RU2826778C1 - Method for prediction of preterm delivery in women with threatening late miscarriage and recurrent miscarriage - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, particularly to obstetrics and gynaecology, and can be used for prediction of premature delivery in pregnant women with threatening late miscarriage at 16–21 weeks and 6 days of gestation and recurrent miscarriage. Prediction is carried out by determining in peripheral venous blood serum content of homologous to lymphotoxins inducible factor, which competes with glycoprotein D of herpes simplex virus for mediator of herpes virus penetration (LIGHT). If LIGHT is equal to 44.5 pg/ml or less, premature delivery is predicted in the patient.

EFFECT: method is simple, accessible, has high accuracy, enables well-timed preventive measures and reduces perinatal complications for mother and child.

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Description

The invention relates to medicine, namely to obstetrics and gynecology, and can be used to predict premature birth in pregnant women with a threatened late miscarriage at a gestation period of 16 - 21 weeks and 6 days and habitual miscarriage.

The relevance of the proposed method is determined by the high risk of premature birth in women with recurrent miscarriage. The frequency of premature births in this group is 35-36% [Obstetrics: national guidelines / edited by G.M. Savelyeva, G.T. Sukhikh, V.N. Serov, V.E. Radzinsky. - 2nd ed., revised and enlarged. - M .: GEOTAR-Media, 2019. - 608 p.]. The problem of premature birth remains one of the most significant and currently unresolved [Preterm birth is an unsolved problem of the 21st century / V.E. Radzinsky, A.A. Orazmuradov, I.V. Savenkova et al. // Kuban Scientific Medical Bulletin. - 2020. - Vol. 27, No. 4. - P. 27-37]. Despite the high achievements of medicine at the present stage, premature births determine a high frequency of neonatal morbidity and mortality, the development of disabling somatic and neuropsychiatric pathology in the child in subsequent periods of life [Evsyukova I.I. Premature babies: current problems of nursing and prevention of adverse effects // Journal of obstetrics and women's diseases. - 2021. - Vol. 70, No. 3. - P. 93-102].

Thus, predicting premature birth in women with threatened late miscarriage and habitual miscarriage will allow timely implementation of treatment and preventive measures in order to reduce perinatal complications for mother and child.

A method for predicting premature birth in patients with primary habitual miscarriage is known [Patent No. 2485896 Russian Federation. Method for predicting premature birth in patients with primary habitual miscarriage / Vinokurova E.A., Bashmakova N.V., Mazurov A.D. // Inventions. Utility models. - 2013. - No. 18]. The essence of the method is that in patients with primary habitual miscarriage, the presence of a regressing pregnancy in the anamnesis is revealed, during the examination, signs of undifferentiated connective tissue dysplasia (UCTD), chronic intrauterine infection (CIUI) not treated before pregnancy are revealed, the severity of preeclampsia is diagnosed, an ultrasound is performed at 20-24 weeks of pregnancy to detect placental insufficiency, the presence of intrauterine growth retardation of the fetus and the prognostic index F is calculated using the formula:

F=0.631·X1+0.692·X2+0.857·X3-1.538·X4-2.182·X5-0.465·X6-0.465;

where X1 is a history of frozen pregnancy syndrome: if present - 1, if absent - 0;

X2 - signs of UCTD in mothers: if present - 1, if not - 0;

X3 - presence of placental insufficiency: if present - 1, if not - 0;

X4 - presence of IUGR of the fetus: if present - 1, if not - 0;

X5 - severe preeclampsia: if present - 1, if not - 0;

X6 - chronic intrauterine infection, treated during pregnancy: if present - 1, if not - 0;

and with F less than 0, a high probability of premature birth is predicted, and with F more than 0, there is no threat of premature birth.

The method has the following disadvantages:

1. The method allows predicting premature birth only in patients with primary habitual miscarriage, which limits its use in women with secondary habitual miscarriage;

2. There is no data on the possibility of using this method in women with habitual miscarriage and threatened late miscarriage.

3. The method is labor-intensive, as it involves evaluating a large number of indicators with subsequent mathematical calculations;

A method for assessing the risk of premature birth in women with habitual miscarriage is known [Patent No. 2701109 Russian Federation. Method for assessing the risk of premature birth in women with habitual miscarriage / Redkov O.V., Dadabaev V.K., Stolnikova I.I., Dosova S.Yu. // Inventions. Utility models. - 2019. - No. 27]. The essence of this method is that at the gestation period of 8-10 weeks the level of interleukin 1 (IL 1), interleukin 6 (IL 6), inflammation marker - tumor necrosis factor ligand, apoptosis-inducing (TRAIL) are determined in the peripheral blood serum, with subsequent entry of these data into the equation for calculating the risk of premature birth: p (preterm birth) = 1/1 + eZ, where p (preterm birth) is the risk of premature birth, e is the base of natural logarithms (Euler's number = 2.718), and the z value is calculated using the formula: z = 1.207 * Π1 + 0.723 * Π2 - 1.823 * Π3 + 5.361, where 1.207, 0.723 and -1.823 are the coefficient values for IL 1, IL 6 and TRAIL, 5.361 is the equation constant, and P1, P2 and P3 are the values of IL 1, IL 6 and TRAIL levels in the blood serum, and if the p value (preterm birth) is greater than 0.470, the risk of preterm birth is considered high, and if it is less than 0.470, the risk of preterm birth is considered low.

The method has the following disadvantages:

1. There is no data on the possibility of using this method in women with habitual miscarriage and threatened late miscarriage;

2. The method is labor-intensive, as it involves evaluating a large number of indicators with subsequent mathematical calculations;

3. The method is less sensitive (80.8%) and specific (69.8%) in contrast to the claimed method.

The closest to the claimed method is the method for predicting premature birth in women with a threatened early miscarriage and a history of habitual miscarriage [Patent No. 2755269 Russian Federation. Method for predicting premature birth in women with a threatened early miscarriage and a history of habitual miscarriage / Malyshkina A.I., Sotnikova N.Yu., Kroshkina N.V., Grigushkina E.V., Talanova I.E. // Inventions. Utility Models. - 2021. - No. 26]. The essence of the method is that the relative content of IL-4 positive monocytes is determined in the peripheral venous blood of pregnant women with a threatened miscarriage and a history of habitual miscarriage at 5-12 weeks of gestation before prescribing conservation therapy. If its value is 26.7% or less, premature birth is predicted.

Disadvantages of the method:

1. The method allows predicting premature birth only in patients with threatened early miscarriage, which limits its use in women with threatened late miscarriage;

2. To implement the method, equipment is required that is only available in level 3 clinical diagnostic laboratories [“On approval of the Rules for conducting laboratory tests”: Order of the Ministry of Health of the Russian Federation dated May 18, 2021 No. 464n. https://legalacts.ru/doc/prikaz-minzdrava-rossii-ot-18052021-n-464n-ob-utverzhdenii/], which limits the availability and widespread use of this method.

The technical result is aimed at expanding the arsenal of prognostic tools and increasing the availability of predicting premature birth in women with a threatened late miscarriage at a gestation period of 16 - 21 weeks and 6 days and habitual miscarriage.

The claimed technical result is achieved by determining the serum content of the lymphotoxin-homologous inducible factor that competes with the glycoprotein D of the herpes simplex virus for the mediator of herpes virus penetration (LIGHT) in the peripheral venous blood of pregnant women with habitual miscarriage and with a threatened late miscarriage at a gestation period of 16-21 weeks and 6 days before the start of maintenance therapy, and at a value of 44.5 pg/ml or less, premature birth is predicted with an accuracy of 90.0%.

The method is carried out as follows.

1. In a pregnant woman with habitual miscarriage and threatened late miscarriage diagnosed at gestation periods from 16 to 21 weeks and 6 days, at the same stages of pregnancy before the start of maintenance therapy, 1 ml of peripheral venous blood is collected from the cubital vein into a dry test tube.

2. The blood is left to settle until a clot forms, then centrifuged for 10 minutes at 1500 rpm to obtain blood serum.

3. Add 100 µl of standards and serum samples to the wells of the strips with antibodies to LIGHT immobilized on the inner surface. Incubate the strips at 25 ^° C for 2.5 hours with gentle shaking on a microplate shaker. After incubation, wash the wells of the strips four times with wash buffer.

4. Add 100 µl of biotinylated antibodies to each well. Incubate the strips at 25 ^° C for 1 hour with shaking on a microplate shaker. After incubation, wash the strip wells four times with wash buffer.

5. Add 100 μl of HRP-streptavidin conjugate to each well and incubate the strips at 25 ^° C for 45 min with gentle shaking on a microplate shaker. After incubation, wash the strip wells four times with wash buffer.

6. Add 100 µl of TMB substrate solution to each well. Incubate the strips for 30 min at room temperature on a microplate shaker, avoiding direct sunlight.

7. To stop the enzymatic reaction, add 50 µl of Stop Reagent to all wells.

8. The optical density in the wells is measured at a wavelength of 450 nm using a Multiscan FC microplate reader (Labsystems, China).

9. Based on the calibration curve constructed by the Multiscan FC microplate reader (Labsystems, China), the serum content of the lymphotoxin-homologous inducible factor that competes with the glycoprotein D of the herpes simplex virus for the mediator of herpes virus entry (LIGHT) is determined, and at a value of 44.5 pg/ml or less, premature birth is predicted in women with threatened late miscarriage at a gestation period of 16 - 21 weeks and 6 days and habitual miscarriage.

The novelty of the claimed method is that for the first time it is proposed to predict premature birth in pregnant women with a threatened late miscarriage at 16-21 weeks and 6 days of gestation and habitual miscarriage by determining the serum content of LIGHT in the peripheral blood.

LIGHT is a member of the tumor necrosis factor (TNF) superfamily. It binds to lymphotoxin beta receptors (LTbetaR), herpes virus entry mediator (HVEM), and decoy receptor 3 (DcR3). LIGHT, through interaction with LTbetaR and HVEM, is able to induce either cell death or activation of the transcription factor NF-kappaB [Steinberg M.W., Cheung T.C., Ware C.F. The signaling networks of the herpesvirus entry mediator (TNFRSF14) in immune regulation // Immunological reviews. - 2011. - Vol. 244, No. 1. - P. 169–187. doi: 10.1111/j.1600-065X.2011.01064.x.; Comprehensive characterization of TNFSF14/LIGHT with implications in prognosis and immunotherapy of human gliomas / M. Han, Y. Sun, W. Zhao et al. // Front. Immunol. - 2022. - Vol. 13. - P. 1025286. doi: 10.3389/fimmu.2022.1025286]. It has been shown that LIGHT is a proinflammatory cytokine that modulates the activity of the T-link of immunity, promotes increased cell proliferation, production of proinflammatory cytokines and activation of endothelial cells. LIGHT is also a potent initiator of T-cell costimulatory signals leading to cytotoxic T-lymphocyte-mediated rejection of tumors and grafts. [Granger S.W., Rickert S. LIGHT-HVEM signaling and the regulation of T cell-mediated immunity // Cytokine Growth Factor Rev. – 2003. – Vol. 14, № 3–4. – P. 289–296. doi: 10.1016/s1359-6101(03)00031-5]. It was established that dysregulation of LIGHT activity leads to disruption of T-cell homeostasis and ultimately to disruption of peripheral tolerance. [Wang J., Fu Y.X. The role of LIGHT in T cell-mediated immunity // Immunol. Res. – 2004. – Vol. 30, № 2. – P. 201–214. doi: 10.1385/IR:30:2:201; Immunoregulation by tumor necrosis factor superfamily member LIGHT / Y. Wang, M. Zhu, M. Miller, Y. X. Fu // Immunological reviews. – 2009. – Vol. 229, No. 1. – P. 232–243. doi:10.1111/j.1600-065X.2009.00762.x].

Apparently, a decrease in the concentration of LIGHT in the blood serum in the second trimester of pregnancy leads to a disruption in the formation of a normal T-cell and innate immune response during pregnancy, a disruption of immunoregulation, which can lead to premature birth.

Distinctive features of the method: in women with habitual miscarriage with the development of a threatened late miscarriage at a gestation period of 16 - 21 weeks and 6 days, the serum content of the inducible factor homologous to lymphotoxins, competing with the glycoprotein D of the herpes simplex virus for the mediator of herpes virus penetration (LIGHT), is determined, and if its value is 44.5 pg/ml or less, premature birth is predicted.

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

Example 1.

Female D., 31 years old, fourth pregnancy, wanted, occurred spontaneously. History of 3 spontaneous miscarriages at 5-6, 6-7 and 8-9 weeks of gestation with curettage of the uterine cavity. At 16 weeks of pregnancy, the woman was hospitalized with complaints of nagging pain in the lower abdomen, bloody discharge from the genital tract. Vaginal examination: external genitalia are developed correctly, the vagina is nulliparous, the cervix is conical, long, the external os is closed. The body of the uterus is enlarged to 16 weeks of pregnancy, round, dense, mobile, painless. Bloody discharge in insignificant amounts. According to ultrasound data, the gestational age is 16 weeks, fetal heartbeat is registered, uterine hypertonicity is noted, there is no evidence of retroplacental hematoma, cervicometry shows that the length of the cervical canal is 30 mm, the shape of the internal os is T-shaped.

Diagnosis: Pregnancy 16 weeks. Threatened late miscarriage. Habitual miscarriage.

Before prescribing maintenance therapy according to the stated method, the serum LIGHT content was determined to be 18.36 pg/ml.

Conclusion: prognosis – premature birth.

Conservative therapy was performed with a positive effect. The woman was discharged from the hospital with a progressive pregnancy at 18 weeks.

At 33 weeks 2 days pregnant woman was hospitalized in the obstetric clinic with complaints of cramping pains in the lower abdomen, increased mucous discharge from the genital tract. Vaginal examination revealed: cervix up to 2 cm long, unevenly softened, external os allows fingertip, uterus enlarged to 33 weeks of pregnancy. Effective tocolytic therapy was started, prevention of fetal respiratory distress syndrome was performed. At 34 weeks of pregnancy, active labor began, premature birth occurred. A live premature girl was born weighing 1800 g, 45 cm tall, Apgar score 4/5 points, the child was transferred to the pediatric intensive care unit.

Diagnosis: first premature birth at 34 weeks of pregnancy. Habitual miscarriage.

The forecast for the claimed method was confirmed.

Example 2.

Patient S., 32 years old, third pregnancy, wanted, occurred spontaneously, in her first marriage. History includes non-viable pregnancy at 6 weeks and spontaneous miscarriage at 8 weeks. At 18 weeks and 3 days of pregnancy, she was hospitalized in the gynecology department with complaints of nagging pain in the lower abdomen and bloody discharge from the genital tract. Vaginal examination revealed that the external genitalia are developed correctly, the vagina is nulliparous, the cervix is conical, long, the external os is closed. The body of the uterus is enlarged to 18 weeks of pregnancy, dense, mobile, painless. Bloody discharge. Ultrasound examination revealed hypertonicity of the uterus; fetal heartbeat is recorded, there is no data for retroplacental hematoma, cervicometry shows the length of the cervical canal is 28 mm, the shape of the internal os is T-shaped.

Diagnosis: Pregnancy 18 weeks 3 days. Threatened late miscarriage. Habitual miscarriage.

Before prescribing maintenance therapy according to the proposed method, the serum LIGHT content was determined, which was 44.5 pg/ml.

Conclusion: prognosis – premature birth.

Conservative therapy was performed with a positive effect. The woman was discharged from the hospital with a progressive pregnancy at 21 weeks.

At 26 weeks of pregnancy, the patient was admitted to the obstetric clinic with symptoms of threatened premature labor, prelabor rupture of membranes, after which labor developed, the first premature birth through the natural birth canal occurred. A live premature girl was born, weighing 900 g, 33 cm tall, with an Apgar score of 3/5 points, and was transferred to the pediatric intensive care unit.

Diagnosis: first premature birth at 26 weeks of pregnancy. Prelabor rupture of membranes. Habitual miscarriage.

The forecast for the claimed method was confirmed.

Example 3.

Patient D., 31 years old, fourth pregnancy, wanted, occurred spontaneously, in her first marriage. The anamnesis includes one timely natural birth through the birth canal, without complications, one spontaneous miscarriage at 15-16 weeks and one non-viable pregnancy at 10-11 weeks with curettage of the uterine cavity. At 21 weeks and 2 days of pregnancy, she was hospitalized in the gynecology department with complaints of nagging pain in the lower abdomen, increased mucous discharge from the genital tract. Vaginal examination: external genitalia are developed correctly, the vagina is that of a woman who has given birth, the cervix is cylindrical, up to 2 cm, the external os is closed. The body of the uterus is enlarged to 21 weeks of pregnancy, dense, mobile, painless. Discharge is mucous. According to the ultrasound examination, hypertonicity was detected along the anterior wall of the uterus, fetal heartbeat was recorded, there was no data for retroplacental hematoma, cervicometry showed the length of the cervical canal was 21 mm, the shape of the internal os was Y-shaped.

Diagnosis: Pregnancy 21 weeks and 2 days. Threatened late miscarriage. Habitual miscarriage.

Before prescribing maintenance therapy according to the proposed method, the serum LIGHT content was determined, which was 70.34 pg/ml.

Conclusion: prognosis – timely delivery.

Conservative therapy was performed with a positive effect. The woman was discharged from the hospital with a progressive pregnancy at 23 weeks.

At 40 weeks of pregnancy, the patient was admitted to the maternity hospital with the development of labor. The second timely birth through the natural birth canal occurred, a live full-term boy was born weighing 3650 g, 53 cm tall, with an Apgar score of 8/9 points, the child was transferred to the children's department. On the 4th day, the woman and the child were discharged home.

Diagnosis: second timely birth at 40 weeks of pregnancy. Habitual miscarriage.

The forecast for the claimed method was confirmed.

Using the specified method, 30 women with threatened late miscarriage at gestation periods of 16–21 weeks and 6 days and habitual miscarriage were examined.

The results of the study are presented in Table No. 1.

Table 1.

Indicator Number of examined Total women 30 true positive result 15 false positive result 3 true negative result 12 false negative result 0 Accuracy 90.0% Sensitivity 100.0% Specificity 80%

Advantages of the claimed method:

1. The wide availability of the method is due to the possibility of its implementation in level 1 clinical diagnostic laboratories [“On approval of the Rules for conducting laboratory tests”: Order of the Ministry of Health of the Russian Federation dated May 18, 2021 No. 464n. https://legalacts.ru/doc/prikaz-minzdrava-rossii-ot-18052021-n-464n-ob-utverzhdenii/].

2. The method can be used as a screening method, as it is simple to perform, has high accuracy, sensitivity and specificity, and involves the study of one indicator;

3. The method allows for the prediction of premature birth in women with habitual miscarriage and threatened late miscarriage at 16–21 weeks and 6 days of gestation in order to carry out timely treatment and preventive measures and reduce perinatal complications for mother and child.

Claims (1)

A method for predicting premature birth in women with habitual miscarriage and threatened miscarriage by immunological examination of peripheral venous blood before the start of conservation therapy, characterized in that in women with habitual miscarriage, when a threatened late miscarriage develops at a gestation period of 16-21 weeks and 6 days, the serum content of the inducible factor homologous to lymphotoxins, competing with the glycoprotein D of the herpes simplex virus for the mediator of herpes virus penetration (LIGHT), is determined, and if its value is 44.5 pg/ml or less, premature birth is predicted.