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FERTILITY AND STERILITY
Copyright 2001 American Society for Reproductive Medicine
Printed on acid-free paper in U.S.A.
Apolipoprotein E alleles in women with
polycystic ovary syndrome
Seppo Heinonen, M.D.,a Seija Korhonen, M.D.,a Maritta Hippela¨inen, M.D.,aMikko Hiltunen, M.Sc.,b Arto Mannermaa, Ph.D.c and Seppo Saarikoski, M.D.a
Kuopio University Hospital, Kuopio, Finland
To investigate the frequency of apoE alleles among women with polycystic ovary syndrome.
Retrospective case-control study.
University-based endocrinology/infertility clinic.
Healthy fertile women (n ϭ 91) and women with a diagnosis of polycystic ovary syndrome (n ϭ
Main Outcome Measure(s):
The presence of the three most common apoE alleles (⑀2, ⑀3, and ⑀4)
determined by polymerase chain reaction–restriction fragment length polymorphism in the two groups and in
the general population in our area.
The frequency of the apo ⑀4 allele was 17.2% among women with polycystic ovary syndrome and
was 18.7% among healthy fertile women, which is close to the rate in the general population in our area (19%).
None of the apoE genotypes (Fisher’s exact test; P
ϭ.71) or alleles (P
ϭ.78) was significantly overrepresented,
and the homozygous genotype ⑀4 was not associated with the clinical disease.Conclusion(s):
The observed profiles of allele and genotype frequencies confirm the equilibrium state
between apoE polymorphism and polycystic ovary syndrome and suggest that apoE does not play a major role
in the development of hyperlipidemia in the group of women with polycystic ovary syndrome. (Fertil Steril
2001;75:878 – 80. 2001 by American Society for Reproductive Medicine.)
Apolipoprotein E, polycystic ovary syndrome, gene polymorphism
Polycystic ovary syndrome (PCOS) is asso-
often found in this patient group. Accordingly,
ciated with hyperinsulinemia and peripheral in-
Talbott et al. (8) compared cardiovascular dis-
sulin resistance, both of which have been
linked to dyslipidemia (1, 2). Insulin resistance
control subjects matched for body mass index
appears to play a critical role in the pathophys-
and documented higher levels of total choles-
iology of PCOS, as evidenced by highly cor-
terol, low-density lipoprotein C, and triglycer-
related levels of ovarian vein androgens and
ides and lower levels of high-density lipopro-
insulin (3). When serum androgen concentra-
tein C in the study group. This suggests that
tions are reduced by means of a gonadotropin-
releasing hormone agonist, insulin resistance
remains in women with PCOS (4). On the other
hand, suppression of serum insulin concentra-
Apolipoprotein E (apoE) is an established
and Gynecology, KuopioUniversity Hospital, 70211
tions by diazoxide reduces serum testosterone
genetic marker for dyslipidemia and athero-
sclerosis, and it plays an important role in lipid
This suggests that the direction of causation
metabolism (9). Cholesterol absorption effi-
is from insulin to androgens, and collectively,
ciency from the intestine increases, whereas
these metabolic abnormalities result in varying
bile acid secretion by the liver falls in the
degrees of glucose intolerance, dyslipidemia,
following allelic order: ⑀2 Ͻ ⑀3 Ͻ ⑀4 (10). The
central obesity, and hypertension (6). However,
aim of the current study was to investigate the
von Eckardstein et al. (7) reported that dyslip-
possible role of apoE in the dyslipidemia seen
in PCOS by determining the frequencies of
with PCOS, independently of the excess weight
apoE alleles and genotypes, using the polymer-
Clinical characteristics of the PCOS women.
Apolipoprotein E genotype and allele frequencies in womenwith polycystic ovary syndrome; in healthy, fertile controls;
PCO ϭ polycystic ovary; BMI ϭ body mass index.
d Or free plasma testosterone Ͼ40 pmol/L, evaluated using SHBG.
Heinonen Apolipoprotein E and PCOS. Fertil Steril 2001.
Heinonen Apolipoprotein E and PCOS. Fertil Steril 2001.
ase chain reaction (PCR) combined with restriction fragment
The clinical characteristics of the patients are presented in
length polymorphism (RFLP) analysis.
Table 1. Serum concentrations of total cholesterol and tri-glycerides were above the normal range in 12% and 24%,
MATERIALS AND METHODS
respectively, of the women with PCOS.
Written approval for the study was obtained from the
Genomic deoxyribonucleic acid (DNA) was extracted
Ethics Committee of Kuopio University Hospital, and the
from peripheral blood lymphocytes using a standard phenol-
protocol was approved by the investigation review board.
chloroform extraction method (13) and subjected to analysis
Informed consent was obtained from all subjects and docu-
by PCR and gel electrophoresis of the products. For detec-
tion of ApoE alleles, polymerase chain reactions of genomicDNA and Hha
I digestions of PCR products were carried out
Information was collected retrospectively from 58 women
as previously reported by Tsukamoto et al. (14). Each set of
with PCOS seen in the endocrinology/infertility clinic at
reactions was run with positive and negative controls and a
Kuopio University Hospital and from 91 nonhirsute, fertile
blank. From the PCR results, the genotypes and allele fre-
control women with regular cycles and normal ovaries who
quencies were calculated. Statistical analysis was conducted
delivered at our institution between January 1999 and De-
using Fisher’s exact test (run on SPSS, version 9.0, Chicago,
cember 1999. In the study group, the indications for referral
IL), and the level of statistical significance was defined as
were menstrual cycle disturbances, infertility, and symptoms
Ͻ.05. ApoE genotypes were found to be in Hardy-Wein-
berg equilibrium in both patient and control groups.
The diagnosis of PCOS was based on information ob-
tained by clinical examination (hirsutism and ovulatory dis-
orders with an intermenstrual interval of Ͼ36 days), obser-vation of polycystic ovaries in ultrasonography (11),
The genotypes and frequencies of apoE alleles are pre-
laboratory testing revealing androgen excess (serum total
sented in Table 2. Population frequencies were derived from
testosterone concentration Ͼ2.5 nmol/L), and an elevated
the same geographical area, and they were based on more
LH/FSH ratio (Ͼ2). In this study, PCOS was defined as the
than 1,500 tested subjects in the general population. The
presence of polycystic ovaries plus either of the classical
distribution of apoE alleles and genotypes was equal in
symptoms and either of the above-mentioned biochemical
affected and unaffected cases (P
ϭ.71 and P
disturbances. Furthermore, we excluded other causes of
tively). The frequency of the apo ⑀4 allele was 17.2% among
anovulation and hirsutism, such as late-onset congenital ad-
women with PCOS and was 18.7% among healthy fertile
renal hyperplasia and prolactin and thyroid function abnor-
women (nonsignificant), which in turn corresponded well
malities. Hirsutism was defined by the presence of excessive
with the population frequency (19%), as expected. None of
body hair in an androgen-dependent pattern, with a modified
the apoE genotypes was significantly overrepresented. Af-
Ferriman-Gallwey score of 8 or more (12).
fected women were then divided into subgroups according to
FERTILITY & STERILITY
the number of ⑀4 alleles: no ⑀4 allele (genotypes 2/3, 3/3),
results of the current study suggest that there is no link
one ⑀4 allele (genotypes 2/4, 3/4), and two ⑀4 alleles (geno-
between apoE genotype and the hyperlipidemia seen in the
type 4/4). The frequency of the 3/4 genotype, although
group of patients with PCOS. Waterworth et al. reported that
somewhat higher in affected women, was not significantly
susceptibility to PCOS maps to a variable number of tandem
different from that in the unaffected controls (P
repeats located upstream of the insulin gene, which also
thermore, using the general population as a reference, the 3/4
supports the theory that PCOS is, in part, caused by an
genotype was not associated with an increased risk of PCOS.
alteration in insulin production (17). The finding suggests
Women having the ⑀4/4 genotype constituted 3.4% of all
that there is a mechanistic link between type 2 diabetes and
affected women and 7.7% of the control group. The clinical
PCOS, which in turn would affect lipid metabolism and the
course of PCOS as regards the severity of the disease (pa-
risk of coronary disease. Overall, the altered pattern of sex
tients with oligoamenorrhea of Ͼ3 months compared with
hormones and insulin plays a major role in the dyslipidemia
those with mild cycle abnormalities) among affected women
associated with PCOS, and the role of the apoE genotype is
having the ⑀4/4 genotype was comparable with that in the
of minor importance. Exercise and weight loss are the ways
remaining PCOS women having other genotypes. Collec-
to reverse insulin resistance primarily and, secondarily, the
tively, an equilibrium state between apoE alleles and geno-
metabolic disturbances associated with it (18). The current
types was established in both PCOS women and controls.
results do not affect the clinical management of plasma lipiddisturbances in PCOS but provide some evidence against
familial hypercholesterolemia, which in turn is treated withlipid-lowering drugs in selected cases.
There is evidence that PCOS is clustered in families and
is inherited as a complex trait, like osteoporosis or hyper-
tension, where environmental factors interact with genetic
1. Barbieri RL, Smith S, Ryan KJ. The role of hyperinsulinemia in the
susceptibility to produce the phenotype. However, studies in
pathogenesis of ovarian hyperandrogenism. Fertil Steril 1988;50:197–212.
families have revealed that even within individual PCOS
2. Conway GS, Agrawal R, Betteridge DJ, Jacobs HS. Risk factors for
pedigrees, the clinical phenotype is very heterogeneous in
coronary artery disease in lean and obese women with polycystic ovarysyndrome. Clin Endocrinol 1992;37:119 –25.
females, and in males, obligate carriers appear to have vary-
3. Nagamani M, Dinh TV, Kelver ME. Hyperinsulinemia in hyperthecosis
ing degrees of premature male-pattern baldness (15). Family
of the ovaries. Am J Obstet Gynecol 1986;154:384 –9.
4. Geffner ME, Kaplan SA, Berch N, Golde DW, Landaw EM, Chang RJ.
studies and studies among twins are valuable in identifying
Persistence of insulin resistance in polycystic ovarian disease after
susceptibility candidate genes that predispose women to
inhibition of ovarian steroid secretion. Fertil Steril 1986;45:327–33.
5. Nestler JE, Barlascini CO, Matt DW, Steingold KA, Plymate SR, Clore
JN, et al. Suppression of serum insulin by diazoxide reduces serumtestosterone levels in obese women with polycystic ovary syndrome.
New therapies may be designed based on knowledge of
J Clin Endocrinol Metab 1989;68:1027–32.
gene function, and disease-associated genetic markers may
6. Reaven GM. Role of insulin resistance in human disease. Diabetes
be used to identify women at high risk of developing the
7. von Eckardstein S, von Eckardstein A, Bender HG, Schulte H, Ass-
disease. Finns, who are derived from a small group of
mann G. Elevated low-density lipoprotein-cholesterol in women withpolycystic ovary syndrome. Gynaecol Endocrinol 1996;10:311– 8.
founders, are more genetically homogeneous than many
8. Talbott E, Guzick D, Clerici A, Berga S, Detre K, Weimer K. Coronary
other populations and are thus ideal for large-scale linkage
heart disease risk factors in women with polycystic ovary syndrome.
Arterioscler Thromb Vasc Biol 1995;15:821– 6.
disequilibrium–mapping studies (16). Accordingly, familial
9. Ilveskoski E, Perola M, Lehtima¨ki T, Laippala P, Savolainen V, Paja-
genetic predisposition can be investigated in association
rinen J, et al. Age-dependent association of apolipoprotein E genotypewith coronary and aortic atherosclerosis in middle-aged men: an au-
studies. Multiallele polymorphisms have been identified in
topsy study. Circulation 1999;100:608 –13.
the apoE gene, which could also be of biologic significance
10. Miettinen T. Impact of apoE phenotype on the regulation of cholesterol
metabolism. Ann Med 1991;23:181– 6.
in PCOS. In the current study, we used a multiallele poly-
11. Adams J, Polson DW, Franks S. Prevalence of polycystic ovaries in
morphism approach to evaluate whether this gene plays any
women with anovulation and idiopathic hirsutism. BMJ 1986;293:355–9.
12. Hatch R, Rosenfield RL, Km MH, Tredway D. Hirsutism: implications,
role in the development of the hyperlipidemia seen in pa-
etiology, and management. Am J Obstet Gynecol 1981;140:815–30.
tients with PCOS, and we found that the frequency of the ⑀4
13. Vandenplas S, Wiid I, Grobler-Rabie A, Brebner K, Ricketts M, Wallis
allele does not appear to be significantly higher among
G, et al. Blot hybridization of genomic DNA. J Med Genet 1984;21:164 –72.
women with PCOS than among healthy fertile women or the
14. Tsukamoto K, Watanabe T, Matsushima T, Kinoshita M, Kato H,
general population. Accordingly, the results of the study
Hashimoto Y, et al. Determination by PCR-RFLP of apoE genotype ina Japanese population. J Lab Clin Med 1993;121:598 – 602.
confirm that there is an equilibrium state between the apoE
15. Carey AH, Chan KL, Short F, White D, Williamson R, Franks S.
genotypes and alleles in both healthy fertile women and
Evidence for a single gene effect causing polycystic ovaries and malepattern baldness. Clin Endocrinol 1993;38:653– 8.
women with PCOS. The frequency of the apo ⑀4/4 genotype
16. Peltonen L, Jalanko A, Varilo T. Molecular genetics of the Finnish
was not significantly increased among affected women.
disease heritage. Hum Mol Genet 1999;8:1913–23.
17. Waterworth DM, Bennett ST, Gharani N, McCarthy MI, Hague S,
An association between apoE isoforms and plasma lipid
Batty S, et al. Linkage and association of insulin gene VNTR regulatorypolymorphism with polycystic ovary syndrome. Lancet 1997;349:986 –
levels has been reported in several populations, and the
influence of PCOS on lipid metabolism is similar to the
18. Paquali R, Antenucci D, Casimirri F, et al. Clinical and hormonal
characteristics of obese amenorrheic women before and after weight
cholesterol-raising effect of the ⑀4 allele (8). However, the
loss. J Clin Endocrinol Metab 1989;68:173–9.
Heinonen et al.
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