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Notes on the study of mitochondria in the human amnion.

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Resumen por el autor, Ludwig A. Emge
Notas sobre el estudio de las mitocondrias en el amnios humano.
Los grhulos mitocondriales aparecen en las c6lulas del amnios
humano en todos 10s estados de la gestaci6n. Bajo el punto de
vista morfol6gico son semejantes a las mitocondrias que se
encuentran en otros 6rganos en todos sus rasgos, con excepci6n
de su afinidad por las materias tint6reas, que es menor que la de
las mitocondrias en otros tejidos. Su disposici6n estA influida
por la forma de la chlula, pero es constante para grupos definidos
de ciertos tipos de c6lulas. El period0 de’ gestaci6n no influye
sobre el tipo, forma, tamafio, disposicih o nGmero de las mitocondrias, con la excepci6n de 10s amnios muy j6venes que el
autor ha estudiado.
Translation by J o d F. Nonidez
Cornell Medical College, New York
Division of Obstetrics and Gynecology, Stanford University School of Medicine,
San Francisco, Colijornia
The search for mitochondria in the human amnion was undertaken for purely anatomical reasons, in order to prove or disprove
their presence and to establish such morphologica1 variations
which may facilitate future investigations. It is well known
that certain types of these granules exist in other units of the
gestation product. Van Cauwenberghe found mitochondria in
the cells of the chorion and the placental villi during the first
half of gestation. Later, Kervily demonstrated them also in
the same structures during the second half of pregnancy, laying
Van Cauwenberghe’s failure to find them during the second half
to insuficient chroniization of the tissues studied. Kervily
concluded that morphologically these granular structures were
alike at the various periods of gestation. While studying the
amnion I have had occasion to verify Kervily’s findings, and
can confirm his contention that mitochondria of quite similar
appearance are present in the cells of the chorion and the placental villi at all stages of gestation. On the other hand, our own
technical observations do not confirm Kervily’s assumption that
Van Cauwenberghe’s failure was due to imperfect chromization,
since we saw these granular structures equally often if chromization was materially shortened.
The study of the material includes twenty human amnions
fully mature and five younger amnions of 13, 4, 5, 7 , and 8
months of age, respectively. Of the first group five were obtained
at caesarean sections before the membranes had ruptured.
The location of the tissue removed from all membranes was
carefully noted, and during the early part of the study various
locations were studied in each amniotic sac. This latter procedure has proved t o be of greatest importance, since there is
a definite difference in the appearance of groups of cells within
one amniotic sac. The greatest uniformity is present in the
area covering approximately the inner two thirds of the placenta.
For a comparative study of human amnions this is the only area
which will give sufficiently uniform results to permit conclusions,
and this rule for selecting tissues should be observed for all
amnions more than five months old.
Since Kervily emphasized prolonged chromization of the tissues
in order to arrive at the best results, small pieces of amnion
obtained directly at birth were fixed in Regaud's neutral
formaldehyde-bichromate mixture, which was changed daily
for six days. The tissues were then chromicized in 3 per cent
potassium bichromate for one week. (Osmic acid could not be
obtained at the time of this study.) All tissues were collected
in body-warm fluids and the containers kept in an ice-box for
twenty-four hours. They were then transferred to a dark locker
and kept at room temperature for the remainder of the period.
The exclusion of light, and especially sunlight, is of importance
in obtaining even penetrations with fixing agents containing
potassium bichromate. I arrived at this conclusion by purely
physicochemical reasoning. As a rule, such fixing fluids remain
clear in the ice-box, and to some degree when light is excluded at
room temperature, while, when they are exposed to bright light
or sunlight or when they are kept in the incubator at 37"C., the
fluids show a heavy precipitate. This means that certain rays
and certain temperatures rapidly reduce the potassium bichromate. When this occurs, one finds a heavy water-insoluble
layer around the tissue which prevents or materially retards the
further process of fixing and mordanting. Since I have observed
this occurrence, I have strictly adhered t,o fixing under the exclusion of direct light, and I am convinced that I have obtained
tissues more uniformly penetrated by these fixing agents.
In the course of this study the period of fixing and chromicizing
was gradually reduced to twenty-four and forty-eight hours,
respectively, and it was found that, this time is entirely sufficient
to obtain satisfactory results. I, therefore, cannot join Kervily
in his contention that Van Cauwenberghe’s failure was due to
an insufficient period of chromization. In this laboratory we
practice secondary chromization as advocated by Bensley if
we feel that poor staining results are due to imperfect mordanting.
This method of chromicizing the mounted section for a few
minutes before staining has proved very valuable in our hands.
Nevertheless, staining difficulties will be encountered continually
when it comes to staining the amniotic cell. This holds true
for both vital and other stains. The only explanation that can
be offered is that the plasma of this cell has a low chemical or
physical affinity for the conventional fixing agents as well as for
the mitochondria1 stains. The prohibitively high price of other
metals used in fixing fine cell structures has kept me from looking
further into this question. The staining of the amniotic cell
for mitochondria, either in the vital or fixed state, is a difficult
problem indeed. I have not been able to discover the reason,
but it remains a fact that this cell as well as all of its finer details
have a poor affinity for dyes.
After trying out various methods, it was found that modified
acid fuchsin mixtures, such as described by Altmann and by
Bensley, would give the best results to demonstrate mitochondria,
although even these ‘best results’ were not very satisfactory.
It is of no use to describe the modifications, as they vary with
each individual piece of tissue and depend upon the ingenuity of
the morphologist in knowing how to adapt staining methods.
Vital dyeing was found t o be unusually difficult. The vital
dyes diffuse the cell plasma so quickly that it is often impossible
to make out individual structures. To obtain a clear cell picture
is a matter of luck in spite of the greatest care in not allowing the
tissues to dry and in attempting to keep them at an even temperature. The saline solutions which were used as solvents for the
dyes were varied in their composition, but this did not materially
affect the end results. There was no better success when amni-
otic fluid was used as a solvent. After finding thatalldyesrelated
to neutral red and methylene blue were useless on account of
their tendency to stain granules of various nature, Janus
green B in 1:4000 Ringer’s solution was used as a criterion.
I n tissues exposed to this dye, basal striations, apparently made
up of mitochondria, could be seen occasionally in certain types
of cells; in other types, masses of what seemed to be mitochondrial substance were observed. But the really finer types of
granules could not be made out on account of the difficulty stated
above. The only definite statement that one can make in
regard to vitally stained amniotic tissue is that mitochondria1
granules are present at all stages of gestation described here.
A comparative study between fresh and fixed tissue beyond the
question of existence of these granules in the amnion proved to
be fruitless.
Among twenty-five amnions studied there were twenty mature
membranes, five obtained at caesarean sections, before rupture,
and one each from a pregnancy terminated therapeutically at
I+, 4, 5, 7, and 8 months. The outstanding factor is that there
are several types of mitochondria in the cells of all these membranes, but that there is no essential differencein the morphological appearance of the individual types of granules in corresponding types of cells at the various periods quoted. A slight
variation is found only on comparing the granules of the earliest
to the fully mature membranes. Wide the relative number of
mitochondria in the given cells of these membranes is apparently
proportionally the same, one does notice a greater uniformity in
the size and the shape of these granules as a whole in the earliest
three amnions described here. The explanation for this may be
the greater uniformity of amniotic cells as such at these periods.
In the second half of gestation groups of cells within one amnion
vary a good deal in appearance, which finding is most marked
‘at term.’ In the mature membrane there are found, side by
side, groups of high and low columnar and cuboidal cells, each
group varying somewhat in the detail architecture of its individual cells.
Nevertheless, after allowing for these slight variations, one
can establish a mitochondrial criterion which is applicable to
practically all groups of cell types in the individual membranes.
I n the first half of pregnancy the globular and cocci-shaped
mitochondrial granules are the most common forms present.
In the second half rod-like and filamentous shapes become more
conspicuous. and in the fully mature membrane masses suggesting mitochondrial substance are common. While the types of
niitochondria are fairly uniform under similar conditions, the
distribution of these granules varies within individual cells of
the same type. Only in the earliest amnion studied a uniform
distribution of the mitochondrial granules was found throughout
the individual cell. I n the cells of all other amnions the distribution depends definitely upon the shape of the cell and its
location in regard to certain points of pressure.
I call attention to the fact that certain locations in the amnion
do not represent the true general type of the amniotic cells and
its internal architecture. For instance, each membrane has
certain areas in which cell life is either interrupted or actually
at its end, if one may judge from morphological appearances.
The extreme of this trophic disturbance is best seen in an area
varying from 8 to 10 or more cm. in diameter, which apparently
represents the point of greatest amniotic pressure. Here the
cell structures have lost most of their staining affinity. If one
may use the expression, it is only the shell of the cell that accepts
some dye. TJsually the cells of this area are flattened out. Their
nuclei are indistinct or necrotic. Mitochondria1 structures are
commonly absent or exceedingly scarce, and in the latter case
will not hold the dye. M y first impression was that this area
was always located over the internal 0s of the cervix uteri, since
it was found here in all of the unruptured membranes obtained
a t caesarean sections. That this assumption is not correct was
demonstrated later by a comparative study, when it was found
that this area may be situated at higher levels and that it corresponds to the site of the spontaneous rupture of the membrane.
Usually there are several of these areas demonstrable which vary
in degree of trophic changes. It is true that in multiparous
22, NO. 5
women such an area is commonly present over the internal 0s of
the cervix, although this may not be the future point of rupture,
and therefore at least two atrophic areas may be encountered in
one membrane. I n primiparous women the location of these
areas may be found anywhere. The presence of these areas is
constant in all membranes and suggests that the spontaneous
rutpure occurs at the point of greatest disintegration of cells.
Since the outline of this area or areas is irregular, it was found
advisable t o secure tissues a good distance away from the point
of rupture. The best and uniform tissues were obtained from
that part of the amnion which covers the inner half of the placenta. I have dwelt so lengthily on this factor because if this
rule is not observed future studies rriust end in conflicting
The distribution of mitochondrial structures in the membranes
of the second half of gestation is relatively uniform in corresponding types of cells, as pointed out above. Here one often
notices a distinct bipolar distribution of mitochondria with a
rather marked difference in the appearance of the granules at
each pole. The largest amount of mitochondria is seen in the
basal third of the cell regardless of what shape the cell may be.
The closer the granular structures approach the actual base of
the cell, the oftener they are seen to assume a formation of basal
striation. This is most pronounced in the high columnar and
piriform cells. This striation is made up of short parallel thin
rods of equal length. The individual rods which bulge slightly
at their midpoints rarely traverse more than one-sixth of the
entire length of the cell and most commonly occupy about onetenth of the long diameter of the cell. When the cell approaches
the lower types of columnar shape, the striation begins to move
away from the base. At the same time the staining intensity
decreases and some of its definition is lost until, when the cell
becomes very low, it is lost entirely. Mitochondria1 structures
are then replaced by a conglomerate mass that has all the staining
characteristics, vital and fixed, of mitochondrial substance.
Janus green B is taken up more readily by this substance than
it is by defined mitochondrial structures. When exposed to any
of the mit,ochondrial solvents t8hemass disappears, which leads
me to believe that it is either a massing of mitochondria prior
to dissolution or that it is a direct and very early mitochondrial
derivative. This mass also has a marked affinity for fat dyes
Fig. 1 Photomicrograph with oil-immersion lens. Represents the rod-shaped
mitochondria of the columnar cell types. Note the regularity with which they
are arranged in basal striation. The cupolas of the cells are conncrted by fine
plasma bands. Few spherical mitochondria can be seen in several cell cupolas.
Fig. 2 Photomicrograph with oil-immersion lens. Represents the low columnar and cuboidal cell type in which the mitochondrial substance has lost its
definition and forms a conglomerate mass away from thc base of the cell.
about in the same proportion as mitochondria have when they
undergo dissolution. Such masses are rarely seen in the high
types of cells and are also uncommon in the very lowest types.
Most commonly they are seen in the cuboidal and low columnar
Towards the mid level of the cell mitochondrial structures
become very scarce. The only exception to this is an occasional
very slender cell standing distinctly alone and supported only
by very delicate plasma strands bridging to adjacent cells. In
this type of cell long, slender filamentous mitochondria traverse
the entire length of the cell. Individual filaments gracefully
embrance the nucleus, leaving a very narrow clear zone in it,s
direct vicinity, and enter the cell cupola to form a densely interlaced labyrinth. In all other cells there is, as a rule, a fairly
wide nuclear zone which is free from mitochondria. This zone
varies in size and position with that of the nucleus. It is widest
if the long axis of the nucleus is parallel to that of the cell and
narrowest if the two axes intersect at right angles. A fair appreciation of this can be gained only if the cell is seen in its true
profile. Most often this zone occupies the upper end of the
middle third of the cell, but it may actually move into the upper
third and even enter the cell cupola.
While in the basal third of the cell rod-shape types of mitochondria are the outstanding structures with the globular and
filamentous forms in the minority, the reverse is true of the upper
third of the cell. In fact, here rods are so rare that they are
negligible and filamentous forms occur onIy in the certain slender
cells described. Most commonly, one finds the spherical, small,
cocci-like types, but in contradistinction to mitochondria in the
basal third these globular and cocci-like structures are ill-defined.
They are few in number, vary considerably in size, usually are
located in the joints of the cellular network, which renders them
still more indistinct, and tends to form a very thin conglomerate
layer directly under the membrane of the cupola. Occasionally,
one sees strands of these conglomerates enter the intercellular
Aberrant forms of mitochondrial structures may be found in
any type of cell, but their number appeared to be so small that
it seemed to be insignificant to describe them. No attempt has
been made at this time to make a very close comparison of the
earlier amnions in regard to mitochondria, because the material
is too scanty to obtain definite comparative pictures. Thc
35 1
description of these, therefore, has been confined to the most
general statements made in the first part of the report.
In conclusion iGis stated:
That mitochondria1 granules occur in the cells of the human
amnion at all stages of gestation;
That, with the exception of their staining affinity, which is
less than in other tissues, they morphologically resemble in every
respect mitochondria found elsewhere;
That their arrangement is influenced by the shape of the cell,
but that this arrangement is quite constant for definite groups of
certain types of cells;
That, with the exception of the very earliest amnion studied,
the period of gestation has no influence on the type, shape, size,
arrangement, or number of mitochondria seen.
I wish to express my appreciation to Miss Ella Wing, who
looked after the cutting and mounting of the material studied.
Stanford University Hospital,
&n Francisco. California.
MICHELDE 1916 Les mitochondries du syncytium dee villositka
placentaires chez la femme. Cpt. Rd. de la SOC.
de Biol., T. 79, p. 226.
A. 1907 Recherches s u r l e r61e du ayncytium dans la
nutrition embryonnaire dans la femme. Arch. d e Biol., T. 23, p. 13.
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