Method for predicting hollow follicles syndrome

FIELD: medical engineering.

SUBSTANCE: method involves determining luteinizing hormone concentration, follicle-stimulating hormone concentration, estradiol concentration, progesterone concentration, testosteron concentration in blood according to program ECO during the first days of menstrual cycle before superovulation stimulation. Prognostic criterion is calculated using the obtained data PC=ехр(K)/(1+ехр(K)), where Ехр(х) is the exponential function, K is the intermediate variable calculated from formula K=0.423+0.02хLG- 0.189хfSG-0.421хE2+0.3хP1-0.987хТs, where LG is the luteinizing hormone concentration in blood, IU/l; fSG is the follicle-stimulating hormone concentration in blood, IU/l; E2 is the estradiol concentration in blood, nmole/l; P1 is the blood progesterone concentration, nmole/l; Ts is the testosteron concentration in blood, ng/ml. Empty follicles syndrome is predicted using calculated PC value less than 0.5.

EFFECT: high accuracy of forecasting; simplified method.

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The invention relates to medicine, namely to gynecology and reproduction, and can be used in the IVF and embryo transfer.

The development of embryology in the last quarter of a century marked the beginning of successful infertility treatment using the method of in vitro fertilization (Ansina M.B., 2002). At the same time, reached a certain threshold of efficiency of use of the method of in vitro fertilization (35-40%) does not meet the needs of embryologists in achieving further reduction in the frequency of infertility in marriage (Kulakov V.I. and others, 2001). Given the fact that the Foundation of a successful IVF programs is to obtain a large number of full pre-ovulatory oocytes, to implement these programs use various schemes to drug stimulation of superovulation. The validity of the appointment of ovulation inductors is connected with the fact that the probability of maturation of several follicles simultaneously in spontaneous ovulatory cycles is no more than 5-10%, while in stimulated cycles, the development of two or more follicles reached in 35-60% of cases.

However, despite sufficient knowledge of processes folliculo and oogenesis, inexplicable or unexplained to date are the phenomena of "poor or inadequate response of the ovaries, the extreme manifestation of which are the two who is the syndrome of "empty follicles (MV Amanova and others, 2004; Krasnopolsky C.V. and others, 2004; Zech N., and others, 1998; Kosterov NR. and others, 1999). A distinctive feature of it is the absolute absence of the possibility of obtaining oocytes with a puncture "sonography full follicles in IVF programs. A characteristic feature of this syndrome is that neither hormonal examination or by ultrasound monitoring of follicle in cycles of superovulation stimulation to predict its development was not possible (Carolyn Coulam C. and others, 1986; Galache P. Vega and others, 1989; Filippo Ubaldi and others, 1997).

Due to the lack of convincing data on the mechanisms of formation of this pathological condition by the author who first described this syndrome was proposed the term syndrome empty follicles (PCF). The frequency of occurrence of this syndrome, according to foreign literature, is 2-7%. At the same time, the analysis of its own data Center for human reproduction at the Rostov Institute of obstetrics and Pediatrics revealed a syndrome empty follicles in 6.1% of patients IVF programs (Shamanova KU and others, 2002).

Fertility specialists have expressed considerable interest in relation to the etiology of the syndrome, its diagnosis and correction, but at the same time, there is little information about the methods of forecasting and further tactics of patients with SPF (Ashkenazi J. and others, 1987; Awoniyi Awonuga and others, 1998).

The occurrence of empty follicles were recorded with the same frequency in the cases of full and partial follicular rupture, and when the syndrome popularmusic follicles (Thomas W. Hilgers et al., Fertil. Steril. - 1992; Robert S. Howe et al., Fertil. Steril. - 1993). Syndrome empty follicles were established by them in the presence or absence of Cumulus - oozing complex. In their opinion, it is typical ultrasonic picture - hyperemotional rim in the form of the moon with internal sonolucent (translucent ultrasound) plot. But in 1993, Robert S. Howe expressed doubts about the validity of the received data, explaining that not all the cases considered sonography was syndrome empty follicles, because the study did not provide further aspiration of oocytes from the observed follicles. You must admit that up to the present time to diagnose PCF to the puncture of follicles is not possible.

Ndukwe G. et al. (Fertil. Steril. - 1996. - Vol.66. - P.845-847) proposed a test for predicting SPF before attempting to perform catheterization of the oocyte. The authors show the Lee, there is no significant difference between the hormonal and sonographic parameters used in the monitoring of follicles in the induction of superovulation in between treatments ended syndrome empty follicles, and courses, during which oocytes were able to aspirate by follicle puncture. It was concluded that this condition is more sporadic than a recurrent phenomenon.

Has been proposed a method for predicting this syndrome by measuring the level of b-hCG (human chorionic gonadotropin) in blood after 36 hours after administration of hCG. In their study, they showed that the threshold level of b-hCG 10 IU/l after 36 hours after administration of an ovulatory dose of hCG, gives 100%visibility SPF. However, this claim was challenged by Anwoniyi et al. (1998), who described the cycles of IVF which failed to aspirate oocytes despite the normal biological activity of b-hCG in the blood of patients within 36 hours after administration of hCG.

Thus, in relation to forecasting SPF opinions of scientists are extremely contradictory. Obviously, there are no reliable prognostic markers, allowing you to safely draw a conclusion about the presence of SPF on the stage of the clinical examination of the patient.

All of the above was the basis for the search for prognostic and diagnostic the definition of criteria for the development of this syndrome and the development of differentiated tactics of patients in order to identify opportunities for further treatment cycles of IVF and embryo transfer.

The task of the invention is the development of reliable and available for wide application of the method for predicting syndrome empty follicles.

The problem is solved in that the patient within 1-3 days of the menstrual cycle prior to the stimulation of superovulation for IVF produce the hormone composition of the blood levels of luteinizing hormone, the level of follicle-stimulating hormone, estradiol, progesterone, testosterone, and using the obtained data, calculate a prognostic criterion PC=exp (K)/(1+exp(K)),

where Exp(x) exponential function,

It is an intermediate variable, calculated by the formula: K=0,423+0,02×LH-0,189×FSH-0,421×E2+0,3×P1-0,987×Ts,

where LH is the level of luteinizing hormone in the blood, IU/l;

FSH level of follicle-stimulating hormone in the blood, IU/l;

E2 - estradiol levels in the blood, nmol/l;

P1 is the level of progesterone in the blood, nmol/l;

Ts - the level of testosterone in the blood, ng/ml

and if the calculated value of PC is less than 0.5, predict the development of the syndrome of empty follicles.

Intermediate variable K is introduced to simplify the notation the basic formula for calculating a prognostic criterion for the PC.

This formula allows for a certain level of gonadotropic and steroid hormones (LH, the IG, estradiol, progesterone, testosterone) in the blood serum of the total number of patients of art (assisted reproductive technologies) to allocate risk for the development of the syndrome empty follicles.

For the development of diagnostic rules for separation of groups of patients with high probability PCF) method was used a logit regression" (the validity of which was not a Gaussian distribution of the source data used in the work), implemented in ...STATISTICA 6 (Rebrov DU, 2003). The critical level of confidence the null statistical hypothesis (about the absence of differences and influences) was taken equal to 0.05, and the assessment of species distribution is 0.01.

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 choice of the investigated group of steroid and gonadotropin-releasing hormone due to the mathematical processing of the received reliably distinguishable by their content in the blood in patients with full follicles and patients with SPF. As you know, they play an extremely important role in the processes folliculo and oogenesis and generally in the whole reproductive system. Their concentration in the blood changes depending on the stage of follicular development, starting with the release of follicle from the resting state to release Mature egg in ovulation, with the subsequent development of the yellow body. The content of these hormones in the blood was determined using immunofluorescence method research, with application specific test systems. When calculating formula PC and getting a value less than 0.5 with a probability of 71% to predict the development of this syndrome. Comparative analysis of the results of immunofluorescence studies with further mathematical processing using formulas To calculate and PC, backed up data is mi visual evaluation of the aspirate obtained from the dominant pre-ovulatory follicles, suggest more informative of the above methods to identify patients with the syndrome empty follicles and develop further tactics in their art programmes depending on the received data. Diagnostic rule: if the value of the PC is less than 0.5, then this particular patient in the near art cycle high risk of developing the syndrome empty follicles. When the value of the PC more than 0.5 development SPF unlikely.

Our studies have shown that the syndrome of "empty" follicle - independent dishormonal disease, manifested by a disorder of the Central and local ovarian regulations.

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.

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

Example 1.

Patient R-VA, case history No. 6959/556 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 10 years. From the anamnesis it is known that the patient in 1989 he moved right tubectomy and Subtotal the second resection of the left ovary about tubal pregnancy right in 1992 resection of right ovary about his cystic changes.

During the survey, with the aim of estimating the initial hormonal status of the reproductive system, the patient is in 1-3 days of the menstrual cycle prior to the stimulation of superovulation for IVF spend determining the concentration level of hormones in the blood - steroid hormones: estradiol (E2), progesterone (P1), testosterone (Ts), and protein hormones: luteinizing hormone (LH), follicle-stimulating hormone (FSH). The concentration of hormones in blood serum define immunofluorescence method using enzyme immunoassay systems kits are used (Wallac Oy, Turku, Finland). In the studies in this patient were obtained following the initial levels of blood hormones: LH - 20,9 IU/l; FSH - 3,21 IU/l; E2 - 0.23 nmol/l; P1 is 0.99 nmol/l; Ts - 1.54 ng/ml.

In order to identify the patients at risk for the development of the syndrome empty follicles obtained data is processed using the above formula and the calculated intermediate variable K: K=0,423+0,02×20,9-0,189×3,21-0,421×0,23+0,3×0,99-0,987×1,54=(-1,082765), and then a prognostic criterion PC=exp(-1,082765)/(1+exp(-1,082765))=0,25298.

According to the calculated value PC=0,25298 (less than 0.5), this patient was classified in the group on the development of the syndrome empty follicles to clarify the diagnosis, and to receive the deposits cohort of oocytes she was appointed to the stimulation of ovulation according to the following scheme.

Stimulation of ovulation was performed with 2 days of the menstrual cycle using inductors of superovulation Protocol klomifentsitrat 100 mg daily for 5 days (total dose was 500 mg) and human menopausal gonadotropin (Humegon ED+ED) 2-4 capsules per day (the dose of an administered drug is determined individually depending on the response of the patient-controlled daily, starting with 5-6 days of stimulation, ultrasound and hormonal studies), the total dose of 8 capsules. When U3 is the study determined the growth of the leading follicle or cohort of follicles and endometrial thickness in accordance with the Protocol.

In this patient was registered at the time of puncture 2 leading follicle in the right ovary. Endometrial thickness was 8.5 mm

When Mature 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 secosteroids transvaginal puncture using a vaginal probe with a frequency of 5 Mz ultrasound Aloka SSD-500.

By follicle puncture had not received a single oocyte.

Example 2.

Patients To the VA, the case history No. 1535/149 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).

Within 1-3 days of the menstrual cycle prior to the stimulation of superovulation in IVF program conducted a blood test for the study of hormones (FSH, LH, estradiol, progesterone, testosterone) in order to identify or exclude criteria, indicating the possibility of development of a syndrome of empty follicles. Vyyavleny levels of hormones: LH - 7,0 IU/l; FSH - 15,MU/l; E2 - 1.3 nmol/l; P1 - 0,56 nmol/l; Ts - 0.2 ng/ml, calculated intermediate criterion K=0,423+0,02×7,0-0,189×15,2-0,421×1,3+0,3×0,56-0,987×0,2=(-2,885221) and predictive criterion PC=exp(-2,885221)/(1+exp(-2,885221))=0,05289.

As can be seen from the results of the calculations in this patient, the value of a prognostic criterion for the PC is less than 0.5, that svidetelstvovat a high probability of the development of the PCF.

Stimulate the superovulation was conducted by Gumagana ED+ED - 10 vials with 3 to 7 day MC, clostilbegyt 100 mg per day 3 to day 7 MC (total dose 500 mg). When Mature follicles on day 11 MC was appointed Pregna dose of 10000 IU.

U3-a study 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 which was confirmed in this cycle of stimulation syndrome empty follicles.

Example 3.

The patient P, the case history No. 8653/712 enrolled in the Center for human reproduction in order to conduct her to the IVF program and PE. The patient complains of infertility II within 2 years. From the anamnesis it is known that the patient in 1999, 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 was not detected.

Blood sampling for the study of hormones (FSH, LH, estradiol, progesterone, testosterone) were performed on day 1 of the menstrual cycle to address the question, whether the patient is threatened by the development of the syndrome of empty follicles. Identified levels of hormones: LH is 9.3 IU/l; FSH - 8 IU/l; E2 - 0.45 nmol/l; P1 - 0.78 nmol/l; Ts - 0,ng/ml Calculated intermediate criterion K=0,423+0,02×9,3-0,189×8-0,421×0,45+0,3×0,78-0,987×0,98=(-1,824658), then the prognostic criterion PC=exp(-1,824658)/(1+exp(-1,824658))=0,13888.

The calculated prognostic criterion PC less than 0.5 svidetelstvovat that this patient is a high probability of the development of the PCF.

Stimulation of superovulation was conducted by Puregon - 15 vials of 2 to 7 day MC (total dose : 750 ME), Diphereline - 11 vials of 2 on day 11 MC (total dose of 1.1 mg). When Mature follicles on day 12 MC was appointed Pregna at a dose of 5000 IU.

U3-a study conducted at the time of follicle puncture, allowed to register 3 leading follicle in the left ovary and 1 in the right. Endometrial thickness of 11.4 mm

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

Example 4.

Patient-to, history No. 8114/815 enrolled in the Center for human reproduction in order to conduct her to the IVF program and PE. The patient complains of infertility I for 15 years. From the anamnesis it is known that the patient in 1995 was made wedge resection of both ovaries.

At the stage of examination before the IVF program identified chlamydia conducted anti-inflammatory treatment (effect).

on the first day of the menstrual cycle extracted hormones blood, using enzyme immunoassay using a specific test systems were obtained with the following concentrations: LH - 9,4 IU/l; FSH - 2,2 IU/l; E2 - 0.08 nmol/l; P1-4 nmol/l; Ts - of 0.56 ng/ml.

Produced by calculating an intermediate outcome according to the formula: K=0,423+0,02×9,4-0,189×2,2-0,421×0,08+0,3×4-0,987×0,56=(0,8092483), then the prognostic criterion PC=exp(0,8092483)/(1+exp(0,8092483))=0,68372.

The calculated prognostic criterion PC more than 0.5 suggests that this patient, the likelihood of SPF unlikely.

Stimulation of superovulation was conducted by Puregon - 20 vials of 2 on day 8 MC (total dose of 1000 IU), Diphereline - 11 vials of 2 to 10 day MC (total dose : 0.7 mg). When Mature follicles on day 14 MC was appointed Pregna dose of 10000 IU.

U3-a study conducted at the time of follicle puncture, allowed to register 2 leading follicle in the left ovary and 3 in the right. Endometrial thickness 10,4 mm

Follicle puncture and aspiration of their contents clinically confirmed adequate ovarian response to superovulation stimulation the patient presents, in this program, as the study revealed punctate 3 full oocyte.

Thus, our studies revealed no effect of different protocols and schemes stimulation of superovulation on the syndrome "empty the follicles. Developed diagnostically important criteria for the selection of patients at risk for the development of PCF.

To achieve these objectives 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 study were divided into 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.

We carried out a comparative analysis of the content of gonadotropic and steroid hormones in the blood of patients in art programmes syndrome empty follicles and with a full response in ovarian stimulation superou is Azii (table 1).

The results of the study of hormones in the blood of women surveyed primary group indicate no significant differences in the level of LH, prolactin, progesterone, estradiol, compared with similar indices in patients of the comparison group, as confirmed by literature data (Valeeva E.M., 1997).

Despite significant differences in levels of the hormones FSH and Ts, important is the fact that as in the case of FSH and Ts, and in the case with the rest of the studied hormones concentrations in their blood did not exceed the standard values. This fact allows us to consider the syndrome empty follicles, as a state, other than wasting syndrome ovarian syndrome and resistant ovarian cancer. The absence of oocytes in patients with the syndrome of "empty" follicles while maintaining physiological levels of LH and FSH in the blood, adequate ovarian steroidogeneza and normal development of a pool of dominant follicles allows you to think about idiopathic dysfunction of ovaries.

Thus, for practical health substantiated the importance of identifying patients in the peripheral blood (1-3 days of the menstrual cycle prior to the stimulation of superovulation for IVF) content of FSH, LH, progesterone, testosterone and estradiol, although this level can fluctuate within the normal range in order to identify high-risk groups for the development of PCF.

The inventive method is more effective in comparison with the known and has a number of advantages to the substance:

- ensures high accuracy of the forecast;

retraumatized 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 is tested on a sufficient volume of clinical material and can be recommended for use in gynecologic hospitals and clinics that use of art.

A method for predicting syndrome empty follicles by examining the blood, wherein the patient in 1-3 days of the menstrual cycle prior to the stimulation of superovulation for IVF determine blood levels of luteinizing hormone, the level of follicle-stimulating hormone, estradiol, progesterone, testosterone, and using the obtained data, calculate a prognostic criterion PC=exp(K)/(1+exp(K)),

where Exp(x) exponential function,

It is an intermediate variable, calculated by the formula

K=0,423+0,02×LH-0,189×FSH-0,421×E2+0,3×P1-0,987×Ts,

where LH is the level of luteinizing hormone in the blood, IU/l;

FSH level of follicle-stimulating hormone in the blood, IU/l;

E2 - estradiol levels in the blood, nmol/l;

P1 is the level of progesterone in the blood, nmol/l;

Ts - the level of testosterone in the blood, ng/ml

and if the calculated value of PC is less than 0.5, predict the development of the syndrome of empty follicles.