RU2535011C2 - Differential diagnostic technique for primary placental insufficiency - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to pathologic anatomy, and enables providing the morphological differential diagnosis of trophoblastic, endometrial and mixed forms of primary placental insufficiency in spontaneous miscarriage in the first trimester. The technique consists in performing an immunohistological analysis of the villous chorion and decidual cells of the gravidary endometrium to evaluate a vascular endothelial expression index (VEEI) and the transforming growth factor (TGF-β2). If the VEEI villous chorion expression index is 1.5 standard units or less, while the TGF-β2 expression index is 1.4 standard units or less, the trophoblastic form of primary placental insufficiency is diagnosed. If the VEEI decidual cell expression index is 1.6 standard units or less, while TGF-β2 is 2.7 standard units or more, the endometrial form of primary placental insufficiency is diagnosed; if observing the above values simultaneously, the mixed form of primary placental insufficiency is diagnosed.

EFFECT: presented method is high-informative, increases the quality of a pathology report, promotes an individual approach to the further diagnosing, treatment and rehabilitation of the female reproductive health depending on the pathogenetic form of placental insufficiency.

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Description

The invention relates to medicine, namely to pathological anatomy, and allows morphological differential diagnosis of trophoblastic, endometrial and mixed forms of primary placental insufficiency during spontaneous abortion in the first trimester.

Relevance

The relevance of the proposed method is determined by the fact that primary placental insufficiency is the main reason for spontaneous abortion in the first trimester, the specific share of which, according to various authors, is 75-80% of the total number of reproductive losses. The application of the proposed method allows not only to conduct a morphological diagnosis of primary placental insufficiency, but also in each case to establish its pathogenetic form in order to develop an individual approach to further diagnosis and correction of disorders of the reproductive function of women.

A known method for the diagnosis of chronic placental insufficiency, according to which at a level of placental growth factor in the blood 126-315 PCG / ml; level of thermostable alkaline phosphatase 67-233 U / l; the content of lymphocytes SD95 + 31-74%; TNFα concentrations of 220-1710 pg / ml diagnose chronic placental insufficiency (Patent No. 2313795. Russian Federation. Method for the diagnosis of chronic placental insufficiency. / Lipatov I.S., Melnikov V.A., Tezikov Yu.V., et al. // Inventions, Utility Models. - 2007. - No. 36). However, the method is designed for chronic placental insufficiency, developing in the third trimester of pregnancy. It is a non-morphological method, the presence of placental insufficiency is judged indirectly, by factors determined in the peripheral blood. The pathogenetic heterogeneity of the syndrome is not taken into account.

There are several methods for diagnosing placental insufficiency using ultrasound. A method for early diagnosis of placental insufficiency, based on dopplerometry of the spiral arteries in pregnant women at 20-24 weeks of gestation. When the ratio of resistance indices in the spiral arteries of the peripheral to the central part of the placenta is less than 1.0, placental insufficiency is diagnosed (Patent No. 2257850. Russian Federation. Method for early diagnosis of placental insufficiency. / Posiseeva LV, Zimina SI, Peretyatko L.P. ., etc. // Inventions. Utility models. - 2005. - No. 22). A method for the diagnosis of fetoplacental insufficiency by assessing systemic and placental blood flow during ultrasound dopplerometric examination. Calculate the fetal-maternal hematological index, on the basis of which they judge the presence of placental insufficiency (Patent No. 2402271. Russian Federation. Method for the diagnosis of placental insufficiency. / Trivozhenko OV, Evtushenko ID // Inventions. Utility models. - 2010. - No. 30). The disadvantages of ultrasound methods are: the use of expensive equipment, a chronological framework that does not allow the diagnosis of primary placental insufficiency in the first trimester.

Closest to the technical solution is the method used in pathological practice for differential diagnosis of primary placental insufficiency in the first trimester during spontaneous abortions (Morphological characteristics of the chorion in spontaneous abortions of chromosomal etiology / M. Faltsev, Yu.V. Gorbacheva, I.N. Voloshchuk and dr. // Archive of Pathology. - 2004. - No. 6. - S.11-15). The method is carried out by immunohistochemical study of villous chorion with determination of the expression level of Ki-67 protein (proliferation marker) and p53 protein (apoptosis marker). If the extravillous cytotrophoblast proliferation index is less than 4.2 and the villous cytotrophoblast apoptosis index is above 26.2, spontaneous abortion with a pathological karyotype is diagnosed, with other values of proliferation and apoptosis indices, spontaneous abortion with a normal karyotype. The method has several disadvantages: non-specific factors are evaluated (proliferation and apoptosis), does not take into account the state of gravidar endometrium, differential diagnosis of other forms of primary placental insufficiency is not carried out.

These disadvantages of the above methods are proposed to eliminate in the inventive method.

The claimed technical result is the development of a method of morphological differential diagnosis of trophoblastic, endometrial and mixed forms of primary placental insufficiency during spontaneous abortion in the first trimester. The technical result is achieved by the fact that in an immunohistochemical study of the villous chorion and decidual cells of the gravidar endometrium, the expression index of vascular endothelial (VEGF) and transforming (TGF-β2) growth factors is evaluated. When the expression index in the villous chorion of VEGF is equal to or less than 1.5 cu, and TGF-β2 equal to or less than 1.4 cu, the trophoblastic form of primary placental insufficiency is diagnosed; when the expression index in decidual cells, VEGF equal to or less than 1.6 cu, and TGF-β2 equal to or more than 2.7 cu, diagnose the endometrial form of primary placental insufficiency; while identifying the above criteria, a mixed form of primary placental insufficiency is diagnosed.

Distinctive features of the method

The differential diagnostic parameters of expression of VEGF and TGF-β2 in the villous chorion and decidual cells of the gravidary endometrium were established. When the expression index in the villous chorion of VEGF is equal to or less than 1.5 cu, and TGF-β2 equal to or less than 1.4 cu, the trophoblastic form of primary placental insufficiency is diagnosed; when the expression index in decidual cells, VEGF equal to or less than 1.6 cu, and TGF-β2 equal to or more than 2.7 cu, diagnose the endometrial form of primary placental insufficiency; while identifying the above criteria, a mixed form of primary placental insufficiency is diagnosed.

The novelty of the method

For the first time, a method is proposed for the morphological differential diagnosis of trophoblastic, endometrial and mixed forms of primary placental insufficiency, based on the evaluation of the results of an immunohistochemical reaction with antibodies to VEGF and TGF-β2. Differential diagnosis of primary placental insufficiency during spontaneous abortion in the first trimester will contribute to the development of an individual approach to further treatment and rehabilitation of a woman's reproductive health, depending on the pathogenetic form of placental insufficiency.

Growth factors play one of the leading roles in the regulation of implantation, placentation and early embryogenesis in the first trimester of pregnancy. Vascular endothelial growth factor (VEGF) is found in high concentrations both in the peripheral blood of pregnant women and in the tissues of the developing placenta and endometrium (Immunological riddle of pregnancy / Edited by N. Sotnikova - Ivanovo: MIK Publishing House, 2005. - 276 p.). Its production is carried out by placental cells (syncytio- and cytotrophoblast, villus stromal cells), as well as decidual cells and endotheliocytes (Localization of angiogenic growth factors and their receptors in the human placental bed throughout normal human pregnancy / Schiessl B., Innes BA, Bulmer JN , et al. // Placenta. - 2009. - Vol.30, No. 1. - P.79-87). Not only vascularization of the villous chorion is controlled by VEGF. It was shown that the above factor stimulates the growth of placental tissue, controls the processes of differentiation and proliferation of cytotrophoblast, activates cytotrophoblastic invasion in the gravidar endometrium (The effects of angiogenic growth factors on extravillous trophoblast invasion and motility / Lash GE, Cartwright JE, Whitley G. StJ. Et. al. // Placenta. - 1999. - Vol.20, No. 8. - P.661-667). The latter process is key for the early and subsequent stages of pregnancy, since the invasion of cytotrophoblast cells leads to gestational remodeling of the spiral arteries of the uterus (endometrial and myometrial segments), transforming them into uteroplacental arteries, i.e. ensures the establishment of an adequate utero-placental blood flow (A. Milovanov, A. K. Kirichenko. Cytotrophoblastic invasion is a key mechanism for the development of a normal and complicated pregnancy. - Krasnoyarsk: Litera-print, 2009. - 188 p.). Data on the nature of VEGF production in placental insufficiency and miscarriage are contradictory: there are studies demonstrating how to reduce the expression level of VEGF (Expression of angiogenesis - and apoptosis-related genes in chorionic villi derived from recurrent pregnancy loss patients / Choi HK, Choi BC, Lee SH , et al. // Molecular Reproduction and Development. - 2003. - Vol.66, No. 1. - P.24-31), and, conversely, increase (Krukier I.I. Pogorelova T.N. endothelial growth factor and endothelin in the placenta and umbilical cord during normal and complicated pregnancy. // Bulletin of experimental bio ogii and medicine -. 2006. - T.141, №2 -. S.177-179). The differences, in our opinion, are due to a number of reasons: the method used, the gestational age, the test material (peripheral blood, placental tissue, endometrium).

Transforming growth factor (TGF-β2) is currently considered as the main immunosuppressive cytokine that provides the mother’s immune tolerance to foreign fetal antigens (Nagaeva O., Jonsson L., Mincheva-Nilsson L. Dominant IL-10 and TGF-beta mRNA expression in gammadeltaT cells of human early pregnancy decidua suggests immunoregulatory potential // American Journal of Reproductive Immunology. - 2002. - Vol. 48, No. 1. - P.9-17). Regarding the production of TGF-β2, regulatory T-lymphocytes are isolated from immune cells. However, in addition to lymphocytes, TGF-β2 is found in syncytio and cytotrophoblast, as well as stromal decidual endometrial cells (Transforming growth factor factor expression in human placenta and placental bed during early pregnancy / Simpson H., Robson SC, Bulmer JN, et al. / / Placenta. - 2002. - Vol.23, No. 1. - P.44-58). Another equally important function of TGF-β2, carried out in the placental area of the gravidar endometrium, is the restriction of cytotrophoblastic invasion (Graham CH Effect of transforming growth factor-beta on the plasminogen activator system in cultured first trimester human cytotrophoblasts // Placenta. - 1997. - Vol. .18, No. 2-3. - P.137-143). Studies have shown that TGF-P2 blocks the activity of matrix metalloproteinases of cytotrophoblastic cells by increasing the production of a tissue inhibitor (TIMP-1) (Cytokine-mediated regulation of 92-kilodalton type IV collagenase, tissue inhibitor or metalloproteinase-1 (TIMP-1), and TIMP -3 messenger ribonucleic acid expression in human endometrial stromal cells / Huang HY, Wen Y., Irwin JC, et al. // Journal of Clinical Endocrinology and Metabolism. - 1998. - Vol. 83, No. 5. - P.1721- 1729). Data on the nature of changes in the expression of TGF-β2 in pregnancy pathology are also contradictory. Based on the decrease in the level of TGF-β2 in the blood serum of women in the second trimester of pregnancy, miscarriage is predicted (Patent No. 2340899. Russian Federation. A method for predicting miscarriage in the second trimester of pregnancy. / Levchenko MV, Orlov VI // Inventions. Useful models. - 2008. - No. 34). With an increase in the level of TGF-β2, starting from the 5th week of gestation, the development of placental insufficiency is predicted (Patent No. 2238567. Russian Federation. Method for predicting fetoplacental insufficiency. / Orlov AV, Krukmer II, Pogorelova TN and others // Inventions. Utility models. - 2004. - No. 29). Studies conducted with the threat and spontaneous abortion in the first trimester showed an increase in the spontaneous production of TGF-β2 by lymphocytes of both the peripheral blood and the basal decidual membrane, however, the total level of TGF-β2 in extracts of the decidual tissue is reduced compared with the uncomplicated pregnancy (Immunological The riddle of pregnancy / Under the editorship of Sotnikova N.Yu. - Ivanovo: MIK Publishing House, 2005. - 276 p.).

Since in the process of placentation in early pregnancy there is a close interaction and integration of developing extra-embryonic structures and the basal endometrium with the formation of the placental placenta and placental bed in the subsequent formation of the placenta, it is likely that morphofunctional failure as a trophoblast is the cause of the development of primary placental insufficiency during spontaneous abortion, and endometrium.

Trophoblastic insufficiency is characterized by a pronounced growth retardation and differentiation of the villous chorion with a predominance of avascularized villi and a reduction in villous cytotrophoblast, as well as mild trophoblastic invasion into the endometrium. A sharp decrease in the expression index of VEGF (equal to or less than 1.5 cu) and TGF-β2 (equal to or less than 1.4 cu) in the villous chorion reflects, in our opinion, the functional insufficiency of trophoblast due to its primary damage, associated primarily with genetic abnormalities. We suggest using the above values of the expression index of both growth factors as a diagnostic criterion for the trophoblastic form of primary placental insufficiency.

Endometrial failure with spontaneous abortion in the early stages is associated primarily with hormonal disorders and, in particular, with insufficiency of the gravidar corpus luteum. The morphological substrate of endometrial failure is incomplete decidualization of the endometrial stroma, defective secretory transformation of the endometrial glands, weak spiralization of the endometrial arteries. Decidual endometrial cells, through the production of growth factors and other cytokines, have a regulatory effect on the differentiation of the villous chorion and cytotrophoblastic invasion. In our opinion, immunohistochemical signs of endometrial insufficiency during spontaneous abortions in the first trimester combine a decrease in expression of VEGF by deciduocytes, which has a stimulating effect on vascularization, growth, differentiation of the villous chorion and remodeling of the spiral arteries, with a simultaneous increase in the expression of TGF-β2, which was said to be inhibitory above , cytotrophoblastic invasion of the endometrium. According to the results of the study, the criteria for the endometrial form of primary placental insufficiency are the value of the VEGF expression index in decidual cells equal to or less than 1.6 cu, and the TGF-β2 expression index equal to or more than 2.7 ± 0.08 .e.

In some cases of spontaneous abortion in the presence of, as a rule, basal and parietal endometritis and impaired differentiation of the villous chorion with a predominance of sclerosed villi, the expression profile of VEGF and TGF-β2 in the villi of the chorion and decidual cells combines the signs of trophoblastic and endometrial insolvency, which allows studies and the above criteria to diagnose a mixed form of primary placental insufficiency.

Thus, an immunomorphological study of the villous chorion and gravidar endometrium with the study of the expression of two growth factors (VEGF and TGF-β2) during spontaneous abortions in the first trimester allows differentiating three forms of primary placental insufficiency that are different in the pathogenesis: trophoblastic, endometrial and mixed.

The method is as follows.

Material obtained by curettage of the uterine cavity during spontaneous abortion in the first trimester is fixed in 10% neutral buffered formalin for 24 hours. After histological posting, pieces of tissue are poured into paraffin, sections are made with a thickness of 5 μm containing fragments of villous chorion and gravidar endometrium. The immunohistochemical reaction is carried out using primary antibodies of Santa Cruz Biotechnology (monoclonal anti-VEGF antibodies (clone C-1) in a working dilution of 1: 200 and polyclonal anti-TOR-β2 antibodies in a working dilution of 1: 400) and the LSAB + System detection system HRP (Dako) according to the following protocol:

1. Dewaxing.

2. Unmasking antigenic structures by boiling in a pressure cooker in citrate buffer pH 6.0 for 10 minutes, cooling to room temperature.

3. Blocking endogenous peroxidase - incubation with 3% hydrogen peroxide for 20 minutes in a humid chamber at room temperature.

4. Incubation with primary antibodies for 20 hours (at night) in a humid chamber at a temperature of + 4 ° C.

5. Incubation with secondary biotinylated antibodies for 20 minutes in a humid chamber at room temperature.

6. Incubation with streptavidin-peroxidase complex for 20 minutes in a humid chamber at room temperature.

7. Reaction with the DAB chromogen for 3 minutes at room temperature.

8. Dyeing of nuclei with hematoxylin, conclusion of sections. After each step, the sections are washed in pH 7.4 phosphate buffer.

Evaluation of the immunohistochemical reaction is carried out by calculating the expression index per 100 chorionic villi and 100 decidual cells in 10 fields of view of the microscope with magnification × 100 and x400, respectively.

Preliminarily evaluate the intensity of the immunohistochemical reaction in points according to the following algorithm:

0 points - no reaction;

1 point - weak or moderate staining of only the cytotrophoblastic layer of the epithelial villi / weak staining of the cytoplasm of decidual cells;

2 points - mild or moderate staining of the villous syncythio- and cytotrophoblast / moderate staining of the cytoplasm of decidual cells;

3 points - pronounced staining of syncytio-, cytotrophoblast and villus stroma / pronounced staining of the cytoplasm of decidual cells.

The expression index of the studied factors is calculated by the formula:

Expression Index = Σ P (i) · i / 100,

where i is the reaction intensity in points from 0 to 3;

P (i) is the percentage of structures (chorionic villi or decidual cells) stained with different intensities.

When the expression index of VEGF and TGF-β2 in the villous chorion is equal to or less than 1.5 c.u. and equal to or less than 1.4 cu accordingly, the trophoblastic form of primary placental insufficiency is diagnosed; with an expression index of VEGF in decidual cells equal to or less than 1.6 cu, and TGF-β2 equal to or more than 2.7 cu, diagnose the endometrial form of primary placental insufficiency; while identifying the above criteria, a mixed form of primary placental insufficiency is diagnosed.

The essence of the method is illustrated by the following examples.

Example 1

Woman G-va, 32 years old. Beginning of a spontaneous miscarriage in a period of 9 weeks. A history of 2 pregnancies: 1 artifact abortion, 1 spontaneous miscarriage in the early stages. In the hospital, curettage of the uterine cavity was performed.

Histological examination revealed the predominance of large avascularized villi with stromal edema, the epithelial cover was thinned as a result of the almost complete absence of the cytotrophoblastic layer. In the compact layer of the gravidar endometrium, intermediate decidual cells, scanty diffuse infiltration by lymphocytes predominate. In the spongy layer, elongated and sawtooth endometrial glands with a light cylindrical epithelium. In the placentation zone, small scattered fibrinoid deposits, single cells of the invasive cytotrophoblast in the surface parts of the compact layer are determined.

An immunohistochemical study was performed. The expression index in the chorionic villi was: VEGF - 1.3 cu, TGF-β2 - 1.4 cu The expression index in decidual cells was: VEGF - 2.3 cu, TGF-β2 - 2.1 cu According to the claimed method, the trophoblastic form of primary placental insufficiency is diagnosed. Recommendations are given for a genetic examination of a married couple.

Example 2

Woman B-va, 26 years old. Beginning of a spontaneous miscarriage in a period of 9 weeks. The first pregnancy. Performed curettage of the uterine cavity.

Histological examination revealed a predominance of hypovascularized villi with incomplete dichotomous division with the formation of syncytial kidneys and free symplasts. The cytotrophoblastic layer in the epithelium of individual villi is reduced. Signs of differentiation of stem villi are absent. In the stroma of the gravidar endometrium, spindle-shaped pre-caudal cells predominate, endometrial arteries are weakly spiralized, glands are elongated and rounded with a smooth inner contour.

When conducting an immunohistochemical study, the expression index in the chorionic villi was: SEFR - 1.9 cu, TGF-β2 - 2.1 cu The expression index in decidual cells was: VEGF - 1.6 cu, TGF-β2 - 2.8 cu According to the claimed method, an endometrial form of primary placental insufficiency is diagnosed. Recommendations are given on a priority study of the hormonal function of the hypothalamus-pituitary-ovary system.

Example 3

Woman Q-VA, 20 years old. Beginning of a spontaneous miscarriage in a period of 12 weeks. Pregnancy second, 1st - artifact abortion. Performed curettage of the uterine cavity.

A histological examination diagnosed a violation of the differentiation of the villous chorion with a predominance of sclerotic avascularized villi. Gravid endometrium with focal necrosis and diffuse infiltration of polymorphonuclear leukocytes in a compact layer. Focal periglandular inflammatory infiltrates with a predominance of lymphocytes were found in the spongy layer. Decidualization of the endometrial stroma is insufficient: intermediate decidual and pre-residual cells predominate.

When conducting an immunohistochemical study, the expression index of VEGF in the chorionic villi was: - 1.2 cu, TGF-β2 in the chorionic villi - 1.1 cu The expression index in decidual cells was: VEGF - 1.4 cu, TGF-β2 - 2.7 cu According to the claimed method, a mixed form of primary placental insufficiency is diagnosed. Recommendations are given for the diagnosis of genital infections and for antibiotic and anti-inflammatory therapy.

The advantages of the method

1. It is an objective immunomorphological method for the differential diagnosis of pathogenetic forms of primary placental insufficiency: trophoblastic, endometrial and mixed.

2. A direct assessment is made of the state of the villous chorion and decidual cells of the gravidar endometrium at the local level.

3. In the diagnosis, growth factors specific for the villous chorion and decidual cells are used that directly control the processes of placenta development in the first trimester.

4. Improves the quality of the histological conclusion and allows you to individually determine the priority measures for the diagnosis and rehabilitation of reproductive disorders.

5. The claimed method is highly informative and easy to evaluate results.

Claims (1)

A method for the morphological differential diagnosis of forms of primary placental insufficiency during spontaneous abortion in the first trimester by immunohistochemical examination of the villous chorion, characterized in that the decidual cells of the gravidary endometrium are additionally examined, while the expression index of vascular endothelial and transforming growth factors is evaluated and the expression index in villous chorion of vascular endothelial growth factor equal to or less than 1.5 cu, and tra n formative growth factor equal to or less than 1.4 cu, diagnose the trophoblastic form of primary placental insufficiency; when the expression index in decidual cells of a vascular endothelial growth factor equal to or less than 1.6 cu, and a transforming growth factor equal to or more than 2.7 cu, diagnose an endometrial form of primary placental insufficiency; while identifying the above criteria, a mixed form of primary placental insufficiency is diagnosed.