The effect of methotrexate on fertility parameters in men with immune mediated diseases

Methotrexate (MTX) is the cornerstone in the treatment of several immune
mediated diseases, such as Inflammatory Arthritis, Psoriasis and Crohn*s
Disease. Besides being teratogenic, MTX may also have a negative impact on
sperm quality. Recommendations for the use of MTX in men wishing to conceive
are inconclusive, mainly because of the lack of studies about the impact of MTX
on semen and spermatozoa.

To determine whether MTX can be quantified in seminal plasma and spermatozoa of
MTX-naïve and chronic-MTX users, and to compare the concentration of MTX in
seminal fluid and spermatozoa between MTX-naïve users and chronic-MTX users and
to evaluate the effect of MTX concentration on parameters of sperm quality,
including the DNA fragmentation index (DFI). In addition, sperm and blood
samples from healthy controls will be processed to measure the concentration
and enzymatic activity of Gamma-glutamyl hydrolase (GGH) and other enzymes
involved in the intracellular metabolism of MTX.

This study consists of three parts:

Part I has been completed. This was a validation phase. Two validation-control
groups were recruited for the validation of the quantification of MTX in blood
and in semen.

Part II consists of a case-control study where two different groups of patients
and one group of healthy controls will be recruited:

1. MTX-naïve: Twenty five male patients with immune mediated diseases who will
start treatment with MTX.
2. Chronic-MTX users: Twenty five male patients with immune mediated diseases
who have been under treatment with MTX uninterruptedly for at least 1 year.
3. Healthy controls: Twenty five healthy men.

The MTX concentration in blood, seminal plasma and spermatozoa will be measured
before and after MTX exposure in cases from the MTX-naïve group and only after
exposure in cases from the chronic-MTX users group. Cases from both groups and
study-controls will go through the same andrological evaluation, consisting of
a physical examination, medical history, endocrine evaluation, semen quality
analysis and DFI.

Part III is the last part of the study. Blood and sperm samples from 10 healthy
men will be processed to measure the concentration and the activity of GGH and
other enzymes involved in the metabolism of MTX.

All study procedures (incl. signing of the informed consent) except the
analysis of the sperm morphology slides and the enzyme measurements, will be
performed during 1 study visit for controls, 2 study visits for chronic-MTX
cases and during 3 study visits for MTX-naïve cases. These visits will take
place at the Erasmus MC. The sperm morphology slides will be assessed at the
Radboudumc and the enzymes at the Amsterdam UMC.

* Part I of the study (already completed)
Semen validation-controls donated one semen sample obtained by masturbation (in
the hospital). Blood validation-controls donated one blood sample (4 ml) during
a blood draw for routine usual care (i.e. no extra venapuncture required).

* Part II of the study (participants are currently being included)
- Cases in the MTX-naïve group will visit the hospital 3 times. During the
first and second visit (approximately 90 min per visit) one semen sample
(obtained by masturbation) and one blood sample (21 ml) will be collected.
During these visits MTX-naïve cases will undergo a physical examination and
fill in questionnaires. During the third study visit (approximately 30 min) one
semen sample will be obtained and a questionnaire about medication use will be
filled in.
- Cases in the chronic-MTX users group will visit the hospital 2 times. During
the first visit (approximately 90 minutes) one semen sample (obtained by
masturbation) and one blood sample (21 ml) will be collected. During this visit
chronic-MTX users will undergo a physical examination and fill in
questionnaires. During the second study visit (approximately 30 min) one semen
sample will be obtained and a questionnaire about medication use will be filled
in.
- Study-controls will visit the hospital once (45-60 minutes). During the visit
one semen sample and one blood sample (21 ml) will be obtained, a physical
examination is performed and the men will fill in questionnaires.

* Part III of the study
Enzyme-controls will visit the hospital once (approximately 30 min). During the
visit one semen sample and one blood sample (8 ml) will be obtained.

Participants will not benefit from taking part in this study. The results will
provide information about the effects of MTX on male fertility and contribute
to a more evidence based advise on stopping or continuing MTX in males who want
to conceive.