Method for selecting female patients with the syndrome of "empty" follicles for carrying out ecf and et program with donor oocytes

FIELD: medicine, gynecology, reproductology.

SUBSTANCE: it is necessary to determine the content follicle-stimulating hormone (FSH) in follicular liquid in female patients with the syndrome of "empty" follicles (SEF), insulin-like growth factor (IGF-1) and human chorionic gonadotropin (hCG) to calculate differential value (DV) by the following formula: DV=exp(IV)/1+expIV), where IV - intermediate variable calculated by the following formula: IV=1.821+1×FSH-0.18×IGF-1+0.136×hCG, Exp - exponential function and at DV being below 0.5 the development of SEF is of absolute nature and a female patient should be recommended to have donation of oocytes, at DV being above 0.5 the development of SEF is of sporadic character and a patient should be recommended to undergo repeated curative ECF cycles. The innovation enables to elaborate the method for predicting the procedure of extracorporal fertilization and embryo transfer.

EFFECT: higher efficiency.

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The invention relates to medicine, namely to gynecology and Reproductology, and will find application in IVF and embryo transfer to increase the pregnancy rate.

Accumulated over the last 25 years experience in the treatment of infertility with IVF and PE proved its high efficiency. If in the 70's-early 80-ies were conducted description only a few cases of successful treatment of patients with IVF, at the present time it must be recognized that due to its implementation in practice has a real opportunity to effectively deal with almost all known forms of female and male infertility (Ovsyannikova T.V., et al., 1998; V.I. Orlov et al., 2000). In medical science there are single treatments, which are so rapidly underwent evolutionary changes aimed at overcoming global problems that occurred at the stage of their formation. For all the non-repudiation evidence of the success and viability of in vitro fertilization and transfer of embryos into the uterine cavity after the successful birth of the first child, the reproduction was faced with serious difficulties, limiting widespread method (Ansina M.B., 1995; V.I. Kulakov et al., 2000; Ailamazyan E.K. et al., 2000).

Almost two decades have passed since then, as the first Coulam et al., (1986) was described a new syndrome is - syndrome empty follicles (PCF). By definition, the author, is a condition characterized by the inability aspiration of oocytes from pre-ovulatory follicles in cycles of in vitro fertilization because of their absence. The occurrence of this syndrome are sporadic, and currently cannot be diagnosed (no endocrinologically or sonography) on some ovarian response to stimulation of superovulation (Penarrulia J, et al., 1999; Zreik THUS, et al., 2000).

There are divergent views in relation to etiological factors, mechanisms of development, diagnosis and treatment of this syndrome. Despite the huge interest from reproduction to this "phenomenon", he remains by far poorly known.

At a relatively low frequency of occurrence SPF practically unresolved issues are associated with the choice of further tactics of treatment of patients, the feasibility of subsequent IVF programs and the need for the use of donor oocytes. The development of oocytes in a growing pool of follicles is largely determined by the composition of the follicular fluid, which are absorbed from the blood or are secreted by the granulosa cells hormones and growth factors (Svetlakov A.V. et al., 2002; Burlew V.A. et al., 1998). In addition, when studying the biochemical composition of follicular fluid is trecena cyclical changes of antral environment according to the stages of follicle development (boyar KU, 2002; Burlew V.A. et al., 1999; Chernukha G. et al., 1996). Biologically active compounds contained in the antral cavity of the follicle, constitute the microenvironment in which the developing oocyte. Both excess and deficiency of these compounds in follicular fluid may have a pronounced negative effect on the development of the oocyte (fesikov A.M., 1990; Vorobyev, O.A. et al., 1998; Potin V.V. et al., 1993). Despite the importance obtained in the present data, namely the possibility of forecasting syndrome empty follicles due to the hormonal composition of the blood, in particular treatment cycle, no confidence in his re-development in other cycles of stimulation.

The lack of clear tactics of patients with SPF creates precedents multiple unsuccessful stimulation of the ovaries, which negatively affects both health and psycho-emotional state of women and the family's financial situation. In addition, the lack of clear guidance from the doctors about the appropriateness of the use of donor oocytes lays the necessary programs for an indefinite period and, as a rule, is solved independently by the patient after a series of unsuccessful IVF attempts.

In cases where religious, religious and ethnic beliefs contradict the use of donor oocytes, the patient clicks the repenteth "long wanderings" in the medical centers at various levels. All of the above was the basis for this syndrome, research and development of differentiated tactics of patients that have defined the purpose and objectives of our research.

In the available scientific and medical and patent literature we have not identified techniques that allow a reasoned selection of women with the syndrome SPF for inclusion in the following cycles of IVF and embryo transfer. This was the basis to consider the claimed invention does not have a prototype.

The objective of the invention: development objective and available for General use, allowing a differentiated selection of patients with "absolute" and "sporadic" forms of the syndrome empty follicles that will justify the holding of such patients, the program "oocyte" with the aim of obtaining the desired pregnancy.

The problem is solved in that the patient with the syndrome of "empty" follicles produce research follicular fluid, which determine the level of follicle-stimulating hormone, the level of insulin-like growth factor-I, human chorionic gonadotropin and expect differential indicator PD according to the formula: PD=exp(PP)/(1+exp(PP)), where:

Exp (x) exponential function,

PP - intermediate variable, calculated by the formula:

PP=1,81+1× FSH-0,18×IPFR-I+0,136+hCG, where:

FSH level of follicle-stimulating hormone in the follicular fluid, mIU/ml;

IPFR-I - the level of insulin-like growth factor in follicular fluid, ng/ml;

hCG - human chorionic gonadotropin in follicular fluid, IU/ml,

if the resulting value DP is less than 0.5, then this particular patient syndrome "empty follicle is a consequence of disturbed processes folliculo and oogenesis and is "absolute" in nature, so it is recommended the oocyte, and a value of DP greater than 0.5 the development of PCF is random "sporadic" nature and may be repeated treatment cycles of IVF, with the aim of obtaining their own oocytes.

The intermediate variable is introduced to simplify the notation of the basic diagnostic formulas.

To conduct differential diagnostics is carried out comparative analysis of the content of local vnutridomovyh regulators antral fluid using the above formulas, received using a logit regression, according to the non-Gaussian distribution of the original data. The use of mathematical processing has allowed us to allocate a specific syndrome empty follicles significantly significant factors vnutriigrovoy regulation (FSH folliculitis yuushi hormone hCG - human chorionic gonadotropin and IPFR-I - insulin-like growth factor). Our studies have shown that there are two forms of the syndrome empty follicles: "sporadic" and "absolute".

Thus, our data reveal a clear correlation between blood levels of FSH, LH, estradiol, progesterone, testosterone, and in antral fluid - FSH, hCG and IPFR-I, in adequate quantity, and a full follicle Genesis. The differences seem to be associated with providing the most favorable conditions for the development of oocytes in the dominant follicle in connection with the important role of the above controls in the processes of follicle Genesis. The inclusion of these controls in the formula is of particular biological significance. According to the literature (Enien W.M. et al., 1998; Roche J.F., 1996) follicle-stimulating hormone, immunoreactive human chorionic gonadotropin, and insulin-like growth factor-I, responsible for the full selection of the dominant follicle and the preparation of the Cumulus-oozing complex subsequent to ovulation. The concentration of these regulators was determined by immunofluorescence using specific test systems. Comparative analysis of the results of immunofluorescence studies further mathematics is a processing indicate more informative of the above methods to select patients with the syndrome of "empty" follicles: "absolute" and "sporadic" forms and depending on the received data to develop further differentiated tactics for keeping them in art programs.

The coincidence of prognostic and diagnostic criteria with a negative result, even a single puncture (no egg) is evidence of the "absolute" form of SPF. When the differential values of the index, calculated on follicular markers less than 0.5 missing the expediency of further gonadotropic stimulation and demonstrates the use of donor oocytes. However, the lack of diagnostically significant confirmation "absolute" form SPF creates the possibility of continuing treatment cycles of art.

Statistical analysis of complex data was conducted using the data Analysis and set of mathematical and statistical functions (Excel 2003), and using standard packages of applied statistical analysis Statistica 6.0 and MegaStat. Reliability differentiated selection of patients with "absolute" form of SPF is 66,1%.

The analysis of the accuracy of the differential metric DP below:

where: PI is the number of true positive cases in the sample,

IO is the number of true-negative cases,

PL is the number of false-positive cases,

LO is the number of false-negative cases.

The performance of the proposed method is confirmed by the following clinical examples.

Example 1.

Patient R-VA, case history No. 2341/222 enrolled in the Center for human reproduction in order to conduct her to the IVF program and PE. The patient complains of infertility II for 8 years. From the anamnesis it is known that the patient in 1987, he moved right tubectomy and Subtotal resection of the left ovary about tubal pregnancy on the right, in 1992 resection of right ovary about his cystic changes.

With the aim of obtaining cohort of oocytes she was appointed to the stimulation of ovulation. When ULTRASOUND was determined growth leading follicle or cohort of follicles and endometrial thickness in accordance with the Protocol. In this patient was registered at the time of the PU which functions 2 the leading follicle in the right ovary. Endometrial thickness was 8.5 mm

To achieve the maturity of the follicles (based on the ultrasound and hormonal monitoring) the patient has an ovulatory dose of HCG (Pregna") in the amount of 5000-10000 IU. The time of the appointment of CG depends on the time to achieve the leading follicle diameter not less than 18 mm

The aspiration of oocytes and follicular fluid produced after 36 hours after administration of an ovulatory dose of HCG separately from the right and left ovary. With the aim of obtaining oocytes from ovarian follicles and follicular fluid produced transvaginal puncture using a vaginal probe with a frequency of 5 MHz ultrasound Aloka SSD-500.

By follicle puncture had not received a single oocyte. In antral fluid aspirated from the follicles, determined the level of FSH, hCG and IPFR-I immunofluorescence method using test kits: kits are used (Wallac Oy, Turku, Finland), "hCG-IVF-TEST" (000 "Diatech-EM", Russia), DSL-10-2 800 (group of companies Biohimik, Russia), respectively. Concentrations of these vnutrimolekulyarnykh regulators: FSH and 5.6 mIU/ml; IPFR-I - 105,7 ng/ml; hCG - 38 IU/ml, with the objective of avoiding the accident revealed syndrome empty follicles of the examined patient mathematical processing of the received data, using the formula of calculation of PP and PD:

PP=1,8211× 5,6-0,18×105,7+0,136×38=(-6,30726).

Differential indicator PD=exp(-6,30726)/(1+exp(-6,30726))=0,001819708.

According to the obtained differential indicator (0,001819708), which is less than 0.5 in this patient, the "absolute" form of the syndrome empty follicles and therefore it was proposed with the aim of obtaining the desired pregnancy program "oocyte".

Example 2.

The patient In society, history No. 1389/158 enrolled in the Center for human reproduction in order to conduct her to the IVF program and PE. The patient complains of infertility II for 7 years. From the anamnesis it is known that the patient in 1997, he moved right tubectomy about tubal pregnancy on the right, in 1999 left tubectomy about tubal pregnancy left.

At the stage of examination before the IVF program identified ureaplasmosis, bacterial vaginosis, conducted anti-inflammatory treatment (effect). With the aim of obtaining cohort of oocytes she was appointed to the stimulation of ovulation. ULTRASONIC research conducted at the time of follicle puncture, allowed to register 9 leading follicles. Endometrial thickness of 7.1 mm

The patient was made follicle puncture and aspiration of their contents, the study of which has not revealed a single oocyte, on the basis of what was clinically confirmed in this cycle of stimulation syndrome empty follicles. The production is prohibited determination of the levels of biologically active substances in antral fluid: FSH the 2.8 mIU/ml; IPFR-I - 90 ng/ml; hCG - 45 IU/ml of The examined patient mathematical processing of the received data, using the formula of calculation of PP and PD:

PP=1,821+1×2,8-0,18×90+0,136×45=(-5,60152).

Differential indicator PD=exp(-5,60152)/(1+exp(-5,60152))=0,003678681.

According to the obtained differential indicator (0,003678681), which is less than 0.5 in this patient, the "absolute" form of the syndrome empty follicles and therefore it was proposed with the aim of obtaining the desired pregnancy program "oocyte".

Example 3.

Sick With society, history No. 769/56 enrolled in the Center for human reproduction in order to conduct her to the IVF program and PE. The patient complains of infertility II for 5 years. From the anamnesis it is known that the patient in 2000, moved or left tubectomy about tubal pregnancy left, in 2001 the plastic right pipe of uterine tubal pregnancy on the right.

At the stage of examination before the IVF program comorbidity is not revealed. With the aim of obtaining cohort of oocytes she was appointed to the stimulation of ovulation. ULTRASONIC research conducted at the time of follicle puncture, allowed to register 5 of the leading follicle in the left ovary and 2 in the right. Endometrial thickness of 11.2 mm

Follicle puncture and aspiration of their content is clinically confirmed the existence of at present is by the patient, in this program, SPF, as the study punctate did not detect a single oocyte.

To clarify further tactics of treatment of this patient made a determination of the levels of biologically active substances obtained in antral fluid: FSH - 3 mIU/ml; IPFR-I - 78,2 ng/ml; hCG - 70 IU/ml, the examined patients mathematical processing of the received data, using the formula of calculation of PP and PD:

PP=1,821+1×3-0,18×78,2+0,136×70=(0,20268).

Differential indicator PD=exp(0,20268)/(1+exp(0,20268))=0,550496268.

Revealed differential rate more than 0.5 testified to the existence in this patient "sporadic" forms of the syndrome empty follicles, so in the future, it is recommended to repeat treatment cycles of IVF, with the aim of getting their own oocytes.

In this study we examined 158 couples attending the Center for human reproduction at Rostov scientific research Institute of Obstetrics and Pediatrics, for the period from 1996 to 2004, with the purpose of carrying out programs included in the list of assisted reproductive technologies (art). Age of women from 158 couples included in this study, in both groups ranged from 21 to 46 years. The average age of the women in group 1 was 32,12±4,59, 2 groups of 31,18±3,69. All patients included in the survey were under the s 2 clinical groups. The first group (control) was 82 women, carried out according to standard methods in the art, with the full response of the ovaries to stimulation of superovulation. The second (main) group included 76 patients diagnosed syndrome empty follicles at the time of follicle puncture and aspiration of their content in therapeutic cycles of art, using standard management protocols. Of the 76 patients with the syndrome of "empty" follicles in 46 women in any of the subsequent attempts of gonadotrophic stimulation aspirating oocytes failed ("absolute" form SPF), while the 30 surveyed still managed to get the oocytes in later art ("sporadic" form SPF).

Given the fact that the composition of the follicular fluid, to a certain extent, reflects the hormonal composition of the blood (due to gonadotropin-releasing hormone within the ovary with blood flow and steroid hormones produced in the ovary), was expected differences found in the antral part of the environment of the examined control and main groups.

The results of the study of hormones in follicular fluid of women surveyed primary group indicate no significant differences in the level of LH, FSH, progesterone, estradiol, testosterone, compared with analogion the mi indices in patients in the comparison group (table 1).

Despite the lack of significant differences, higher concentrations of LH, progesterone and estradiol, on the background of relatively reduced performance FSH and testosterone are logged in patients with complete follicular apparatus. However, we also examined the comparative analysis of the levels intrafollicular regulators folliculo and oogenesis (epidermal growth factor, insulin-like growth factor I, inhibin a). The results of the study indicate the inequality of their distribution in empty and full follicles (table 2).

The results of this study emphasize the importance of determining the content of such biochemical substrates antral environment, as hCG and IPFR-I.

Table 3 shows the indicators intrafollicular regulators in patients with negative results follicle puncture for all subsequent art programmes (2-subgroup) and effective cycles (by follicle puncture in repeated treatment cycles of IVF were able to aspirate oocytes) (2-b subgroup).

As can be seen from the data presented in table 3, the concentration of luteinizing hormone and estradiol in follicular fluid aspirated from patients with positive puncture, was significantly higher, and testosterone, however, significantly lower than those surveyed in ineffective cycles. These differences apparently reflect pathological conditions, since it is known that follicular growth and maturation of oocytes depends on a complex chain of hormonal factor and the regulation of reproductive function at various stages of their progressive transformation. Antral environment in which the developing follicle undergoes cyclic transformation-dependent phase m is strannogo cycle and stages of follicle development.

Thus, based on the study intrafollicular regulators folliculo and oogenesis proposed diagnostic criteria of "absolute" and "sporadic" forms of the syndrome empty follicles, as well as the indications for the conduct of the "oocyte" to develop differentiated tactics of treatment of patients in art cycles. The program "oocyte" is shown patients even with a single negative result follicle puncture (absence of oocytes in IVF programs, when the differential values of the index, calculated on follicular markers, less than 0.5.

For practical health substantiated the importance of identifying patients with the syndrome of empty follicles in follicular fluid - FSH, hCG and IPFR-I for the diagnosis of "absolute" forms to reduce the material cost ineffective attempts of art.

Thus, the inventive method is more effective in comparison with the known and has a number of advantages:

- ensures high accuracy of the forecast;

- reliable and simple in execution;

has no complications and contraindications;

- can be widely used in the centers of human reproduction in the IVF and embryo transfer.

The method of selection of patients with the syndrome of "empty" follicles for IVF program with PE with IP is the use of donor oocytes characterized in that the patient with the syndrome of "empty" follicles in follicular fluid determine the level of follicle-stimulating hormone, the level of insulin-like growth factor, human chorionic gonadotropin and expect differential indicator DP by the formula

DP=exp(PP)/(1+exp(PP)),

where exp is the exponential function,

PP - intermediate variable, calculated by the formula

PP=1,821+1×FSH-0,18×IPFR-I+0,136×hCG,

where FSH level of follicle-stimulating hormone in the follicular fluid, mIU/ml;

IPFR-I - the level of insulin-like growth factor in follicular fluid, ng/ml;

hCG - human chorionic gonadotropin in follicular fluid, IU/ml,

and if the resulting value DP is less than 0.5, then the syndrome "empty follicle is "absolute" and the patient is recommended the oocyte, and a value of DP greater than 0.5 the development of SPF is "sporadic" nature and the patient is recommended repeated treatment cycles of IVF, with the aim of obtaining their own oocytes.