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The microscopic anatomy of the hymen.

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The Microscopic Anatomy of the Hymen
The Department of Obstetrics and Gynaecology and the Department of
Histology, A i n Shams University, Faculty of Medicine, Cairo, U.A.R.
The hymen at different stages of life from birth to menopause was
examined under the microscope. Each section was stained with hematoxylin and
eosin, Van Gieson, Best’s carmine, Orcein, PAS, Mallory’s and modified Wilder technique. The modified Bodian’s technique was used to stain the nerve fibers and the
nerve cells.
The main bulk of the hymen is formed of fibrous connective tissue, partly
elastic and partly collagenous fibers. Both surfaces are covered by stratified squamous
epithelium which lacks any evidence of cornification. The epithelium is thicker at
the attached edge. Glycogen granules are equally present on the vaginal and on the
vulvar surface.
The connective tissue papillae are more numerous and more branching a t the free
edge and on the vaginal surface. There is no trace of glandular or muscle element.
The hymen is not richly supplied with nerve fibers. No nerve cells and fibers
are present at the free edge of the hymen. Nerve cells are bipolar, small i n size, oval,
spindle or kidney shaped. The terminal nerve fibers penetrate the epithelium in
between the connective tissue papillae.
In the newborn the hymen is vascular and the epithelium is thick. During
pregnancy the epithelium is very thick and very rich i n glycogen. At menopause the
epithelium is thin and certain areas are cornified.
The literature is nearly silent about the
hymen except when it is imperforate or
when its examination may solve a medicolegal problem. We believe a histological
study using recent techniques is important
for better understanding of its pathology.
The latter is important particularly in relation to the problem of introitus dyspareunia when pain or difficulty during
sexual intercourse is particularly experienced at the vaginal introitus. A histological study may also cast a helpful light on
its development, which is still a point of
The material used, whether the hymen
or the carunculae hymenalis, was taken
from newborn, adult virgins, nullipara,
multipara, pregnant women and menopausal women. The newborn were stillborn females while in virgins they were
two cases of hymenectomy for psychological apareunia.
Specimens were fixed in 10% formol
except those prepared for the modified
Bodian technique.
Sections of each hymen were stained
with hematoxylin and eosin. Sections
ANAT. REC., 149: 313-318.
were stained with Van Gieson, Orcein and
Weigart’s elastic stain for demonstration
of elastic tissue. Periodic acid-Schiff was
used for the demonstration of the mucopolysaccharides and the basement membrane. Modified Wilder technique was
used to stain the reticular fibers (Romeis,
’48; Ashton, ’61). The modified Bodian
technique was used to stain the nerve
fibers and the nerve cells (Ziesmer, ’52).
Modified Bodian technique
Fixation: Formol 40 %
Alcohol 80%
Glacial acetic acid
5 ml
90 ml
5 ml
1. Bring the section down to water.
2. Leave the section in a solution of 1%
protargol to which 4-5 g of metallic copper
are added. This is left in a n oven 38°C
for 24 hours.
3. Wash four times i n distilled water.
4. Transfer to the following mixture for
five minutes:
Solution A 5% silver nitrate.
Solution B hydroquinone
1.95 g
Citric acid
1.95 g
Distilled water
500 ml
The mixture is composed of 4 ml of
(A and 20 ml of ( B ) .
5. Wash four times in distilled water.
6. Transfer to Negrobrome for ten
7. Wash four times in distilled water.
8. Tone in 1% gold chloride to which
are added few drops of acetic acid.
9. Wash four times in distilled water.
10. Transfer to 2% oxalic acid.
11. Wash four times in distilled water.
12. Fix in 10% sodium thiosulphate.
13. Wash in running water.
14. Dehydrate, clear and mount (Ziesmer, '52).
The main bulk of the hymen is formed
of fibrous connective tissue covered on both
vulval and vaginal surfaces by stratified
squamous epithelium (fig. 1 ) . The following is the description of the intact adult
The epithelium lacks any evidence of
cornification. Its thickness is variable. It
is thicker towards the attached base to the
vaginal wall. The number of layers varies
between 30 and 50.
The basal palisade layer is columnar.
This is followed by 4 or 5 polygonal layers
with relatively large rounded or oval nuclei. The remaining thickness is formed
of a definitely larger clearer polygonal
cells. Their size, however, decreases towards the surface and their shape, becomes flatter. The cells forming the main
thickness of the epitheium are characterized by their clear cytoplasm and pyknotic
nuclei. This clarity is probably due to
rich glycogen content which disappeared
during preparation of the section. When
stained with Best's carmine, glycogen
could be easily demonstrated (fig. 2).
It was equal on both vulval and vaginal
surfaces. The epithelium rests upon a
definite basement membrane. No glands
could be demonstrated in any examined
Connective tissue
The connective tissue is formed of a
dense central lamina separated on either
side by a loose papillary layer. The connective tissue papillae are few and elongated at the vulval surface, but at the free
edge of the hymen and on the vaginal
surface they are numerous and the elongated ones branch many times. In sections
stained with Van Gieson and Orcein it is
clear that the amount of elastic tissue is
quite variable both in the papillary and
laminar layer. In sections impregnated
with silver to show the distribution of the
reticulin fibers the laminar connective
tissue part appears to be very rich in the
argyrophil network. The papillary part
however on either side is very poor in the
reticulin content except at the basement
membrane of the epithelium (fig. 3 ) .
Blood supply
The vascular supply of the hymen is
rich. Stained by H. and E. and Mallory
stain the fibrous tissue core is rich in blood
vessels especially near the free edge where
vessels are dilated and engorged. Multiple
capillaries were seen penetrating the covering epithelium and reaching its superficial layers (fig. 4).
Nerue supply
The hymen is not richly supplied with
nerve fibers. Examination of step sections
stained by the modified Bodian technique
was necessary to detect the nerve tissue
in the hymen. It was found that the nerve
supply is relatively rich in the attached
part, detected with difficulty in the middle
part and absent in the free edge where no
cells or fibers could be demonstrated after
meticulous search.
Nerve cells are bipolar, small in size and
taking different shapes, oval, spindle shape
and kidney shape (fig. 5). The axons and
dendrites take a wavy course among the
fibrous tissue.
The terminal nerve endings were demonstrated penetrating the epithelium in
between the connective tissue papillae
(fig. 6 ) . These nerve endings are similar
to those demonstrated by Hamilton, '57 in
the buccal mucous membrane.
T h e h y m e n at different stages o f life
( 1 ) The h y m e n o f the newborn. It is
formed of a thin fold of connective tissue
covered with stratified squamous epithelium. The epithelium is thick and shows
a picture very near to that described before
in the adult virgin. The connective tissue,
vaginal orifice. It is placed horizontally
when the subject is erect. It has two surfaces, a n inferior surface towards the
vulva and a superior surface towards the
vagina. The latter surface shows to a certain extent the continuation of the rugae
of the vagina. It has two borders: a n
attached or adherent border and a free
border around the hymenal orifice. The
free border is usually irregular, festonn6
or dentell6 or divided into multiple fingers
(Testut, '01). The hymenal orifice may
take different shapes. It may be semilunar,
falciform, cubiform or septate.
More than one theory has been suggested for the development of the hymen.
The theory that the hymen develops for
the Wolffian ducts is no longer accepted.
The hymen is the partition which persists
to a varying degree between the dilated
canalized fused sinovaginal bulbs and the
urogenital sinus proper. It is thus formed
passively by the invagination of the posterior wall of the urogenital sinus due to
expansion of the lower end of the vagina.
It is thus composed of vaginal cells derived
from the sinovaginal bulbs and a n external
layer of the urogenital sinus epithelium
and with mesoderm between (Browne and
Browne, '55).
The microscopic picture of the hymen
has not been reported in detail in recent
literature. According to McLeod and Read
('55), it is composed of vascular connective tissue covered on both vaginal and
vulvar surfaces by stratified squamous epithelium. It was described by Testut ('01)
as a reduplication of the mucosa with a
cushion of tissue very rich in elastic fibers.
This fibroelastic cushion is continuous
around the blood vessels of the hymen
and their ramifications. We found that the
hymen is rich in reticular fibers which are
condensed to form a definite basement
membrane upon which the epithelium
rests. We found that the amount of elastic
fibers is variable. Some hymens were rich
in elastic fibers while others were mainly
formed of collagenous fibers. This supports the clinical observation that the
hymen is of variable consistency and
elasticity. Sometimes it tears easily while
The hymen is a membrane attached to on certain occasions it is elastic allowing
the posterior half or two-thirds of the a sexual intercourse without being torn.
however, is less dense and less fibrous.
The number of blood vessels, however, is
greater per fold than at maturity. The
thick epithelium may be the result of
female sex hormones transmitted to the
fetus during pregnancy (fig. 7 ) .
( 2 ) The h y m e n in a nullipara. This is
typical of the adult virgin, microscopically,
in every respect (figs. 8, 9 ) .
( 3 ) During pregnancy. There is marked
irregularity and branching of the fibrous
tissue papillae i n a n arborized manner.
Each fold is mainly formed by the epithelial element with relatively thin connective tissue core.
There are exaggerated thin parallel
connective tissue intra-epithelial papillae
which are apparent a s islands when cut
The epithelium is very thick and the
cells are vacuolated, being rich in glycogen. The papillae when cut transversely
show central blood vessels surrounded by
cellular loose connective tissue (fig. 10).
( 4 ) T h e h y m a n of a multipara. The
connective tissue core is dense and more
vascular. The connective tissue papillae
are long, thin, parallel, and extend near
the superficial layers of the epithelium.
The epithelium is relatively diminished in
comparison to the connective tissue. The
cells are deeply basophilic and not so clear
as in the virgin hymen (fig. 11).
( 5 ) Hymenal remnants at menopause.
The epithelium which is thin is divided
into two zones :
( a ) A narrow deep zone of polygonal
cells with relatively large dark nuclei surrounded by a thin rim of cytoplasm.
( b ) A thick superficial zone of polygonal cells which are elongated in a transverse direction. The nuclei are relatively
small, oval or pyknotic; the cytoplasm is
Certain areas of the epithelium show a
tendency to the formation of linear, scaly
cornified cells acidophilic in staining but
still contain flat nuclei (fig. 12).
The papillary layer of connective tissue
is still apparent but less marked than in
the adult hymen.
It was suggested at one time that the
hymen contained muscle fibers which are
continuous with the vaginal muscle fibers
(Testut, ’01). The presence of muscular
elements has never been admitted by histologists. There was no trace of muscle
fibers in our specimens.
The hymen does not contain glandular
tissue, which explains why it is very rare
to get hymenal cysts. If they develop they
must arise from aberrant glandular elements. The hymen in our specimens is
particularly vascular. Multiple capillaries
reach the superficial layer of the epithelium. The vascularity is the cause of the
severe bleeding which occasionally accompanies defloration injuries.
The microscopic anatomy of the nerve
supply of the hymen and its distribution
has not been discussed before. We found
that the free border of the hymen is without nerve fibers while the attached border
is relatively rich. The absence of nerve
fibers from the free border is quite physiological, otherwise defloration would have
been a very painful process. I n fact it is
very painful when the tears are extensive
and in these cases defloration is usually
accompanied with bleeding. This is not
the only place in the genital tract where
the distribution of the nerve endings serves
a physiological need. The functioning layers of the endometrium are devoid of nerve
fibers, otherwise menstruation would always be a very painful process.
Further study of the nerve supply of the
hymen may reveal microscopic changes
in cases of introitus dyspareunia seen in
early reproductive life and near menopause.
We gratefully acknowledge the helpful
advice, encouragement and direction given
by Prof. A. Ammar and Prof. I. A. Naga.
Browne, F., J. Browne and J. C. Brown 1955
Postgraduate Obstetrics and Gynaecology. Butterworth & Co., Ltd., London. Chap. 6, 110-112.
Draeger, M., and J. Konrad 1958 Praktikum
fur Med-Techn. Assistenten, Veb Verlag Voik
und Gesundheit, Berlin, page 92.
Hamilton, L. 1957 Die Nervenausbreitungen
In Der Uvula. Acta Anat., 3 1 : 66-75.
McLeod, H., and D. Read 1955 Gynaecology,
J. & A. Churchill Ltd., London, Chap. I., 17-18.
Romeis 1948 Mikroskopische Technik, R. Oldenbourg, Miinchen. Chap. 1936, 357.
Testus, L. 1901 Traite d’Anatomie Humaine,
Octove Doin, Editeur, Paris.
Ziesmer, C. 1952 Zentrablatt fur Microskopische Forschung und Methodik, 7 : 415.
Rich reticulin condensed at the basement membrane stained by Modified Wilder. X 100.
Nerve cells, bipolar and kidney shaped, stained by Modified Bodian stain. X 240.
Nerve fibers penetrating the deep layers of the epithelium stained by Modified Bodian.
4 Dilated capillaries penetrating the superficial layers of the epithelium stained by Mallory stain. x 100.
The vulvar surface of the hymen showing glycogen granules in the epithelium stained by Best Carmine.
x 6.
x 240.
1 The virgin hymen showing stratified squamous epithelium on both surfaces with fibrous tissue in between.
Maher Mahran and A. M. Saleh
9 The
10 The
11 The
12 The
hymen of the newborn showing the well developed epithelium. X 240.
virgin hymen. X 240.
hymen i n a nullipara is similar to the virgin hymen. x 100.
hymen during pregnancy showing very thick epithelium rich i n glycogen. X 240.
hymen of a multipara. The epithelium is relatively diminished in comparison to the connective tissue.
thin epithelium at menopause. x 240.
Maher Mahran and A. M. Saleh
x 240.
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anatomy, hymen, microscopy
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