Koncor1_2014.PM6


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IJMMR 2015 Vol. 1 No. 1

ULTRASTRUCTURAL RESEARCH OF THE ENDOMETRIUM
RECEPTIVITY IN THE CONDITIONS OF PRE�CONCEPTIONAL PREPARATION
IN REFRACTORY PREGNANCY LOSS

I. O. Marinkin, D. L. Nepomnyashikh, V. M. Kuleshov,
N. A. Ilizarova, T. O. Semchenko, S. V. Aidagulova

NOVOSIBIRSK STATE MEDICAL UNIVERSITY,  NOVOSIBIRSK, RUSSIA

Background. Refractory pregnancy loss (RPL) is a multifactoral pathology in women of reproductive age
which treatment represents great difficulties. Great role in RPL pathogenesis is stipulated by chronic endometritis
resulting from disturbances of implantation followed by gestational sac malfunction, which is not less significant
than progesterone deficiency.

Objective. The aim of research was to study the dynamics of ultrastructural changes of endometric epithelial
cells in the conditions of pre-conception preparation in refractory pregnancy loss.

Methods. 63 samples of endometrium bioptates obtained from female patients of reproductive age with
insufficient middle luteal phase of the cycle were studied. Light and electronic microscopy was used to study
the morphological changes in endometric epithelial cells.

Results. Pre-conceptional cyclic hormone therapy (1 mg 17�-estradiol and 20 mg of didrogesteron) in
comparison with monotherapy of 20 mg of didrogesteron during 3 months contributed to intracellular regeneration
and restoration of secretor phenotype of endometrium epithelial cells corresponding to the status of receptivity
with “opened window of implantation”.

Conclusions. Cyclic hormonotherapy in combination with complex metabolic therapy as a pre-conceptional
preparation contributing to pregnancy illustrates much higher effectiveness of pathogenetically proved pre-
conceptional hormonotherapy

KEY WORDS: refractory pregnancy loss, endometrium receptivity, hormone therapy, ultrastructure.

Introduction
Refractory pregnancy loss (RPL) is a multi-

factoral pathology in women of reproductive age,
which treatment represents great difficulties [1, 3,
4]. In the majority of cases the termination of
pregnancy in PRL is due to inadequate reaction to
endogenic progesterone and disturbance of
secretory transformation of endometrium that results
in the disturbance of blastocyst receptivity.

Great role in RPL pathogenesis is stipulated by
chronic endometritis resulting from disturbances of
implantation followed by gestational sac malfunction,
which is not less significant than progesterone
deficiency [11]. Weak receptive sensitivity of the
endometrium in maintaining normal level of hormo-
nes is connected with a wide prevalence of chronic
infectious and inflammatory processes accompanied
by high content of cytotoxic cells CD16+ and CD56+
and their products such as anti-inflammatory
cytokines that, in their turn, contribute to activation
of prothrombinase with following thrombosis and
placental infarctions [5, 8]. Besides of all mentioned

causes of RPL and chromosomal fetus pathologies,
there are idiopathic misbirths (approximately 15–
25 %), pathogenesis of which is not clear and
empirically progesterone is used [4].

Hormone therapy, as well as drugs possesing
direct or indirect action on the steroid metabolism,
are used to recover endometrium receptivity in case
of correction of luteal insufficiency in RPL [12].
However, a great percentage of misfortune in the
recovery of endometrium receptivity and achieve-
ment of fertility in RPL needs development of new
approaches to the therapy and improvement of
schemes of pre-conceptional preparation [9].

Aim of the research: the research was aimed to
study the dynamics of ultrastructural changes of
epithelial cells of the endometrium in the conditions
of pre-conceptional hormone therapy in RPL.

Methods
Morphological studying of endometrium sam-

pling bioptates in 63 women of reproductive age with
RPL was performed. The patients were empirically
divided into 2 groups (31 and 32 female patients
27.2±1.2 and 28.3±0.9 years old, respectively)
depending on the scheme of pre-conceptional

Address for correspondence: Igor Marinkin, State Budget
Educational Institution of Higher Professional Education
“Novosibirsk State Medical University” of the Ministry of Public
Health,  Krasny Prospect, 52, Novosibirsk, 630091, Russia

I. O. Marinkin et al.

International Journal of Medicine and Medical Research
2015, Volume 1, Number 1, p. 35-38
copyright © 2015, TSMU, All Rights Reserved



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IJMMR 2015 Vol. 1 No. 1

hormone therapy performed during 3 menstrual
cycles.

The first scheme is a cyclic hormone therapy, in
which femostin was administered 1/10 (1 mg
17�-estradiola and 10 mg didrogesterone) from the
1st to the 28th day of the cycle with additional
supplementation of dodrogesterone (dufastone) in
the dosage 10 mg per 24-hours from the 16th till 25th

day. It’s important to note that in comparison with
recommended schemes [4], the 17�-estradiol
dosage was decreased twice to stimulate insig-
nificantly proliferative and differentiated activity of
cellular endometrium populations. The first scheme
included administration of 20 mg of didrogesterone
(dufastone) per 24-hours from the 16th to 25th days
of cycle. In both groups hormone therapy was used
on the background of cyclic metabolic therapy
including 100 mg of vitamin E in combination with
0.012 g of lipoic acid and 0.1 g of calcium panto-
thenate from the 5th to 15th days of the cycle, and
with 1 mg of folic acid and 0.5 mg potassium orotate
from the 16th to 25th days, respectively.

Sampling bioptates of endometrium were
obtained according to the indications on the 22-24th

day of the menstrual cycle till pre-conceptional
preparation. Bioptates were fixed in 4 % solution of
paraform. Paraffin sections colored with haema-
toxylin and eosin, as well as semithin sections
colored with Schiff’s reagent and toluydine blue, were
studied using microscope Axio Scope.A1 with
camera AxioCam MRc5 and software ZEN blue
(C.Zeiss). Ultrathin sections of epithelial cells of
endometrium contrasted with uranyl acetate and lead
citrate were studied with electronic microscope JEM-
100S. Written agreement on performing examination
and treatment of RPL was taken in female patients.

Results
Before pre-conceptional hormone therapy the

complex of structural changes of endometrium in
female patients with RPL in the middle secretory
stage of menstrual cycle in light-optical studying of
paraffin and semithin sections corresponded to
general pathological agreement about severe
dystrophy and atrophy. Endometrial glands were
reduced in the amount, being shortened and
polymorphic according to the maturity: secretory
glands did not prevail and were desynchronized with
the phase of menstrual cycle, with dystrophy and
atrophy of epithelium. Subepithelial stroma of
endometrium was characterized by weak signs of
decidualization of cellular elements and the presence
of simple subepithelial lymphatic cells (fig. 1), that
didn’t correspond to the severe inflammatory
changes in chronic endometritis.

Electronic microscopic examination of epithelial
cells of endometrium revealed ultrastructural signs

of a considerable reduction of the level of biosynthetic
processes. The decreasing of height of epithelial
cells, apical membrane of which didn’t form pinopods,
associated with severely decreased amount of
nucleolus and components of canalicular system in
nucleus and was accompanied with the reduction of
elements of granular cytoplasmatic net in the
absence of mega mitochondria. The decrease of syn-
thesized protein and energetic potentials of epithelial
cells was due to the accumulation of only singular
glycogen grains.

Thus, ultrastructural equivalents of the dis-
turbance of endometrium receptivity in RPL were
insufficiently expressed by elements of protein
synthesized complex of the nucleus and cytoplasm
that in combination with little content of glycogen and
absence of mega mitochondria confirmed insuffi-
ciency of plastic and energetic material for providing
of secretory transformation. Disbalance of hormone-
dependent cellular differentiation of epithelial cells
of endometrium in cyclic stage in female patients
with RPL is possible to be interpreted as systemic
manifestation of universal syndrome of regenerative
and plastic insufficiency [7], which essence consists
of the disturbance of processes of intracellular
regeneration and forming of plastic deficiency in
different organs and tissues undergoing chronic
unfavorable influences.

The use of the 1st scheme of hormone therapy
during 3 menstrual cycles has stimulated intracellular
regenerative reactions of epithelial cells since an
adequate stage of the cycle by means of diffe-
rentiation and expression of secretor phenotype of
endometrium (fig. 2) more than proper level of re-
productive health with “opened window of implan-
tation” in comparison with the 2nd group (fig. 3).
Gigantic mega mitochondria contributing to hormo-
ne-induced cytological differentiation were revealed
in the epithelial cells of endometrium. There were
large karyosomes with clasters of regular tubular
structures, those membranes were appropriate to
the tubules of granular cytoplasmatic net with
numerous polysomes in the nucleus of the majority
of cells (fig. 4); gross deposits of glycogen were
localized in the cytoplasm diffusively and like super
nuclear aggregates. High polymorphic protrusions
of apical plasmolemma as pinopods were formed in
47-63 % of epithelial cells (fig. 5), other cells had
polymorphic microvillus and cilia on their apexes.

Discussion
As a result of using the 2nd scheme of pre-

conceptional preparation during three cycles, it was
noted the growth of proliferative activity of epithelial
cells which corresponds to the luteal phase of the
menstrual cycle – the glands of stellar and serrated
forms with wide lumens, and folded counters lined

I. O. Marinkin et al.



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IJMMR 2015 Vol. 1 No. 1

by prismatic epithelium with ultrastructural signs of
adequate cytodifferentiation. However, taking into
account the complex of clinical data we can observe
that the given scheme in most cases did not provide
the stimulation expression level of plastic and energy
potential of endometrial epithelial cells necessary to
“open a window to implantation”.

 Normally the epithelium and the endometrium
stroma are rapidly renewing tissues under the
influence of a particular combination of reproductive
hormones and should be exposed to remodeling for
the normal receptivity of blastocytes. The basis of
luteal phase deficiency is a reduced level of
progesterone receptor expression as a consequence
of inadequate estrogen content [6]. However, the
morphological “immaturity” of endometrium can be
observed even during the insufficient progesterone
production as a result. What is important is not only
the level of steroids in the organism, but also the
adequate realization of hormonal effect [8, 10].

Fig 1. Refactory pregnancy loss. Endometrial biopsy sample
on the 23rd day of menstrual cycle. Dystrophy of epithelial
cells, absence of pinopods; isolated stromal cells. Semifine
section, coloring with Schiffs reagent and toluidine blue.
Magnification 1050.

Fig. 2. Refactory pregnancy loss after cyclic hormonotherapy
during 3 months. Endometrium biopsy sample on the 23d
day of menstrual cycle. Numerous pinopods on the apical
pole of epithelial cells. Semifine section, coloring with Schiffs
reagent and toluidine blue. Magnification 1050.

Fig. 3. Refactory pregnancy loss after dydrogesterone
hormonotherapy during 3 months. Endometrium biopsy
sample on the 24th day of menstrual cycle. Isolated pinopods
on the epical poles of epithelial cells. Semifine section,
coloring with Schiffs reagent and toluidine blue. Magnification
1050.

 Fig. 4. Refactory pregnancy loss after cyclic hormonotherapy
during 3 months. Fragments of endometrium of granulocytes
on the 23rd day of menstrual cycle: the nucleolus in the
nucleus is with the clusters of granular tubular structures;
the cytoplasm contains free and attached polysoms. Electron-
diffraction pattern. Magnifying power 10 000.

Fig. 5. Refactory pregnancy loss after cyclic hormonotherapy
during 3 months. High pinopods on the apical poles of
endometrium epithelial cells on the 23rd day of menstrual
cycle: numerous exosomes and vesicles. Electron-diffraction
pattern. Magnifying power 15 000.

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IJMMR 2015 Vol. 1 No. 1

In our trial the low doses of estrogens at the stage
of pre-conceptional preparation of patients with
refectory pregnancy loss contributed both to
proliferation and hormone-mediated epithelium
differentiation with receptors expression both to
estrogens and to progesterone with the increased
dosages to the 16th day of a cycle inducing secretory
transformation. In other words, the cyclic hormo-
notherapy, providing the adequate supply of re-
productive steroid hormones, consequently induced
the intracellular regeneration of endometrium
epithelial cells with the restoration of the necessary
level of the receptor apparatus to estrogens and
progesterone. The result of this process was the
realization of status of endometrium receptivity
corresponding to the indexes of reproductive health.

Mucous membranes including endometrium play
a leading role in the adaptation processes; complex
morphological studies of mucous membrane based
on endoscopic biopsies demonstrate the per-
manence in their reaction that allows to formulate
the conception of “reaction unity of mucous mem-
branes” frequently realized by the syndrome of
regenerative and plastic insufficiency based on the
reducing of the processes of intracellular rege-
neration [2]. This syndrome can become a funda-
mental basis to create principally new approaches
to the therapy of chronic diseases especially of
endometrium by means of remedies inducing
regenerative reactions in opposition to aggressive
antibiotic therapy [3].

Conclusions
Thus, the obtained results allow us to distinguish

a new aspect in morphogenesis of endometrium
pathology in case of refractory pregnancy loss. In
the complex of interrelated pathologic phenomena,
there are the endocrine disorders, thrombophilia, and
chronic endometritis. One of the important positions
is concerned with endometrium dystrophy, which is
evident in the reduction of intracellular regenerative
epithelium reactions. The ultrastructural equivalents
of reducing the endometrium receptivity is the
reduction of protein synthesizing nuclear and
cytoplasmic organelles in epithelial cells that underlie
the discrepancy of the structure of luteal phase cycle
with a reduction of secretory activity and «closed
door to implantation».

The strategy basis of refractory pregnancy loss
treatment is the intensification of regenerative
reactions in cellular endometrium populations that
contributed to the induction of biosynthetic reactions
with endometrium receptivity and fertility. Cyclic
hormonotherapy in combination with the complex
metabolic therapy as a pre-conceptional preparation
contributed to pregnancy in 16 (51.6 %) women of
the 1st group and 13 (40.6 %) women in the 2nd group;
full-term children with Apgar scores 8-9 were born
in the 1st group – in 10 women (32.3 %), in the 2nd

group – in 8 women (25.0 %) demonstrating higher
effectiveness of pathogenetically proved pre-
conceptional hormonotherapy.

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Received: 2014.06.09

I. O. Marinkin et al.