RU2309411C2 - Method for selecting female patients with the syndrome of "empty" follicles for carrying out ecf and et program with donor oocytes - Google Patents

Abstract

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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Description

The invention relates to medicine, namely to gynecology and reproductology, and will find application in IVF and PE programs in order to increase the frequency of pregnancy.

The experience gained over the past 25 years in the treatment of infertility using IVF and PE has been shown to be highly effective. If in the 70s and early 80s only single cases of successful treatment of patients with IVF were described, now it must be recognized that thanks to its implementation in practice, there is a real opportunity to effectively overcome almost all known forms of female and male infertility (Ovsyannikova T.V. et al., 1998; Orlov V.I. et al., 2000). In medical science, single treatment methods are known that underwent evolutionary changes so rapidly aimed at overcoming global problems that arose at the stage of their formation. Despite the irrefutable 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, reproductologists faced serious difficulties limiting the widespread use of the method (Anshina MB, 1995; Kulakov V.I. et al., 2000 ; Ailamazyan E.K. et al., 2000).

Almost two decades have passed since the first time Coulam et al., (1986) described a new syndrome - the syndrome of "empty" follicles (SPF). According to the author’s definition, this condition is characterized by the impossibility of aspiration of oocytes from preovulatory follicles in in vitro fertilization cycles due to their absence. The occurrence of this syndrome is sporadic, and at present it cannot be diagnosed (either endocrinologically or sonographically) by any specific reaction of the ovaries to stimulation of superovulation (Penarrulia J et al., 1999; Zreik T.O. et al., 2000 )

There are conflicting opinions regarding the etiological factor, developmental mechanisms, diagnosis, and methods of treating this syndrome. Despite the great interest on the part of reproductologists in this “phenomenon,” it remains poorly understood to date.

With a relatively low incidence of SPF, the issues related to the choice of further management tactics for patients, the appropriateness of subsequent IVF programs, and the need to use donor oocytes are practically unresolved. The development of oocytes in a growing pool of follicles is largely determined by the composition of the follicular fluid, where hormones and growth factors are adsorbed from the blood or secreted by granulosa cells (Svetlakov A.V. et al., 2002; Burlev V.A. et al., 1998). In addition, when studying the biochemical composition of follicular fluid, cyclical changes in the parameters of the antrum environment are noted depending on the stages of follicle development (Boyarsky K.Yu., 2002; Burlev V.A. et al., 1999; Chernukha G.E. et al., 1996). Biologically active compounds contained in the antrum of the follicle make up the microenvironment in which the oocyte develops. Both the excess and the lack of these compounds in the follicular fluid can have a pronounced negative effect on the development of the oocyte (Feskov A.M., 1990; Vorobyova O.A. et al., 1998; Potin V.V. et al., 1993) . Despite the significance of the data currently obtained, namely, the possibility of predicting the syndrome of “empty” follicles by the hormonal composition of the blood in a specific treatment cycle, there is no certainty in its re-development in other stimulation cycles.

The lack of a clear management strategy for patients with SPF creates precedents for repeated unsuccessful stimulation of the ovaries, which negatively affects both the health and psycho-emotional state of women and the financial situation of the family. In addition, the lack of timely clear instructions from doctors on the advisability of using donor oocytes postpones the necessary program for an indefinite period and, as a rule, is decided independently by the patient after a series of failed IVF attempts.

In cases where religious, religious and ethnic beliefs contradict the use of donor oocytes, the patient is doomed to “long-term wanderings” in medical centers of various levels. All of the above was the basis for the study of this syndrome, the search and development of differentiated tactics of patient management, which determined the purpose and objectives of our research.

In the available scientific, medical and patent literature, we have not identified methods that allow for the well-reasoned selection of women with SPF syndrome for inclusion in the following cycles of IVF and PE programs. This served as the basis to consider the claimed invention not having a prototype.

The objective of the invention: the development of an objective and affordable, for widespread use, allowing for differentiated selection of patients with the "absolute" and "sporadic" forms of the syndrome of "empty" follicles, which will justify the implementation of the program "Oocyte donation" in this group of patients in order to obtain a desired pregnancy.

The problem is solved in that in a patient with the syndrome of "empty" follicles, a follicular fluid is studied, in which the level of follicle-stimulating hormone, the level of insulin-like growth factor-I, the level of human chorionic gonadotropin are determined and the differential DP is calculated by the formula: DP = exp (PP ) / (1 + exp (PP)), where:

Exp (x) is an exponential function,

PP - an intermediate variable calculated by the formula:

PP = 1.821 + 1 × FSH-0.18 × IPFR-I + 0.136 + hCG, where:

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

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

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

if the obtained DP value is less than 0.5, then in this particular patient the development of the syndrome of “empty” follicles is a consequence of disturbed processes of follicle and oogenesis and is “absolute” in nature, therefore she is recommended donation of oocytes, and with a value of DP more than 0.5 development SPF is random “sporadic” in nature and it is possible to carry out repeated IVF treatment cycles in order to obtain your own oocytes.

An intermediate variable is introduced to simplify the recording of the main diagnostic formula.

In order to conduct differential diagnostics, a comparative analysis of the content of local intra-ovarian regulators of antral fluid is carried out using the above formula obtained using the "logistic regression" method according to the non-Gaussian distribution of the source data. Using mathematical processing allowed us to identify reliably significant factors of intra-ovarian regulation specific for the syndrome of “empty” follicles (FSH-follicle-stimulating hormone, hCG - human chorionic gonadotropin and IPFR-I - insulin-like growth factor). Our studies have proved that there are two forms of the syndrome of "empty" follicles: "sporadic" and "absolute".

Thus, the data we obtained show a clear relationship between the content of FSH, LH, estradiol, progesterone, testosterone in the blood, and FSH, hCG and IPFR-I, in the required amount, in adequate amounts, and full folliculogenesis. The revealed differences are obviously 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-mentioned regulators in the processes of folliculogenesis. The inclusion of these regulators in the formula also has a certain biological meaning. According to the literature (Enien W.M. et al., 1998; Roche J.F., 1996), follicle-stimulating hormone, immunoreactive chorionic gonadotropin, as well as insulin-like growth factor-I, are responsible for the selection of a complete dominant follicle and preparation of the cumulus-oocyte complex for the subsequent. The concentration of these regulators was determined by the immunofluorescence method using specific test systems. A comparative analysis of the results of immunofluorescence studies with their further mathematical processing indicates a more informative method described above for the purpose of selecting patients with the syndrome of "empty" follicles: with its "absolute" and "sporadic" forms and, depending on the data obtained, develop further differentiated tactics for their management in ART programs.

The coincidence of prognostic and diagnostic criteria with a negative result even of a single ovarian puncture (absence of oocytes) is evidence of the “absolute” form of SPF. When the values of the differential indicator calculated by follicular markers are less than 0.5, there is no feasibility of further gonadotropic stimulations and the use of "donor" oocytes is shown. However, the absence of a diagnostically significant confirmation of the “absolute” form of SPF makes it possible to continue treatment cycles of ART programs.

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 reliability of the differential selection of patients with an “absolute” form of SPF is 66.1%.

An analysis of the accuracy of the differential index of DP is given below:

ACCURACY (PI + IO) / (PI + IO + LP + LO) LP / (LP + IO) 66.10% SENSITIVITY SP / (SP + LO) LO / (IP + LO) 68.57% SPECIFICITY OF IO / (IO + PL) PI (PI + LP) 62.50% FALSE POSITION OF DIAGNOSIS IO / (LO + IO) 37.50% FALSE NEGATIVE DIAGNOSIS SHARE 31.43% ACCURACY OF POSITIVE RESULTS 72.73% ACCURACY OF NEGATIVE RESULTS 57.69%

where: PI - 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 performance of the proposed method is confirmed by the following clinical examples.

Example 1

Patient R-va, case history No. 2341/222 was admitted to the Center for Human Reproduction in order to conduct her IVF and PE programs. The patient complains of infertility II for 8 years. From the anamnesis it is known that the patient in 1987 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.

In order to obtain an oocyte cohort, she was prescribed ovulation stimulation. An ultrasound study determined the growth dynamics of the leading follicle or cohort of follicles and the thickness of the endometrium 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 MHz of an Aloka SSD-500 ultrasound apparatus.

During follicular puncture, not a single oocyte was obtained. In antral fluid aspirated from follicles, the levels of FSH, hCG and IPFR-I were determined by the immunofluorescence method using test systems: Delfia (Wallac Oy, Turku, Finland), “hCG-ECO-TEST” (000 “Diatech-EM”, Russia), DSL-10-2 800 (group of companies BioHimMak, Russia), respectively. The resulting concentrations of these intrafollicular regulators: FSH-5.6 mIU / ml; IPFR-I - 105.7 ng / ml; hCG - 38 IU / ml, in order to exclude the randomness of the revealed syndrome of "empty" follicles in the examined patient, the mathematical processing of the obtained data was carried out using the formula for calculating PP and DP:

PP = 1.821 + 1 × 5.6-0.18 × 105.7 + 0.136 × 38 = (- 6.30726).

Differential index DP = exp (-6.300726) / (1 + exp (-6.300726)) = 0.001819708.

According to the differential indicator (0.001819708), which is less than 0.5, this patient has the “absolute” form of the syndrome of “empty” follicles and therefore she was offered the program “Donation of Oocytes” with the aim of obtaining a desired pregnancy.

Example 2

Patient V-va, case history No. 1389/158 was admitted to the Center for Human Reproduction with the aim of conducting an IVF and PE program. 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). In order to obtain an oocyte cohort, she was prescribed ovulation stimulation. An ultrasound 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 clinically confirmed in this stimulation cycle. The levels of biologically active substances in the antrum were determined: FSH - 2.8 mIU / ml; IPFR-I - 90 ng / ml; hCG - 45 IU / ml. The examined patient underwent mathematical processing of the obtained data using the formula for calculating the PP and DP:

PP = 1.821 + 1 × 2.8-0.18 × 90 + 0.136 × 45 = (- 5.60152).

Differential index DP = exp (-5.60152) / (1 + exp (-5.60152)) = 0.003678681.

According to the differential indicator (0.003678681), which is less than 0.5, this patient has the “absolute” form of the syndrome of “empty” follicles and therefore she was offered the program “Donation of Oocytes” with the aim of obtaining a desired pregnancy.

Example 3

Patient S-va, case history No. 769/56 was admitted to the Center for Human Reproduction in order to conduct her IVF and PE programs. The patient complains of infertility II for 5 years. From the anamnesis it is known that the patient in 2000 underwent a left-sided tubectomy for tubal pregnancy on the left, in 2001, plastic surgery of the right uterine tube for tubal pregnancy on the right.

At the examination stage, before the IVF program, no concomitant pathology was detected. In order to obtain an oocyte cohort, she was prescribed ovulation stimulation. An ultrasound study carried out at the time of follicular puncture made it possible to register 5 leading follicles in the left ovary and 2 in the right. The thickness of the endometrium is 11.2 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.

In order to clarify the further management tactics of this patient, the determination of the levels of biologically active substances in the obtained antral fluid was made: FSH - 3 mIU / ml; IPFR-I - 78.2 ng / ml; hCG - 70 IU / ml, the examined patient underwent mathematical processing of the obtained data using the formula for calculating PP and DP:

PP = 1.821 + 1 × 3-0.18 × 78.2 + 0.136 × 70 = (0.20268).

Differential index DP = exp (0.20268) / (1 + exp (0.20268)) = 0.550496268.

The revealed differential index of more than 0.5 testified to the existence of a “sporadic” form of the syndrome of “empty” follicles in this patient, therefore, in the future she was recommended to conduct repeated treatment cycles of IVF in order to obtain her own oocytes.

In the course of this study, 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, in order to carry 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. Of the 76 patients with empty follicles syndrome, 46 women failed to aspirate oocytes in any of the subsequent attempts at gonadotropic stimulation (“absolute” form of SPF), while 30 patients managed to get oocytes in subsequent ART programs (“ sporadic "form of SPF).

Given the fact that the composition of the follicular fluid, to a certain extent, reflects the hormonal composition of the blood (due to gonadotropic hormones that enter the ovary with blood flow and steroid hormones produced in the ovary), the differences revealed by us in the composition of the antrum in the subjects were expected control and main groups.

The results of the study of hormones in the follicular fluid of the examined women of the main group indicate the absence of significant differences in the levels of LH, FSH, progesterone, estradiol, testosterone compared with similar indicators in patients of the comparison group (table 1).

Table 1.
Hormone content in ovarian follicular fluid in 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 1.7
(1.2-2.2) 1,6
(1.0-2.0) 0.631018 Follicle-stimulating hormone (FSH), mIU / l 2,8
(2.2-3.7) 2.9
(2.4-4.4) 0.258362 Prolactin, mIU / L 855.0
(673.3-1124.0) 466.5
(400.0-640.0) 0.000036 Chorionic gonadotropin (hCG), mIU / ml 184.0
(128.6-275.3) 58.9
(40.0-90.5) 0.000001 Progesterone, nmol / L 11370,0
(10655.0-11940.0) 11080,0
(10530.0-11890.0) 0.290423 Estradiol, nmol / L 1654.0
(1385.5-1780.0) 1610.0
(1428.5-1714.5) 0.756587 Testosterone ng / ml 22.9
(22.0-23.6) 23.8
(20.9-25.0) 0.117182

Despite the absence of significant differences, higher concentrations of LH, progesterone and estradiol, against the background of relatively low levels of FSH and testosterone, are recorded in patients with a full follicular apparatus. At the same time, we also performed a comparative analysis of the levels of intrafollicular regulators of follicular and oogenesis (epidermal growth factor, insulin-like growth factor I, inhibin A). The results of the study indicate the unevenness of their distribution in the "empty" and full follicles (table 2).

Table 2.
The content of intrafollicular regulators in the antrum of the ovaries in patients of clinical groups Intrafollicular Regulators Control group n = 82 The main group n = 76 Error Probability (p) The content of intrafollicular regulators (median and interquartile range) IPFR-I 131.0
(117.2-156.4) 91.6
(72.0-104.2) 0.000008 EGF 64.8
(47.1-83.2) 56.1
(43.0-65.2) 0.132372 Inhibin A 76400.0
(51050.0-94875.0) 72,380.0
(26925.0-101550.0) 0.763414

The results of the studies underline the particular importance of determining the content of such biochemical substrates of the antrum environment as hCG and IPFR-I.

Table 3 shows the indicators of intrafollicular regulators in patients with negative results of follicle puncture for all subsequent ART programs (2nd subgroup) and with resultant cycles (when the follicle puncture was repeated in IVF treatment cycles, oocytes were aspirated) (2-subgroup).

Table 3.
Hormone levels in follicular fluid in patients with empty follicles syndrome in a series of ART programs Hormones 2nd subgroup n = 46 2-b
subgroup n = 30 Error Probability (p) Hormone content (median and interquartile range) Luteinizing hormone (LH), mIU / L 1.3
(0.9-1.6) 1.9
(1,5-3,1) 0,025188 Follicle-stimulating hormone (FSH), mIU / l 2.9
(2.4-4.3) 2,8
(2,3-4,2) 0.086265 Prolactin, mIU / L 585.5
(439.3-761.0) 500,0
(400.0-792.0) 0.082359 Chorionic gonadotropin (hCG), mIU / ml 65,4
(54.0-90.1) 55.0
(40.0-80.7) 0.118135 Progesterone, nmol / L 11090.0
(9963.5-11920.0) 10685.0
(6845.5-11587.5) 0.211949 Estradiol, nmol / L 1471.0
(1330.0-1535.3) 1616.5
(1469.5-1654.8) 0.010117 Testosterone ng / ml 25.3
(23.2-25.7) 20.6
(17.7-22.5) 0,024871

As can be seen from the data presented in table 3, the concentration of luteinizing hormone and estradiol in the follicular fluid aspirated in patients with a positive puncture result was significantly higher, and testosterone, on the contrary, was significantly lower than that of the subjects in ineffective cycles. These differences, apparently, reflect the essence of the pathological condition, since it is known that the growth of follicles and the maturation of oocytes in them depend on the complex chain of hormonal and factor regulation of reproductive function at various stages of their translational transformation. The antral environment in which the follicle develops undergoes a cyclic transformation, depending on the phase of the menstrual cycle and the stage of development of the follicle.

Thus, based on the study of intrafollicular regulators of folliculo- and oogenesis, diagnostic criteria for the “absolute” and “sporadic” forms of the syndrome of “empty” follicles are proposed, as well as indications for the program “Donation of oocytes” with the aim of developing differentiated tactics of managing patients in ART cycles . The program "Donation of oocytes" is shown to patients even with a single negative result of puncture of the follicles (absence of oocytes) in IVF programs, with values of the differential indicator calculated by follicular markers less than 0.5.

For practical public health, the importance of determining the “empty” follicles in the follicular fluid — FSH, hCG and IPFR-I — for diagnosing its “absolute” forms in order to reduce material costs for ineffective ART attempts, is substantiated.

Thus, the claimed method is more efficient compared to the known and has several advantages:

- provides high accuracy of the forecast;

- reliable and simple to execute;

- has no complications and contraindications;

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

Claims (1)

A method for selecting patients with empty follicles syndrome for an IVF and PE program using donor oocytes, characterized in that a patient with empty follicles syndrome in follicular fluid determines the level of follicle-stimulating hormone, the level of insulin-like growth factor, the level of human chorionic gonadotropin and calculate the differential indicator of DP by the formula DP = exp (PP) / (1 + exp (PP)), where exp is an exponential function, PP - an intermediate variable calculated by the formula PP = 1.821 + 1 × FSH-0.18 × IPFR-I + 0.136 × hCG, where FSH is the level of follicle-stimulating hormone in the follicular fluid, mIU / ml; IPFR-I — level of insulin-like growth factor in follicular fluid, ng / ml; hCG - the level of human chorionic gonadotropin in the follicular fluid, IU / ml, and if the obtained value of DP is less than 0.5, then the development of the syndrome of “empty” follicles is “absolute” and the patient is recommended oocyte donation, and if the value of DP is more than 0.5, the development of SPF is “sporadic” and the patient is recommended to repeat IVF treatment cycles , in order to obtain their own oocytes.