Ó 2012 Wiley Periodicals, Inc. Birth Defects Research (Part A) 00:000–000 (2012) Teratogen Update: Methotrexate Sara C. Hyoun,1 Sarah G. Obicˇan,2 and Anthony R. Scialli2,3* 1George Washington University, School of Medicine and Health Sciences, Washington, D.C 2Department of Obstetrics and Gynecology, George Washington University Medical Center and Reproductive Toxicology Center, Washington, D.C 3Tetra Tech Sciences, Arlington, Virginia Received 9 December 2011; Revised 9 January 2012; Accepted 13 January 2012 Methotrexate and aminopterin are folic acid antagonists that inhibit dihydrofolate reductase, resulting in a block in the synthesis of thymidine and inhibition of DNA synthesis. Methotrexate has been used for the treatment of malignancy, rheumatic disorders, and psoriasis and termination of intrauterine pregnancy. Recently, methotrexate has become a standard treatment for ectopic pregnancy. The misdiagnosis of an intrauterine pregnancy as an ectopic pregnancy can result in exposure of a continuing pregnancy to dose levels of methotrexate of 50 mg/m2 (maternal body surface area). Experimental animal studies have associ- ated methotrexate therapy with embryo death in mice, rats, rabbits, and monkeys. Structural malformations have been most consistently produced in rabbits at a maternal dose level of 19.2 mg/kg. Abnormalities in rabbits include hydrocephalus, microphthalmia, cleft lip and palate, micrognathia, dysplastic sacral and cau- dal vertebrate, phocomelia, hemimelia, syndactyly, and ectrodactyly. Based on human case reports of metho- trexate exposure during pregnancy, a methotrexate embryopathy has been described that includes growth deficiency, microcephaly, hypoplasia of skull bones, wide fontanels, coronal or lambdoidal craniosynostosis, upswept frontal scalp hair, broad nasal bridge, shallow supraorbital ridges, prominent eyes, low-set ears, maxillary hypoplasia, epicanthal folds, short limbs, talipes, hypodactyly, and syndactyly. This syndrome may be associated with exposures between 6 and 8 weeks after conception and dose levels of 10 mg/week or greater. More recent case reports of methotrexate exposure for the misdiagnosis of ectopic pregnancy involve treatment before 6 weeks after conception and have raised the suggestion of a distinct syndrome due to such early exposures. Tetralogy of Fallot and perhaps other neural crest cell-related abnormalities may be features of this early syndrome. A disproportionality analysis of methotrexate and aminopterin case reports and series provides support for pulmonary atresia, craniosynostosis, and limb deficiencies as reported more often than expected in methotrexate-exposed children. Denominator-based data will be welcome to better define elements of a methotrexate embryopathy and possibly to distinguish an early expo- sure syndrome from anomalies traditionally associated with methotrexate exposure. Birth Defects Research (Part A) 00:000–000, 2012. Ó 2012 Wiley Periodicals, Inc. Key words: methotrexate; aminopterin; dihydrofolate reductase; malformations; teratology; ectopic preg- nancy; abortion INTRODUCTION has also been used with misoprostol for voluntary abor- tion (Moreno-Ruiz et al., 2007) and in the treatment of ec- Methotrexate is a folic acid analog that inhibits dihy- topic pregnancy (American College of Obstetricians and drofolate reductase, thereby decreasing the availability of Gynecologists, 2008; Barnhart, 2009). Typical doses for tetrahydrofolate (Fig. 1). Because tetrahydrofolate is an the treatment of psoriasis or rheumatoid arthritis are 7.5 important cofactor in thymidylate synthesis and de novo to 20 mg/week. The methotrexate dose used in the treat- purine synthesis, methotrexate inhibits the synthesis of DNA and has antiproliferative activity. *Correspondence to: Anthony R. Scialli, Tetra Tech Sciences, 2200 Wilson Methotrexate has been used clinically in the treatment Blvd, Suite 400, Arlington VA 22201. E-mail: [email protected] of malignancy, psoriasis, rheumatoid arthritis, and other Published online in Wiley Online Library (wileyonlinelibrary. com). autoimmune and inflammatory disorders. Methotrexate DOI: 10.1002/bdra.23003 Birth Defects Research (Part A): Clinical and Molecular Teratology 00:000–000 (2012) 2 HYOUN ET AL. resultsonaperfetusbasisanddidnotreportstatistical analy- ses.Despitetheselimitations,itappearsthatintheratandmon- key, embryolethality was more common than teratogenesis, which was relatively unusual. Mouse and rabbit produced clearmalformationsyndromes,albeitatexposurelevelsmuch higheronamilligramperkilogrambasisthandoselevelsused inhumantherapy. Mouse Skalko and Gold (1974) administered methotrexate in- traperitoneal (ip) to ICR mice on gestation day (GD) 10 (plug 5 GD 0) at dose levels of 0.3 to 50 mg/kg. There was an increase in resorptions at 10 mg/kg, with no reported adverse effect at 5 mg/kg. Malformations were increased at 25 and 50 mg/kg and consisted predomi- nantly of ectrodactyly and cleft palate. Darab et al. (1987) also produced median facial clefts in fetuses after treat- ment of pregnant C57Bl/6J mice with methotrexate 20 mg/kg ip on GD 9 (plug 5 GD 0). Elmazar and Nau (1992) treated Han:NMRI mice with ip methotrexate on GD 8 (24 hours after detection of vaginal plug 5 GD 0) at dose levels of 1.25 to 20 mg/kg. There was increased embryolethality at 2.5 mg/kg, but no increase in exence- phaly or any other external malformation at any dose level. Skeletal and visceral malformations were not eval- Figure 1. Methotrexate blocks the reduction of dihydrofolate to uated. tetrahydrofolate, a step in the cycle that results in transfer of a Sutton et al. (1998) used a transgenic mouse with a methyl group from serine to deoxyuridine monophosphate mutation in dihydrofolate reductase that did not bind to (dUMP) to produce deoxythymidine monophosphate (dTMP), methotrexate and was, therefore, not inhibited by it. This which is necessary for DNA synthesis. The bottom panel shows 5 10 dihydrofolate reductase mutation resulted in protection that N ,N -methylene tetrahydrofolate serves as a shuttle for from methotrexate teratogenicity. Methotrexate was the transfer of a methyl group (dashed circle) from serine to administered at 30 mg/kg ip on GD 10 (plug 5 day 0 or dUMP to produce dTMP day 0.5, depending on time of mating) to pregnant mice af- ter hemizygous mating. Some fetuses in each litter carried 2 resistant dihydrofolate reductase, whereas other fetuses ment of ectopic pregnancy is 50 mg/m , often given had the wild type dihydrofolate reductase. Fetuses were intramuscularly and repeated at intervals depending on evaluated on GD 14, with the exception of two litters that the protocol used and the patient response. A 60-kg (132- were permitted to go to term. Facial clefts and limb defects, lb) woman who is 168 cm (5 feet, 6 inches) tall has a 2 the only malformations evaluated, occurred in 37% of body surface area of 1.67 m . Her methotrexate dose for transgenic fetuses compared with 77% of nontransgenic ectopic pregnancy will be 83.5 mg or 1.4 mg/kg. siblings. Resorptions were not genotyped. Methotrexate therapy is associated with nausea, vomit- None of the mouse studies showed an increase in ing, mucositis, immunosuppression, alopecia, pneumoni- malformations at ip methotrexate dose levels lower than tis, and liver injury. Cotreatment with leucovorin (also 20 mg/kg. called citrovorum factor and 5-formyltetrahydrofolate) is used in some treatment regimens to reduce the toxicity of methotrexate. One of the accepted protocols for ectopic Rat pregnancy treatment includes the use of methotrexate Wilson (1971) reported that methotrexate given ip to (1 mg/kg) on days 1, 3, 5, and 7 with administration of an unspecified rat strain on GD 9 (GD 0 not defined) leucovorin on days 2, 4, 6, and 8 (American College of caused 64% resorptions and 30% malformations. Addi- Obstetricians and Gynecologists, 2008). tional details were not provided. In a subsequent publi- The therapy of ectopic pregnancy with methotrexate cation from Jordan et al. (1977), Wistar rats treated on results in the administration of this medication to a GD 9 (positive smear 5 GD 0) with ip methotrexate were woman known to be pregnant. The misdiagnosis of ec- described as showing an increase in resorptions at a dose topic pregnancy in a woman with an intrauterine preg- level as low as 0.1 mg/kg, although effects were not eval- nancy or treatment of a woman with a coexisting ectopic uated statistically. Treatment with methotrexate (2.5 mg/ and intrauterine pregnancy can result in methotrexate ex- kg) before implantation on GD 4 had little effect on im- posure of a continuing pregnancy. plantation failure or resorption, but embryo death or resorption increased with treatment on each subsequent EXPERIMENTAL ANIMAL STUDIES day until GD 6, when 100% resorptions occurred after treatment. Malformations occurred in 75% of surviving Embryotoxicityhasbeendescribedaftermethotrexatetreat- fetuses after treatment with methotrexate (0.3 mg/kg on ment in mice, rats, rabbits, cats, and monkeys; however, the GD 9). The nature of the malformations was not dis- studies reporting these effects predate modern embryofetal cussed, but in another publication (Wilson et al., 1979) toxicity study design. These older studies typically reported methotrexate 0.3 mg intravenously (iv) on GD 11 in Wis- Birth Defects Research (Part A) 00:000À000 (2012) METHOTREXATE 3 tar rats produced malformations confined largely to the treatments resulted in defects primarily confined to the caudal vertebrae.