RU2842306C1 - Method for postpartum morphological differential diagnosis of full-term and premature pregnancy in women with recurrent pregnancy loss - Google Patents

Abstract

FIELD: medical science.

SUBSTANCE: invention refers to medicine, namely to pathological anatomy, and can be used for postpartum morphological differential diagnosis of full-term and premature pregnancies. A biomaterial representing placental chorionic villi syncytiotrophoblast of a woman suffering recurrent pregnancy loss, who gave birth in the third trimester, is sampled not later than 2 hours from the moment of delivery of the placenta. A staining intensity of the immunohistochemical reaction of placental chorionic villi syncytiotrophoblast sections is assessed in 10 microscope visual fields in 100 cells of the preparation at magnification x400. A VEGFR2 expression index is determined. If the VEGFR2 expression index is more than 0.54, the pregnancy is considered to be full-term and corresponds to the gestational age of 37 weeks or more. If the VEGFR2 expression index is less than 0.54, the pregnancy is considered to be premature and corresponds to the gestational age of 36 weeks 6 days or less.

EFFECT: method provides more objective and accurate postpartum morphological differential diagnosis of the full-term and premature pregnancies in the patients with recurrent pregnancy loss in the anamnesis ensured by determining the VEGFR2 expression index in placental chorionic villi syncytiotrophoblast.

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Description

The invention relates to medicine, namely pathological anatomy, and can be used for postpartum differential diagnostics of full-term and premature pregnancy in women with habitual miscarriage.

Habitual miscarriage (RMP) is a woman’s history of two or more consecutive spontaneous abortions before 37 weeks [Clinical guidelines “Habitual Miscarriage” of the Russian Ministry of Health 2022; 46 p.]. According to literature, the incidence of habitual miscarriage in Russia and the world varies from 5 to 20% and even tends to increase further [Egerup P., Kolte A.M., Larsen E.C., Krog M., Nielsen H.S., Christiansen O.B. Recurrent pregnancy loss: what is the impact of consecutive versus non-consecutive losses? Human Reproduction 2023; 31(11):2428-34]. Women with habitual miscarriage for various reasons may find themselves without medical monitoring of the course of pregnancy, which will not allow establishing the exact gestational age. According to Rosstat data for 2023 [Information on medical care for pregnant women, women in labor and women in labor (form N32; OKUD 0609364)], a total of 3,572 cases of home births were registered in the Russian Federation; 1,218,353 people were not registered for pregnancy at a antenatal clinic and were admitted to maternity institutions directly during childbirth. The above-described cases, including patients with RPL, account for 12% of the total number of births in the Russian Federation and require verification of whether the pregnancy was full-term or the birth was premature. Establishing this fact is necessary to resolve both medical issues - choosing the tactics for managing the newborn and the woman in the postpartum period, and legal issues - conducting a forensic medical examination and an examination of the quality of medical care, establishing the duration of the sick leave, etc.

Thus, the development of a method for postpartum morphological differential diagnostics of full-term and premature pregnancy in patients with habitual miscarriage will allow objectively establishing the fact of full-term or premature pregnancy in order to choose the tactics of postpartum management of the mother and newborn child, as well as resolving controversial legal issues regarding the timing of delivery.

There is a known method for determining the gestational age by the date of the last menstrual period, which is used as the starting point for the pregnancy [Obstetrics: National Guide / edited by E.K. Ailamazyan, V.I. Kulakov, V.E. Radzinsky, G.M. Savelyeva. - M.: GEOTAR - Media, 2011. - 1200 p.].

The method has a number of disadvantages:

1) is subjective: a woman cannot always accurately remember the date of the start of her last menstruation;

2) in the presence of a threatened early miscarriage, a woman may regard bloody discharge from the genital tract as menstruation, and then the starting point for the onset of pregnancy will be erroneous;

3) after childbirth, a new pregnancy can occur before menstruation;

4) pregnancy can occur before the first menstruation (menarche).

There is a known method for determining the gestational age using ultrasound examination (US), in which, at a gestational age of 11 weeks 0 days - 13 weeks 6 days, the gestational age is determined by the crown-rump length (CRL) of the fetus, and at later stages of pregnancy (with CRL> 84 mm) - by the size of the fetal head circumference. If there is a discrepancy between the gestational age determined by the date of the last menstruation and by ultrasound performed at 11 weeks 0 days - 13 weeks 6 days, the gestational age is established based on the ultrasound data [Normal pregnancy: clinical guidelines / N.V. Dolgushina, R.G. Shmakov, I.I. Baranov et al. - M., 2023. - 90 p.].

The disadvantage of the method is that it cannot be used in women who have not been examined and have not been registered for pregnancy at a women's clinic.

The closest to the claimed method is a method for determining the gestational age, namely, full-term pregnancy, based on the verification of terminal villi with a diameter of 30 to 80 µm, dominant over other types of chorionic branches, and the rearrangement of syncytiotrophoblast nuclei, accompanied by the formation of syncytial nodes and syncytiocapillary membranes[Ozhiganova I.N. Pathology of the placenta (to the Federal clinical guidelines) / ed. prof. G. B. Kovalsky // Library of the pathologist. Scientific and practical journal named after N.N. Anichkov. - St. Petersburg: State Budgetary Healthcare Institution "GPAB", 2017. - Issue 180. - 136 p.].

The disadvantages of the method are the subjectivity of the assessment of the morphological substrate, which does not exclude diagnostic errors in an inexperienced specialist, and the impossibility of using the method without additional training of a pathologist in placentaology.

The technical result of the claimed method is aimed at increasing the objectivity and accuracy of postpartum morphological differential diagnostics of full-term and premature pregnancy in patients with a history of habitual miscarriage.

The technical result is achieved by determining the expression index of vascular endothelial growth factor receptor-2 (VEGFR2) in the syncytiotrophoblast of the chorionic villi of the placenta of a woman with habitual miscarriage, delivered in the third trimester, and if its value is more than 0.54 conventional units, the pregnancy is full-term and corresponds to a gestation period of 37 weeks or more, and if the value is less than 0.54 conventional units, the pregnancy is premature and corresponds to a gestation period of 36 weeks 6 days or less.

The method is carried out as follows: within 2 hours after delivery, the placenta of a woman with habitual miscarriage who gave birth in the third trimester is delivered to the pathological anatomy department for morphological examination using the generally accepted method [Methodological principles of studying placental tissue and optimization of pre-preparation modes of material / N.V. Nizyaeva, Yu.S. Volkova, S.M. Mullabaeva, A.I. Shchegolev // Obstetrics and Gynecology. 2014. - No. 8. - P. 10-18.]. Upon completion of the macroscopic examination, fragments of the villous chorion in combination with the basal plate measuring 0.5 cm x 0.5 cm x 0.5 cm are excised from the central and paracentral areas of the placenta and, after fixation in 10% buffered neutral formalin and routine wiring, embedded in paraffin.

To conduct immunohistochemical studies, paraffin sections with a thickness of 4–5 μm are used; polyclonal rabbit antibodies to VEGFR2 Ab-AF6281 in a working dilution of 1:100 (Affinity) are used as primary antibodies.

After deparaffinization of the sections, antigen is unmasked by boiling twice in citrate buffer for 5 minutes in a microwave oven at a power of 650 W.

Endogenous peroxidase is blocked with 1% hydrogen peroxide solution with sequential incubation in a humid chamber with primary and biotinylated secondary antibodies. Between incubations, sections are washed in three changes of phosphate buffer.

Visualization of antigen-antibody complexes is performed using the Ultra Vision Quanto Detection System HRP (Thermoscientific, USA). The sections are then stained with DAB chromogen and finished with Mayer's hematoxylin.

In order to exclude the possibility of obtaining false-negative and false-positive results, each immunohistochemical study is carried out with the establishment of positive and negative controls. The negative control excludes specific staining of the preparations. The positive control confirms the specificity of the immunohistochemical reaction with these antibodies [Petrov S.V., Raikhlin N.T. Guide to Immunohistochemical Diagnostics of Human Tumors. - Kazan: Titul, 2004. - 456 p.].

The intensity of VEGFR2 expression in the syncytiotrophoblast of the chorionic villi of the placenta is assessed using a Micros MS-300 light microscope (Austria) in a semi-quantitative manner [Pathology of the Breast / F.A. Tavassoli et al. - Stamford: Appleton-Lange, 1999], the result is expressed in points: 0 - no expression, 1 - weak staining, 2 - moderate staining, 3 - maximum staining. The immunohistochemical reaction is assessed in 10 fields of view of the microscope at a magnification of x400 in 100 cells of the preparation, and the biomarker expression index is calculated using the histological scoring formula:

Expression index = ∑P(i) ⋅ i/100 conventional units,

where i is the intensity of staining of the immunohistochemical reaction of sections of syncytiotrophoblast of chorionic villi of the placenta of a woman with habitual miscarriage who gave birth in the third trimester, assessed in 10 fields of view of the microscope in 100 cells of the preparation at a magnification of x400, expressed in points from 0 to 3; P(i) is the percentage of cells stained with different intensity. The result is denoted by arbitrary units. [Pathology of the Breast / FA Tavassoli et al. - Stamford: Appleton-Lange, 1999] .

and if the value of the VEGFR2 expression index in the syncytiotrophoblast of the chorionic villi of the placenta is more than 0.54 conventional units, the pregnancy is full-term and corresponds to a gestational age of 37 weeks or more, and if the value is less than 0.54 conventional units, the pregnancy is premature and corresponds to a gestational age of 36 weeks 6 days or less.

Novelty of the method.

For the first time, a method for postpartum morphological differential diagnostics of full-term and premature pregnancy in women with recurrent miscarriage, delivered in the third trimester, was proposed based on determination of the VEGFR2 expression index in the syncytiotrophoblast of placental chorionic villi. It is known that VEGFR2 is expressed in the endothelial lining of blood vessels and the stroma of chorionic villi and has a proangiogenic effect. By binding to the endothelial growth factor (VEGF) and activating it, VEGFR2 promotes proliferation and growth of endothelial cells and formation of new vessels [Pavlov K.A., Dubova E.A., Shchegolev A.I. Fetoplacental angiogenesis in normal pregnancy: the role of vascular endothelial growth factor // Obstetrics and Gynecology. - 2011. - No. 3. - P. 11-16]. Our study has established that in the third trimester of pregnancy, VEGFR2 is also expressed in the syncytiotrophoblast, which is combined with the peripheral location of the sinusoid capillaries in the stroma of the chorionic villi. Probably, VEGFR2 located in the syncytiotrophoblast indicates the direction of migration of endothelial cells, and its interaction with VEGF promotes the formation of sinusoid capillaries involved in the formation of extended syncytiocapillary membranes, which are markers of placental maturity in full-term pregnancy [Shchegolev A.I. Modern morphological classification of placental damage // Obstetrics and Gynecology. - 2016. - No. 4. - P. 16-23].

Distinctive features of the method: that the expression index of the vascular endothelial growth factor receptor-2 (VEGFR2) is determined in the biomaterial, which is the syncytiotrophoblast of the chorionic villi of the placenta of a woman with habitual miscarriage who gave birth in the third trimester, according to the formula:

Expression index = ∑P(i) ⋅ i/100 arbitrary units,

where i is the intensity of staining of the immunohistochemical reaction of sections of the syncytiotrophoblast of the chorionic villi of the placenta, assessed in 10 fields of view of the microscope in 100 cells of the preparation at a magnification of x400, expressed in points from 0 to 3;

P(i) is the percentage of cells stained with different intensity,

and if the value of the VEGFR2 expression index in the syncytiotrophoblast of the chorionic villi of the placenta of a woman with habitual miscarriage who gave birth in the third trimester is more than 0.54 conventional units, the pregnancy is full-term and corresponds to a gestational age of 37 weeks or more, and if the value is less than 0.54 conventional units, the pregnancy is premature and corresponds to a gestational age of 36 weeks 6 days or less, and the biomaterial is collected no later than 2 hours from the moment of delivery of the placenta.

The essence of the claimed method is explained by the following examples :

Example 1.

Patient S., 37, leads an asocial lifestyle, was registered for pregnancy monitoring in the second trimester of pregnancy, finds it difficult to name the date of the first day of her last menstrual period. According to the woman, she has a history of 3 spontaneous miscarriages before 22 weeks, and has not been examined for habitual miscarriage. The woman was admitted to the obstetric clinic in the second stage of labor. During the objective examination, the following was revealed: height of uterine standing (HUP) 36 cm, abdominal circumference (AC) 100 cm, fetal heartbeat is clear, below the navel, rhythmic, 142 beats per minute. The fetal position is longitudinal, the fetal head is with the large segment at the entrance to the small pelvis. The contraction ring is 10 cm above the pubis. The third birth occurred in cephalic presentation of the fetus. A boy was born weighing 2550 g, body length 48 cm, with an Apgar score of 7/8 points.

According to the proposed method, the VEGFR2 expression index in the syncytiotrophoblast of the chorionic villi of the placenta was determined to be 0.85 conventional units, which corresponds to a full-term pregnancy of 37 weeks of gestation or more.

Conclusion: the pregnancy is full-term and corresponds to a gestational age of 37 weeks or more.

Example 2.

Patient P., 33, had a late spontaneous miscarriage at 21 weeks ten months ago. According to the patient, her menstrual function did not recover after the miscarriage. She was registered for pregnancy monitoring in the second trimester. The woman was delivered to the maternity hospital by an ambulance team 40 minutes after a home birth. According to the woman, her medical history includes two full-term and one premature birth, then two spontaneous miscarriages. The woman had her fourth home birth. A boy was born weighing 2738.0 g, with a body length of 47.0 cm.

According to the proposed method, the VEGFR2 expression index in the syncytiotrophoblast of the chorionic villi of the placenta was determined to be 0.36 conventional units, which corresponds to a premature pregnancy of 36 weeks 6 days of gestation or less.

Conclusion: the pregnancy is premature and corresponds to a gestational age of 36 weeks 6 days or less.

A morphological study was conducted on 30 placentas of women with a singleton pregnancy and a history of habitual miscarriage, who delivered in the third trimester: at gestational periods of up to 36 weeks 6 days - 15 placentas, 37 weeks and more - 15 placentas.

All patients were registered for pregnancy at the antenatal clinic before 12 weeks and were regularly monitored by an obstetrician-gynecologist. The gestation period was calculated from the first day of the last menstrual cycle, confirmed by obstetric examinations and ultrasound examination of pregnant women. The study did not include placentas with gross shape anomalies, extensive macroscopic foci of placental damage, as well as with signs of decompensated placental insufficiency. A review histological study of the placentas was carried out, which in all cases confirmed the correspondence of the villous chorion structure to the diagnosed gestation period. As a result of the assessment of the physical development of the newborns, it was found that the anthropometric parameters and physical development of all newborns corresponded to the gestation period.

An immunohistochemical study of placentas was performed to determine the VEGFR2 expression index in the syncytiotrophoblast of the chorionic villi of the placenta. According to the ROC analysis, a high VEGFR2 expression index in the syncytiotrophoblast of the chorionic villi of the placenta is associated with full-term pregnancy, and a low one is associated with preterm pregnancy. The threshold value was chosen to be the VEGFR2 expression index equal to 0.54 conventional units. The area under the curve was 1.0. The sensitivity and specificity of the model in the threshold value area were 100%. Thus, the VEGFR2 expression index in the syncytiotrophoblast of the chorionic villi of the placenta of more than 0.54 conventional units is associated with a full-term pregnancy, which corresponds to a gestational age of 37 weeks or more, and less than 0.54 conventional units - with a preterm pregnancy, which corresponds to a gestational age of 36 weeks 6 days or less (sensitivity 100%, specificity 100%) (Fig. 1).

Advantages of the proposed method.

1. The method has high sensitivity (100%) and specificity (100%).

2. The method is an objective, highly informative and reliable method of postpartum morphological differential diagnostics of full-term and premature pregnancy in women with habitual miscarriage, delivered in the third trimester.

2. The method increases the reliability of the histological conclusion based on the results of the forensic medical examination.

3. The method is easily reproducible and easy to evaluate the results; its implementation does not require special training of the pathologist.

4. The method will allow the use of a personalized approach in the tactics of managing mothers and newborns in the postpartum period.

5. The method will allow resolving controversial legal issues regarding the timing of delivery.

Claims (5)

A method for postpartum morphological differential diagnostics of full-term and premature pregnancy, characterized in that the expression index of vascular endothelial growth factor receptor 2 (VEGFR2) is determined in a biomaterial representing the syncytiotrophoblast of the chorionic villi of the placenta of a woman with habitual miscarriage who gave birth in the third trimester, according to the formula Expression index = ∑P(i) ⋅ i/100, conventional units, where i is the intensity of staining of the immunohistochemical reaction of sections of the syncytiotrophoblast of the chorionic villi of the placenta, assessed in 10 fields of view of the microscope in 100 cells of the preparation at a magnification of x400, expressed in points from 0 to 3; P(i) is the percentage of cells stained with different intensity, and if the value of the VEGFR2 expression index in the syncytiotrophoblast of the chorionic villi of the placenta of a woman with habitual miscarriage who gave birth in the third trimester is more than 0.54 conventional units, the pregnancy is full-term and corresponds to a gestational age of 37 weeks or more, and if the value is less than 0.54 conventional units, the pregnancy is premature and corresponds to a gestational age of 36 weeks 6 days or less, and the biomaterial is collected no later than 2 hours from the moment of delivery of the placenta.