Inflammatory bowel diseases (IBDs) comprise ulcerative colitis (UC) and Crohn's disease (CD). The incidences of both diseases peak during the reproductive age.1 Consequently, many male patients will father children after they have been diagnosed with IBD, while receiving drugs that can potentially influence male fertility.2
Methotrexate (MTX) is a folate analog3 which has steroid-sparing effects in CD.4,5 Similar to its effects in rheumatoid arthritis, MTX reduces the immunogenicity to biologic therapies such as infliximab.6 In UC, the recent French METEOR study (Controlled, Randomized, Double-Blind, Multicenter Study Comparing Methotrexate versus Placebo in Steroid Dependent Ulcerative Colitis)7 found MTX to be nonstatistically superior to placebo in inducing combined clinical and endoscopic remission after 16 weeks, although more patients achieved steroid-free clinical remission with MTX than with placebo. An ongoing study (MEthotrexate Response In Treatment of UC—MERIT-UC, NCT01393405) is investigating the efficacy of MTX to maintain steroid-free remission in UC. Despite evidence of clinical efficacy in CD, MTX has not gained widespread acceptance in clinical practice in adults.8 In Europe, the use of MTX in CD is restricted to a second or third line therapy,9 whereas U.S. guidelines advocate either thiopurines or MTX to maintain steroid-induced remission and acknowledge the lack of trials that investigate the safety and dosage use in CD patients.10 However, because of the recognized risk of lymphoma related to thiopurines and, in particular, the risk of hepatosplenic T cell lymphomas in young men, there has been a gradual evolution beginning with pediatric gastroenterologists to preferentially use MTX as a steroid-sparing agent.11
MTX is actively transported across cell membranes by the reduced folate carrier 1.12 Inside cells, MTX undergoes polyglutamination that entraps the long-lived MTX polyglutamates (MTXpg) that competitively inhibit the enzyme dihydrofolate reductase and de novo synthesis of purines and pyrimidines.12 High-dose MTX (HD-MTX; ≥500 mg/m2) is used in oncologic regimens to inhibit cellular proliferation because of reduced DNA and RNA synthesis leading to (malignant) cell death. In inflammatory diseases, the inhibition of DNA and RNA synthesis does not explain the anti-inflammatory effects observed with low-dose MTX (<50 mg/m2), where the proposed mechanisms of action include increased adenosine levels that lead to decreased neutrophil chemotaxis and the inhibition of lymphotoxin synthesis from polyamines.13
MTX is contraindicated during pregnancy and lactation (U.S. Food and Drug Administration category X).14 Indeed, MTX is used as an abortifacient for voluntary abortions and ectopic pregnancy.15 In pregnant women who are exposed to low-dose MTX, the risks of spontaneous abortions and major birth defects in the offspring are increased.16 The congenital malformations observed after MTX exposure are described as specific forms of MTX embryopathies that include hypoplasia of the skull bones, wide fontanels, craniosynostosis, prognatism, epicanthus, low-set ears, short limbs, club foot, and hypo- and syndactyly.15 The syndrome has been associated with exposure 6 to 8 weeks postconception during organogenesis at doses as low as 10 mg per week.15,17 Studies in women treated with MTX as a chemotherapeutic agent before conception found no adverse effect on subsequent female fertility.18 However, because of the long half-life of MTXpg, it is advised that female IBD patients stop MTX 3 to 6 months before anticipating pregnancy.19,20 Current IBD guidelines state that MTX is also contraindicated in men aiming to father children,19 and men have been advised to stop 3 to 4 months before conception.2,21 This advice aligns with current rheumatology and dermatology guidelines22,23 and is mainly based on expert opinion and the experience from female exposure.24 Cryopreservation of sperm before initiating MTX treatment, condom use during pregnancy, as well as prenatal screening in case of pregnancy after paternal MTX exposure have also been recommended.25–27 Although advice to prevent teratogenic effects after paternal exposure most often regards MTX,28 a recent patient survey disclosed that only 13 out of 28 men on MTX therapy received advice about contraception use during treatment.29
Human spermatogenesis occurs in distinct developmental phases, i.e., mitosis (spermatogonia), meiosis (primary and secondary spermatocytes), differentiation or spermiogenesis (spermatids), and maturation (spermatozoa), see Figure 1. It lasts approximately 64 days and is followed by 2 to 5 days of transit through the epididymis.30 Specialized junctions between Sertoli cells constitute the blood-testis barrier (BTB) that creates a unique environment for germ cells in the testis.31 Mitotic division and differentiation into primary preleptotene spermatocytes take place outside the BTB, whereas the meiotic division and spermiogenesis take place inside this immunological barrier in the seminiferous tubules.31 The type of germ cells affected by drug exposure can be ascertained by recording the time between drug administration and conception.32 A drug that causes irreversible damage to the chromatin structure in spermatocytes during meiosis or in later stages would most likely affect the progeny conceived up to 40 days later. If the spermatids are affected by a drug after DNA repair mechanisms have ceased, it poses a substantial risk of chromosomal aberrations in the progeny because these sperm cells may still be able to fertilize eggs.33 Drugs that affect DNA formation during the late stages of spermatogenesis may induce more damage than those that affect the early stages where DNA repair mechanisms are still active.34 The BTB, however, restricts the delivery of drugs to the seminiferous tubules by multiple different drug transporters expressed in the Sertoli cells.31 Of note, efflux pumps involved in MTX transport out of the cells, e.g., ATP-binding cassette transporters12 are expressed in human Sertoli and Leydig cells,31 and mRNA of reduced folate carrier 1 which regulates MTX influx has been detected in human testes.35 Potential mechanisms whereby MTX could affect sperm quality are listed in Figure 1.
When evaluating the effects of MTX exposure on male fertility, several questions arise. Does MTX affect sperm quality? Does MTX induce chromosomal damage in the sperm cells? Does paternal MTX exposure affect pregnancy outcomes in the female partner? Animal studies may provide hypotheses but firm conclusions should be drawn from human evidence. The aim of the present review was to review the literature that describes MTX effects on male fertility and pregnancy outcomes after paternal exposure.
MATERIALS AND METHODS
We performed a comprehensive literature search, using the PubMed database (http://www.ncbi.nlm.nih.gov/pubmed/) to identify published literature. We made no language or publication year restrictions. We applied 2 focus areas, “methotrexate” and “male fertility or pregnancy outcome,” the latter including sperm, spermatogenesis, testis, paternal exposure, fetus, and father (see Table, Supplemental Digital Content 1, http://links.lww.com/IBD/B474, with complete search strategy including all MeSH terms and free-text terms). All relevant terms and keywords in each focus area were listed and searched both as MeSH terms and free-text terms combined with OR. Thereafter, the 2 focus areas were combined with AND. A total of 568 records were identified through the PubMed database search. We excluded 486 hits by reviewing the abstract and/or title for nonrelevance to the subject matter. The remaining 82 articles were reviewed in detail for eligibility, and of these, we excluded 36 (16 were review articles with no original data and 2 were articles with data already published). Reviewing the reference lists of pertinent records, we identified 7 additional relevant articles. The last search was performed on November 7, 2016.
For obvious ethical reasons, no randomized controlled trials have investigated the teratogenic effect of paternal MTX or the effects that MTX may have on spermatogenesis. Most available data arise from case reports and small case series as well as animal studies. A total of 53 papers were included in this review, including 17 animal studies and 36 human case reports.
MTX and Sperm Quality
The effect of MTX on spermatogenesis was investigated in several rodent studies. Only studies that reported adverse effects have been published. Germ cell apoptosis in rat testes were demonstrated as early as 2 days after a single injection of low-dose MTX intraperitoneal (IP) (20 μg/kg)36 and a murine study demonstrated a dose- and time-dependent reduction in sperm count and impaired morphology after treatment with 5 to 40 mg/kg MTX for 5 to 10 weeks.37 Histology of the testis revealed gonadal toxicity with decreased germ cell count. The effect was ameliorated by pretreatment with folic acid.38 Similar findings have been reported in other studies, with reduction in sperm count, motility, morphology,39–41 as well as a decrease in the diameter of the seminiferous tubules and increase in the interstitial space.42,43 Koehler et al44 found a reduction in spermatogenic activity and morphological changes after repeated MTX exposure in rabbits and speculated that the effect was because of MTXpg synthesis in the testes. They noticed that the Sertoli cells were all normal, and there were no changes at the level of the cellular junction (i.e., the BTB). Neither the Leydig cells nor the basement membranes were affected. Despite the normal appearance of the endocrinological cells, they measured an increase in follicle-stimulating hormone and proposed that this resulted from a loss of inhibin production from the germinal epithelium.45 Another study showed a duration-dependent reduction in testosterone levels in rats treated with MTX.46 follicle-stimulating hormone and luteinizing hormone levels were not measured. Meistrich47 and Russel48 both demonstrated that the cells primarily affected by HD-MTX were subtypes of spermatogonia, whereas the stem cells or more developed germ cells were not affected.
MTX, Sperm DNA Damage, and Transmission by Ejaculate
Animal studies have shown chromosomal abnormalities in sperm after MTX exposure. Alam et al39 showed a significant increase in chromosomal aberrations in murine spermatocytes after a single dose of IP MTX. The type of aberrations included both hypoploidy, hyperploidy, and univalents. Choudhury et al49 also investigated the influence of a single dose MTX IP to germ cells in male mice. They detected a significantly higher number of aberrant spermatogonial metaphases, mostly chromatid gaps and breaks within 24 hours. Four weeks posttreatment, the percentage of aberrant primary spermatocytes was increased but they could not detect a significant increase in abnormal sperm morphology 8 weeks after exposure. Padmanabhan et al37 assessed DNA damage in the germ cells from mice after repeated drug administration. Besides gonadal toxicity (see above), they also observed significant DNA strand breaks and increased cell death after exposure to MTX. They concluded that the observed morphological changes could be caused by DNA damage, and the mode of cell death was attributed to the inhibition of DNA synthesis.
Riccardi et al50 measured MTX in the interstitial space and the seminiferous tubules of rat testes after 4 hours of MTX infusion. The drug was detected in the interstitial space, i.e., outside the BTB, with levels 2- to 4-fold lower compared with corresponding plasma levels. In the seminiferous tubules, levels 18- to 50-fold lower than in plasma were detected. They concluded that a significant BTB existed at the tubular level. Krakower et al51 examined the in situ polyglutamination of MTX in rat testis after a single MTX 10 mg/kg IP injection. Three hours later, both MTX and MTXpg were detected, and the MTXpg levels increased to 50% of the total measured MTX in the testis after one week. The measurements were performed using supernatants from extracted tissue, and it is not clear from which cell types or compartments the metabolites originated. Other studies have also measured MTX in the supernatant of animal testes, but none have measured it in the ejaculate.45,52
Paternal MTX Exposure and Pregnancy Outcomes
One small animal study found that male treatment with MTX resulted in fewer litters in female mice after mating.40 Successful mating was observed soon after MTX was stopped. The female mice were not examined for biochemical pregnancies, and the authors did not state whether any of the mice miscarried, as only the number of litters was reported. These results are not easily extrapolated to humans.
MTX and Sperm Quality
An adverse effect of MTX on human sperm quality was first postulated in 1959 by Van Scott and Reinertson.53 They described 2 psoriatic patients who were treated with a single intravenous dose of MTX and who had low sperm counts 12 to 14 days later. The exact dose was not reported, but was between 0.5 and 5 mg/kg. They did not evaluate sperm quality before MTX exposure, and no follow-up samples were collected to determine if the effects were reversible. A potential adverse effect on sperm quality was subsequently suggested in 3 other case reports.54–56 A psoriasis patient treated with low-dose MTX (5–20 mg/wk) had increased abnormal sperm cells during treatment, although sperm counts fluctuated between different samples, with reversal of the quality to normal within 3 months after cessation of MTX.54 Hinkes55 reported severe oligospermia, demonstrated by a decreased sperm count, but with normal sperm morphology and volume in a patient with acute leukemia who received maintenance chemotherapy with MTX 2.5 mg/d and cyclophosphamide, an alkylating drug known to cause infertility.33 A follow-up sample 2.5 years after treatment was normal. Other case series where MTX had been given as HD therapy together with other chemotherapeutics, including cyclophosphamide, also found a transient adverse effect on sperm quality.57–60
Sussman et al56 investigated sperm samples from a patient with psoriasis and psoriatic arthritis treated with low-dose MTX (15 mg/wk). During the initial 3 months after cessation of treatment, they documented oligospermia that reverted to normal, but recurred within 3 weeks after the drug was resumed because of worsening of disease. Morphologic examination revealed an increase in immature germ cells in the ejaculate. The rapid adverse effect on spermatogenesis suggested that MTX mainly had an effect on the later stages of development, namely spermiogenesis. At the same time, there was no change in luteinizing hormone, follicle-stimulating hormone, or testosterone. Shafik61 compared sperm samples before, during, and after treatment of a bilateral testicular seminoma treated with repeated intratunical injections of HD-MTX. Sperm samples showed a significant drop in count, motility, and normal morphology during treatment, but returned to pretreatment values 3 months after discontinuation of treatment.
In contrast to the above-cited studies, several reports described no or minimal impact of MTX on spermatogenesis. Gunther62 and subsequently De Luca et al63 found no impact of treatment before, during, or after low-dose MTX on sperm count, morphology, or motility in 11 and 10 psoriasis patients, respectively. Grunnet64 compared sperm samples from 10 psoriatic patients on long-term MTX (1–9 years) with those from 10 patients treated with topical corticosteroids for the same disease and reported that men on MTX had significantly better sperm quality compared with patients treated with steroids. However, only 4 patients on MTX had normal sperm samples according to the authors' criteria despite the 6 patients with impaired quality having not received a higher cumulative dose of MTX. They did not identify a predominance of specific sperm abnormalities and no pretreatment samples were collected for comparison. Subsequently, El Behery et al65 reported 26 patients with psoriasis in whom sperm quality was assessed before treatment and 70 days after treatment with MTX 25 mg/wk orally. There was, however, no sperm analysis during MTX treatment. They found no difference in sperm count, morphology, and motility. Furthermore, 5 patients had testicular biopsies and spermatogenic activity tests performed before and after treatment, and no adverse effects were detected.
Follow-up studies of the fertility in men treated with HD-MTX in combination with other chemotherapeutics showed reassuring results with only limited or no long-term effect on gonadal development, spermatogenesis, or fertility.58,66–69 The long-term effect on spermatogenesis seen in some studies was most likely related to treatment with alkylating agents and radiotherapy.57,60,70
MTX, Sperm DNA Damage, and Transmission by Ejaculate
Human semen consists of spermatozoa and seminal plasma. The latter originates from the seminal vesicles (60%), the prostate (30%), and other glands (10%).71 When male patients are treated with MTX around conception, it could potentially affect the conceptus' DNA, either through a direct genotoxic effect on the spermatozoa or by its presence in the seminal fluid. To date, no studies have measured MTX in human ejaculates, but many other drugs have been detected in similar concentrations as in plasma.72 Three modes of delivery of drugs in semen to the conceptus have been suggested: passage of drug-containing semen from the vagina to the uterus; transfer via the placenta after absorption from the vagina to the maternal circulation; and bound on or in the spermatozoa fertilizing the egg.72 However, seminal fluid does not enter the uterus, and drug transfer to the fetus via the maternal circulation seems negligible. The remaining mechanism, if MTX bound in or on spermatozoa affects the egg without exerting an effect on sperm DNA, is yet to be investigated in humans, but has been shown to occur for metals and cocaine.72
Very few human studies have evaluated the potential genotoxic effect of MTX on sperm cell DNA. Melnyk et al73 investigated spermatocytes from a testis biopsy taken from a man with psoriasis treated with low-dose MTX for 5 months. Screening of meiotic chromosomes revealed both extra and missing bivalents, early dissociation of sex chromosomes, and polyploid cells. Although there was no control, the authors concluded that the findings were similar to those in normal individuals. A study of sperm samples from 4 rheumatoid arthritis patients demonstrated that low-dose MTX did not induce significant chromosome breakage.74 Another case report found no increase in numerical or structural chromosomal abnormalities in sperm from a patient treated 3 years previously with chemotherapy that included MTX.75
For a direct toxic effect of MTX on the germ cells to take place, they must be exposed to MTX during spermatogenesis. Only one human study has measured MTX metabolites in testis tissue from a patient who died from osteosarcoma after 6 months treatment with HD-MTX.76 The total MTX amount detected was 167 ng/mg protein. The analysis was done on supernatant from a testis specimen, so the exact localization of the drug (i.e., blood vessel, the interstitial space, or the seminiferous tubules) could not be determined.
Paternal MTX Exposure and Pregnancy Outcome
A total of 284 pregnancies with known paternal MTX exposure during or 3 months before conception have been published (Table 1).25,28,29,77–86 They resulted in 248 live births, 19 spontaneous abortions, 16 elective terminations of pregnancy (ETOP), and one pregnancy with unknown outcome. Thirteen congenital malformations (including 3 ETOP) were reported. Amniocentesis was performed in 18 cases, and one chromosomal aberration was diagnosed (trisomy 16, the most common chromosomal abnormality, representing 30% of all trisomies87).25,83 The malformations were manifold and not specific for MTX embryopathy (Table 1). Studies that compared exposed individuals with control groups found no increased risk of major congenital malformations,81–83 spontaneous abortions, or ETOP.83 Furthermore, studies reporting exposure during the organogenesis found no increased risk of congenital abnormalities during this sensitive period with a potential risk of exposure through contaminated seminal fluids.25,83 The gestational age at delivery and birth weight were comparable with control groups.82,83 Most ETOP were performed for personal reasons with only a limited number of cases having had a prenatal diagnosis been made before the procedure. Weber-Schoendorfer et al83 speculated that the high number of ETOP observed in their cohort could be explained by fear of MTX teratogenicity. The results from the largest prospective cohort study to date were reassuring, and the authors concluded that a 3-month interval between drug cessation and conception may not be necessary and that amniocentesis in established pregnancies is not required.83
In addition, children who were conceived more than 3 months after cessation of MTX given in combination with other chemotherapeutic agents did not have an increased frequency of congenital malformations.55,67,70,88
An important limitation to most outcome studies is the lack of information about the parents' age and other exposures such as smoking, medical treatment of the mother, and diseases which may influence pregnancy outcomes and cause congenital malformations.89 The high background incidence of major congenital malformations (2.4% in Western countries90) makes it difficult to establish a causal relation between paternal MTX exposure and reproductive outcomes, and it would require thousands of births to rule out a 50% increased relative risk of congenital malformations after paternal MTX exposure. It should be anticipated that malformations would occur in MTX-exposed fetuses, even if MTX was not the cause. Although a retrospective study to investigate the views of immunosuppressive and biological treatments in relation to reproduction and pregnancy outcomes by Ostensen et al29 identified 2 congenital malformations among 9 live births, the study was not designed to examine the association between MTX exposure and pregnancy outcomes.
Another limitation to the published studies is that the study designs do not allow assessment of the risk of preclinical (i.e., between the time of implantation and the menses) or early pregnancy (i.e., in the first trimester) losses potentially caused by paternal MTX toxicity. In general, only 30% of all pregnancies develop into live births, up to 60% are lost before clinical detection of pregnancy and another 10% result in clinical miscarriage. This makes it extremely difficult to investigate a theoretical association between paternal MTX exposure and early pregnancy loss in humans.87 Although the published data are reassuring and most likely only represent a fraction of all pregnancies conceived during paternal MTX exposure, long-term follow-up data to evaluate the risk of cancer, behavioral deficits, and impaired reproductive capacity in the offspring are lacking.
Most of the published studies are case reports and small case series with no control groups. The multifactorial nature of male infertility complicates the interpretation of anecdotal reports,91 and the translation of data from animal studies to humans is not straightforward. There is a paucity of studies exploring the effect of MTX on sperm DNA integrity. The heterogeneity in the types of studies that investigated reproductive outcomes after paternal MTX exposure did not allow us to derive pooled estimates of adverse pregnancy events. Most studies were not designed to examine the correlation between exposure and outcome. This underpins the importance of continuous sharing of cases that expand our knowledge about the potential impact of paternal MTX treatment on pregnancy outcomes.
Animal studies clearly indicate an adverse effect of MTX on spermatogenesis, primarily affecting the germ cells. Only a small number of human studies have investigated the effects of low-dose MTX therapy on sperm quality and male fertility in patients receiving no concomitant treatment. There is a need for more studies to clarify the effects of MTX on sperm quality in man. Reassuringly, all human studies found a normalization of sperm quality within 3 months after treatment cessation. This indicates that the human testes stem cells are not affected by MTX treatment, and that any potential adverse effect is transient and implies that cryopreservation before initiation of treatment is not necessary. Although there is a theoretical risk of DNA damage after paternal periconception MTX exposure causing adverse pregnancy outcomes, the published studies indicate no such risk. A prospective study investigating the effect of MTX on male fertility and subsequent associations with adverse reproductive outcomes would be helpful to provide evidence of a causal link between paternal MTX exposure and outcome. For obvious ethical reasons, such a study is not feasible. Hence, evidence on reproductive outcomes must be retrieved from epidemiological studies. It is reassuring that studies comparing pregnancy outcomes with a control group found no increased risk of adverse events after paternal MTX use. Given the limited data, a conservative approach would be to advise withdrawal of MTX 70 days before conception in men wanting to father children. If continuation is crucial for disease control, the decision should be made on a case-to-case basis keeping in mind the limited literature in this field, with mainly positive clinical outcomes. The use of condoms during pregnancy seems unnecessary, because the potential risk to the fetus is considered negligible. In case the female partner achieves pregnancy during male MTX treatment, amniocentesis is not mandatory and should be weighed against the risk of complications to the procedure.
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