RU2273031C1 - Method for predicting ecf and et program results - Google Patents

Abstract

FIELD: medicine, gynecology.

SUBSTANCE: in follicular liquid one should detect the content of tumor necrosis factor-α (TNF-α) and β-fibroblast growth factor (β-FGF). It is necessary to calculate prognostic index (PI) by the following formula: PI=0.0027*X+Y*(+8.9626*10^-7*Y-3.0827*10^-6*X)-0.1448, where X - the quantity of TNF-α, Y - the quantity of β-FGF. The result being below 0.3038 illustrates favorable result for in-vitro fertilization. At its value being equal or above 0.3038 one should predict unfavorable result of in-vitro fertilization. The innovation enables to increase information value of predicting the results of extracorporal fertilization (ECF) and embryo transfer (ET) program.

EFFECT: higher accuracy of prediction.

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Description

The invention relates to medicine, namely to gynecology, and will find application for predicting in-vitro fertilization in order to increase the effectiveness of the method of in vitro fertilization and embryo transfer (IVEC).

The problem of infertility treatment is currently gaining not only medical, socio-demographic, but also economic importance (Kulakov V.I., Leonov B.V., 2004). The frequency of infertility in Russia is 10-15%, and in some regions it exceeds the 15% level defined by the WHO problem group as critical, affecting demographic indicators (V. Kulakov, 1999). Today, a method for the treatment of infertility through in vitro fertilization and transfer of crushing embryos into the uterine cavity of the patient is becoming more widespread in our country and around the world. If in 1987-1990 the effectiveness of the method was 10-15% per embryo transfer, then at present this indicator has increased to 25-35% of cases. At the same time, in clinics that use the IVF and PE method, the search for new approaches to increasing the effectiveness of the method continues.

The IVF and PE program is complex due to the fact that it is multi-stage, but not all stages can be objectively monitored. To a certain extent, part of the unsuccessful IVF outcomes lies in the very nature of the human reproductive process, which is about 20% for a married couple at the age of 20 (Nikitin A.I., 1995). In this regard, the idea arose of stimulating superovulation in order to obtain a larger number of eggs through the use of gonadotropins. However, an increase in the number of eggs entails another negative factor - some of them may turn out to be inferior, with impaired oogenesis or chromosomal pathology, while others may take the path of reverse development in the process of folliculogenesis. In order to improve oocyte quality and prevent premature maturation due to the release of endogenous luteinizing hormone, gonadotropin releasing hormone agonists are included in the stimulation scheme of superovulation (Fleming R. et al., 1990).

A very important factor for the outcome of IVF is the state of male germ cells. At the same time, significant progress associated with the introduction into practice of the method of intracytoplasmic sperm injection allows offspring to men with severe forms of the disease, previously doomed to absolute infertility (Leonov et al., 1999).

Endometrial readiness is an essential condition for successful implantation of an embryo transferred into the uterus (Tabibzadeh S. et al., 1995). If there is a suspicion of disturbances in the cyclic transformation of the endometrium, a number of authors recommend that preliminary therapy be carried out with drugs containing progesterone and estradiol (Ffrindler S. et al., 1992).

A very important factor affecting the success of embryo implantation is the process of its transfer to the uterus (Tomar et al., 2002). But at present, there are various types of catheters that make this procedure atraumatic (Mansour R.T. et al., 2002). In addition, hysteroscopy before the IVF program allows you to evaluate the condition of the walls of the cervical canal, endometrium and, in the presence of pathology, carry out appropriate surgical interventions.

A significant factor affecting the success of embryo implantation is the age of the woman. However, given the use of donor oocytes, the success rates of IVF and PE programs do not differ between patients of early and late reproductive age (Craft J., 1990).

Particular attention should be paid to infectious diseases affecting the reproductive system, which can lead to impaired in vitro fertilization and pregnancy, and to prevent the birth of healthy offspring (Steyaert C.R., et al., 2000). Therefore, an essential moment in the implementation of the IVF and PE program is the timely detection and treatment of infections in the couple that are closely related to the reproductive system, included in the so-called TORCH complex (toxaplasmosis, rubella, cytomegalovirus and herpetic infections, chlamydia, syphilis, hepatitis A and B , gonococcal infection, listeriosis) (Kuzmichev L.N., 1998).

The determining factor for a positive outcome of the IVF and PE program is the state of the embryo, which determines the success of P implantation (Lenz J.V. et al., 1996). At the same time, in order to obtain embryos of good quality, it is necessary that the development of the preovulatory oocyte is not impaired in vitro fertilization.

Recent studies confirm the hypothesis that many immunoactive cytokines are involved in implantation and placental development. The change in the maternal T-helper (Th) cellular response necessary for normal pregnancy to occur sharply changes from the Thi- to Th2-type response. On the contrary, the persistence of the Th1-type response is characteristic of early pregnancy failure (Hill J.A. et al., 1995). This opinion is supported by many clinical studies (Marzi M. et al., 1996; Raghupathy R. et al., 2000; Makhseed M. et al., 2001), which have convincingly shown that increased production of peripheral Th1 lymphocytes of cytokines is observed in women with unfavorable results of the IVF and PE program, in contrast to the dominance of the Th2 reaction characteristic of a normal pregnancy.

At the same time, evidence was obtained that cytokines, in particular growth factors contained in the follicular fluid, are important modulators of intraovarial processes that regulate oocyte development (Kane MT Morgan PM Coonan C. Peptide growth factors and preimplantation development // Hum Reprod. Upd.- 1997-Vol. 3 - 137-157).

Reduced content of insulin-like growth factor (IGF) in the follicular fluid was detected in a group of infertile patients with endometriosis, ovarian dysfunction and low IVF and PE results (Cunha-Filho JSL, Lemos NA, Freitas FM, Kiefer K, Faller M., Passos M. EP // Insulin-like growth factor (IGF) -l and IGF binding protein-1 and -3 in the follicular fluid of infertile patients with endometriosis Hum. Reprod. - 2003-18-423-428).

Some researchers have linked reduced concentrations of vascular endothelial growth factor (VEGF) in follicular fluid to produce better quality oocytes and embryos and increased pregnancy rates (Friedman CI, Seifer DB, Kennard EA, et al .. Elevated level of follicular fluid vascular endothelial growth factor is a marker of diminished pregnancy potential // Fertil. Steril. - 1998-70-836-839; Barroso G., Barrionuevo M., Rao P. et al Vascular endothelial growth factor, nitric oxide, and leptin follicular fluid levels correlate negatively with embryo quality in IVF patients // Fertil. Steril.-1999-V.72-P.1024-1026).

It is known that higher concentrations of transforming growth factor (TGF-b1) in follicular fluid are significantly associated with successful preembryonic development, increased levels of embryo implantation and pregnancy (van Rooij IAJ, M. Broekmans FJ, te Velde ER Serum anti-Mullerian hormone levels : a novel measure of ovarian reserve // Hum. Reprod. - 2002-17-3065-3071).

Hammadeh M. E. et al. (2002) found no significant changes in the concentration of colony-stimulating growth factor in follicular fluid in a comparative study of patients with various infertility factors (tubal, endometriosis, infertility of unknown origin and male factor) (Hammaden ME, Ertan AK, Zeppezauer M. et al. Immmunoglobulins and Cytokines Level in Follicular Fluid in Relation to Etiology of Infertility and their.//AJRI 2002-47-82-90).

However, the common drawbacks of the above works are: determination of growth factors in follicular fluid in groups of patients with various etiological factors of female and male infertility, in other words, there are no criteria for inclusion and exclusion in the study groups on the etiological principle. The works also lack information on indications for the IVF and PE program. Also, a significant drawback of the above works should be recognized as the lack of information on the content of growth factors in follicular fluid in cases of stopping fertilization of oocytes or embryos.

Thus, to date, certain information has been accumulated that indicates the possible forecasting of the results of the IVF and PE program based on the determination of the content of growth factors in the follicular fluid.

At the same time, it is known that in the group of patients with tubal infertility during the IVF program, in vitro absence of egg fertilization is noted in 9% of cases, and embryo development is stopped in 8.4% of cases (Witkin, SS et al., 1996 ) It should be emphasized that in Russia for the vast majority of patients the main indication for the IVF and PE program is tube infertility.

In this connection, further development of methods is needed that allows for the prediction of in-vitro fertilization, especially for this category of patients, which is especially relevant in cases of using expensive methods of infertility treatment, which include IVF and PE.

The prototype of the invention selected a method for assessing the development of oocytes by examining markers in the follicular fluid obtained during follicle puncture and aspiration of their contents during the IVF and PE program, in order to determine the outcome of the program. The essence of this study is that higher levels of insulin-like growth factor (IGF) and low interleukin-1 are determined in follicular fluid, which indicates a favorable prognosis for pregnancy. (Mendoza S., Ruiz-Requena E., Ortega E., Cremades N., Martinez F., Bemabeu R., Greco, E., Tesarik, J. Follicular fluid markers ofoocyte developmental potential.// Hum. Reprod.- 2002-17-107-1022).

The disadvantages of the prototype are: firstly, the lack of clear criteria for which values of the studied markers should think about the adverse outcome of the IVF program, which reduces the accuracy and information content of this method; secondly, this study was conducted in a group of patients without pathology from the reproductive system, the indication for the IVF program was the male factor of infertility, in connection with which in-vitro fertilization was carried out using intracytoplasmic injection of sperm, while the main indications for in vitro fertilization programs is tubal infertility; thirdly, there is no information about patients who did not receive embryo transfer due to impaired in vitro fertilization.

These disadvantages are eliminated in the present invention. The objective of the invention is to improve the accuracy of the method and its information content.

The problem is solved in that after the puncture of the follicles, aspiration of their contents and isolation of oocytes, the average concentrations of TNF-α and β-FGF are determined. The predictive index (PI) is obtained when calculating the formula: PI = 0.0027 · X + Y · (+ 8.9626 · 10 ^-7 · Y-3.0827 · 10 ^-6 · Х) 0.1448, where X is TNF -α, Y-β-FGF. A PI value of equal to or greater than 0.3038 indicates that in-vitro fertilization will be impaired. Reliability of the obtained formula - P-value: 2,299e-8

The technical result obtained as a result of predicting the outcome of the IVF and PE program in this way is that:

1) the accuracy and information content of the forecast of the outcomes of the IVF and PE program at the early stages of its implementation is significantly increased;

2) it is possible to conduct a study of the material obtained during puncture (follicular fluid and the cells contained in it) in order to timely examine the patient;

3) increases the effectiveness of the following IVF programs by conducting etiotropic therapy of the patient, taking into account the results of the examination;

4) a timely decision can be made on intracytoplasmic sperm injection in order to improve the in vitro fertilization result;

5) the psychological load on the patient is reduced, awaiting information about the results of in-vitro fertilization and the day of embryo transfer due to early warning of a possible unfavorable outcome of fertilization.

6) the drug load on the patient is reduced due to its exclusion from the IVF and PE program.

The criterion for success of one of the most important stages of the IVF program is the receipt of good quality embryos. At the same time, in-vitro embryo development is directly related to the state of the oocyte and the introvarial processes that ensure its development, which occur, as noted above, with the direct participation of growth factors, the determination of the content in the follicular fluid of which can help predict the outcome of the IVF and PE program in the early stages of its implementation.

In order to develop a method for predicting the results of the IVF and PE program, we studied the content of growth factors — TNF-α and β-FRF in follicular fluid in 637 follicles in 75 patients with tubal infertility factor who applied to the Rostov Research Institute of Obstetrics and Pediatrics. Of 36 patients (group 1), in-vitro fertilization was impaired at the stage of egg development in 61.2% (22) cases, and at the stage of development of embryos in 38.8% (14). Embryo transfer in this group was not performed. The second group of the study included 39 patients whose in-vitro fertilization proceeded normally and ended with embryo transfer. The prognostic formula (PI) was obtained on the basis of a comparative analysis of the average concentrations of TNF-α and β-RFF in groups of patients with in-vitro fertilization and lack of in-vitro fertilization. Mathematical analysis was performed using PolyAnalist 3.5 software (Megaputer Intelligence).

An analysis of the medical, scientific and patent literature revealed that only a few studies have been devoted to the study of in-vitro fertilization disorders, which are aimed at determining the adverse effect of the fluid contained within the hydrosalpinx on the development and implantation of an embryo in an experiment (Arrighi CV et aL, 2001, Carrasco I .et al., 2001, Ajonuma LC et al., 2003). In other studies, impaired fertilization during the cultivation of oocytes is associated with cellular or genetic disorders, which are called the “oocyte factor”, and, as the authors themselves emphasize, these findings require further clarification (Levran D. et al., 2002).

At the same time, most patients suffering from tubal infertility have a chronic inflammatory process that causes them to have adhesive disease of the pelvic organs. Adhesions of the pelvic organs can be a consequence of reconstructive plastic surgery or tubectomy. The administration of gonadotropins during controlled stimulation of superovulation may be one of the factors contributing to the exacerbation of the chronic inflammatory process of the uterine appendages, which leads to the appearance of cellular and plasma mediators of the inflammatory process - cytokines, which include growth factors TNF-α and β-FRF.

TNF-α and β-RFF contained in the follicular fluid can be secreted by granulosa cells and leukocytes: B-lymphocytes, monocytes, natural killer cells, the number of which is increased in inflammatory diseases of the pelvic organs. Theoretically, they can also enter the follicles from the peritoneal fluid due to diffusion through the ovarian capsule in cases of inflammatory process of the pelvic organs, however, these data are currently not available in the literature. However, a study by Cheong Y.C. et al. (2002) indicates high concentrations of TNF-α in the peritoneal fluid with adhesive disease of the pelvic organs, which may also have a negative effect on the processes of oogenesis. Growth factors can affect follicle cells in various ways: autocrine, paracrine, intracrine and endocrine (Mandrup-Poulsen T. et al., 1995; Vinatier D. et al., 1995). TNF-α can cause a decrease in ovarian function due to the inflammatory process due to the inhibition of gonadotropin-stimulated steroidogenesis in undifferentiated ovarian cells due to decreased secretion of adenylyl cyclase and c-ATP (Terranova P.F., Rice V.M., 1997). In addition, elevated concentrations of TNF-α in the follicular fluid lead to apoptosis (Matsubara H. et al, 2000).

Currently, evidence has been obtained indicating inhibition of estrogen production of TNF-α (Pottratz S.T. et al., 1994). Moreover, it was found that TNF-α inhibits the production of progesterone by the corpus luteum and causes follicular atresia (Buscher U. et al., 1999, Nash MAet al., 1999; Yeh J. and Adashi EY, 1999; Doraiswamy V. et al , 2000). An increase in the concentration of TNF-α in the follicular fluid can adversely affect ovulation and in vitro fertilization (Cianci A. et al., 1996) and have an embryotoxic effect (Sung L. et al., 1997). On the other hand, it is known that the ovary is an organ with an extremely high level of angiogenesis (Redmer, D.A. and Reynolds, L.P., 1996), and the growth of the vascular part of the follicle tissue tissue is regulated by β-RFF (Schams D. et al., 1996). There is also a hypothesis that low levels of estrogen in the blood serum, insufficient for cell proliferation, depend on various growth factors, including β-RFF (Hermenegildo S., Sapo A., 2000). There is also evidence of the importance of β-RGF for preimplantation development of the embryo (Chai N. et al., 1998).

Studies have established the relationship between the concentrations of TNF-α and β-FGF in the follicular fluid and in-vitro fertilization. Determination of the content of these growth factors allows us to predict the result of in-vitro fertilization in the early stages of the IVF and PE program.

A detailed description of the method and examples of its specific application.

To implement the method, the following equipment is used: a standard set of equipment and equipment for IVF and PE laboratories, a photometer (Victor, 1420 Multilabel counter, Wallac, Finland). The level of concentration of cytokines in the follicular fluid was determined using commercial test systems TNF-α (CytElisa) and β-RFF (ACCUCYTE) (CYTIMMUNE, Science INC., USA).

Superovulation is stimulated according to the “short protocol” using an analog of gonadoliberin (Diferelin, Bofur Ipsen) in a daily dose of 0.1 mg, which is prescribed on the first day of the menstrual cycle after ultrasound examination. The next day, stimulation of folliculogenesis begins with the introduction of recombinant follicle-stimulating hormone preparations (Puregon, Organon) in a daily dose of 150-250 ME. Stimulation is carried out until the day the leading follicles reach a diameter of 18 mm, determined by transvaginal ultrasound, and then an injection of an “ovulatory dose” of chorionic gonadotropin is prescribed (Pregnil, Organon).

35-36 hours after the injection of chorionic gonadotropin, transvaginal puncture of the follicles and aspiration of preovulatory oocytes are performed. Transvaginal puncture is performed in the gynecological chair using an ultrasound machine "ALOKA SAL500" (Japan). The crotch and vagina are disinfected. The patient is covered with sheets, leaving the crotch open. Using a vaginal transducer with a special nozzle for a puncture needle installed on it, an ultrasound examination of the pelvic organs is performed to select the optimal position for the location of the puncture trajectory. After determining the correct direction of the puncture path through the lumen of the nozzle of the vaginal ultrasound probe, the contents of the follicles are punctured with a needle. The follicles are aspirated using vacuum suction at a pressure of 80 mmHg to ensure that the aspirate enters the sterile tubes. The resulting follicular fluid is passed to the embryologist for identification and isolation of oocytes from it. Sperm preparation is carried out using a swim-up technique in Mtdium B1 medium (CCD, France). Aspirated oocytes are inseminated and cultured in B2 INRA MEDIUM medium (CCD, France). Samples of follicular fluid are centrifuged at a speed of 400 g for 10 minutes and the pure fraction is used to determine TNF-α and β-RFF in it. The procedure for determining TNF-α and β-FRF is described in the manufacturer's instructions for the commercial kit.

The obtained values of TNF-α and β-FGF are substituted in the formula for calculating PI = 0.0027 · X + Y · (+ 8.9626 · 10 ^-7 · Y-3.0827 · 10 ^-6 · X) -0.1448, where X is TNF-α, Y is β-RFF, and if the PI value is equal to or greater than 0.3038, then an unfavorable outcome of IVF and PE is predicted, which is an indication to exclude a woman from the program and send her for an additional examination.

We present clinical examples confirming the efficiency of the method.

Example 1

Patient P-va L.V., 35 years old, medical history No. 582/60, date of follicle puncture 06/14/01.

Diagnosis: Chronic bilateral salpingitis. Condition after salpingo-ovariolysis, salpingostomy. Fitz Hugh Curtis Syndrome. Secondary infertility

Average concentrations were TNF-α = 20.5 pg / ml, β-FRF = 167.5 ng / ml.

After substituting the obtained data into the formula, the value PI = -0.0501 was obtained, which indicated a favorable outcome of IVF. As a result of in vitro cultivation of oocytes, embryos were obtained and carried out.

Example 2

Patient Ch-va N.V. 28 years old, medical history No. 4094/441, date of follicle puncture 06/19/02

Diagnosis: Condition after 2-sided tubectomy. Adhesive disease of the pelvic organs. Secondary infertility

Average concentrations were TNF-α = 315.1 pg / ml, β-FRF = 1210.0 ng / ml.

After substituting the obtained data into the formula, a PI value of 1.1933 was obtained, which indicated an unfavorable outcome of IVF. As a result of culturing oocytes, in-vitro fertilization did not occur; embryos were not received.

Example 3

Patient Sh-k I.V., 33 years old, medical history No. 2529/286, date of follicle puncture 04/13/02

Diagnosis: Condition after 2-sided tubectomy. Adhesive disease of the pelvic organs. Secondary infertility

Average concentrations were TNF-α = 87.0 pg / ml, β-FRF = 482.3 ng / ml.

After substituting the obtained data into the formula, the value PI = 0.3038 was obtained, which indicated an unfavorable outcome of IVF and PE. As a result of culturing oocytes, in-vitro fertilization did not occur; embryos were not received.

Based on the materials of the proposed method for predicting the IVF and PE program, we examined 152 patients who applied for in vitro fertilization for tubal infertility at the Rostov Research Institute of Obstetrics and Pediatrics. The age of the examined patients ranged from 24 to 36 years, averaging 29.4 ± 2.2 years. Of these, 59% were diagnosed with primary infertility, in 41% - secondary. The duration of infertility ranged from 6 to 14 years, averaging 7.4 ± 2.1 years. In 11 patients, the PI values were more than 0.3038; in-vitro fertilization did not occur in these cases, which served as the basis for their further examination. In the remaining patients, the PI values were less than 0.3038, in-vitro fertilization in these cases occurred and the embryo was transferred.

The inventive method is based on the use of standard laboratory equipment, does not require technical modification. The use of this technique allows the doctor to predict the result of fertilization on the first day of oocyte cultivation in order to develop further tactics for the patient and conduct additional laboratory tests in order to clarify the causes of impaired fertilization. The claimed method can be used in gynecological hospitals using the IVF and PE program.

Claims (1)

A method for predicting the outcome of an IVF and PE program by examining a follicular fluid, characterized in that the follicular fluid is quantitatively determined for the content of TNF-α and β-RFF, and the prognostic index (PI) is calculated by PI = 0.0027 · X + Y · (+ 8.9626 · 10 ^-7 · Y-3.0827 · 10 ^-6 · X) -0.1448, where X is the amount of TNF-α, Y is the amount of β-FRF, and with a PI value less than 0.3038, a favorable in-vitro fertilization outcome is predicted, and with a value equal to or greater than 0.3038, an unfavorable prediction in vitro fertilization outcome.