of hormones is Study visits another important function of the testicles. Therefore, the eval- A study visit consisted of three parts. First, the demographic uation of the male reproductive endocrine axis (ie, testosterone, and medical history were obtained from an interview, followed luteinising hormone (LH), follicle stimulating hormone (FSH)) by a concise physical evaluation. Second, a blood sample was should also be considered an important outcome. obtained. Finally, participants provided a semen sample. Two Another novel outcome of interest that reflects the integrity study visits were required from participants from the MTX- or damage of the sperm DNA and that can be evaluated is the starters group, one before exposure to MTX (pre- exposure) and sperm DNA fragmentation index (sDFI). Sperm DNA integrity another one at least 13 weeks after their initial MTX exposure is indispensable for the birth of healthy offspring and sperm (postexposure). Participants from the MTX-chronic and healthy DNA damage has been strongly associated with male infertility control groups were just required to complete one study visit. and a higher risk of miscarriages. Sperm DNA damage can be induced by either the pharmacological exposure itself or the Demographic characteristics, medical history and physical oxidative stress and inflammation states associated with a diag- evaluation nosis of IMID. Participants answered a questionnaire that included questions Finally, it is known that the pharmacological efficacy of MTX concerning their demographic characteristics, medical history critically depends on the intracellular bioactivation of MTX to (including reproductive history) and their current and previous MTX- polyglutamates (MTX- PG). This process is catalysed by medication exposure. the enzyme folylpolyglutamate synthetase (FPGS). Lower FPGS A physical evaluation was performed to determine height, activity and subsequent inefficient polyglutamylation is a well- weight and testicular volume (using an orchidometer). Disease known phenomenon associated with a rapid efflux of MTX from activity was calculated using validated scores (DAS28 for RA, cells. MTX-PG have been detected in several human cells such BASDAI for SpA, DAPSA for PsA and PASI for psoriasis). as erythrocytes or peripheral blood mononuclear cells (PBMCs) and low levels of MTX- PG have been associated with increased 12 13 Blood sample resistance to therapy. In this regards, recent molecular Blood from all participants was obtained between 09:00 and studies have shown that a significant reduction of FPGS activity 11:00 hours by venipuncture. The concentration of testosterone, is associated with aberrant pre- mRNA splicing of this enzyme, LH, FSH, inhibin B, sex hormone binding globulin (SHBG) and including partial retention of intron 8 (8PR) and a higher ratio C reactive protein were evaluated. Erythrocytes and PBMCs of FPGS 8PR over FPGS wild type (WT). Whether intracel- 12 15 were isolated using the protocol previously described. lular MTX-PG can be detected in the spermatozoa and whether the spermatozoa have the enzymatic capabilities of forming and Semen sample collection retaining intracellular MTX- PG has never been studied before. Semen samples were delivered by masturbation. To ensure a To establish whether MTX can be safely used by men diag- timely analysis of the samples, participants provided the sample nosed with an IMID and a wish to conceive, we aimed to prospec- in the hospital. All samples were evaluated within 30 min of tively evaluate the testicular toxicity profile of MTX focusing on production. The fresh semen sample was analysed for semen several markers of male fertility such as semen parameters, the volume, sperm concentration and motility. Slides were prepared male reproductive endocrine axis and the sDFI. Furthermore, as for the morphology analysis and sent to a specialised centre a secondary outcome, we aimed to evaluate whether MTX-PG (Radboud UMC, Nijmegen The Netherlands). can be detected in spermatozoa and seminal plasma and to eval- Thereafter, the semen samples were individually processed to uate the FPGS enzymatic activity in spermatozoa. prepare pellets for further analysis. First, samples were processed for future sDFI measurement following the steps previously METHODS published. Second, the semen samples were centrifuged and Study design and patient selection washed out three times with PBS and spermatozoa were isolated The iFAME-MTX study was a prospective cohort study for future MTX- PG concentration, FPGS catalytic activity and conducted in the Erasmus University Medical Center, Rotterdam, mRNA expression measurements. All samples were stored at The Netherlands. All participants were aged 18–55 years and −80°C. 2Perez- Garcia LF, et al. Ann Rheum Dis 2023;0:1–8. doi:10.1136/ard-2023-224032 Treatment Table 1 Demographic characteristics MTX- naïveMTX- naïve Pre- exposure (n=20)Post- exposure (n=18) Healthy controls (n=25) MTX chronic§ (n=5) P value General information Age years, mean (95% CI) 35.2 (31.4 to 39.1) 34.7 (32.9 to 36.7) 36.6 (32.1 to 41.1) NS Smoking, n (%) 4 (20) 4 (20) 6 (24) 1 (20) NS BMI %, mean (95% CI) 27.1 (24.8 to 29.2) 26.8 (24.5 to 29.1) 25.5 (24.2 to 26.8) 25.5 (21.4 to 29.6) NS Testicular volume, mean (95% CI) 22.9 (21.4 to 24.3) 22.6 (21.3 to 23.8) 22.6 (21.3 to 23.8) NS Inflammatory arthritis Diagnosis: RA, n (%) 7 (35.0) 6 (33.3) - 2 (40.0) PsA, n (%) 8 (40.0) 7 (38.9) - 1 (20.0) SpA, n (%) 1 (5.0) 1 (5.6) - 2 (40.0) Psoriasis, n (%) 4 (20.0) 4 (22.2) - 0 (0.0) Age at diagnosis, mean (SD) 27.4 (22.1–32.6) 28.5 (24.5–34.5) – 30.3 (18.5–42.1) Disease duration, mean (SD) 6.9 (2.1–11.8)5.6 (- 5.5–16.8) MTX dose (mg/week), mean (95% CI) – 16.0 (13.6 to 18.4) – 18.3 (15.6 to 21.1) Prednisone exposure, n (%) 2 (10.0) 6 (33.3) -- 1 (20) NS TNFa inhibitor exposure, n (%) 3 (15.0) 5 (27.8) -- 2 (40) NS C reactive protein mg/dL, median (IQR) 2.1 (0.6–5.0) 1.4 (1.0–3.2) 0 (0.0–0.9) 1.1 (0–1.8) * p=0.011 † p=0.008 Disease activity scores VAS general health mm, 42 (19 to 67) 20 (12 to 36) 17.7 (10.5 to 25- 5) 20.20 (0.3 to 40.1) * p=<0.001 mean (95% CI) ‡ p=0.008 VAS pain mm, mean (IQR) 42 (5.5–75) 14 (4–36) -- 11 (4–34) VAS activity mm, mean (IQR) 68 (51–78) 27 (16–49) -- 10 (4–52) RA: DAS28, mean (IQR) 2.7 (2.4–2.9) 2.55 (1.34–3.40) -- 2.3 (1.4–2.7) PsA: DAPSA, mean (IQR) 24.1 (20.5–33.3) 17.8 (11.2–22.7) -- 1.1 (1.1–1.1) Psoriasis: PASI, mean (IQR) 1.9 (1.3–5.1) 1.1 (0.7–3.1) -- 0.8 (0.3–1.2) *Statistically significant difference between pre- exposure and healthy controls. †Statistically significant difference between post- exposure and healthy controls. ‡Statistically significant difference between pre- exposure and post- exposure. §Presented only for descriptive purposes, no statistical analyses were conducted. BMI, body mass index; DAPSA, disease activity index for psoriatic arthritis; DAS28, disease activity score 28; MTX, methotrexate; PsA, psoriatic arthritis; RA, rheumatoid arthritis; SpA, spondyloarthritis; TNFa, tumour necrosis factor alpha; VAS, visual analogue scale. Sperm DNA fragmentation index for 45 min. After washing, the pellets were incubated in fresh Assessment of sDFI was performed using the terminal deosy- permeabilisation solution (0.1% sodium citrate, 0.1% triton nucleotidyl transferase dUTP nick end labeling (TUNEL) assay X–100, both Sigma-Aldrich, Belgium) for 5 min at 4°C. The described by Mitchell et al. In short, the spermatozoa pellets positive control samples were treated with 5 µl of DNase I were, after thawing, washed in phosphate buffered saline and (Qiagen, Germany) 1500 Kunitz Units for 30 min. The assay was incubated in 2 mM dithiothreitol (Sigma- Aldrich, Belgium) performed using the fluorescein In Situ Cell Death Detection Table 2 Conventional semen parameters and sperm morphology MTX- naïveMTX- naïve Pre- exposure (n=20)Post- exposure (n=18) Healthy controls (n=25) MTX chronic* (n=5) P value Conventional semen parameters Sperm concentration x10 /mL, median (IQR) 57.0 (35.0–90.5) 54.0 (41.0–82.0) 60.0 (37.0–111.0) 37.0 (32.0–59.9) NS Progressive motility* %, 63.2 (55.4 to 70.9) 60.1 (49.5 to 70.6) 56.9 (51.1 to 62.8) 50.4 (34.8 to 65.9) NS mean (95% CI) Semen volume mL, median (IQR) 2.4 (1.6–3.2) 3.0 (1.5–3.2) 3.0 (2.0–4.0) 2.0 (1.6–2.4) NS Sperm morphology evaluation Normal morphology %, mean (95% CI) 6.4 (4.5 to 8.3) 7.1 (5.6 to 8.4) 6.3 (4.7 to 7.9) 5.9 (2.6 to 9.1) NS Teratozoospermia index, mean (95% CI) 1.2 (1.2 to 1.3) 1.3 (1.2 to 1.4) 1.2 (1.2 to 1.3) 1.2 (1.1 to 1.4) NS Excess residual cytoplasm, median (IQR) 2.0 (0.7–4.3) 2.0 (1.0–4.5) 2.0 (1.0–4.0) 2.0 (1.0–4.0) NS Abnormalities in head (%), mean (95% CI) 92.8 (90.8 to 94.7) 93.0 (91.2 to 94.6) 92.7 (91.0 to 94.3) 92.3 (88.0 to 96.5) NS Abnormalities in middle piece (%), mean (95% CI) 19.2 (14.2 to 24.1) 24.5 (18.6 to 30.2) 19.9 (15.3 to 24.5) 22.9 (5.7 to 40.0) NS Abnormalities in tail (%), mean (95% CI) 7.1 (3.8 to 10.5) 7.3 (3.7 to 11.1) 6.6 (3.9 to 9.3) 4.6 (1.4 to 7.7) NS *Presented only for descriptive purposes, no statistical analyses were conducted. MTX, methotrexate. Perez- Garcia LF, et al. Ann Rheum Dis 2023;0:1–8. doi:10.1136/ard-2023-224032 3 Treatment Kit (Roche Diagnostics, Mannheim, Germany) with an Accuri polyglutamylation in acute lymphoblastic leukaemia and RA C6 flow cytometer (BD Sciences, Erembodegem, Belgium). For patients. To evaluate if alterations in FPGS pre-mRNA splicing each sample, 5000–10 000 events were recorded at a flow rate levels in spermatozoa could explain our findings, an additional of 35 µL/min. experiment was performed. Shortly, RNA was isolated from PBMCs and spermatozoa according to the manufacturer’s protocol (BD Biosciences). RNA (250 ug) was reverse transcribed MTX polyglutamates quantification in erythrocytes, to cDNA using Moloney Murine Leukaemia Virus (M- MLV; spermatozoa and seminal plasma Thermo Fisher Scientific, Waltham, Massachusetts) in a reaction Procedure EDTA erythrocyte cell pellets buffer containing random hexamer primers (Roche, Basel, Swit- Considering that MTX-PG have never been measured in sper - zerland), Deoxynucleotide triphosphates (dNTPs) (Roche) and a matozoa, we opted to measure MTX-PG in erythrocytes as a ribonuclease inhibitor RNasin (Promega, Madison, Wisconsin). control measure for MTX adherence. The erythrocyte cell pellets Primer sequences (see online supplemental figure 1) and methods are measured accordingly to our previously described validated used to quantify the levels of FPGS 8PR, FPGS WT are described LC- MS/MS method using custom- made stable isotopes of MTX- 12 15 elsewhere. pg1- 7 as internal standards. This method with minor alter- ations was also used to measure MTX-PG in spermatozoa and Statistical analysis seminal fluid. Comparisons between the pre- exposure and postexposure MTX- starters groups and healthy controls were tested. Because of the Procedure spermatozoa cell pellets low number of participants included in the MTX-chronic group, Spermatozoa cell pellets were kept at −80°C until analysis. we present their data in this article only for descriptive purposes. To avoid polyglutamate deconjugation activity by γ-glutamyl Categorical variables were presented as number (percentage), hydrolase (GGH), semen cells were placed on ice and denatured and continuous variables are reported as mean±SD, or medi- immediately. A volume of 320 µL perchloric acid (16%) was an±IQR, as appropriate. Continuous variables were compared added to the pellets and mixed immediately to denature. After using a one-way analysis of variance, Tukey post hoc test, paired denaturation samples were supplemented with 200 µL saline t- test, Mann- Whitney test and Wilcoxon signed- rank. Categor- and 200 µL internal standards mixture (pg1–pg7). Supernatants ical variables were compared using χ tests and Fisher’s exact were transferred two times into clean tubes after centrifugation tests. For linear correlation analysis, we used the Pearson correla- (Hettich micro 220R, 10 min, 21 250×g, 5°C) and used for tion coefficient. The level of significance was set as a two-tailed measurements of MTX-PG. p≤0.05, and statistical analyses were completed using Stata V.15 (StataCorp- LP). Procedure for seminal fluid Seminal fluid samples were kept at −80°C until analysis. To Ethics avoid GGH activity, seminal fluids were shortly thawed on ice This study was approved by the ethic review board of the before denaturation. A sample volume of 200 µL of previously Erasmus University Medical Center in compliance with the isolated seminal fluid was used. Because of its high viscosity, Declaration of Helsinki (NL64218·078·18). All participants samples were denatured with 320 µL acidic methanol (16% gave their informed consent. perchloric acid in methanol) and mixed immediately to dena- ture the seminal plasma. After denaturation, 200 µL of internal Patient and public involvement standard mixture (pg1–pg7) was added. Supernatants are trans- Three male patients diagnosed with inflammatory arthritis and ferred two times into clean tubes after centrifugation (Hettich who are active members of the research advisory board from the micro 220R, 10 min, 21 250×g, +5°C.) Samples were dried Department of Rheumatology of the Erasmus University Medical under nitrogen flow (Evaporex EVX-192,+50°C) and dissolved Center were involved in the design of the questionnaire and the in 720 µL purified water (Millipore) and mixed (IKA MTS4, invitation letter. Together, we carefully assessed the burden on 10 min, 600 rpm, room temperature (RT)). Samples were filtered participating patients. We intend to share the results to partic- (Whatmann mini-uniprep, UN203NPUPP) before being used for ipating patients and will appropriately disseminate the results. MTX- PG measurements. RESULTS FPGS activity in PBMCs and spermatozoa Between February 2019 and January 2022, a total of 118 (46 FPGS catalytic activity in PBMCs (as positive control) and sper- MTX- starters, 49 healthy controls and 23 MTX- chronic) men matozoa of healthy controls and MTX-starters were analysed in were invited to participate in the study. In total, 50 men agreed 10 µg protein extracts and assay mixtures containing 250 mmol/L to participate (20 MTX- starters, 25 healthy control and 5 MTX- MTX, and 4 mmol/L N- labelled L-glutamic acid as subtract chronic). Most men who did not participate in the study provided concentrations as described by Muller et al. FPGS activity their reasons not to do so (no time for study visits (n=23), is reported as pmol MTX- PG - N formed/hour/mg protein. unwilling to provide semen samples (n=16), COVID- 19 lock- In addition, cell extracts of CCRF-CEM and CEM/R30dm down (n=12), no interest in the topic (n=8), Erasmus University leukaemia cells were used a positive and negative controls for Medical Center being too far away (n=2), reason not provided FPGS activity, respectively. (n=7). The demographic and clinical characteristics of these men are presented in table 1. mRNA expression profiles of folate genes in spermatozoa An important mechanism of loss of FPGS activity and subse- quent inefficient polyglutamylation can occur due to aber- Conventional semen parameters and sperm morphology rant pre- mRNA splicing of FPGS. We recently identified a There were no statistically significant differences in the median partial retention of FPGS intron 8 (8PR) as a prominent splice sperm concentration, semen volume, sperm motility and sperm variant conferring FPGS dysfunction and decreased MTX morphology parameters between MTX- starters and healthy 4Perez- Garcia LF, et al. Ann Rheum Dis 2023;0:1–8. doi:10.1136/ard-2023-224032 Treatment Table 3 Sperm DNA fragmentation index MTX- naïveMTX- naïve Pre- exposure (n=20)Post- exposure (n=18) Healthy controls (n=25) MTX chronic* (n=5) P value Sperm DNA fragmentation index %, median (IQR) 24.3 (7.1–30.7) 13.1 (9.5–19.9) 13.5 (8.7–20.2) 13.5 (13.3–26.1) NS *Presented only for descriptive purposes, no statistical analyses were conducted. MTX, methotrexate. controls. Only one case of oligospermia (<15 million sperma- MTX- PG2- N formed/hour/mg protein) was statistically signif- tozoa/mL) was observed in an MTX- starter (pre- exposure and icant higher in PBMCs (183 pmol MTX- PG2- N formed/hour/ postexposure samples, table 2). mg protein) compared with spermatozoa from healthy controls (15 pmol MTX- PG2- N formed/hour/mg protein) and sper- matozoa from MTX- starters (5 pmol MTX- PG2- N formed/h/ Sperm DNA fragmentation index mg protein) (figure 1D). Remarkably, FPGS activity in sperma- The median sDFI was higher in the pre- exposure samples from tozoa is even lower than a control cell line (CEM/R30dm) with the MTX- starters (22.0% (IQR 10.7–30.7), but this was not acquired resistance to MTX due to loss of FPGS activity and statistically significant different when compared with the post- impairment of MTX-PG ( figure 1D). exposure sample (13.1% (IQR 9.5–16.3), p=0·247) and to the healthy controls (13.5% (IQR 8.7–20.2), p=0.257). See table 3. mRNA expression profiles of folate genes in spermatozoa Male reproductive endocrine axis To explore whether molecular alterations underlie the extremely The median serum concentrations of testosterone, SHBG, LH low FPGS activity in spermatozoa, mRNA expression profiles and FSH were not statistically significant different between the of FPGS and other folate genes were evaluated in spermatozoa groups. The median serum concentration of inhibin B was statis- from four healthy controls and six MTX-starters (see online tically significant lower in the pre-exposure (132.5 ng/L (IQR supplemental figure 1). Of note, a relatively high FPGS/8PR and 101.5–179.5)) and postexposure samples of the MTX-starters FPGS 8PR/WT ratio was observed in spermatozoa compared group (123.0 ng/L (IQR 116.0–179.0)) compared with the with PBMCs (results are not shown). healthy controls (189.0 ng/L (170.0–236.0)). See table 4. DISCUSSION MTX polyglutamate quantification The iFAME-MTX study is the largest study to date that prospec- MTXpg1–5 were detected in erythrocytes from all partic- tively evaluated the potential impact of MTX on many important ipants (figure 1A), consistent with MTX- pg accumulation markers of testicular toxicity and to evaluate the potential under- profiles in erythrocytes of patients under MTX therapy and lying mechanisms explaining why MTX does not impair sperm thus confirming their intake of MTX. In seminal fluid, quality. It shows that MTX is not associated with conventional mainly MTX- pg1 was detected with a median concentration semen analysis abnormalities, disturbances in the male repro- of 184 nmol/L (IQR 39–265), whereas MTXpg2,3 were barely ductive endocrine axis or with increased sperm DNA damage. noticeable (figure 1B). In spermatozoa, mainly MTXpg1 was Furthermore, to the best of our knowledge, our study reports detected (median 0.26 fmol/10 spermatozoa, IQR 0.16–0.69), for the first time that the enzyme responsible for intracellular whereas MTXpg2,3 levels were at the lower limit of detection polyglutamylation and hence the bioactivation of MTX, that is, (figure 1C). Total MTXpg levels in spermatozoa were approxi- FPGS, has an extremely low activity in spermatozoa. Ultimately mately 18- fold lower than in erythrocytes (0.32 fmol/10 sper- resulting in very low concentrations of intracellular MTX-PG in matozoa vs 5.8 fmol/10 erythrocytes, respectively, figure 1A/C). spermatozoa. Our results provide long-waited answers to important clin- FPGS activity in PBMCs and spermatozoa ical questions. First, studies that evaluated the effect of MTX To determine whether the marginal MTX- pg accumulation on semen parameters and/or the male reproductive endo- is related to FPGS catalytic activity, this enzyme activity was crine axis have resulted in conflicting results. Most of these measured in PBMCs and spermatozoa from four healthy controls studies included a small number of patients, lacked prospec- and 10 MTX-starters (postexposure). FPGS activity (pmol tively collected samples, did not have a control group or did Table 4 Male reproductive endocrine axis MTX- naïveMTX- naïve Pre- exposure (n=20)Post- exposure (n=18) Healthy controls (n=25) MTX chronic‡ (n=5) P value Testosterone (nmol/L) median (IQR) 14.6 (11.3–16.2) 13.4 (12.0–15.6) 14.1 (12.8–16.7) 16.3 (16.3–17.1) NS SHBG (nmol/L) median (IQR) 26.6 (22.6–34.6) 28.8 (22.5–34.6) 32.6 (25.7–41.9) 35.4 (34.1–38.7) NS LH (U/L) median (IQR) 3.1 (2.3–3.9) 2.7 (2.2–3.2) 2.9 (2.2–3.4) 4.10 (4.0–4.1) NS FSH (U/L) median (IQR) 4.6 (3.5–5.3) 4.2 (3.2–5.0) 3.7 (3.0–4.5) 4.1 (4.0–4.1) NS Inhibin B (ng/L) median (IQR) 132.5 (101.5–179.5) 123.0 (116.0–179.0) 189.0 (170.0–236.0) 92.2 (87.0–203.0) * p=<0.001 p=<0.001 *Statistically significant difference between pre- exposure and healthy controls. †Statistically significant difference between post- exposure and healthy controls. ‡Presented only for descriptive purposes, no statistical analyses were conducted. FSH, follicle stimulating hormone; LH, luteinising hormone; MTX, methotrexate; SHBG, sex hormone binding globulin. Perez- Garcia LF, et al. Ann Rheum Dis 2023;0:1–8. doi:10.1136/ard-2023-224032 5 Treatment Figure 1 MTX- polyglutamate (PG) accumulation in erythrocytes, seminal fluid and spermatozoa of RA patients and FPGS activity in spermatozoa. Please note that the y axis varies between figures. (A) Individual and total MTX- PG accumulation in erythrocytes of male RA patients (n=17) on MTX therapy for >12 weeks. Data are depicted as fmol MTX- PG/106 erythrocytes and presented in box plots with the sample median and IQR. (B) MTX-PG concentrations (in nmol/L) in seminal fluid of RA patients (n=17) on MTX therapy. (C) Individual and total MTX- PG accumulation in spermatozoa of RA patients (n=17) on MTX therapy. Data are depicted as fmol MTX-PG/106 spermatozoa and presented in box plots with the sample median and IQR . (D) FPGS catalytic activity in spermatozoa of healthy donors (n=4) and RA patients (n=10) for>12 weeks on MTX therapy, and for comparison in PBMCs of healthy individuals (n=4). For comparative and analytical controls, FPGS data are shown for proliferative CCRF-CEM leukaemia cells (n=24) and CEM/R30dm cells (n=15), a subline of CCRF- CEM with 1% residual FPGS activity and with acquired resistance to MTX due to impairment of MTX- PG formation. Data for FPGS catalytic activity are depicted as pmol MTX-PG2- 15N formed/hr/mg protein and presented in box plots with the sample median and IQR. FPGS, folylpolyglutamate synthetase; MTX, methotrexate; MTX-PG , methotrexate polyglutamate; RA, rheumatoid arthritis. not correct for relevant confounders (ie, disease activity or high FPGS gene (8PR) over the WT transcript. Regarding seminal dose glucocorticoids). Recently, Kazutaka et al evaluated the fluid, similar to the recent findings of Grosen et al, we detected testicular toxicity profile of MTX in a cross-sectional study that predominantly MTX- pg1. included 14 patients mainly diagnosed with Crohn’s disease. The iFAME-MTX study provides a strong scientific basis to Although this was not a prospective study, their findings are consider that MTX is safe for men with an active wish to become similar to ours, as they report that MTX therapy is not associ- a father. Our study showed that exposure to MTX did not result ated with abnormalities in semen parameters or the male repro- in abnormalities in semen parameters and other male fertility ductive endocrine axis. outcomes. Furthermore, although this study was not designed to Second, sperm DNA integrity is essential for producing evaluate the potential teratogenic effect of paternal MTX, three normal spermatozoa and DNA damage has been associated with pregnancies that were exposed to paternal MTX were reported male infertility. Based on the known effects of MTX on DNA by MTX- starters (conception within 1 year after their first study synthesis, we were concerned that MTX could result in sperm visit). No negative pregnancy outcomes or congenital malforma- DNA damage. Reassuringly, we did not find a negative impact of tions were reported. This goes in line with our data that shows MTX on sDFI. Noteworthy, the median pre-exposure sDFI of that MTX is not associated with sperm DNA damage and that 24.3% in MTX- starters may be a reflection of a negative impact polyglutamylation is inefficient in spermatozoa is reassuring. of disease activity on spermatogenesis. Although not statisti- In this regard, the risk of birth defects associated to paternal cally significant, after exposure to MTX, the sDFI decreased to MTX has been evaluated before in more than 250 men and it 13.5%. This may be caused by a reduction of disease activity. was concluded that MTX was not associated with an increased 21 24 Third, another concern of patients and healthcare profes- risk of birth defects. sionals is that it was not known whether MTX could be detected Other secondary findings from our study warrant further in spermatozoa. Therefore, we aimed to measure the concentra- discussion. Inhibin B is secreted by the Sertoli cells and is consid- tion of MTXpg, the active forms of MTX in spermatozoa and in ered a marker of Sertoli cell function and spermatogenesis. seminal fluid. Reassuringly, we detected only MTXpg1 in very Sertoli cells are one of the most important cells necessary for low concentrations in spermatozoa and barely detected longer sperm production in men. Comprehensive evaluation of the retained MTXpg2,3. Furthermore, the findings of our comple- reproductive axis revealed statistically significant lower serum mentary experiments are reassuring, as we report a very low concentrations of inhibin B in the MTX-starters before expo- activity of FPGS in spermatozoa, indicating that MTX polyglu- sure to MTX. Lower serum concentrations of inhibin B have tamylation in spermatozoa is limited. Mechanistically, the low also been reported in men diagnosed with ankylosing spondy- 26 27 FPGS activity in spermatozoa may be associated with a higher litis and systemic lupus erythematosus. These findings further ratio of mRNA expression of an alternatively spliced form of the support the evidence that autoimmunity and inflammation can 6Perez- Garcia LF, et al. Ann Rheum Dis 2023;0:1–8. doi:10.1136/ard-2023-224032 Treatment 22 28 29 Consejo Nacional de Ciencia y Tecnologia (CONACYT) (project number 601574). All result in Sertoli cell dysfunction. Further research is needed are non- profit organisations. to corroborate these findings. Contributors All authors met the authorship criteria, they had a substantial Furthermore, both findings (higher sDFI and lower inhibin contribution to the conception or design of the work (LFPG, ER, BPK, GRD, RJEMD) B before exposure to MTX) go in line with the conclusion of or the acquisition (LFPG, LJCKvK, RvA, MBAvD, RJEMD), analysis (LFPG, RJEMD, our recent study where we reported that inflammatory arthritis ML, GJ, EAS, PHG, RdJ) or interpretation of data for the work (all authors) and were might impair male fertility. Inflammation, especially via mech- involved in revising a draft of this work, gave final approval of this version to be anisms associated with oxidative stress, was considered as a published and are accountable for all aspects of the work in ensuring accuracy and integrity. LFPG and RJEMD accept full responsability for the work and/or conduct of potential contributor to these findings. Whether inflammation the study, had access to the data, and controlled the decision to publish. secondary to IMIDs such as IA results in an increased oxidative Funding The funder had no role in study design, data collection, data analysis, or stress state in the testicles (or elsewhere) with the potential to data interpretation; writing the report; or the decision to submit this manuscript for disrupt the required homeostasis for normal spermatogenesis publication. remains unknown and warrants further research. Altogether, Competing interests RJEMD has received fees from Galagapos and UCB this may imply that in men with a wish to conceive, treating unrelated to this work and research support from UCB. LFPG has received fees the disease with immunosuppressive drugs (without known from Galapagos unrelated to this work. All the other authors declare no competing testicular toxicity profiles) while aiming at lower disease activity interest related to this work. states, may improve their chances of a successful pregnancy. Patient and public involvement Patients and/or the public were involved in the Our study has several strengths. It is the first prospective study design, or conduct, or reporting, or dissemination plans of this research. Refer to the Methods section for further details. that included cases and healthy controls and that was specifically designed to evaluate the impact of MTX on several markers of Patient consent for publication Consent obtained directly from patient(s) testicular toxicity. Our low loss to follow-up rate maximises the Ethics approval This study was approved by the ethics review board of the validity of our data. Furthermore, our results were corroborated Erasmus University Medical Center in compliance with the Declaration of Helsinki. All patients gave their informed consent (Erasmus MC - Ethics Committee: by the results of our complementary experiments that reported NL64218.078.18). Participants gave informed consent to participate in the study for the first time that polyglutamylation of MTX is very limited before taking part. in spermatozoa, leading to very low concentrations of the active Provenance and peer review Not commissioned; externally peer reviewed. forms of MTX in seminal fluid and spermatozoa. Our study has Data availability statement Data are available upon reasonable request. important limitations. First, our results are significant and repre- sentative for the MTX- starters group and not necessarily of the Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have MTX- chronic group. Second, we only have one semen sample been peer-reviewed. Any opinions or recommendations discussed are solely those per study visit and not the ideal two (with an average value of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and reported). Thus, the wide known variability of sperm concentra- responsibility arising from any reliance placed on the content. Where the content tion might have influenced our results. includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, In conclusion, treatment with MTX is not associated with terminology, drug names and drug dosages), and is not responsible for any error testicular toxicity in men diagnosed with an IMID. It can also and/or omissions arising from translation and adaptation or otherwise. be concluded that the concentration of intracellular MTX-PG in Open access This is an open access article distributed in accordance with the seminal fluid and spermatozoa is very low. Therefore, therapy Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits with MTX can be safely started or continued in men diagnosed others to copy, redistribute, remix, transform and build upon this work for any with an IMID and with an active wish to become a father. purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. See: https://creativecommons.org/ licenses/by/4.0/. Author affiliations Department of Rheumatology, Erasmus Medical Center, Rotterdam, Netherlands 2 ORCID iDs Department of Rheumatology, Sint Franciscus Vlietland Group, Rotterdam, Luis Fernando Perez- Garcia http://orcid.org/0000-0002-8958-9493 Netherlands Esther Röder http://orcid.org/0000-0003-2139-3838 Department of Rheumatology, IJsselland Hospital, Capelle aan den IJssel, Pieter H Griffioen http://orcid.org/0000-0002-1299-2866 Netherlands Department of Rheumatology, Maasstad Ziekenhuis, Rotterdam, Netherlands Department of Clinical Chemistry, Erasmus Medical Center, Rotterdam, Netherlands REFERENCES Centre for Reproductive Medicine, University of Antwerp, Antwerpen, Belgium 1 Sammaritano LR, Bermas BL, Chakravarty EE, et al. American college of Department of Laboratory Medicine, Amsterdam University Medical Center, rheumatology guideline for the management of reproductive health in rheumatic and Amsterdam, Netherlands musculoskeletal diseases. 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Journal

Annals of the Rheumatic DiseasesBritish Medical Journal

Published: Aug 1, 2023

Keywords: Methotrexate; Arthritis; Antirheumatic Agents

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