Method of predicting spontaneous onset of pregnancy in women with i and ii stage of external genital endometriosis

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to a method of predicting spontaneous onset of pregnancy in women with I and II stage of external genital endometriosis. The essence of the invention consists in the fact that before treatment in peripheral blood of women with infertility, associated with I and II stage of external genital endometriosis determined is a relative quantity of IL- IL-1β + monocytes, and if the value of the said index is 50.0% and higher in the monocytic gate the spontaneous onset of pregnancy within a year after carrying out the surgical treatment of endometriosis is predicted.

EFFECT: application of the claimed method makes it possible to predict with high accuracy the spontaneous onset of pregnancy in the women with infertility in case of I and II stage of external genital endometriosis within a year after therapeutic laparoscopy, which makes it possible to select optimal tactics of the patients' management and estimate the necessity of administering them methods of assisted reproductive technologies.

1 tbl, 3 ex

 

The invention relates to medicine, namely to gynecology, and can be used to predict spontaneous pregnancy in one year in women with stages I and II of external genital endometriosis after surgical treatment of infertility.

The relevance of the proposed method is determined by the fact that there are currently no effective methods of treatment of infertility in women with stages I and II (the so-called "small forms") of external genital endometriosis. The "gold standard" of treatment of endometriosis is currently the surgical removal of the lesions in combination with hormonal therapy (Budinetz T, Sanfilippo JS Treatment of endometriosis: a hormonal approach Minerva Ginecol., 2010, 62 (4): 373-80; Ozkan S, Murk W, Arici A Endometriosis and infertility: epidemiology and evidence-based treatments Ann N Y Acad Sci. 2008; 1127: 92-100). However, a combined surgical and hormonal treatment used in women with infertility only in the common forms of endometriosis (Ge CX, Zhu XH, Tang XQ. Efficacy of conservative laparoscopic surgery combined with goserelin in treatment of 206 patients with severe ovarian endometriosis at short-term and long-term follow-up. Zhonghua Fu Chan Ke Za Zhi. 2012 Aug; 47 (8): 603-7), whereas in patients with I-II stage of endometriosis is carried out only surgical removal of endometriotic lesions. Although therapeutic laparoscopy and has a positive impact on reproductive function (Jacobson TZ, Duffy JM, Barlow D, Farquhar C, Koninckx R, Olive D. Laparoscopic surgery for subfertility associated with endometriosis. Cochrane Database Syst Rev. 2010 Jan 20; (1): CD001398. dot: 10.1002/14651858.CD001398.pub2), but according to the literature, surgical treatment of infertility in women with "small" forms of endometriosis can achieve pregnancy only 1 out of 12 of the operated women (Diwadkar GB, Falcone T. Surgical management of pain and infertility secondary to endometriosis. Semin Reprod Med. 2011 Mar; 29(2): 124-9). To improve the efficiency of surgical treatment of infertility with "small" forms of external genital endometriosis is recommended after the surgical treatment of endometriosis to include women in IVF protocols (Chen ML, Lee KS, Yang CT, Hung KH, Wu MH. Simultaneous laparoscopy for endometriotic women undergoing in vitro fertilization. Taiwan J Obstet Gynecol. 2012 Mar; 51(1): 66-70). In this regard, the physician needs to properly assess the chance of spontaneous conception after therapeutic laparoscopy. Predicting the effectiveness of laparoscopic treatment will help the doctor adjust the tactics of management of patients and to assess the need for such costly method like IVF.

Previously it was proposed to predict the onset of pregnancy in infertile patients with endometriosis on the EFI index (index of fertility endometriosis, which is calculated based on anamnestic data (age, duration of infertility and obstetrical history) and data obtained during hysteroscopic and laparoscopic�komicheskom survey (the condition of the pipes, ovary, to attack the right and left, the prevalence of endometriosis by classification AFS), wherein the gradation of each element is assessed on a scale from 0 to 4 points and as a result determine the total score, which can range from 0 to 10. If the value of the EFI index of 0, the occurrence of pregnancy within 1-3 years after surgical removal of foci of endometriosis predict in 10% of cases, and when the index value of EFI, is $ 10, pregnancy through 1 year after surgery predict in 56% of cases after 2 years - 72%, after 3 years in 75% of cases (Adamson G. D., D. J. Pasta Endometriosis fertility index: the new, validated endometriosis staging system // Fertility and Sterility. - 2010. - Vol.94. - No. 5. - P. 1609-1615).

This method has several disadvantages:

- used many signs evaluated by the physician subjectively, which reduces the diagnostic value of the index;

- calculation of the index is time-consuming;

the method has relatively low prediction accuracy (70%).

One of the analogues of the claimed method is a method of predicting pregnancy rates in women with external genital endometriosis after holding them therapeutic laparoscopic operations by definition they have to treat the content in the embryonic serum antibodies using ELI-P test. In cases where performance indicators reac�s ELI-P test are 41 CONV.ed. and more in women with endometriosis predict the onset of pregnancy in 57.4% of cases, rejecting all antigens of the test system in the direction of decrease to 50% of the standard indicators of prognosis of the pregnancy is 28.4%, while in the case of reducing all antigens of the test system more than 50% from normal values predicted 100% no pregnancy (Borovkova L. V. Reproductive function in patients with genital endometriosis: author. dis. M. D. M., 2004. - 34 p.) However, in this method is by using the ELISA method, which has a fairly low reproducibility and requires setting of samples in duplicate, which significantly increases the cost of the method and time of the meeting. Also, this method has low accuracy.

The closest technical solution to the claimed method is a method of predicting pregnancy rates in women with external genital endometriosis stage I-II by immunological studies of peripheral venous blood before surgery, characterized in that in the blood determine the relative number of lymphocytes with the phenotype CD20+CD5+ and its value is 2.6% or less, predict the onset of pregnancy within one year after the surgical treatment of endometriosis (Patent No. 2318215 Russia. Method prog�of Osinovaya the effectiveness of surgical treatment of infertility in women with external genital endometriosis / L. V. Posiseeva, N. Yu. Sotnikov, D. N. Shishkov, etc. / / the Invention. Utility models. - 2008. - №6).

This method was selected as the prototype method has an accuracy of 77.8 percent.

The technical result is to expand the Arsenal of technical means, as well as improving the accuracy and specificity of the method for the prediction of spontaneous pregnancy in women with stages I and II of external genital endometriosis. The technical result is achieved in that before the surgery in the peripheral blood of women with I-II stage of endometriosis determine the relative amount of IL-1β + monocytes and its value is equal to or more than 50%, in the monocyte gate predicts spontaneous pregnancy within one year after surgical treatment.

The novelty of the proposed method lies in the fact that the first proposed to predict spontaneous pregnancy in women with stages I and II of external genital endometriosis after surgical treatment of infertility by definition of the relative content in peripheral blood monocytes, producing intracellular IL-1β.

Earlier this indicator was used to evaluate the response of monocytes on stimulation by lipopolysaccharides in patients with hypercholesterinemia (Moutzouri E., C. C. Tellis, Rousouli K., Liberopoulos E. N., H. J. Milionis, Elisaf M. S., A. D. Tselepis Effect of simvastain or its combination with ezetimibe on Toll-like receptor expression and lipopolysaccharide - induced cytokine production in monocytes of hypercholesterolemic patients // Atherosclerosis.- 2012.- Vol. 225. - N. 2. - P. 381-387). It was also shown that the number of IL-1β - producing monocytes was increased in Alzheimer's disease (Torres de Lima KC, de Lima GS, Fiamoncini CM, de Rezende VB, de Araujo Pereira P, Bicalho MA, de Moraes EN, Romano-Silva MA. Increased frequency of cluster of differentiation 14 (CD14+) monocytes expressing interleukin 1 beta (IL-1β) in Alzheimer's disease patients and intermediate levels in late-onset depression patients // Int J Geriatr Psychiatry. 2013 May 14. doi: 10.1002/gps.3973. [Epub ahead of print]). In pregnant women with preeclampsia in the peripheral blood revealed increased amounts of IL-1β + monocytes (Manie S. C. the Role of the proinflammatory cytokine interleukin-1β in the pathogenesis of gestosis // author. Diss. Cand. honey. Sciences. - Ivanovo, 2006, 24 p.). For prediction of pregnancy in women with infertility associated with external genital endometriosis after surgical treatment of infertility, this indicator was not used.

It is known that Il-1β plays an important role in the regulation of reproductive processes, in particular in the provision of endometrial receptivity (Bulletti C, Flamigni C, de Ziegler D. Implantation markers and endometriosis // Reprod Biomed Online. - 2005. - Vol.11. - N. 4. - P. 464-468). It is shown that IL-1β is involved in the development of immunological tolerance in the placenta and contributes to a successful pregnancy by regulating the activity of nuclear factor NF-κ (Geisert R., Fazleabas A., Lucy M., Mathew D. Interaction of the coneptus and endometrium to establish pregnancy in mammals: role of interleukin 1β // Cell Tissue Res. - 2012. - Vol.349. - N. 3. - P. 825-838). It was also reported that production of IL-1β at the system level correlates with the onset of pregnancy in women participating in the program of in vitro fertilization, with high values of serum IL-1β was observed in women with successful pregnancy occurred (T. C. Bonetti, R. Salomao, Bruniaiti M., Braga D. P., E. Borges Jr., Silva I. D. Cytokine and hormonal profile in serum samples of patients undergoing controlled ovarian stimulation: interleukin-1βeta predicts ongoing pregnancy // Hum Reprod. - 2010. - Vol.25. - N. 8. - P. 2101-2106). Reduced intracellular production of IL-1β by monocytes of the blood of infertile women with stage I-II external genital endometriosis may be the cause of disrupting implantation. Therefore, only surgical removal of foci of endometriosis in patients with low IL-1R+ monocytes inefficient for the recovery of reproductive function and requires the appointment of additional therapeutic interventions.

The method is as follows:

1) To 3 ml of heparinized blood from the cubital vein add 3 ml of RPMI 1640 and produce an enriched population of mononuclear cells using a standard method of high-speed centrifugation (Bourn A. Isolation of rnononuclear cells and granulocytes from human blood and bone marrow // Scand. J. Clin. Invest - 1968. - v.21. - #97. - p.77).

2) a Suspension of mononuclear cells washed twice with saline, adjusted the cell concentration to 1×106 cells/ml. Then perform the procedure intracellular staining of cells using a commercial kit Fixative Solution IOTest®3 10x Concentrate (Beckman Coulter, USA) and monoclonal antibody to IL-1β, conjugated with fluorescein isothiocyanate (eBioscience, USA) according to instructions of the manufacturer. To this end, 50 μl of cell suspension, add 100 ál of fixation buffer And from the set of Fixative Solution IOTest®3 10x Concentrate and cells were incubated for 15 min at room temperature in the dark. Next, cells were washed off the retaining buffer and add 100 ál permeabilizing buffer and 2 µl of monoclonal antibody to IL-1β labeled fluoresceinisothiocyanate (FITC). Cells were incubated for 15 min at room temperature in the dark, washed and fixed in 0.5 ml of fixative CellFix™ (BD Biosciences, Belgium).

3) Determine the relative amount of IL-1β cells in the monocyte gate on flow cytometer. When the value is equal to 50% or more, predict spontaneous pregnancy in one year.

The distinguishing features of the method: a prognostic parameter of the relative content of IL-1β + monocytes, the value of which is 50.0% or more in the monocyte gate predicts spontaneous pregnancy.

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

Example 1. Patient I., 37 l�t, asked about secondary infertility for 3 years. History 1 medical abortion without complications and 1 spontaneous miscarriage in early pregnancy; obesity is treated. Vaginal examination: cervix without pathology, conical shape; the uterus is normal size, movable, painless. The appendages are not enlarged, painless on both sides. Palpation Sacro-uterine ligaments painless. Examined for infertility: cycle - a two-phase tests of functional diagnostics and histological examination of the endometrium, according to hysterosalpingography-the uterine cavity is not deformed, the lumen of the fallopian tubes has an unmodified form, fill pipe throughout, contrast flows freely into the abdominal cavity. Husband examined the semen without deviation from the norm. Postcoital test is positive.

Clinical diagnosis: secondary infertility. Suspicion of external genital endometriosis.

Made a determination in peripheral blood relative content of IL-1β+ monocytes, which was 66.2%.

Conclusion: it is predicted spontaneous pregnancy within one year after operative laparoscopy and destruction foci of endometriosis.

On day 9 of the menstrual cycle produced by laparoscopy, during which the DEA�hidden: the abdominal cavity free, the uterus is normal size. The fallopian tubes are not changed freely passable by chromopertubation. The ovaries are not changed. On the peritoneum of the right uterosacral ligament discovered single superficial endometriotic lesions measuring less than 3 cm2. Produced coagulation foci of endometriosis monopolar coagulator. Performed biopsy of the peritoneum. Histological examination of tissue removed - endometriosis of the peritoneum.

Diagnosis at discharge: secondary Infertility. External genital endometriosis stage I-II.

Two months later, after surgery, pregnancy, culminating in timely delivery.

Forecast according to the claimed method was confirmed.

Example 2. Patient P., 33, with secondary infertility for 4 years. A history of 3 medical abortion, he joined the Department of endosurgery to clarify the causes of infertility. Married, gynecological diseases denies. Complains of constant nagging pain in the abdomen, not associated with the menstrual cycle, physical activity, sexual intercourse, defecation and poorly stoped taking nonsteroidal anti-inflammatory drugs. During previous surveys revealed the presence of two-phase menstrual cycle (on tests of functional diagnostics and histological examination of the endometrium), according to hysteresis�salpingography the uterus is not changed, fallopian tubes moderately tortuous, fill all over, the contrast in the abdominal cavity comes from both sides. Spouse examined - nothing abnormal detected. Postcoital test is positive. Vaginal examination: the uterus is normal in size, movable, painless, appendages are not enlarged on both sides, painless on palpation. Tenderness to palpation in the region of the Sacro-uterine ligaments and the posterior fornix.

Clinical diagnosis: secondary infertility. Suspicion of external genital endometriosis.

Made a determination in peripheral blood relative content of IL-1β+ monocytes, which is 33.5%.

Conclusion: the predicted lack of spontaneous pregnancy within one year after the laparoscopy.

At laparoscopy: free abdominal cavity, the uterus is normal size. The fallopian tubes are not visually changed when hromogidrotubatsii are filled with colors. The ventral Sacro-uterine ligaments, superficial and deep endometriotic lesions >3 cm2. The ovaries are of normal size, in the right - postovulatory "stigma".

Performed excision and destruction of foci of endometriosis. Endometriosis was verified histologically. Diagnosis at discharge: secondary Infertility. External genital endometriosis stage I-II.

In t�during the year of regular sexual intercourse without contraception pregnancy does not occur.

Forecast according to the claimed method was confirmed. The patient is recommended in vitro fertilization.

Example 3. Female patient, 26 years. Appealed with complaints of pain in the lower abdomen before menstruation and mild to moderate pain during intercourse. Infertility within the marriage for 3 years. Is the first marriage, gynecological diseases in history notes.

At the preoperative examination set two-phase menstrual cycle (on tests of functional diagnostics and histological examination of the endometrium). According hysterosalpingography uterine cavity has the correct form, the fallopian tubes are filled with a contrast all over, not dilated; the contrast in peritoneal cavity comes from both sides. Husband examined normozoospermia. Postcoital test is positive. On examination: the uterus is normal size, movable, painless. Appendages are not defined. Sharply painful back arch. Mucous, moderate. Aimed at laparoscopy to clarify the causes of infertility.

Diagnosis: primary infertility. Suspicion of external genital endometriosis.

Made a determination in peripheral blood relative content of IL-1β+ monocytes, which amounted to 50,0%.

Conclusion: it is predicted to achieve a pregnancy within one year after the operating�Oh laparoscopy and destruction foci of endometriosis.

On day 7 of the menstrual cycle produced by laparoscopy, which found: free abdominal cavity, the uterus is normal size. The fallopian tubes are not changed freely passable by chromopertubation. The ovaries are not changed. The ventral posterior leaves of the broad uterine and uterosacral ligaments revealed multiple superficial and deep endometriotic lesions larger than 3 cm2. Produced by coagulation and excision of endometriosis monopolar coagulator. Performed biopsy of the peritoneum. Histological examination of tissue removed - endometriosis of the peritoneum.

Diagnosis at discharge: primary Infertility. External genital endometriosis stage I-II.

After 6 months of pregnancy, culminating in timely delivery.

Forecast according to the claimed method was confirmed.

In this way we examined 30 women with infertility associated with external genital endometriosis stage I-II. Research data shown in the table.

IndicatorThe number of surveyed
Total females30
a true-positive result4
false positive4
a true-negative result21
a false-negative result1
Total: the accuracy of the proposed method - 83,3%
the sensitivity of the proposed method - 80,0%
the specificity of the proposed method - 84,0%

The advantages of the proposed method:

1. High accuracy - 83,3%, sensitivity - 80,0%, a specificity of 84.0%.

2. The simplicity in the interpretation of the survey results.

3. The method allows to correctly estimate the chance of spontaneous conception after therapeutic laparoscopy, adjust the tactics of management of patients and to assess the need for such costly method like IVF.

A method for predicting spontaneous pregnancy in one year in women with stages I and II of external genital endometriosis after surgical treatment of infertility by immunological studies of peripheral venous blood before surgery, characterized in that determine the relative amount of IL-1β + monocytes and �th value equal to 50.0% or more in the monocyte gate predicts spontaneous pregnancy in one year in women with stages I and II of external genital endometriosis.



 

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