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Introduction
Hormone replacement therapy (HRT) has been
widely used in postmenopausal women and is thought to be beneficial in
protecting against the development of both cardiovascular disease and osteoporosis. However, there are intense disputes
about whether HRT can reduce the incidence of Alzheimer's disease (AD) in postmenopausal women. AD is a neurodegenerative
disease characterized by progressive dementia with very complicated etiology. It is regarded to be as a result of interactions
of multiple factors, including genetics, metabolism, and environment. Now it has been confirmed that the
apolipoprotein E (apoE) ε4 allelomorphic gene located on chromosome 19, is one of the major genetic risk
factors[1_3]. Many studies reported that women with the
ε4 allelomorphic gene have greater risk of suffering from dementia than
men[4,5]. The relationship between apoE gene type and estrogen is a hot topic in the
pathogenesis of AD[6_8]. Many studies have already found
that exogenous estrogen has protective effects on the
development of dementia and the prevention of cognitive
impairment[9,10]. However, not all the estrogen therapies,
especially in prospective studies, showed consistent effects. It
is thought that HRT can only improve the memory function
of non-apoE ε4-carriers[11_14]. An observation from a study
with a large sample of 7500 people by the Women's Health
Initiative Memory Study showed that estrogen or
progesterone treatment did not effectively prevent the decline of
cognitive function in women 65 years old or older. On the
contrary, this treatment increased the risk of
dementia[12].
Our previous study showed that long-term and low-dose
hormone replacement therapy could reduce bone loss and
the incidence of bone pain[15]. Meanwhile, the serum levels
of total cholesterol, low density lipoprotein 2 cholesterol,
apoB, apoCIII, and apoE decreased obviously in the
postmenopausal women receiving low-dose and long-term HRT.
The decrease of blood lipid levels may be protective for the
cardiovascular system and therefore reduces the risk of
cardiovascular diseases[16]. This prompted us to conduct this
retrospective study of female medical staff at Peking Union
Medical College Hospital (PUMCH; Beijing, China) to
evaluate the effects of long-term, low-dose HRT treatment on the
hippocampus and cognition. We examined the sex hormone
level, cognitive function, brain hippocampus volume, and
biochemical changes in the anterior cingulate cortex and
hippocampus among the apoE carriers.
The reports about the effects of HRT on cognition are
controvertible. Part of the reason, as supported by the latest
reports[17_20], is due to the different designs of studies,
including the initial use of HRT, type and dose of hormones,
ratio of estrogen/progestogen, duration of HRT, individual
difference, and whether minute cognitive difference can be
detected in a dementia screening assay (ie sensitivity of the
tests). In this study, besides the cognitive assessment tests,
we also detected the effect of HRT on the morphology of the
hippocampus based on brain imaging observations provided
by proton magnetic resonance spectroscopy.
Materials and methods
Study population and design In this study, 983 (94.6%)
of the 1039 female medical staff at PUMCH aged over 40
years old, either still working or retired, were interviewed via
telephone. Seven hundred and fourteen of the interviewees
were postmenopausal. Among these 714 women, 255
(35.7%) received HRT. According to our study criteria of inclusion
and exclusion, only those who never had any of the
following diseases, including stroke, AD, breast cancer, or uterine
tumors, were included. Eighty three of the 255 women, aged
66.3±8.3 years and had been taking HRT for at least 4 years
by the time of interview, were enrolled in the HRT group.
Another 99 interviewees, who had matched age (67.1±7.6
years), education level, economic and social status, and had
never taken any kind of HRT, were enrolled in the control
group. The HRT paradigms of the participants were various
and were based on the participants' needs, risk evaluation,
and the availability of drugs. Different types of preparation,
dosage, route of absorption, and combinations of HRT were
used. The dosage of HRT was adjusted according to
individuals' symptoms and specific needs during the course of
HRT. Furthermore, during the course of HRT, some
individuals were allowed to stop from a couple of weeks to a
month for different reasons and then resumed their HRT.
The hormones used included estradiol valerate, conjugated
equine estrogens (Premarin), tibolone, and
medroxyproges-terone acetate for women with an intact uterus. However, all
of them used a lower dose of HRT. Among the 83 participants,
9 used half a dosage, 52 used one-quarter of a dosage, and
22 used less than one-quarter of a dosage based on the
manufacturer's recommendation. All the participants were
at postmenopausal stage at the time of the tests. This study
was approved by the institutional review board of the Ethics
Committee of PUMCH, and all participants gave written
informed consent.
Blood sample preparation Fasting blood (10 mL) was
collected from each participant by vein puncture and placed
into prepared tubes containing
EDTA·K2. The blood samples were centrifuged to separate blood content into the plasma,
blood cell pellet (red and white blood cells), and platelet.
The separated blood content was frozen immediately and
stored at -80 oC until use.
Plasma sex hormone concentration assay The plasma
levels of estradiol (E2) and progesterone (P) were assayed by
Immulite assay according to the manual of the kit
[diagno-stics products corporation (DPC) Immulite-1,
chemilumine-scent immunoassay system kit, Genzyme-Techne,
Minnea-polis, MI, USA]. The testosterone (T) level was assayed by
enzyme immunosorbent assay (Model 550, Bio-Rad, Hercules,
CA, USA) using 1 mL plasma.
Genotyping of participants Genomic DNA was
isolated from peripheral lymphocytes according to the
instructions of the reagent manual (Takara, Dalian, China). ApoE
genotyping was performed by polymerase chain
reaction-restriction fragment length polymorphism (PCR-RFLP).
10 µL of the PCR product was loaded onto a 1.5% agarose gel
containing ethidium bromide (0.3 mg/L) for electrophoresis
at 90 V for 30 min; the gels were photographed for the
analysis of the apoE alleles.
Assay of the hippocampus volume Magnetic resonance
imaging (MRI, GE Signa 3.0 T VHIEXITE3, General Electric
Medical Systems, Milwaukee, WI, USA) was performed to
determine the volume of the brain hippocampus. The
scanning protocol included a T1 weighted image [T1 FLAIR,
oblique coronal, vertical to the hippocampus, echo time (TE)
24 ms, repetition time (TR) 2500 ms, TI 2250 ms, slice
thickness 3 mm without gaps, and including the whole body of
the hippocampus] and a 3-D [fast spoiled GRASS (SPGR),
TE 3.3 ms, TR 400 ms, flip angle 15 °, slice thickness: 1.6 mm]
image of the whole brain. The volume of the bilateral
hippocampus and the whole brain were manually measured
using the GE workstation (Advanced Workstation 4.2_07, GE
Healthcare, Chicago, IL, USA) by first drawing the contour
of the hippocampus in each slice of the oblique coronal
T1-weighted image, adding all the areas, and then multiplying
slice thickness to calculate the volume of the hippocampus.
The ratio of the hippocampus volume to the whole brain
volume was then calculated.
Assessment of the brain biochemical indexes in apoE
ε3 and apoE ε4 carriers The peaks of NAA, tCr, and mI
were measured in apoE ε3 and apoE ε4 carriers by
1H MRS respectively. Proton magnetic resonance spectroscopy
(1H MRS) was also acquired by 3.0T MRI with the following
parameters: single voxel, 20×20×20 mm; TE 35 ms; TR 1500
ms; number of excitations 8; and total acquired times 128.
Three locations were examined, including the hippocampus,
posterior cingulated cortex (indicating gray matter), and
periventricular white matter (indicating white matter). Using
post-processing software Sage 11.0 (GE Medical Systems,
Chicago, IL, USA), the values of NAA/tC and mI/tCr were
then calculated.
Evaluation of the total score of cognition
Cognitive ability was assessed by comprehensive neuropsychological
tests, including the California verbal learning test, logical
memory, figure complex, verbal fluency, constructional praxis,
and digit span. The sum of these scores was used to
evaluate the overall cognitive ability and was manifested by least
squares means (LSMEAN, the LSMEAN of total cognition).
Statistical analysis All data obtained in the study were
analyzed by SPSS 13.0 for Windows (SPSS, Chicago, IL,
USA). The t-test and variant analysis were used to compare
values of normal distribution in the 2 groups. The
non-parametric test was used to analyze data of non-normal
distribution in the 2 groups. P-values lower than 0.05 were
considered to be statistically significant between the 2
groups.
Results
Participants In the HRT group, the mean age of
beginning HRT was 54.91±5.75 years and the average time of
using HRT was 11.67±5.81 years (4_33 years). Among the 83
participants, 30 (36%) used HRT for 4_9 years, 31 (37%) for
10_14 years, 15 (18%) for 15_19 years, and 7 (9%) exceeded
20 years.
Genotype of apoE After digestion of apoE PCR products,
6 genotypes were obtained, ε2/ε2, ε2/ε3, ε2/ε4,
ε3/ε3, ε3/ε4, and ε4/ε4. The distribution of the apoE alleles and the
frequency in both the HRT and control groups are shown in
Table 1. ApoE ε4 carriers in the HRT and control groups
were 15 and 16, respectively. ApoE ε3/ε3 was the most
common gene type among all of the participants. There was no
significant difference between the 2 groups in the frequency
of allele distribution (Table 1).
Hormone, cognitive ability, and volume of the
hippocampus The plasma E2 concentration in the HRT group was
significantly higher than that of the control group
(P=0.0001, Table 2). Figure 1 depicts the
E2 concentration in every age group. No significant difference was observed in plasma P
and T concentrations and the total cognitive ability score
between the 2 groups (P>0.05, Table 2).
Furthermore, the brain hippocampus MRI showed a trend
of the hippocampus volume in the 2 groups decreasing with
age. In the participants older than 60 years of age, the
hippocampus volume decreased sharply in the control group
and mildly in the HRT group (Figure 2), but the difference
was not statistically significant in each age stage between
the two groups.
Further analysis in participants carrying different apoE
alleles Due to the small size of the study, no statistical
analysis could be done with the ApoE ε2 carriers and
therefore it was not included in this study. The number of apoE
ε4 carriers in the HRT and control groups was 15 and 16,
respectively. The number of apoE ε3 carriers in the HRT
and control groups was 68 and 72, respectively. However,
not all the participants were willing to take MRI detection.
As a consequence, only 25 apoE ε4 allele carriers (14 from
the HRT group and 11 from the control group) and 126 apoE
ε3 allele carriers (54 from the HRT group and 72 from the
control group) were further studied by MRI and
1H MRS. There were significant differences in the plasma
E2 concentration (P=0.026), but not in P and T
(P=0.359, P=0.897, respectively) between the 2 groups in the ε4 allele
carriers (Table 3). Similar results were found in the
ε3 allele carriers; the E2 level in the HRT group was significantly higher
(P=0.001) than that of the control group, while there were no
significant differences in the P and T levels
(P=0.252, P=0.281, respectively). It is noteworthy that the cognitive scores
of the 2 groups in the ε4 allele carriers were comparable
(P=0.892, Table 3). We also noticed that all the participants had
good educational background, with the average education
duration being 14.22±2.63 years in the control group and
14.36±3.11 years in the HRT group (P=0.964). However, for
the ε3 allele carriers, the cognition score of the HRT group
was also obviously higher than that of the control group.
HRT had a dramatic effect on the volume of the
hippocampus of apoE ε4 carriers (Table 3). In the apoE
ε4 carriers of the HRT group, the ratio of the left and right hippocampus
volume to the left and right brain hemisphere was
significantly higher than those of the apoE ε4 carriers of the
control group (0.186±0.016, 0.183±0.016, respectively,
and P=0.0083, P=0.0097,
respectively). Furthermore, the average of the total volume of the hippocampus of the apoE
ε4 alleles carriers in the HRT group (0.406±0.028) was significantly
larger than that of the apoE ε4 alleles carriers in the control
group (0.369±0.031, P=0.0046). A morphological comparison
of the hippocampus of 2 participants both aged 65 years
¡ªone from the HRT group (Figure 3B) who had been receiving
HRT for 12 years and the other from the control group (Figure
3A) who had never received HRT ¡ª was made. The
1H MRS analysis in these apoE ε4 carriers showed that there were no
differences of NAA/tCr and mI/tCr in either white or gray
matters between the 2 groups. However, in the hippocampus,
NAA/tCr increased significantly (1.54±0.08,
P=0.031) in the HRT group, compared with that of the control group (1.45±
0.13). Although mI/tCr decreased in the HRT group, no
statistical difference was found (P>0.05; Table 4).
With the apoE ε3 carriers, there was no significant
difference of the volumes of each side of the hippocampus and
the whole hippocampus between the 2 groups. The
1H MRS analysis in these apoE ε3 carriers showed that there
were no differences of NAA/tCr and mI/tCr in either white or gray
matters or the hippocampus between the 2 groups.
Discussion
The present study is a retrospective, cross-sectional
study on cognition and brain imaging in postmenopausal
women who were medical staff at PUMCH. Our results
supported the hypothesis that long-term, low-dose HRT had
protective effects on the hippocampus of postmenopausal
women. In our study, the mean age of beginning HRT was
54.9 years and the mean HRT duration was over 12 years. No
severe HRT-related side effects, such as breast cancer or
uterine tumors, were observed before and during this study.
The HRT and control groups were well equilibrated and
comparable, which enhanced the reliability of the results.
The effects of low-dose HRT on the prevention of
postmenopausal osteoporosis were well studied in China in the
1980s. It has been shown that low-dose HRT helped to
maintain genital health and postponed age-related
atherosclerosis of the cardiovascular system in women receiving HRT for
5_31 years[21]. In this study, we found that despite the
diversity of estrogen regimens, all the doses of HRT used were
only half does, one-quarter of a dose, or even less, based on
the manufacturer's recommended dosage. All of these doses
of HRT increased E2 levels effectively at all ages within the
HRT group, although it was still within the postmenopausal
range, indicating the reliability of the treatment regimen.
The hippocampus is a critical for retaining memories. Its
pathological changes are often used for diagnosis of
AD[22,23]. The volume ratio of the hippocampus to the whole brain is
closely related to function of learning and
memory[24]. There-fore, in this study, we detected the changes of this ratio to
evaluate the degree of brain atrophy and found that the mean
hippocampus volume in both groups had a trend of
decreasing with age, especially after the age of 65 years. In addition,
there was a sharp decrease of the hippocampus volume in
the control group and a relatively mild decrease in the HRT
group, but there was no statistical difference of the ratio
between the 2 groups. However, further analysis of the
volume ratio of the hippocampus to the whole brain of 25 cases
carrying AD risk factor, the apoE ε4 allelic gene, in the 2
groups found that the mean ratio in the HRT group was
significantly greater than that of the control group,
indicating that long-term, low-dose HRT might be beneficial for
reducing the risk of AD development in vulnerable,
postmenopausal women.
To some extent, the level of NAA can reflect the
metabolism and functions of the neurons. A decrease of NAA
indicates damage or reduction of population of the neurons,
whereas an increase of NAA implies enhancement of the
neuron metabolism and therefore the effectiveness of
therapy[25]. Increased mI is often found in
neurodege-nerative diseases[26]. tCr is mainly composed of
phosphocreatine (PCr) and creatine
(Cr)[27]. Under normal conditions, the tCr concentration (Cr+PCr) in the brain is very stable and
therefore can be used to normalize other indexes, such as
NAA/tCr and mI/tCr. In the hippocampus of apoE ε4 carriers,
NAA/tCr was significantly higher in the HRT group than it
was in the control group, while mI/tCr decreased in the HRT
group compared to the control group. These results indicate
that HRT may help to improve brain metabolism, maintain the
population of the hippocampus neurons, and reduce glia cell
proliferation in apoE ε4 carriers who are vulnerable to AD.
These observations were in agreement with the imaging
findings from MRI, which showed that the volume of the
hippocampus of apoE ε4 carriers in the HRT group was larger
than that of the control group.
The total scores of cognition by blind test on the
participants in this study showed that there was no significant
improvement of cognitive function in postmenopausal women
receiving low-dose HRT compared to the women in the
control group who had never used HRT. The absence of
cognitive changes at the presence of metabolic changes may be
explained by high education attainment among the
participants in this study, which is known to be able to
compensate for mild cognitive
impairment[28]. With these apoE ε4 carriers, the discrepancy between imaging and cognition
tests might be due to the compensation capability of the
brain, which might not enable impairment to cognitive
difference when brain atrophy does not reach a critical level. It
has been reported that the structural changes in the cortex
usually precede cognitive impairment by at least 2
years[29,30]. Among the apoE ε3 carriers, HRT showed little effect on
the volume of the hippocampus and the value of NAA/tCr
and mI/tCr. However, the cognition score of the apoE
ε3 carriers in the HRT group was significantly higher than that
of the apoE ε3 carriers in the control group, which is
consistent with a previous report that HRT could improve
cognition in apoE ε3 carriers[11_14]. The different cognition
results in the ε3 and ε4 carriers may be due to the small-scale
study; large-scale research will provide a clear answer in the
future.
In summary, our data showed that although long-term,
low-dose HRT had no effect in brain metabolism and the
hippocampus volume in general, it did improve these indexes
in apoE ε4 carriers, indicating that long-term, low-dose HRT
regimen may be beneficial in reducing the risk of AD
development in vulnerable, elderly women. In addition, our
results provide a valuable clue for studying the relationship
among the apoE gene type, estrogen level, and the delay in
the pathogenesis of AD. However, because of limitations
due to the small sample size of apoE ε4 carriers in this study,
a randomized, double-blind, placebo-controlled longitudinal
study needs to be done in the future.
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