Seizure 2003; 12: 621627

doi:10.1016/S10591311(03)00056-6

Gonadal morphology and sex hormones in male and

female Wistar rats after long-term lamotrigine
treatment
LINE SVEBERG RSTE , ERIK TAUBLL , JOUKO I.T. ISOJRVI , AASMUND BERNER , KJELL
ANDERSEN BERG , ARTO J. PAKARINEN
, ILPO T. HUHTANIEMI , MIKAEL KNIP
& LEIF GJERSTAD
Department

of Neurology, Rikshospitalet, University of Oslo, Oslo, Norway; Department of

Neurology, University of Oulu, Oulu, Finland; Department of Pathology, The Norwegian Radium
Hospital, University of Oslo, Oslo, Norway; The Norwegian School of Veterinary Science, Oslo,
Norway;
Department of Clinical Chemistry, University of Oulu, Oulu, Finland; Department of
Physiology, University of Turku, Turku, Finland; Department of Pediatrics, Medical School, Tampere
University Hospital, University of Tampere, Tampere, Finland

Correspondence to: Dr Line Sveberg Rste, Department of Neurology, Rikshospitalet, 0027 Oslo, Norway.
E-mail: line.sveberg.roste@rikshospitalet.no

Drug-induced disturbances in reproductive hormones and gonadal morphology have been observed both in patients with epilepsy
and in non-epileptic animals. Less is known about the influence of newer antiepileptic drugs including lamotrigine on reproduction. Lamotrigine is now increasingly used both in epilepsy and psychiatric disorders. Sixty-five Wistar rats were fed by gastric
tube either 5 mg kg1 lamotrigine solution (males = 15, females = 20) or 0 (vehicle control, males = 15, females = 15) twice
daily for 90 days. In males, no significant differences were found in body or testicular weight. Testicular atrophy was observed
in one control animal and in two of the rats receiving lamotrigine. No morphological changes were seen in the other organs
investigated (liver, kidney, pancreas, brain, lymphatic tissue, heart). None of the animals showed over-expression of p53. No significant differences were observed between the control rats and the male rats receiving lamotrigine regarding testosterone, FSH
and LH. In females, no changes in ovarian morphology or alterations in other tissues were observed. Serum testosterone, FSH,
LH, insulin and progesterone remained unchanged in the lamotrigine treated animals, while serum estrogen was significantly
reduced.
2003 BEA Trading Ltd. Published by Elsevier Science Ltd. All rights reserved.
Key words: lamotrigine; hormones; gonadal morphology; male; female; rat.

INTRODUCTION
Endocrine problems and reduced fertility affect both
men and women with epilepsy. Drug-induced changes
are one possible explanation16 , but the precise role
of the different antiepileptic drugs in these effects has
been questioned7, 8 . Some studies however, are based
on patients on polytherapy, where the effects of single
drugs are difficult to assess912 . In monotherapy studies, several liver-enzyme inducing drugs have been
found to reduce the levels of sex steroid hormones in
both males and females1316 . On the other hand, valproate which does not have the same effect on liver
10591311/$30.00

enzymes, seems to induce hyperandrogenism, menstrual disturbances and polycystic ovaries in women
with epilepsy46, 17 . Elevated androgens have also
been reported in men on valproate18 . Animal studies
support a drug-specific effect of valproate on ovarian
morphology and endocrinology1922 . In males, case
studies indicate valproate-induced structural gonadal
changes23, 24 , which also gains support from animal studies demonstrating testicular atrophy in rats
treated with valproate25 . Thus, several antiepileptic
drugs may influence endocrine mechanisms and call
for caution in the use for patients of reproductive
age.

2003 BEA Trading Ltd. Published by Elsevier Science Ltd. All rights reserved.

622

Lamotrigine can be an alternative to many of these

older drugs including both enzyme inducers and valproate. There is, however, limited information regarding possible endocrine effects of lamotrigine. In this
study, we assessed the effect of lamotrigine on gonadal
and hormonal function in non-epileptic rats.

L. Sveberg Rste et al.

light microscopic evaluation. For all tissues studied,

a blinded microscopic evaluation of the sections was
made.

Immunohistochemistry
Staining for p53

METHODS
Sixty-five (30 males, 35 females) Wistar rats (Mllegaards Avlslaboratorium, Skensved, Denmark) were
fed perorally through a gastric tube with lamotrigine
mixture or placebo solution twice daily for 90 days.
All animals were at the age of 80 days at the beginning of the treatment. The rats were caged in triplets
in Macrolon III cages at constant temperature and humidity on a 12-hour light and darkness schedule at an
air exchange rate at 18 changes/hour. The rats were
kept on B&K pelleted rat diet and given tap water ad
libitum. The animal ethical committee at the National
Hospital, University of Oslo, Oslo, Norway approved
the study.
The lamotrigine mixture was made by dissolving
lamotrigine (gift from GlaxoSmithKline AS, Norway), in methylcellulose 0.25% in double distilled
water. Male and female rats were divided into two
groups each, receiving either 5 mg kg1 of lamotrigine solution twice daily (n = 20 males; n = 15 females) or control solution (n = 15 males; n = 15 females). The animals receiving lamotrigine were given
half-daily doses the first week. They were weighed
on the first day of the study, and the doses were later
adjusted according to weight changes every week.
After 90 days, all animals were weighed and thereafter killed. This was done by means of exanguination under pentobarbital narcosis by abdominal artery
puncture, 46 hours after their last medication. The
blood was collected and serum frozen at 20 C until
analysed. Each testicle and each ovary was weighed
separately and immediately cut in two pieces. One
half was formaline fixed in 4% buffered formaldehyde for morphological and immunohistochemical
examination. The other half of the testis/ovary was
immediately stored at 70 C until further analysis.

Testicular/ovarian sections (25 m) were mounted

on silane-coated slides, deparaffinized and rehydrated. A mouse monoclonal p53 antibody (Oncogene
Science, Cambridge, MA) was used as primary antibody. The slides were incubated with p53 antibody
diluted 1:100. Staining was performed with labelled
Avidin-Biotin (LAB), Vectastain kit, Vector26, 27 . We
used microwave 2 5 minutes in citrate buffer, pH
6.0 as antigen retrieval technique. Negative control
sections were subject to similar procedure, with exclusion of the primary antibody application. Only
nuclear staining was regarded as positive.

Flow cytometric DNA measurements (FCM)

(male rats)
Nuclear suspensions were prepared from thawed,
fresh frozen testicular specimens that were minced
in slices in 10 mM phosphate buffer and filtered
through a 50 m filter. The preparation and staining of nuclei for FCM was performed according to
the detergent-trypsin method developed by Vindelv
et al.28 using solution A (trypsin 0.003 mg ml1 ), solution B (trypsin inhibitor 0.50 mg ml1 +ribonuclease
A 0.01 mg ml1 ) and solution C (spermin tetrachloride 0.01 mg ml1 +propidium iodide, 0.41 mg ml1 ).
Single cell suspensions from each specimen was
mixed with suspension of chicken and rainbow trout
red blood cells for internal DNA standards.
The nuclear DNA content was measured in a FACSCalibur flow cytometer. An argon ion laser from
which the 488-nm line was used to produce the excitation light. The output signals were sorted by a
256-channel analyser and presented as histograms.
The DNA amount was calculated in relation to two
internal standards, chicken and trout erythrocytes.

Histology
Hormone analyses
In addition to testicular/ovarian tissue, specimens of
liver, pancreas, kidney, lung, heart, lymph nodes and
brain including both hemispheres, brainstem and cerebellum were fixed in 4% neutral buffered formaldehyde. The specimens were routinely processed,
embedded in paraffin, and 5 m thick sections were
cut and stained with haematoxylin and eosin for

Serum testosterone and progesterone concentrations

were measured by the Chiron Diagnostics ACS:180
automated chemiluminescence system using ACS:180
analyzer (Medfield, MA, USA). The sensitivity of
the testosterone assay was 0.4 nmol l1 . The coefficient of intra-assay variation was 6.7% and that of the

Gonadal morphology and sex hormones after long-term lamotrigine treatment

inter-assay variation was 8.6%. The respective values

for the progesterone assay were 0.5 nmol l1 , 4.6 and
7.5%. Serum 17-estradiol concentrations were analysed by radioimmunoassay with kits obtained from
Orion Diagnostica (Turku, Finland) after extraction of
the serum samples with ether/ethyl acetate (9:1). The
sensitivity of the estradiol assay was 5 pmol l1 and
the intra-assay and inter-assay variations were 4.5 and
4.1%, respectively. Serum insulin concentrations in female rats were analysed with a RIA kit for rat insulin
(Linco Research Inc., St. Louis, MO, USA) with a
sensitivity of 15 pmol and intra-assay and inter-assay
coefficients of variation of less than 8%.
Serum levels of FSH and LH were measured using in-house immunofluorometric assays (Delfia,
Perkin-Elmer-Wallac, Turku, Finland) as previously
described2931 . The antibodies used in the FSH assay were a monoclonal antibody against recombinant
human FSH (FSH 56A) and a polyclonal antibody
against recombinant human FSH (R93-2705), both
donated by Organon (Oss, The Netherlands). The
minimum levels of detection for FSH and LH were
0.1 and 0.04 g l1 , respectively, using the NIH-RP-2
rat gonadotropin standards. The intra-assay CV for
both assays was <5%, and all samples to be compared were analysed in the same assay, to eliminate
inter-assay variation.

Lamotrigine serum concentrations

The serum lamotrigine concentrations were analysed
with HPLC (Perking Elmer Co) using a UV-detector
(Diode array Detector 235C). The sensitivity of the
assay was 2 mol l1 , the coefficient of intra-assay
variation was 1.2%, and the inter-assay variation was
7.6%.

Statistics

623

of variances, for several of the parameters studied.

Median and quartile deviations (QD) are given in the
tables. Statistical analyses were performed using the
KruskallWallis test followed by MannWhitney test
with Bonferroni corrections for multiple comparisons
using the SPSS package.

RESULTS
All animals except one tolerated the treatment and the
gastric tube feeding without any signs of discomfort.
One female animal receiving lamotrigine died of oesophageal rupture caused by the gastric tube leading
to intra-thoracic bleeding. There was no reduction in
motor activity or any observed changes in the thriving in the animals, including their body weights. In
male rats, the median serum concentration of lamotrigine were 45 mol l1 , 4 hours after the last dose
and 35 mol l1 , 6 hours after the last dose. The respective median serum concentration of lamotrigine
in female animals was 45.2 and 38.6 mol l1 .
Male rats
There was no significant body weight difference between the lamotrigine treated animals and the control
group (Table 1). Similarly, there was no significant
difference in testicular weights of these animals compared to the controls.
On the basis of the light microscopical findings
in the testicles, the animals were classified into four
groups (Table 1). Group Anormal findings, group
Bslight atrophy, group Cmoderate, diffuse atrophy and group Dextensive diffuse atrophy. Eighteen
of the 20 lamotrigine treated animals were classified into group A, 1/20 into group C and 1/20 into
group D. In the control group, 14/15 were classified
into group A, and one into group B. This difference
between lamotrigine treated and controls was not

Morphological studies

Mean and standard deviation for all analyses were

calculated. The statistical analyses were performed
with a one-way ANOVA technique using a commercially available software package (SPSS Inc.,
Chicago, USA) for all data. Data with a skewed
distribution were analysed with the non-parametric
KruskallWallis test (specified in tables). A chi-square
test was performed when appropriate.
Hormonal studies

Non-parametric statistics were chosen due to lack of

normal distribution and/or large differences in residual variances according to Levenes test of equality

Table 1: Effects of chronic LTG treatment on endocrine

function and testicular morphology in male Wistar rats.

(nmol l1 );

Testosterone
median (QD)
Estrogen (pmol l1 );
median (QD)
FSH (ng ml1 ); median (QD)
LH (ng ml1 ); median (QD)
Animal weight (g); mean (SD)
Gonadal weight (g); mean (SD)
Testicular pathology

Control

Lamotrigine

3.55 (2.15)

4.44 (2.05)

ND

ND

4.91 (0.59)
0.61 (0.20)
440.6 (27.8)
1.9 (0.2)
1/15a

5.63 (0.59)
0.72 (0.25)
422.6 (26.5)
1.7 (0.4)
2/20b

ND, not detectable.

a One animal classified within group B, see text. b Two animals
classified within groups C and D, see text.

624

L. Sveberg Rste et al.

Table 2: Effects of chronic LTG treatment on endocrine function and ovarian morphology in female Wistar rats.

(nmol l1 );

Testosterone
median (QD)
Estrogen (pmol l1 ); median (QD)
Progesterone (nmol l1 ); median (QD)
FSH (ng ml1 ); median (QD)
LH (ng ml1 ); median (QD)
Insulin (pmol l1 ); median (QD)
Animal weight (g); mean (SD)
Gonadal weight (g); mean (SD)
Number of cysts/section; mean (SD)
Number of corpora lutea/section; mean (SD)
Number of secondary follicle/section; mean (SD)

Control

Lamotrigine

1.8 (0.7)
71 (72.5)
25.5 (20.9)
26.2 (2.1)
0.89 (0.65)
70.5 (33.8)
254.5 (18.0)
46.1 (10.0)
0.47 (0.51)
10.7 (3.1)
5.2 (2.4)

1.4 (0.4)
27* (15.5)
43.1 (33.7)
31.2 (3)
0.73 (0.47)
81 (24)
260.8 (25.6)
43.0 (9.9)
0.54 (0.66)
8.4 (4.4)
2.5 (1.8)

Ovarian morphological data described in detail in Reference 19.

* P = 0.024 compared to control.

statistically significant. No pathological changes were

found in the interstitial compartment of the testes in
either of the two groups. Differences in staining distribution and intensity of Leydig cells were not observed
between the treated rats and the control animals. No
significant morphological changes were observed in
the heart, lungs, kidneys, liver, lymph-nodules or
CNS of the lamotrigine treated animals.
To explore a possible carcinogenic effect, we performed flow cytometric analysis and immunohistochemistry for p53 expression. All specimens were
diploid, mean CV, 3.7 (2.75.2) (CV = coefficient of
variation of signals within the main peak, providing a
measurement of the peak width). There were no significant differences between the groups with mean CV
in controls (3.9) and in lamotrigine treated rats (4.2).
Aneuploid cell populations were not detected in either of the two groups. No over-expression of p53 was
seen in the testes of the lamotrigine treated animals.
There were no significant differences between the
lamotrigine treated rats and the rats receiving control solution regarding serum testosterone, FSH or LH
levels (Table 1). The serum estrogen concentrations
were not detectable in either of the two groups. This
reflects that the values are below the detection limit
of this method (5 pmol l1 ), not significantly different from zero. The analysis was unchanged when the
zero-values were replaced with the lowest level of detection.

Female rats
No changes were observed in ovarian weight or ovarian morphology after long-term lamotrigine treatment
(Table 2). Further, there were no alterations in body
weight, body temperature or tissue morphology of the
liver, kidney, pancreas, brain, brainstem, cerebellum,
lymphatic tissue or heart. The morphological findings
in female rats are described in detail elsewhere20 .

There was a significant reduction in the serum estrogen levels in the lamotrigine treated animals compared
with controls (P = 0.024). No significant differences
were found between the lamotrigine treated rats and
the controls in serum concentrations of testosterone,
FSH, LH, insulin or progesterone (Table 2).

DISCUSSION
Male rats
There were no significant changes in testicular morphology between animals treated with lamotrigine or
those receiving control solution. Two of 20 lamotrigine treated animals, however, showed moderate to extensive atrophy of the testis compared to one control
animal with moderate atrophic changes. The two animals in question did not differ from the other animals
in behaviour, body weight or serum concentrations of
the drug. However, the serum concentrations achieved
in lamotrigine treated animals were in general higher
than what is commonly used when treating humans.
Thus, an effect of lamotrigine at high serum concentrations cannot be excluded.
To evaluate whether these morphological findings
had a pre-cancerogenic potential, measurement of p53
expression and flow cytometry was done. The p53
gene is regarded as one of the most frequently mutated genes associated with cancer development. More
than 50% of human primary tumours over-express a
variety of mutant p53 proteins32, 33 . Most seminomatous and non-seminomatous germ cell tumours exhibit increased p53 protein expression evaluated by
immunohistochemistry34 . Neither the two lamotrigine treated animals with different amount of atrophy
nor the rest of the lamotrigine group or the controls
showed any over expression of p53, speaking against
a carcinogenic effect of the drug as evaluated by p53
expression. This is also in accordance with the flow

Gonadal morphology and sex hormones after long-term lamotrigine treatment

cytometric results; cells with abnormal DNA were not

detected.
No significant differences in endocrine hormone levels were found between the rats on long-term lamotrigine treatment and controls. This is in contrast to
the results after long-term treatment of valproate in
which a significant rise in the serum FSH and LH
values was found21 . The majority of these high-dose
valproate treated animals also exhibited extensive testicular atrophy25 .

Female animals
The ovaries of the long-term lamotrigine treated animals showed features indistinguishable from the
controls in terms of morphology, and there were no
differences in ovarian weight. In particular, no significant differences were observed regarding ovarian
cysts, number of corpora lutea or number of secondary follicles between the control rats and the rats
receiving lamotrigine (see Reference 20 for details).
As animals with cystic ovaries have very irregular
oestrous cycles35 , this finding is in support of normal
oestrous cycles in the lamotrigine treated rats.
Regarding endocrinological consequences, no effect
was seen on circulating LH and FSH concentrations,
indicating no centrally mediated effect of the drug.
A peripheral effect could be questioned as an isolated reduction in estrogen concentrations was found
while the levels of progesterone remained unaltered.
One cannot, therefore, exclude the possibility that
lamotrigine reduce the conversion of testosterone to
estrogen. This would, however, usually result in an
increase in testosterone, which was not observed in
our animals. Further, a reduction in the conversion
from testosterone to estrogen would most commonly
imply an effect on aromatization35 . Such an effect
of lamotrigine has not been described to date. Alternatively, an increased metabolism of estrogen might
contribute to a reduced estrogen level. Increased
specific metabolism of estrogen is however most
commonly seen in chronic liver disease. There was
not observed any liver pathology in our lamotrigine
treated rats, lamotrigine might however interfere with
estrogen metabolism in a similar way. On the other
hand, drug related hypermetabolism is normally related to other steroids than estrogen36 . So far, we
have no definite explanation to the reduction in estrogen, which, however, is less than what has been
described in valproate treated female rats20 . In rats
on long-term valproate treatment, barely detectable
estrogen concentrations were found, with median
estrogen levels of 1.5 pmol l1 and 0 in animals receiving 200 or 300 mg kg1 dose1 , respectively. The
median estrogen concentrations in the lamotrigine

625

treated animals on the other hand, was 27 pmol l1 .

The valproate-induced effects were found in animals
having a mean serum concentration 6 hours after last
dose of 331 mol l1 , which is within therapeutic
range for humans20 . Rats treated with lamotrigine had
relatively high serum concentrations of 38.6 mol l1
after 6 hours. Further, the valproate treated rats also
had ovarian morphological changes with a significant
increase in the number of follicular cysts, whereas
no changes were seen in the ovaries of lamotrigine
treated rats. These animal observations of valproate
support the findings in humans46, 12, 37, 38 . As yet,
no clinical observations have disclosed the possibility
of lamotrigine-induced interference with the estrogen
balance3945 . In a recent study by Stephen et al.44 ,
patients on lamotrigine monotherapy did not have
any alterations in reproductive hormones including
gonadotropins, whereas the women on valproate had
significantly higher testosterone levels. Furthermore,
substitution of valproate with lamotrigine has been
observed to reduce the occurrence of polycystic
ovaries/hyperandrogenism, obesity, hyperinsulinemia
and lipid abnormalities in women with epilepsy45 .

CONCLUSION
No significant changes in gonadal morphology were
noticed either in male or in female rats after long-term
treatment with lamotrigine at high concentrations. In
the male animals, no disturbances of reproductive hormones were observed. The female animals showed an
isolated reduction in estrogen level, which is not, however, observed in human patients.

ACKNOWLEDGEMENTS
This study has been supported in part by the Norwegian branches of ILAE and IBE, and the Norwegian
Research Council.

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