RU2302639C1 - Method for predicting hollow follicles syndrome - Google Patents

Abstract

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=exp(K)/(1+exp(K)), where Exp(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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Description

The invention relates to medicine, namely to gynecology and reproductology, and can be used in IVF and PE programs.

The development of embryology in the last quarter of a century marked the beginning of the successful treatment of infertility using the method of in vitro fertilization (Anshina MB, 2002). At the same time, the achieved, certain threshold for the effectiveness of using the in vitro fertilization method (35-40%) does not satisfy the need for embryologists to achieve a further reduction in the frequency of infertility in marriage (Kulakov V.I. et al., 2001). Given the fact that the basis for the successful implementation of IVF programs is to obtain a large number of complete preovulatory oocytes, various schemes of drug stimulation of superovulation are used to implement these programs. The validity of the appointment of ovulation inducers is due to the fact that the probability of maturation of several follicles simultaneously in spontaneous ovulatory cycles is no more than 5-10%, whereas in stimulated cycles the development of two or more follicles is achieved in 35-60% of cases.

However, despite sufficient knowledge of the processes of folliculo- and oogenesis, the phenomena of a “poor” or inadequate ovarian response, the extreme manifestation of which is the syndrome of “empty” follicles (Yamanova M.V. et al., 2004; Krasnopolskaya, are still difficult to explain or inexplicable). K.V. et al., 2004; Zech N. et al., 1998; Kustarov V.N. et al., 1999). A distinctive feature of it is the absolute absence of the possibility of obtaining oocytes during puncture of “sonographically complete” follicles in IVF programs. A characteristic feature of this syndrome is that neither hormonal examination nor ultrasound monitoring of follicles in superovulation stimulation cycles made it possible to predict its development (Coulam Carolyn B. et al., 1986; Galache Vega P. et al., 1989; Ubaldi Filippo et al., 1997).

Due to the lack of convincing data on the mechanisms of the formation of this pathological condition, the author, who first described this syndrome, proposed the term “empty” follicle syndrome (SPF). The frequency of occurrence of this syndrome, according to foreign literature, is 2-7%. At the same time, an analysis of our own data from the Center for Human Reproduction at the Rostov Institute of Obstetrics and Pediatrics revealed the syndrome of “empty” follicles in 6.1% of patients with IVF programs (Sagamonova K.Yu. et al., 2002).

Reproductologists express significant interest in the etiology of the syndrome, its diagnosis and methods of correction, but at the same time there is little information about its prediction methods and further management tactics for patients with SPF (Ashkenazi J. et al., 1987; Awoniyi Awonuga et al., 1998).

A known method of sonographic diagnosis of SPF, proposed in 1989, Thomas W. Hilgers et al. According to the obtained ultrasonic parameters, the incidence of SPF was 50%. There were no differences in the processes of follicular development and ovulation time.

The occurrence of empty follicles was recorded with the same frequency in cases with complete and partial follicular rupture, as well as in the syndrome of neovulating follicles (Thomas W. Hilgers et al., Fertil. Steril. - 1992; Robert S. Howe et al., Fertil. Steril. - 1993). The syndrome of "empty" follicles was established by them by the presence or absence of a cumulus - oocyte complex. In their opinion, he has a characteristic ultrasound picture - a hyperechoic border in the shape of a moon with an internal sonoluminescent (translucent ultrasound) area. But in 1993, Robert S. Howe expressed doubt about the reliability of the data, explaining that not all cases examined sonographically were the syndrome of “empty” follicles, since the study did not provide for further aspiration of oocytes from the observed follicles. It must be recognized that to date, it is not possible to diagnose SPF prior to follicle puncture.

Ndukwe G. et al. (Fertil. Steril. - 1996. - Vol.66. - P.845-847) a test was proposed to predict SPF before attempting to catheterize an oocyte. The authors showed that there is no significant difference between the hormonal and sonographic parameters used to monitor follicles during the induction of superovulation, between treatments that culminate in the syndrome of "empty" follicles, and courses during which oocytes were aspirated during follicular puncture. It was concluded that this condition is more sporadic than a recurring event.

A method has been proposed for predicting this syndrome by measuring the level of b-hCG (human chorionic gonadotropin) in the blood 36 hours after receiving hCG. In their studies, they showed that the limiting level of b-hCG 10 IU / l - 36 hours after taking an ovulatory dose of hCG, provides a 100% chance of predicting SPF. However, this claim has been challenged by Anwoniyi et al. (1998), who described IVF cycles in which oocytes could not be aspirated even despite the normal bioactivity of b-hCG in the blood of patients 36 hours after receiving hCG.

Thus, in relation to attempts to predict the SPF, the opinions of scientists are extremely contradictory. Obviously, there are no reliable prognostic markers that allow with certain confidence to conclude that there is SPF at the stage of clinical examination of the patient.

All of the above was the basis for the search for prognostic and diagnostic criteria for the development of this syndrome and the development of differentiated tactics for the management of patients in order to identify the possibility of continuing further treatment cycles of IVF and PE.

The task of the invention is the development of a reliable and widely available method for predicting the syndrome of "empty" follicles.

The problem is solved in that the patient in 1-3 days of the menstrual cycle before stimulation of superovulation under the IVF program determines the hormonal composition of the blood: the level of luteinizing hormone, the level of follicle-stimulating hormone, the level of estradiol, the level of progesterone, the level of testosterone and using the obtained data, calculate prognostic criterion PC = exp (K) / (1 + exp (K)),

where exp (x) is an exponential function,

K is an intermediate variable calculated by the formula: K = 0.423 + 0.02 × LG-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 - the level of follicle-stimulating hormone in the blood, IU / l;

E2 - the level of estradiol in the blood, nmol / l;

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

Ts - blood testosterone level, ng / ml

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

An intermediate variable K is introduced to simplify the writing of the basic formula for calculating the prognostic criterion for PC.

This formula allows us to identify the risk group for the development of empty follicles syndrome by the level of certain gonadotropic and steroid hormones (LH, FSH, estradiol, progesterone, testosterone) in blood serum from the total number of patients with ART (assisted reproductive technologies) programs.

To develop diagnostic rules for identifying groups of patients with a high probability of SPF, the “logit-regression” method was used (the justification for the use of which was not the Gaussian distribution of the initial data used in the work), implemented in the items of STATISTICA 6 (D. Rebrova, 2003). The critical level of reliability of the null statistical hypothesis (the absence of differences and influences) was taken equal to 0.05, and when assessing the type of distribution - 0.01.

Analysis of the accuracy of the prognostic formula PC ACCURACY (PI + IO) / (PI + IO + LP + LO) LP / (LP + IO) 71.28% SENSITIVITY SP / (SP + LO) LO / (IP + LO) 80.85% SPECIFICITY OF IO / (IO + PL) IP / (IP + LP) 61.70% FALSE POSITION OF DIAGNOSIS IO / (LO + IO) 38.30% FALSE NEGATIVE DIAGNOSIS SHARE 19.15% ACCURACY OF POSITIVE RESULTS 67.86% ACCURACY OF NEGATIVE RESULTS 76.32%

where PI is the number of truly positive cases in the sample;

IO - the number of truly negative cases;

LP - the number of false positive cases;

LO - the number of false negative cases.

The choice of the studied group of steroid and gonadotropic hormones is due to the mathematical processing of the obtained reliably distinguishable blood content in patients with full follicles and patients with SPF. As you know, they play an extremely important role in the processes of folliculo- and oogenesis and in general throughout the reproductive system. Their blood concentrations vary depending on the stage of follicular development, starting from the moment the follicle leaves its resting state and until the mature egg is released into ovulation, followed by the development of the corpus luteum. The content of these hormones in the blood was determined using the immunofluorescence method of research, using specific test systems. When calculating the PC formula and obtaining a value of less than 0.5, it is possible with a 71% probability to predict the development of this syndrome. A comparative analysis of the results of immunofluorescence research methods with their further mathematical processing using the calculation formulas K and PC, supported by visual assessment of the obtained aspirate from dominant preovulatory follicles, indicate that the above methodology is more informative in order to identify patients with empty follicles syndrome and develop further tactics keeping them in ART programs, depending on the data received. Diagnostic rule: if the obtained value of the PC index is less than 0.5, then this particular patient has a high probability of developing an “empty” follicle syndrome in the next cycle of ART. With a PC score of more than 0.5, the development of SPF is unlikely.

Our studies have shown that the syndrome of "empty" follicles is an independent dishormonal disease, manifested by a violation of the central and local ovarian regulation.

Statistical analysis of the obtained data complex was carried out using the “Data Analysis” package and a set of mathematical and statistical functions (Excel 2003), as well as 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 was admitted to the Center for Human Reproduction in order to conduct her IVF and PE program. The patient complains of infertility II for 10 years. From the anamnesis it is known that the patient in 1989 underwent a right-sided tubectomy and subtotal resection of the left ovary due to tubal pregnancy on the right, in 1992 a resection of the right ovary due to his cystic change.

At the stage of the examination, in order to assess the initial hormonal state of the reproductive system, the patient in 1-3 days of the menstrual cycle before stimulation of superovulation under the IVF program determines the concentration 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 the blood serum is determined by the immunofluorescence method using Delfia enzyme-linked immunosorbent assay systems (Wallac Oy, Turku, Finland). As a result of the study, the following initial levels of blood hormones were obtained in this patient: LH - 20.9 IU / l; FSH - 3.21 IU / L; E2 0.23 nmol / L; P1 0.99 nmol / L; Ts - 1.54 ng / ml.

In order to identify patients at risk for the development of the syndrome of "empty" follicles, the data obtained were processed using the above formula and the intermediate variable K was calculated: 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 the prognostic criterion PC = exp (-1.082765) / (1 + exp (-1.082765)) = 0.25298 .

According to the calculated value of PK = 0.25298 (which is less than 0.5), this patient was assigned to the group for the development of the syndrome of “empty” follicles to clarify the diagnosis, and also in order to obtain a cohort of oocytes she was prescribed ovulation stimulation according to the following scheme.

Ovulation was stimulated from day 2 of the menstrual cycle using superovulation inducers according to the clomiphencitrate protocol of 100 mg per day for 5 days (total dose was 500 mg) and human menopausal gonadotropin (Humegon 75ED + 75ED) at 2-4 ampoules per day (dose the administered drug is determined individually depending on the patient’s reaction, controlled daily, starting from 5-6 days of stimulation, ultrasound and hormonal studies), the total dose is 8 ampoules. In the U3 study, the growth dynamics of the leading follicle or cohort of follicles and the thickness of the endometrium were determined in accordance with the protocol.

In this patient, at the time of the puncture, 2 leading follicles in the right ovary were registered. The thickness of the endometrium was 8.5 mm.

Upon reaching maturity by follicles (based on ultrasound and hormonal monitoring data), the patient was prescribed an ovulatory dose of chronic hepatitis C (“Rotted”) in the amount of 5000-10000 PIECES. The time of appointment of HG is determined depending on the period of reaching the leading follicle with a diameter of at least 18 mm.

Oocytes and follicular fluid were aspirated 36 hours after the administration of an ovulatory dose of CG separately from the right and left ovary. In order to obtain oocytes from ovarian follicles and follicular fluid, transvaginal puncture was performed using a vaginal probe with a frequency of 5 Mz of an Aloka SSD-500 ultrasound machine.

During follicular puncture, not a single oocyte was obtained.

Example 2

Patient K., medical history No. 1535/149 was admitted to the Center for Human Reproduction with the aim of conducting her IVF and PE programs. The patient complains of infertility II for 7 years. From the anamnesis it is known that the patient in 1997 underwent a right-sided tubectomy about a tubal pregnancy on the right, in 1999 a left-sided tubectomy about a tubal pregnancy on the left.

At the examination stage, before the IVF program, ureaplasmosis, gardnerellosis was detected, anti-inflammatory treatment was carried out (with effect).

On days 1-3 of the menstrual cycle, before the start of stimulation of superovulation, an IVF blood sampling was performed to study hormones (FSH, LH, estradiol, progesterone, testosterone) in order to identify or exclude criteria indicating the possibility of developing the syndrome of "empty" follicles. The levels of hormones were identified: LH - 7.0 IU / l; FSH - 15.2 IU / L; E2 - 1.3 nmol / l; P1 - 0.56 nmol / l; Ts - 0.2 ng / ml; 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 = calculated (-2.885221) and prognostic criterion PC = exp (-2.885221) / (1 + exp (-2.885221)) = 0.05289.

As can be seen from the calculation result, in this patient the value of the prognostic criterion for PC is less than 0.5, which indicates a high probability of developing SPF.

Superovulation was stimulated by Humegon 75ED + 75ED - 10 ampoules from 3 to 7 days of MC, clostilbegit 100 mg per day from 3 to 7 days of MC (total dose - 500 mg). Upon reaching maturity by follicles on day 11, MC was prescribed Pregnil at a dose of 10,000 units.

The U3 study conducted at the time of follicular puncture allowed 9 leading follicles to be registered. The thickness of the endometrium is 7.1 mm. The patient underwent puncture of the follicles and aspiration of their contents, the study of which did not reveal a single oocyte, on the basis of which the syndrome of “empty” follicles was confirmed in this stimulation cycle.

Example 3

Patient P., medical history No. 8653/712 was admitted to the Center for Human Reproduction in order to conduct her IVF and PE program. The patient complains of infertility II for 2 years. From the anamnesis it is known that the patient in 1999 underwent a left-sided tubectomy about a tubal pregnancy on the left, in 2001 a plastic surgery of the right uterine tube about a tubal pregnancy on the right.

At the examination stage, before the IVF program, no concomitant pathology was detected.

Blood sampling for the study of hormones (FSH, LH, estradiol, progesterone, testosterone) was performed on the 1st day of the menstrual cycle to decide whether this patient is threatened by the development of the syndrome of "empty" follicles. Revealed hormone levels: LH - 9.3 IU / L; FSH - 8 IU / L; E2 - 0.45 nmol / l; P1 - 0.78 nmol / l; Ts - 0.98 ng / ml. The 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) was calculated, then the prognostic criterion PC = exp (-1.824658) / (1 + exp (-1.824658)) = 0.13888.

A calculated prognostic criterion of PC less than 0.5 indicates that this patient is highly likely to develop SPF.

Superovulation was stimulated by Puregon - 15 ampoules from the 2nd to 7th day of MC (total dose of 750 ME), Diferelin - 11 ampoules from the 2nd to 11th day of MC (total dose of 1.1 mg). Upon reaching maturity by follicles on day 12, MC was prescribed pregnil in a dose of 5000 units.

The U3 study conducted at the time of follicular puncture allowed to register 3 leading follicles in the left ovary and 1 in the right. The thickness of the endometrium is 11.4 mm.

Puncture of the follicles and aspiration of their contents clinically confirmed the existence of SPF in the presented patient in this program, since the punctate study did not reveal a single oocyte.

Example 4

Patient S., medical history No. 8114/815 was admitted to the Center for Human Reproduction with the aim of conducting an IVF and PE program. The patient complains of infertility I for 15 years. From the anamnesis it is known that the patient in 1995 had a wedge-shaped resection of both ovaries.

At the examination stage, before the IVF program, chlamydia was detected, anti-inflammatory treatment was carried out (with effect).

On the first day of the menstrual cycle, blood hormones were taken, using enzyme-linked immunosorbent assay using specific test systems, their following concentrations were obtained: LH - 9.4 IU / l; FSH - 2.2 IU / l; E2 - 0.08 nmol / l; P1-4 nmol / L; Ts - 0.56 ng / ml.

The intermediate criterion was calculated using 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 PC prognostic criterion = exp (0.8092483) / (1 + exp (0.8092483)) = 0.68372.

The calculated prognostic criterion of PC of more than 0.5 indicates that this patient is unlikely to develop SPF.

Superovulation was stimulated by Puregon - 20 ampoules from the 2nd to 8th day of MC (total dose 1000 ME), Diferelin - 11 ampoules from the 2nd to 10th day of MC (total dose of 0.7 mg). Upon reaching maturity by follicles on day 14, MC was prescribed Pregnil at a dose of 10,000 units.

The U3 study conducted at the time of follicular puncture allowed to register 2 leading follicles in the left ovary and 3 in the right. The thickness of the endometrium is 10.4 mm.

Puncture of the follicles and aspiration of their contents clinically confirmed an adequate ovarian response to stimulation of superovulation in the patient presented in this program, since the punctate study revealed 3 full-fledged oocytes.

Thus, our studies have established the absence of the influence of various protocols and schemes of stimulation of superovulation on the development of the syndrome of "empty" follicles. Diagnostically significant criteria have been developed for selecting patients at risk for the development of SPF.

To solve the tasks, we examined 158 couples who applied to the Center for Human Reproduction at the Rostov Research Institute of Obstetrics and Pediatrics for the period from 1996 to 2004, with the aim of carrying out programs included in the list of assisted reproductive technologies (ART). The age of women from the 158 couples included in this study ranged from 21 to 46 years in both groups. The average age of women in the 1st group was 32.12 ± 4.59, 2 groups - 31.18 ± 3.69.

All patients included in the examination were divided into 2 clinical groups. The first group (control) consisted of 82 women, performed by the standard method in ART programs, with a full response of the ovaries to stimulation of superovulation. The second (main) group included 76 patients with a diagnosed “empty” follicle syndrome at the time of follicular puncture and aspiration of their contents during treatment cycles of ART programs using standard management protocols.

We carried out a comparative analysis of the content of gonadotropic and steroid hormones in the blood of patients with ART programs with “empty” follicles syndrome and with a complete response of the ovaries to stimulation of superovulation (table 1).

The results of the study of hormones in the blood of the examined women of the main group indicate the absence of significant differences in the levels of LH, prolactin, progesterone, estradiol compared with similar parameters in patients of the comparison group, which is also confirmed by literature data (Vikhlyaeva EM, 1997).

Despite the revealed significant differences in the levels of FSH and Ts hormones, it is important that, both in the case of FSH and Ts, and in the case of the rest of the studied hormones, their concentration in the blood did not exceed the standard values. This circumstance allows us to consider the syndrome of "empty" follicles, as a condition different from the syndrome of ovarian exhaustion and the syndrome of resistant ovaries. The fact of 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 steroidogenesis and the normal development of the pool of dominant follicles allows us to think about idiopathic dysfunction of the ovaries.

Table 1. Basal level of hormones in the blood of patients of clinical groups Hormones Control group n = 82 The main group n = 76 Error Probability (p) Hormone content (median and interquartile range) Luteinizing hormone (LH), mIU / L 7.1
(4.8-13.2) 8.5
(5,6-12,1) 0.123537 Follicle-stimulating hormone (FSH), mIU / l 4,5
(3.2-7.5) 6.3
(4.0-9.0) 0,003826 Prolactin, mIU / L 314.0
(234.0-456.5) 278.0
(234.5-386.0) 0,310987 Progesterone, nmol / L 3.4
(1,0-6,4) 1,2
(0.4-3.6) 0.449125 Estradiol, nmol / L 0.3
(0.1-0.7) 0.4
(0.3-0.6) 0.326789 Testosterone ng / ml 0.3
(0.2-0.4) 0.5
(0.4-1.0) 0,000039

Thus, for practical public health, the importance of determining the levels of FSH, LH, progesterone, testosterone and estradiol in FSH, LH, progesterone, testosterone, and estradiol in patients with peripheral blood (1-3 days of the menstrual cycle before starting superovulation stimulation by the IVF program) is substantiated, although this level can fluctuate within normal limits in order to identify a high-risk group for the development of SPF.

The inventive method is more efficient compared to the known and has several advantages:

- provides high accuracy of the forecast;

- non-traumatic and easy to perform;

- has no complications and contraindications;

- can be widely used in human reproduction centers in IVF and PE programs.

The method was tested on a sufficient volume of clinical material and can be recommended for use in gynecological hospitals and clinics using ART.

Claims (1)

A method for predicting the syndrome of "empty" follicles by a blood test, characterized in that the patient in the 1-3 days of the menstrual cycle before stimulation of superovulation with the IVF program determines the level of luteinizing hormone in the blood, follicle-stimulating hormone level, estradiol level, progesterone level, testosterone level and using the data obtained, the prognostic criterion PK = exp (K) / (1 + exp (K)) is calculated where exp (x) is an exponential function, K is an intermediate variable calculated by the formula K = 0.423 + 0.02 × LG-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 - the level of follicle-stimulating hormone in the blood, IU / l; E2 - the level of estradiol in the blood, nmol / l; P1 is the level of progesterone in the blood, nmol / l; Ts - blood testosterone level, ng / ml, and if the calculated PC value is less than 0.5, the development of the syndrome of "empty" follicles is predicted.