RU2441243C1 - Method for embryo quality prediction in extracorporeal fertilisation in infertile women - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention can be used for embryo quality prediction preceding conducting the extracorporeal fertilisation program in infertile women mediated by tuboperitoneal factor and external genital endometriosis. Peripheral venous blood of an infertile women is examined for the HLA-DR+ MON% value, and if the derived value is 68% or more, transferring a high-quality embryo is predicted, while the value being less shows transferring a low-quality embryo.

EFFECT: examining women suffering tuboperitoneal factor and external genital endometriosis under the declared method shall enable the pre-fertilisation embryo quality prediction and well-timed prescription of adequate therapeutic measures.

1 tbl, 3 ex

Description

The invention relates to medicine, namely to gynecology, and can be used to predict the quality of embryos before conducting an in vitro fertilization (IVF) program in women with infertility due to tubal-peritoneal factor and external genital endometriosis.

The relevance of the proposed method is determined by the fact that in the treatment of infertility in women with tubal-peritoneal factor and with external genital endometriosis, recently new methods of assisted reproductive technologies, including in vitro fertilization (IVF), are widely used. Despite the fact that in vitro fertilization and the subsequent transfer of embryos into the uterine cavity becomes a routine method of treating infertility, the frequency of pregnancy in the IVF program is still low and, according to different authors, ranges from 20% to 40% (V. Grishenko ., Petrushko MP, Pinyaev VI The effectiveness of the IVF program depending on the quantity and quality of transferred embryos // Problems of reproduction. - 2000 - No. 1. - P.44-47.). It is known that the success of IVF is largely determined by the quality of the embryos transferred to the uterine cavity. Many experts believe that the main cause of failed IVF outcomes is an insufficiently accurate assessment of the quality of the embryos, which today is based mainly on morphological criteria (Ilyina A.A., Kalinina I.I., Troshina T.G., Ilyin K.A., Boltovskaya MN, Stepanova II, Anshina MB Follicular fluid as a medium that determines the quality of the oocyte and the outcome of ART programs (literature review) // Problems of reproduction. - 2008. - No. 4. - P.27-38 .). There is an opinion that the quality of embryos obtained during IVF depends on the aspirated follicles and on the composition of the follicular fluid (Barkalina N.V. Follicular fluid and prognosis of outcomes of ART programs // Reproduction Problems. - 2006. - No. 6. - C. 57-63.). However, other factors associated with embryo quality remain poorly understood. In this regard, to increase the effectiveness of IVF, it is of particular interest to develop new laboratory criteria for predicting the quality of embryos, the use of which would allow the attending physician to optimize the patient management tactics before initiating the ovarian stimulation protocol and, if necessary, prescribe a set of additional therapeutic measures.

It was previously proposed to use an indicator of the concentration of lactoferrin in the follicular fluid as an indicator of embryo quality, while high values of this indicator (500.2 ± 35.5 ng / ml) serve as an indicator of high quality of embryos, and low values of the concentration of lactoferrin in the follicular fluid (379 , 7 ± 45.1 ng / ml) are associated with the production of low quality embryos (Yanaihara A., Mitsukawa K., Iwasaki S., Otsuki K., Kawamura T., Okai T. High concentration of lactoferrin in the follicular fluid correlate with embryo quality during in vitro fertilization cycles // Fertil. Steril - 2007. - Vol. 87. - N.2. - P.279-282).

However, the method for predicting the quality of the embryo by these parameters has a number of the following disadvantages.

1. The method has a high risk of complications, since the collection of follicular fluid is possible only with transvaginal puncture of the follicles under general anesthesia and may be complicated by bleeding.

2. The use of the method is possible after the end of the ovarian stimulation protocol and the collection of follicular fluid, ie at that stage when it is no longer possible to adjust the patient management tactics.

3. There is evidence in the literature that with tubal infertility factor, the concentration of lactoferrin in the follicular fluid does not change depending on the success or failure of IVF (Markina L.A., Zorina PM, Zorina V.N., Bazhenova L.G., Arkhipova SV, Ryabicheva TG, Polukarov AN Prevention of reproductive loss and placental insufficiency. Proteins, cytokines and antigen-specific immunocomplexes of follicular fluid and blood serum in women with in vitro fertilization // Questions of gynecology, obstetrics and perinatology. - 2008. - T.7. - No. 5. - S. 29-33.).

4. There is no data on the accuracy of the method.

There is also a method for predicting the quality of embryos, characterized in that the content of inhibin B is determined in the follicular fluid and, at its low values (10.9-103.9 ng / ml), the production of low quality embryos is predicted. (Chang CL, Wang T.N., Horng SG, Wu N.M., Wang HS, Soong YK The concentration of inhibin B in follicular fluid: relation to oocyte maturation and embryo development // Hum.Reprod. - 2002. - Vol.17. - N.7. - P.1724-1728.)

However, this method has several disadvantages.

1. The method has a high risk of complications, since the collection of follicular fluid is possible only with transvaginal puncture of the follicles under general anesthesia and may be complicated by bleeding.

2. It is known that the production of inhibin B in women changes throughout life, starting to decrease after 40 years, which is associated with an age-related decrease in ovarian reserve (Gougeon A. The biological aspects of risks of infertility due to age: the female side // Rev Epidemiol Sante Publique. - 2005. - Vol. 53. - No 2. - P.2S37-45.). IVF is performed in women of a fairly wide age range, therefore, the change in the inhibin B content in the follicular fluid will be largely determined by the age of the patient.

3. There is no data on the accuracy of the method.

The closest technical solution to the claimed method is a method for predicting the quality of the embryo by determining the level of leptin in peripheral blood of women who underwent ovarian stimulation according to the long protocol and at low serum leptin levels (50.05 ± 5.6 ng / ml) high quality embryos are predicted, while at higher serum leptin levels (59.48 ± 7.6 ng / ml, P = 0.047), low quality embryos are predicted (Georgios A., Eleni K., Ioannis S., Vassilios L., Constantinos L., T hemis M., Nikolaos V. Serum and follicular fluid leptin levels are correlated with human embryo quality // Reproduction. - 2005. - Vol. 130 - P.917-921.

This method was chosen by us as a prototype. However, it has several disadvantages.

1. This forecasting method is applicable only to women who have undergone superovulation stimulation according to a long protocol.

2. In the literature there are conflicting data on the possibility of using this indicator to predict the quality of the embryo. A number of researchers indicate a lack of relationship between serum leptin levels and embryo quality in IVF-ICSI cycles (Asimakopoulos B., Koster F., Felberbaum R., Tripsiannis G., Caglar GS, Nikolettos N., Al-Hasani S., Diedrich K Intrafollicular and circulating concentrations of leptin do not predict the outcome in IVF-ICSI cycles // Reprod Sci. - 2009. - Vol.l6. - N.1. - P.113-119.)

3. It is known that leptin is produced by adipocytes and its content correlates with body mass index, so the individual spread of this indicator depending on the body weight of women can affect the accuracy of the prognosis if it is used to predict the quality of the embryo (Alviggi C., Clarizia R. , Castaldo G., Matarese G., Colucci CC, Conforti S., Pagano T., Revelli A., De Placido G. Leptin concentration in the peritoneal fluid of women with ovarian endometriosis are different according to the presence of a 'deep' or 'superficial' ovarian disease // Gynecol Endocrinol. - 2009. - Vol.25. - N.9. - P.610-615.).

4. There is no data on the accuracy, sensitivity and specificity of the method.

These disadvantages are proposed to eliminate in the present method.

The technical result is to increase the accuracy, sensitivity and specificity of the method for predicting the quality of the embryo in women preparing for the IVF program.

The novelty of the proposed method lies in the fact that for the first time it is proposed to predict the quality of embryos obtained during IVF in women with infertility due to tubal peritoneal factor and external genital endometriosis, by determining the relative content of HLA-DR + monocytes in peripheral blood. Previously, this indicator was used to predict the development of secondary septic shock in patients with extensive thermal burns (> 30% of the body surface) (Venet F., Tissot S., Debard AL, Faudot C., Crampé C., Pachot A., Ayala A. , Monneret G. Decreased monocyte human leukocyte antigen-DR expression after severe burn injury: Correlation with severity and secondary septic shock // Crit Care Med. 2007 Aug; 35 (8): 1910-7.) It has also been found that progressive decline the circulating pool of HLA-DR + monocytes is associated with the development of infectious complications in stroke patients (Zhang DP, Yan FL, Xu HQ, Zhu YX, Yin Y., Lu HQ A decrease of human leucocyte antigen-DR expression on monocytes in peri pheral blood predicts stroke-associated infection in critically-ill patients with acute stroke // Eur. J. Neurol. - 2009. - Vol.l6. - N.4. - P.498-505). Low expression of HLA-DR molecules on persistent blood monocytes has also been proposed as a predictor of the risk of pneumonia in children undergoing lung transplantation (Hoffman JA, Weinberg KI, Azen CG, Horn MV, Dukes L., Starnes VA, Woo MS Human leukocyte antigen-DR expression on peripheral blood monocytes and the risk of pneumonia in pediatric lung transplant recipients // Transpl Infect Dis. 2004 Dec; 6 (4): 147-55.). To predict the quality of embryos obtained during IVF in women with infertility due to tubal peritoneal factor and external genital endometriosis, this indicator was not used.

It is known that HLA-DR molecules related to antigens of the main histocompatibility complex of class II are directly involved in the presentation of antigens by phagocytic cells (Yarilin A.A. Fundamentals of immunology: Textbook. - M .: Medicine, 1999. - 608 p.). The low level of expression of HLA-DR molecules can inhibit the interaction of phagocytes with antigens and impair the development of an adequate immune response to antigenic stimuli of various nature. A decrease in the peripheral blood of women with infertility of a circulating pool of functionally active monocytes indicates a significant violation of innate immunity reactions in these patients, which can negatively affect the regulation of various reproductive processes, including oogenesis. It was shown that HLA-DR - positive macrophages are present in the ovaries and are actively involved in the regulation of follicular growth and in the process of ovulation (Wu R., Van der Hoek KH, Ryan NK, Norman RJ, Robker RL Macrophages contributions to ovarian function // Hu. Reprod. Update. - 2004. - Vol.10. - N.2. - P.119-133).

The method is carried out according to the standard method as follows.

1) Add 3 ml of Medium 199 to 3 ml of heparinized blood from a cubital vein and isolate an enriched population of mononuclear cells by standard centrifugation at 1500 rpm in a density gradient of ficoll-urographin (d = 1,078) for 45 min (Boum A. Isolation of mononuclear cells and granulocytes from human blood and bone marrow // Scand. J. Clin. Invest. - 1968. - V.21. - N97. - P.77.

2) A suspension of mononuclear cells is washed twice with saline, the cell concentration is adjusted to 1 × 10 ⁶ cells / ml. To 0.1 ml of the resulting suspension, 20 μl of anti-HLA-DR monoclonal antibodies conjugated with fluorescein isothionate (Sorbent, Moscow) are simultaneously added. Cells are incubated for 30 min at room temperature in the dark, washed in 1 ml of saline.

3) Determine the relative (percentage) number of HLA-DR + cells in a monocytic gate using a flow cytometer.

Using the proposed method allows with high accuracy, 80.0% to predict the quality of the embryo during IVF.

Distinctive features of the method are: a prognostic parameter of the relative content of HLA-DR + monocytes is established, at a value of which 68% or more predict the production of a high quality embryo.

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

Example 1. Patient Ch., 29 years old, was admitted for an IVF program and embryo transfer (PE). It was observed and treated for secondary infertility for 4 years. The cause of infertility is the tubal-peritoneal factor, in marriage II, the husband has a child. A history of 2 ectopic pregnancies, timely delivery in 2000, without complications. Surgical interventions: in 1998, an ectopic pregnancy - laparotomy - left tubotomy; in 2007, diagnostic laparoscopy - fallopian tube plastic, salpingo-ovariolysis; in 2008, an ectopic pregnancy - laparoscopy-tubectomy on the left.

When examining the hormonal status: FSH - 4.0 mU / ml; LH - 6.4 mU / ml; PRL - 386.2 mU / ml; cortisol - 494.1 nmol / l; Inhibin B - 68.44 pg / ml; AMG - 4.420 ng / ml.

According to the USG on the 3rd day of the menstrual cycle (m.c.): the body of the uterus is of a homogeneous structure, 51 × 35 × 40 mm in size. M-echo 4 mm. The right ovary is 34 × 23 × 25 mm, with antral follicles up to 6 in p / sp, max d - 9 mm. The volume of the ovary is 9.8 cm ³ . The left ovary is 35 × 25 × 2 mm, with antral follicles up to 6 in p / sp, max d - 7 mm. The volume of the ovary is 10.5 cm ³ . Both ovaries are of normal structure.

Conclusion of hysterosalpingography (GHA): the left fallopian tube is absent, the right fallopian tube is impassable in the ampullar region.

Blood test: Нb - 138 g / l, erythrocytes 4.0 t / l, color indicator 0.9, leukocytes - 5.9 g / l, stab neutrophils - 0, segmented neutrophils - 64, monocytes - 2, lymphocytes - 34, ESR - 3 mm / h.

Conclusion of the husband's spermogram: asthenozoospermia.

Clinical diagnosis: Infertility II. Tubal peritoneal factor. Condition after tubectomy on the left. Distal occlusion of the right fallopian tube. Adhesive process of the pelvis.

According to the immunological examination according to the claimed method, it was found that the content in the peripheral blood of HLA-DR + monocytes was 85%, which is higher than the claimed parameter (68%).

Conclusion: High embryo quality is predicted.

Superovulation was stimulated using a short protocol (cetrotide + gonal-f). An operation was performed (on day 13 m.ts.) - transvaginal puncture. 14 follicles were punctured, 14 oocytes were obtained, 9 were fertilized - 9 high-quality embryos were obtained on the basis of morphological signs, embryo transfer (PE) on day 3 (2 embryos on 8 blastomeres) and 5 days (1 embryo at the blastocyst stage) of cultivation.

Thus, the forecast for obtaining high quality embryos was confirmed.

Example 2. Patient K., 37 years old. Received for the IVF and PE program. It was observed and treated for secondary infertility for 13 years. The cause of infertility is the tubal-peritoneal factor, external endometriosis, married 1, the husband has no children. In the history of 1 dead pregnancy (after IVF), there were no births. Surgical interventions: in 2000, diagnostic laparoscopy - salpingo-ovariolysis. In 2003, laparoscopy regarding external endometriosis is the destruction of foci of endometriosis, salpingo-ovariolysis.

When examining the hormonal status: FSH - 6.29 mU / ml; LH - 1.99 mU / ml; PRL - 671.8 mU / ml; cortisol - 261 nmol / l; Inhibin B - 36.96 pg / ml; AMG - 0.534 ng / ml.

According to the USG (on day 3 m.ts.): the uterine body is of a homogeneous structure, with dimensions of 61 × 36 × 52 mm M-echo 3 mm. The right ovary is 23 × 33 × 20 mm, with an antral follicle up to 1 in s / sp, max d - 11 mm. The ovary volume is 7.6 cm. The left ovary is 24 × 13 × 16 mm, with an antral follicle up to 1 in s / sp, max d - 8 mm. The volume of the ovary is 2.5 cm ³ . Both ovaries are of normal structure.

GHA conclusion: fallopian tubes are impassable.

Blood test: Нb - 140 g / l, erythrocytes 4.4 t / l, color indicator 0.9, leukocytes - 5.9 g / l, stab neutrophils - 0, segmented neutrophils - 57, monocytes - 6, lymphocytes - 35 , ESR - 5 mm / h.

Conclusion of the husband's spermogram: asthenozoospermia.

Clinical diagnosis: Infertility II. Tubal peritoneal factor. Distal occlusion of both fallopian tubes. External endometriosis.

According to the immunological examination according to the claimed method, it was found that the content in the peripheral blood of HLA-DR + monocytes was 68.4%, which is higher than the claimed parameter (68%).

Conclusion: High embryo quality is predicted. Superovulation was stimulated using a short protocol (cetrotide + gonal-f). An operation was performed (on day 12 m.ts.) - transvaginal puncture. 2 follicles punctured, 1 oocyte obtained, which was fertilized - 1 high-quality embryo was obtained on the basis of morphological characteristics, PE on the 3rd day of cultivation (1 embryo at the morula stage).

Thus, the forecast for obtaining a high quality embryo was confirmed.

Example 3. Patient M., 37 years old. Received for the IVF and PE program. It was observed and treated for primary infertility for 16 years. The cause of infertility is external endometriosis, in marriage I, the husband has a child. Surgical interventions: in 2006, diagnostic laparoscopy - produced: diathermopuncture of the ovaries; in 2008, laparoscopy for chronic salpingitis, external endometriosis, uterine fibroids - produced: fimbriolysis, conservative myomectomy, destruction of foci of endometriosis.

When examining the hormonal status: FSH - 3.7 mU / ml; LH - 15.1 mU / ml; PRL - 85 mU / ml; cortisol - 540 nmol / l.

According to the USG: (on day 3), the uterine body is of a homogeneous structure, with dimensions of 41 × 32 × 41.5 mm. M-echo 3 mm. The right ovary is 25 × 18 × 20 mm, with antral follicles up to 3 in p / sp, max d - 5 mm. The volume of the ovary is 4.5 cm ³ . The left ovary is 27 × 17 × 19 mm, with antral follicles up to 5 in p / sp, max d - 5 mm. The ovary volume is 4.4 cm. Both ovaries are of normal structure.

Blood test: Нb - 136 g / l, erythrocytes - 4.7 t / l, color indicator - 0.9, white blood cells - 4.2 g / l, stab neutrophils - 0, segmented neutrophils - 58, monocytes - 7.5 lymphocytes - 34.5; ESR - 3 mm / h.

Conclusion of the husband's spermogram: asthenozoospermia.

Clinical diagnosis: Infertility I. External endometriosis. Chronic salpingitis. Condition after conservative myomectomy.

According to the immunological examination by the present method it was found that the content in the peripheral blood of HLA-DR + monocytes was 32.7%, which is lower than the claimed parameter (68%).

Conclusion: low embryo quality is predicted. Superovulation was stimulated according to a long protocol (diferelin + gonal-f + menopur). An operation was performed (on day 16 m.ts.) - transvaginal puncture. 3 follicles punctured, 3 oocytes obtained, 1 of them was fertilized - 1 poor quality embryo was obtained on the basis of morphological signs, PE on the 3rd day of cultivation (1 embryo on 6 blastomeres).

Thus, the forecast for low quality embryo was confirmed.

In this way, 35 women with infertility due to tubal-peritoneal infertility and external genital endometriosis were examined. The research data are shown in table 1.

Table 1 Indicator The number of examined Total women 35 true positive result 10 false positive 3 true negative result eighteen false negative four Total: the accuracy of the proposed method is 80.0%, the sensitivity of the proposed method 71.4%, the specificity of the proposed method 85.7%

The advantages of the proposed method:

1) the method is applicable for predicting the quality of the embryo using various protocols of ovarian stimulation in women with infertility due to tubal-peritoneal infertility and external genital endometriosis;

2) high accuracy - 80.0%, sensitivity - 71.4%, specificity - 85.7%;

3) simplicity and accessibility of the methodology;

4) the application of the method allows predicting the quality of the embryo before starting the IVF procedure and timely appointing additional therapeutic measures if necessary.

Claims (1)

A method for predicting embryo quality before an in vitro fertilization (IVF) program in women with infertility due to tubal-peritoneal factor and external genital endometriosis, by immunological examination of peripheral venous blood, characterized in that the relative content of HLA-DR + monocytes and its value are determined equal to 68% or more, a high quality embryo is predicted.