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Male-to-Female Transsexuals Have Female Neuron Numbers in a Limbic Nucleus

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Transsexuals experience themselves as being of the opposite sex, despite having the biological characteristics of one sex. A crucial question resulting from a previous brain study in male-to-female transsexuals was whether the reported difference according to gender identity in the central part of the bed nucleus of the stria terminalis (BSTc) was based on a neuronal difference in the BSTc itself or just a reflection of a difference in vasoactive intestinal polypeptide innervation from the amygdala, which was used as a marker. Therefore, we determined in 42 subjects the number of somatostatin-expressing neurons in the BSTc in relation to sex, sexual orientation, gender identity, and past or present hormonal status. Regardless of sexual orientation, men had almost twice as many somatostatin neurons as women (P < 0.006). The number of neurons in the BSTc of male-to-female transsexuals was similar to that of the females (P = 0.83). In contrast, the neuron number of a female-to-male transsexual was found to be in the male range. Hormone treatment or sex hormone level variations in adulthood did not seem to have influenced BSTc neuron numbers. The present findings of somatostatin neuronal sex differences in the BSTc and its sex reversal in the transsexual brain clearly support the paradigm that in transsexuals sexual differentiation of the brain and genitals may go into opposite directions and point to a neurobiological basis of gender identity disorder.
BSTc neuron numbers. Distribution of the BSTc neuron numbers among the different groups according to sex, sexual orientation, and gender identity. M, Heterosexual male reference group; HM, homosexual male group; F, female group; TM, male-to-female transsexuals. The sex hormone disorder patients S1, S2, S3, S5, S6, and M2 indicate that changes in sex hormone levels in adulthood do not change the neuron numbers of the BSTc. The difference between the M and the TM group (P 0.04) is also statistically significant according to the sequential Bonferonni method if S2, S3, and S5 are included in the M group or if S7 is included in the TM group (P 0.01). Note that the number of neurons of the FMT is fully within the male range. Whether the transsexuals were male oriented (T1, T6), female oriented (T2, T3, T5), or both (T4) did not have any relationship with the neuron number of the BSTc. The same holds true for heterosexual and homosexual men. This shows that the BSTc number of somatostatin neurons is not related to sexual orientation. A, AIDS patient. The BSTc number of neurons in the heterosexual man and woman with AIDS remained well within the corresponding reference group (see Fig. 1), so AIDS did not seem to affect the somatostatin neuron numbers in the BSTc. P, Postmenopausal woman. S1 (& 46 yr of age): adrenal cortex tumor for more than 1 yr, causing high cortisol, androstendione, and testosterone levels. S2 (( 31 yr of age): feminizing adrenal tumor that induced high blood levels of oestrogens. S3 (( 67 yr of age): prostate carcinoma; orchiectomy 3 months before death. S5 (( 86 yr of age): prostate carcinoma; prostatectomy; orchiectomy, and antiandrogen treatment for the last 2 yr. S6 (& 25 yr of age): Turner syndrome (45,X0; ovarian hypoplasia). M2 (& 73 yr of age): postmenopausal status.
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Male-to-Female Transsexuals Have Female Neuron
Numbers in a Limbic Nucleus
FRANK P. M. KRUIJVER, JIANG-NING ZHOU, CHRIS W. POOL,
MICHEL A. HOFMAN, LOUIS J. G. GOOREN, AND DICK F. SWAAB
Graduate School Neurosciences Amsterdam (F.P.M.K., J.-N.Z., C.W.P., M.A.H., D.F.S.), Netherlands
Institute for Brain Research, 1105 AZ Amsterdam ZO, The Netherlands; Department of Endocrinology
(L.J.G.G.), Free University Hospital, 1007 MB Amsterdam, The Netherlands; and Anhui Geriatric Institute
(J.-N.Z.), The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, 230032 China
ABSTRACT
Transsexuals experience themselves as being of the opposite sex,
despite having the biological characteristics of one sex. A crucial
question resulting from a previous brain study in male-to-female
transsexuals was whether the reported difference according to gender
identity in the central part of the bed nucleus of the stria terminalis
(BSTc) was based on a neuronal difference in the BSTc itself or just
a reflection of a difference in vasoactive intestinal polypeptide inner-
vation from the amygdala, which was used as a marker. Therefore, we
determined in 42 subjects the number of somatostatin-expressing
neurons in the BSTc in relation to sex, sexual orientation, gender
identity, and past or present hormonal status. Regardless of sexual
orientation, men had almost twice as many somatostatin neurons as
women (P 0.006). The number of neurons in the BSTc of male-
to-female transsexuals was similar to that of the females (P 0.83).
In contrast, the neuron number of a female-to-male transsexual was
found to be in the male range. Hormone treatment or sex hormone
level variations in adulthood did not seem to have influenced BSTc
neuron numbers. The present findings of somatostatin neuronal sex
differences in the BSTc and its sex reversal in the transsexual brain
clearly support the paradigm that in transsexuals sexual differenti-
ation of the brain and genitals may go into opposite directions and
point to a neurobiological basis of gender identity disorder. (J Clin
Endocrinol Metab 85: 2034 –2041, 2000)
A
NIMAL experiments and observations in human brains
have convincingly shown that sexual differentiation
not only concerns the genitalia but also the brain (1, 2). The
strongly connected and sexually differentiated hypothala-
mus, septum, bed nucleus of the stria terminalis (BST), and
amygdala are implicated in sexually dimorphic patterns of
reproductive and nonreproductive behaviors (2–18).
Gender identity (i.e. the feeling to be male or to be female)
is an important trait of a subject. Transsexuals experience
themselves as being of the opposite sex, despite having the
biological characteristics of one sex (19 –21). In line with the
hypothesis that in transsexuals sexual differentiation of the
brain contrasts with that of the genetic and physical char-
acteristics of sex, our group has recently found that the size
of the central subdivision of the BST (BSTc) was within the
female range in genetically male-to-female transsexuals (22).
In that study the, BSTc was defined on the basis of its va-
soactive intestinal polypeptide innervation, which is prob-
ably mainly derived from the amygdala (23). A crucial ques-
tion resulting from that study was, therefore, whether the
difference according to gender in the BSTc is based on a
neuronal difference in the BSTc itself or rather a reflection of
a difference in innervation from the amygdala. To see
whether the BSTc itself has a neuronal organization that is
opposite to that of the genetic and genitalial characteristics
of transsexuals, we determined the number of somatostatin
(SOM)-expressing neurons in the BSTc, which is the major
neuronal population in this structure (23).
Materials and Methods
Patients
In the present study, 42 brains of patients were analyzed (for an
overview see Table 1). The brains of 34 reference subjects (9 presumed
heterosexual males, 9 homosexual males, 10 presumed heterosexual
females, and 6 male-to-female transsexuals) ranging from 20 –53 yr of
age, together with six brains (three males and three females) of patients
with sex hormone disorders were obtained at autopsy, after the required
permissions had been obtained. Twenty-six of the reference subjects
were the same as used in the earlier study of Zhou et al. (22), whereas
eight new patients (five females, two males, and one homosexual man)
were included because not enough sections were left for the present
study. A Turner syndrome patient (S6) and a castrated (orchiectomized)
male patient (S5) were included in the sex hormone disorder group [n
6; see the legend to Fig. 1; S1, S2, S3, and M2 were also used in the study
of Zhou et al. (22)]. A nontreated individual with strong cross-gender
identity feelings (S7), which were already present since his earliest
childhood, was also analyzed. In addition, we had the exceptional op-
portunity to be able to study the first collected brain ever of a female-
to-male transsexual (FMT). The brains were matched for age, postmor-
tem time, and duration of formalin fixation. Neuropathology of all
subjects was systematically performed by Dr. W. Kamphorst (Free Uni-
versity, Amsterdam, The Netherlands), Dr. D. Troost (Academic Med-
ical Centre of the University of Amsterdam, Amsterdam, The Nether-
lands), or Prof. F. C. Stam (Netherlands Brain Bank, Amsterdam, The
Netherlands). Subjects had no primary neurological or psychiatric
diseases, unless stated otherwise.
Histology
Brains were weighed, generally followed by 37 days of fixation in 4%
formaldehyde at room temperature. The hypothalamic area was sub-
sequently dissected, dehydrated, and embedded in paraffin. Serial 6-
m
Received October 13, 1999. Revised January 11, 2000. Accepted Jan-
uary 11, 2000.
Address all correspondence and requests for reprints to: Frank P. M.
Kruijver, M.D., or Prof. Dick F. Swaab, M.D., Ph.D., Graduate School
Neurosciences Amsterdam, Netherlands Institute for Brain Research,
Meibergdreef 33, 1105 AZ Amsterdam ZO, The Netherlands. E-mail:
F.Kruijver@nih.knaw.nl.
0021-972X/00/$03.00/0 Vol. 85, No. 5
The Journal of Clinical Endocrinology & Metabolism Printed in U.S.A.
Copyright © 2000 by The Endocrine Society
2034
frontal sections were cut on a Leitz microtome, mounted on SuperFrost/
Plus (Menzel-Gla¨ser, Braunschweig, Germany; Art. No. 041300) slides,
and subsequently dried overnight on a hot plate at 58 C.
Immunocytochemistry
Sections were hydrated and rinsed in aquadest 2 5 min and Tris-
buffered saline [TBS; 0.05 m Tris, and 0.9% NaCL (pH 7.6)] for 30 min.
To enhance antigen retrieval [for a review see Shi et al. (24)], sections
were put in a plastic jar [filled with a Citrate 0.05 m (pH 4.0) buffer
solution] and heated to boiling (120 C) for 10 min at 700 W in a micro-
wave oven (Miele Electronic M696, Darmstadt, Germany). After cooling
down for about 10 min, the sections were washed in TBS for 310 min
and preincubated in TBS (pH 7.6) containing 5% nonfat dry milk
(Elk, Campina bv., Eindhoven, The Netherlands) to reduce background
staining. Subsequently, a circle was drawn around the sections with a
Dakopen (Glostrup, Denmark; Code No. S 2002) to prevent the antibody
from diffusing. The sections were: 1) incubated with 300-
L rabbit
antisomatostatin [SOMAAR, 8/2/89; dilution 1:500; for details and spec-
ificity see Van de Nes et al. (25)] in 0.5% Triton X-100 (Sigma, Steinheim,
Germany), 0.25% gelatin, and 5% nonfat dry milk TBS solution [super-
mix-milk (pH 7.6)] overnight at 4 C; 2) washed in TBS-milk 310 min,
followed by a second incubation with goat antirabbit IgG antiserum
(Betsie, NIBR, Amsterdam, The Netherlands; dilution 1:100) in supermix
for 60 min; 3) washed in TBS-milk 3 10 min; 4) incubated with rabbit
TABLE 1. Brain material
NBB patient
number
Age (yr)
Brain
weight (g)
Postmortem
delay (h)
Fixation
time (days)
Clinicopathological diagnosis
Reference men (n 9)
86042 28 1450 24 46 Guillain-Barre´ syndrome
84015 29 1400 13 41 Congenital heart disease; cardiac failure
94040 20 1490 8 82 B-cell lymphoma; viral pneumonia, hemorrhage, heart failure
89042 30 1340 30 26 AIDS; disseminated non-Hodgkin’s lymphoma
84023 37 1370 39 35 Bronchopneumonia
88011 41 1500 21 33 Suicide
92011 47 1520 89 77 Pneumococcen sepsis
95102 53 1383 10 33 Aorta dissection
86048 30 1430 8 35 AIDS, pneumocystic carinii pneumonia, lung tuberculosis,
toxoplasmosis, heroin addiction
Reference women (n 10)
85027 29 1150 13 60 Corrected Fallots’ teratology; cardiac failure, hepatic coma
85041 28 ND 5 44 Cardiogene shock
84025 23 1300 10 35 Acute myeloid leukemia
86032 33 1035 41 20 Adenocarcinoma with metastasis
92037 32 1280 30 45 Bronchopneumonia
88096 34 1400 12 31 AIDS; disseminated histoplasmosis
84002 36 1420 86 51 Multiple fractures; rupture of thoracic aorta
80002 46 1300 3 ND Ovarium carcinoma
89104 49 1260 41 32 Septic shock; lung carcinoma
86039 53 1410 34 17 Myelocytic leukemia; blastomatosis
Homosexuals (n 9)
89031 25 1530 23 28 AIDS; pneumonia
88009 30 1480 5 27 AIDS; cytomegalic infections
87015 30 1640 24 26 AIDS; Pneumocystic carinii pneumonia
87080 39 1320 24 28 AIDS; progressive multifocal leukoencephalopathy
88121 42 1340 19 30 AIDS; cytomegalic meningoencephalitis
86023 43 1260 2 100 AIDS; disseminated Kaposi’s sarcoma and pneumonia
88087 41 1240 12 34 AIDS; bronchopneumonia, cytomegalic infections and
toxoplasmosis
86046 32 1440 49 11 AIDS: pneumocystic carinii pneumonia
89024 21 1430 49 25 AIDS; mycobacterial infections, pneumonia,
cerebrovascular accident
Male-to-female transsexuals (n 6)
84020 (T1) 50 1380 ND 30 Suicide
84037 (T2) 44 1450 ND 34 Cardiovascular death
88064 (T3) 43 1540 ND ND Sarcoma
93042 (T4) 36 1145 21 31 AIDS, pneumonia, pericarditis, cytomegaly in brain
93070 (T5) 53 1500 96 34 Acute fatty liver due to alcohol
95018 (T6) 48 1198 24 36 Cardiovascular death, cardiac arrest
Sex hormone disorder cases (n 6)
83004 (S1) 46 1260 ND 34 Adrenocortical carcinoma; postoperative hemorrhage
89103 (S3) 67 1290 ND 28 Pancreaticocarcinoma; prostate carcinoma; orchidectomy
91044 (S6) 25 1200 13 103 Turner syndrome (XO); related cardiovascular problems;
decompensatio cordis
94090 (S5) 86 1663 3 93 Lung and prostate carcinoma; orchidectomy; septic shock
89077 (M2) 73 1090 48 33 AIDS; pneumonia; epilepsy
91005 (S2) 31 1377 34 35 Feminizing adrenocortex carcinoma
Nontreated male with cross-gender identity feelings (n 1)
96088 (S7) 84 1433 41 38 Lung carcinoma
FMT (n 1)
98138 51 4 32 Cachexia
ND, Not determined.
MALE-TO-FEMALE TRANSSEXUALS 2035
peroxidase-antiperoxidase (dilution 1:1000 in supermix) for 30 min; 5)
rinsed 310 min in 0.05 m Tris-HCL (Merck, Darmstadt, Germany; pH
7.6); 6) incubated in 0.05 mg/mL 3,3-diaminobenzidine (Sigma), 0.25%
nickel ammonium sulphate (BDH, Poole, UK) in 0.05 m Tris-HCL (pH
7.6) containing 0.01% H
2
O
2
(Merck) for 15 min; 7) washed in aquadest
for 10 min; 8) dehydrated in ethanol; and 9) mounted in Entallan.
Morphometry
Every 50th section stained for SOM along the rostro-caudal axis of the
BSTc on one side of the brain (22) was used for analysis with the help of
a specially developed program on an IBAS (Kontron Electronik, Munich,
Germany) image analysis system. The image analysis system was con-
nected to a scanning stage control box (MCU, Carl Zeiss, Oberkochem,
Germany) and had a Sony B/W CCD-camera for image acquisition. Both
the scanning stage and the camera were mounted on a microscope (Carl
Zeiss) equipped with planapo objectives. To provide optimal contrast and
homogenous illumination of the section the voltage of the light source was
set maximally. The light was reduced by neutral gray filters (0.03/0.12/
0.5/Schott; Mainz, Germany) to improve light contrast. For each section, the
analysis consisted of the following steps:
By using the plan 2.5 objective of the microscope, a low magnification
image covering the BSTc area was obtained and loaded into the IBAS image
memory.
In this image the BSTc was outlined manually on the basis of the
distribution of the SOM immunoreactivity in neurons and fibers (see Fig. 3).
Subsequently, the image analyzer covered the outlined area with a grid of
rectangular fields, each with the size of the area displayed by the camera
when the 40 objective was installed.
By a random systematic sampling procedure, 50% of the fields (which were
for at least 80% covered by the outlined area) were selected for analysis.
Taking into account the aberration of the optical axis between the 2.5 and
the 40 objective, the pixel positions of the selected rectangular fields in the
2.5 image were converted into scanning stage coordinates to position the
corresponding areas of the BSTc in front of the camera when using the 40
objective.
After the 40 objective was installed, the image analyzer moved the scan-
ning stage automatically to the coordinates of the selected fields. In each
field, SOM-positive neurons containing a nucleolus were counted manu-
ally, taking into account the exclusion lines according to Gundersen (26).
Neurons with double nucleoli were never seen. The spectrum of neuronal
sizes was equally distributed among the different groups.
The total volume of the BSTc was calculated by rostro-caudal inte-
gration of the outlined areas, taking into account the distance between
the measured sections. The neuronal density was calculated on the basis
of the nucleolus counts in the sample volume. An estimation of the total
number of SOM neurons was obtained by multiplying the total volume
with the mean neuronal density. The finding that the mean BSTc vol-
umes of the various groups are almost twice as large as those found in
the study of Zhou et al. (22) can be explained by the fact that in the present
study another peptidergic system (SOM instead of vasoactive intestinal
polypeptide) was used as a marker and also an antigen retrieval tech-
nique (i.e. microwave tissue pretreatment), which makes the staining
more sensitive (24, 27).
Statistics
Differences among the groups were statistically evaluated by the
nonparametric Kruskal-Wallis multiple comparison test. Differences be-
tween the groups were analyzed two-tailed using the Mann-Whitney U
test with a 5% experiment wise error rate (sequential Bonferroni meth-
od). Throughout this study values are expressed as mean sem.A
significance level of 5% was used in all statistical tests.
Results
Differences among the groups were statistically significant
by the nonparametric Kruskal-Wallis multiple comparison
test (P 0.002 for SOM neuron number). No statistical group
differences were found for age (P 0.090), brain weight (P
0.125), postmortem time (P 0.738), fixation time (P 0.065),
or storage time (P 0.308). To further test whether the
differences in the BSTc between the groups were affected by
possible confounding factors, such as paraffin-embedded
storage time of sections, fixation time, postmortem time, or
brain weight, an analysis of covariance was carried out.
These factors seemed to have no significant effect on the BSTc
SOM neuron numbers (P 0.10).
The number of SOM neurons in the BSTc of heterosexual
men (32.9 3.0 10
3
) was 71% higher than that in hetero-
sexual women (19.2 2.5 10
3
)(P 0.006), whereas the
number of neurons in heterosexual and homosexual men
(34.6 3.4 10
3
) was similar (P 0.83). The BSTc number
of neurons was 81% higher in homosexual men than in het-
erosexual women (P 0.004). The number of neurons in the
BSTc of male-to-female transsexuals was similar to that of
females (19.6 3.3 10
3
)(P 0.83) (see also Figs. 1 and 2).
In addition, the neuron number of the FMT was clearly in the
male range (see Fig. 1). The number of neurons in transsex-
uals was 40% lower than that found in the heterosexual
reference males (P 0.04; see the legend to Fig. 1) and 44%
FIG. 1. BSTc neuron numbers. Distribution of the BSTc neuron num-
bers among the different groups according to sex, sexual orientation, and
gender identity. M, Heterosexual male reference group; HM, homosex-
ual male group; F, female group; TM, male-to-female transsexuals. The
sex hormone disorder patients S1, S2, S3, S5, S6, and M2 indicate that
changes in sex hormone levels in adulthood do not change the neuron
numbers of the BSTc. The difference between the M and the TM group
(P 0.04) is also statistically significant according to the sequential
Bonferonni method if S2, S3, and S5 are included in the M group or if
S7 is included in the TM group (P 0.01). Note that the number of
neurons of the FMT is fully within the male range. Whether the trans-
sexuals were male oriented (T1, T6), female oriented (T2, T3, T5), or both
(T4) did not have any relationship with the neuron number of the BSTc.
The same holds true for heterosexual and homosexual men. This shows
that the BSTc number of somatostatin neurons is not related to sexual
orientation. A, AIDS patient. The BSTc number of neurons in the het-
erosexual man and woman with AIDS remained well within the corre-
sponding reference group (see Fig. 1), so AIDS did not seem to affect the
somatostatin neuron numbers in the BSTc. P, Postmenopausal woman.
S1 ( 46 yr of age): adrenal cortex tumor for more than 1 yr, causing high
cortisol, androstendione, and testosterone levels. S2 ( 31 yr of age):
feminizing adrenal tumor that induced high blood levels of oestrogens.
S3 ( 67 yr of age): prostate carcinoma; orchiectomy 3 months before
death. S5 (86 yr of age): prostate carcinoma; prostatectomy; orchiec-
tomy, and antiandrogen treatment for the last 2 yr. S6 ( 25 yr of
age): Turner syndrome (45,X0; ovarian hypoplasia). M2 ( 73 yr of age):
postmenopausal status.
2036 KRUIJVER ET AL.
JCE&M2000
Vol 85 No 5
lower than that found in the homosexual males (P 0.02).
Including patients S2, S3, and S5 in the male group and S1,
S6, and M2 in the female group or S7 in the transsexual group
to increase the number of their respective gender groups
enhanced the level of significance among the groups (P
0.001 for SOM neuron number). There seemed to be no clear
difference in the BSTc number of neurons between early
onset (T2, T5, T6) and late-onset transsexuals (T1, T3), indi-
cating that their smaller number of neurons is related to the
gender identity per se rather than to the age at which it
became apparent. No indication was found for a relationship
between cause of death and BSTc neuron numbers. Analysis
of the BSTc volumes showed a similar pattern of differences
among the groups with heterosexual men having a BSTc
volume of 4.60 0.28 mm
3
, similar to that in homosexual
men (5.00 0.39 mm
3
)(P 0.76). The BSTc volume of
females (3.38 0.41 mm
3
) and that of transsexuals (3.58
0.19 mm
3
) did not differ either (P 0.50). The volumes of all
males, regardless of sexual orientation, vs. all females or vs.
all genetic male transsexuals were statistically highly signif-
icant (P 0.01). The FMT had a BSTc volume in the male
range (4.80 mm
3
).
Discussion
In the present study, we show regardless of sexual orien-
tation: 1) a sex difference in SOM neuron numbers in the
human BSTc, with males having almost twice as many SOM
FIG. 2. Representative immunocyto-
chemical stainings of the somatostatin
neurons and fibers in the BSTc of a ref-
erence man (a), reference woman (b),
homosexual man (c), and male-to-fe-
male transsexual (d). Note the sex dif-
ference regardless of sexual orienta-
tion. The male-to-female transsexual
has a BSTc in the female range. *, Blood
vessel. Bar represents 0.35 mm.
MALE-TO-FEMALE TRANSSEXUALS 2037
neurons as females; 2) a number of SOM neurons in the BSTc
of male-to-female transsexuals in the female range; and 3) an
opposite pattern in the BSTc of a female-to-male transsexual
with a SOM neuron number in the male range.
Analysis of the total number of SOM neurons of the human
BSTc in individual patients with highly different hormone
levels does not give any indication that changes in sex hor-
mone levels in adulthood change the neuron numbers. Be-
FIG. 3. The image analysis procedure.
a, Illustration of a somatostatin immu-
noreactive BSTc. b, The BSTc is out-
lined manually. c, Outlined BSTc is di-
vided automatically into rectangular
fields. d, Fifty percent of the fields is
selected by a random systematic sam-
pling procedure. e, Higher magnifica-
tion of somatostatin neurons in a field
displayed by the camera when the 40
objective is installed. Only somatosta-
tin-positive neurons with a visible nu-
cleolus were counted (see Morphometry
in Materials and Methods). Bar repre-
sents 40
m. f, Example of a clearly
visible nucleolus in a somatostatin im-
munoreactive neuron.
2038 KRUIJVER ET AL.
JCE&M2000
Vol 85 No 5
cause the transsexuals had all been treated with estrogens, at
least for some time (see Table 2), the reduced neuron num-
bers of the BSTc could theoretically be due to the presence of
high levels of circulating estrogens. Arguments against this
possibility come from the finding that transsexuals T2 and T3
both showed a small BSTc (Fig. 1), despite the fact that T2
stopped taking estrogens about 15 months before her death
because of hyperprolactinemia, and T3 no longer received
hormone treatment when a sarcoma was found about 3
months before she died. T5 continued to take estrogens until
3 months before death and had even more SOM neurons than
T3, whereas T1 and T6 continued to take estrogens until
death and even had higher SOM neuron numbers than T2
and T3 (Fig. 1). Furthermore, a 31-yr-old man (S2), who
suffered for at least 1 yr from a feminizing adrenal tumor that
produced high blood levels of estrogens, still had a BSTc
neuron number in the normal male range (the latest highest
serum estradiol levels before death varied between 577–779
pmol/L; the normal range is 50 –200 pmol/L).
Our results might theoretically also be explained by a lack of
androgens in the transsexual group because all subjects, except
for T4, had been orchiectomized. We, therefore, studied two
nontranssexual men (S3 and S5) who had been orchiectomized
because of prostate cancer 3 months and 2 yr before death,
respectively, and found that the BSTc neuron number of S3 was
close to the mean of the male group and that the BSTc number
of neurons of S5 was even the highest observed (Fig. 1), indi-
cating that orchiectomy did not cause any decrease in SOM
neuron numbers. Not only were five of the transsexuals orchi-
ectomized, they all used the antiandrogen cyproterone acetate
(CPA). However, an effect of CPA reducing the number of SOM
neurons of the BSTc is highly unlikely because S5 had taken
CPA during the last 2 yr of his life and his BSTc neuron number
was at the upper end of the male range, whereas T6 had not
taken CPA for the past 10 yr, and T3 took no CPA during the
last 2 yr before her death, and they still had relatively low
numbers of SOM neurons.
The BSTc SOM neuron numbers of two postmenopausal
women [73- (M2) and 53-yr-old (P)] and of a 25-yr-old
woman with Turner syndrome (S6: complete 45,X0, with
ovarian hypoplasia) were completely within the normal fe-
male range (Fig. 1). If high estrogen levels would have a
reducing effect on BSTc neuron numbers, the opposite effect
(high neuron numbers) would be expected in the postmeno-
pausal women and the Turner syndrome patient due to their
low endogenous sex hormone level status. However, this
was not the case. Noteworthy is that according to the avail-
able clinical data the two postmenopausal women did not
receive any estrogen replacement therapy either. Although
the Turner syndrome patient had been receiving hormone
replacement therapy since she was 16 yr of age, her neuron
numbers were even higher than P, whereas she had almost
the same BSTc neuron number as M2 who did not receive
such a therapy. Again, this argues against the probability of
an estrogen-induced reduction effect on the number of SOM
neurons. Finally, the BSTc neuron number of a 46-yr-old
woman who had suffered for at least 1 yr from a virilizing
tumor of the adrenal cortex (that produced very high blood
levels of androstendione and testosterone) was also clearly
within the lower spectrum of that of other women (Fig. 1; S1:
latest androstendione serum level before death was 48.0 ng/
mL; the normal range for women is 0.4–3.5 ng/mL; the latest
serum testosterone level before death was 26.82 nm/L; the
normal range for women is 1.04–3.30 nm/L). Thus, an in-
creasing effect of testosterone on the BSTc neurons does not
seem likely to be the case either. Furthermore, it should be
noted that the FMT stopped taking testosterone 3 yr before
death while having a BSTc neuron number clearly within the
male range.
In conclusion, estrogen treatment, orchiectomy, CPA treat-
ment, or hormonal changes in adulthood did not show any clear
relationship with the BSTc SOM neuron number. In addition,
we had the unique opportunity to study the brain of an 84-yr-
old man (S7) who also had very strong cross-gender identity
feelings but was never orchiectomized, sex re-assigned, or
treated with CPA or estrogens. Interestingly, this man had also
a low BSTc SOM neuron number that was fully in the female
range (see Fig. 1, S7). This case provides an additional argument
against the view that orchiectomy, CPA, or adult estrogen treat-
ment of the transsexuals would be responsible for the reduced
somatostatinergic neuron numbers. Moreover, studies that in-
vestigated the effects of estrogen treatment on hypothalamic
SOM neurons in (castrated) rats are also not in support of such
an effect. Estrogen treatment does not reduce the amount of
SOM messenger RNA (mRNA) in neurons but even enhances
its neuronal expression (28). Moreover, another animal study
indicates that, although changes occur in the hypothalamic
neuronal expression of SOM mRNA due to castration or tes-
tosterone treatment of male rats, no differences in hypothalamic
SOM neuron numbers are induced at all by either of such
treatments (29). This observation is also in agreement with the
control SOM neuron numbers of the castrated male patients (S3,
S5) and testosterone-exposed (S1) female patient. Together, all
these data clearly indicate that sex hormone-mediated reduc-
tion (or enhancement) effects on transsexual BSTc neurons in
adulthood are extremely unlikely to be the underlying mech-
anism of the observed somatostatinergic BSTc differences.
In short, our findings seem to support the hypothesis that
the somatostatinergic sex differences, the female number of
SOM neurons in the BSTc of the male-to-female transsexual
brain and the male number of SOM neurons in the BSTc of
the FMT are not the result of changes of sex hormone levels
in adulthood. Instead, the neuronal differences are likely to
have been established earlier during development [see also
Zhou et al. (22), and for functional differences see Cohen-
Kettenis et al. (30)]. In line with this reasoning are the de-
velopmental data on the rat BST showing that adult volumes
and neuron numbers of BST subdivisions are orchestrated by
androgen exposure during early brain development (31, 32).
Such a mechanism is also in agreement with data of Breed-
love (33, 34) showing that perinatal androgens but not adult
variations in androgen exposure induce differences in the
total neuron number of the rat spinal nucleus bulbocavern-
osus. Apart from such well known irreversible “organizing”
effects of sex hormones on the developing brain, the possi-
bility of a direct action of genetic factors on sexual differen-
tiation of the brain should not be ruled out (35).
We are aware of the fact that our data are based on post-
mortem brain material derived from a heterogeneous patient
population of which each individual’s clinical status might
MALE-TO-FEMALE TRANSSEXUALS 2039
TABLE 2. Clinicopathological data of subjects with gender identity disorder
Patient no. (NBB) Age (yr)
Age of hormonal
treatment/orchiectomy
Hormone treatment Cause of death
Male-to-female transsexuals
(n 7)
T1 (84020) 50 42/44 Age 42: Stilbestrol 5 mg 1 dd; after 2 months to 5 mg 2 dd;
age 44: CPA 50 mg 2 dd; (treatment lasted 4 yr, stopped
2 yr before death); Ethinyloestradiol 50
g2dd
(treatment lasted 8 yr until death)
Suicide
T2 (84037) 44 35/37 Age 35: stilbestrol 5 mg 3 dd; after 2 months to 5 mg 2 dd;
CPA 50 mg 1 dd; 1977: CPA 50 mg 2 dd; stilbestrol 5 mg
1–2 dd (generally this lasted 7 yr until death; stilbestrol
stopped about 15 months before death)
Cardiovascular death
T3 (88064) 43 36/39 Age 36: received standard CPA treatment (50 mg 2 dd)
until 2 yr before death; At age 39 received standard
ethinylestradiol treatment (50
g 2 dd) that stopped 3
months before death
Sarcoma, right-side temporal
T4 (93042) 36 NA/no orchiectomy, testes atrophy CPA 50 mg 1 dd at least the last 10 months before death;
the patient received estradiol in combination with
hydroxyprogesterone in therapeutical dosages. Exact
period of treatment is not known but based on the
significant testes atrophy she was most probably treated
for a period of about 5 yr or more.
AIDS, pneumonia,
pericarditis, cytomegaly in
brain
T5 (93070) 53 40/50 Age 40: stilbestrol treatment (stopped after 1 yr); at age
43–47: premarin 0.625 mg dd; at age 47–50: premarin
3.75 mg dd; at age 50 –53; premarin 2.5 mg 3 dd; CPA
50 mg 1 dd; topical estrogen cream (estrogen treatment
stopped 3 months before death)
Acute fatty liver due to
alcohol abuse
T6 (95018) 48 35/36 Age 35: spironolactone 100 mg 2 dd; CPA 50 mg 2 dd;
ethinyloestradiol 50
g 2 dd; at age 36 40: CPA 50 mg
2 dd; ethinyloestradiol 50
g 2 dd; at age 40 48;
aldoctone 100 mg 1 dd; ethinyloestradiol 50
g1dd
(treatment lasted until death)
Cardiovascular death
S7 (96088) 84 No orchiectomy or sex
reassignment therapy
Patient did not receive sex hormone replacement therapy Lung carcinoma
FMT (n 1)
FMT (98138) 51 27/28 Age 27: testosterone sustanon 250 mg, twice a month
injections; at age 30 testosterone undecanoaat 40 mg 3
dd. At age 34 testosterone undecanoaat 40 mg 2 dd; At
age 36 testosterone undecanoaat 40 mg 4 dd; At age 44
testosterone sustanon 250, twice a month injections; At
age 47 to 48: testosterone sustanon 250, every 3 weeks;
from the age 48 until the age of death (51), no
testosterone replacement therapy anymore
Cachexia
NBB, Patient number of the Netherlands Brain Bank; CPA, cyproterone acetate; NA, not available; AIDS, acquired immune deficiency syndrome.
2040 KRUIJVER ET AL.
JCE&M2000
Vol 85 No 5
have had an impact on the brain. However, despite that we
were still able to find striking sexual dimorphic differences
(that become even more significant if patients S1, S2, S3, S5,
S6, S7, and M2 are included in their respective gender
groups; see statistics and the legend to Fig. 1). An exciting
additional new finding came from the FMT who revealed a
“masculine” BSTc, which is completely in line with the sexual
brain paradigm (7, 22, 30, 36 40).
Although our collection of male-to-female transsexual
brains is small, it offers new opportunities to explore neu-
robiological correlates of transsexualism, as has previously
been done in relation to sexual orientation (46). The de-
velopment of high resolution imaging techniques may allow
in vivo volume measurements of particular brain areas in
much larger groups of transsexuals, which could extend our
findings in the distant future. Although brain imaging
proved to be useful in visualizing [e.g. septo-hypothalamic
brain injuries leading to hypersexuality or altered sexual
preference (9, 10)], precise neuroanatomical delineation of
small brain structures such as the BSTc or neuronal counts
are, at present, not possible using such techniques.
Taking into account the aforementioned limitations of our
studies, the present study of SOM neurons in the human
BSTc provides unequivocal new data supporting the view
that transsexualism may reflect a form of brain hermaphro-
ditism such that this limbic nucleus itself is structurally sex-
ually differentiated opposite to the transsexual’s genetic and
genital sex. It is conceivable that this dichotomy is just the tip
of the iceberg and holds also true for many other sexually
dimorphic brain areas.
Because the sexually differentiated brain in general (41)
may be the basis of sex differences in the prevalence of many
neurobiological diseases and disorders (7), more studies are
needed to further unravel the potential determinants of the
sexual dimorphic brain and its related clinical disorders.
Acknowledgments
We thank Bart Fisser, Unga Unmehopa, and Joop van Heerikhuize for
their technical help; Henk Stoffels for preparing Fig. 1; and Gerben van
der Meulen for making the photographs. Tini Eikelboom, Wilma Ver-
weij, and Olga Pach are thanked for their secretarial help. Mariann Fodor
is thanked for critically reading the manuscript. Brain material was
provided by the Netherlands Brain Bank (coordinator Dr. Rivka Ravid).
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MALE-TO-FEMALE TRANSSEXUALS 2041
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