Prognostic value of determination of matrix metalloproteinase-1 and tissue inhibitor of matrix metalloproteinase-1 in follicular fluid in patients in vitro fertilization programs

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38​ patients with infertility were examined during period 2013 - 2016 y. Depending​ on the infertility pathogenesis, all patients were divided into 2 groups: first group - 20 patients with tuboperitoneal genesis of infertility, second group - 18 patients with tubo endocrine infertility. Studied groups were formed by the method of continuous thematic sampling. Patients with infertility were served as inclusion criteria, which methods were prescribed as ART treatment for reproductive function restoration. The examination of patients from both groups carried out according to standards of care. The examination included complaint analysis, anamnesis collecting, information about past diseases, past surgery, menstrual and reproductive functions, physical examination, gynecological and rectal examinations, instrumental research methods ( transvaginal, transperineal echography, colposcopy ). Patients’ age is from 27 up to 36 years, 31,03 ± 0,37 years on average. 97% of patients live in city, 66% had a higher education, 78% of women had a registered first marriage. 

Every woman's somatic anamnesis was burdened, also patients with endocrine disorders had damage to several organs of the endocrine system, on average, 2 diseases were detected per patient, their presence greatly aggravated the course of infertility. Patients from 2 group were diagnosed with hyperprolactinemia in 32% of cases, in 22% of cases - PCOS (polycystic ovary syndrome) and hirsutism. 18% of patients had structural changes of the thyroid gland (were detected by ultrasound examination), in 8% of cases – thyroid function abnormalities in the form of hypothyroidism, compensated medicamentally. Out of the total number of patients, having obesity (47%), most women (32%) had tubal endocrine genesis of infertility. In 33% of cases vascular dystonia was diagnosed, 23% of women had liver disease and gastrointestinal tract, 15% had kidney disease and urinary system disease.

Menarche age on average is 13,3 ± 0,21 years. 63% of patients had menstrual dysfunction. The duration of infertility on average is 3,24 ± 0,39 years, and 47% of patients had primary infertility, 53% had secondary infertility. 13% of patients’, with secondary infertility, previous pregnancies came to giving birth, others (87%) had complications with pregnancies (ectopic, frozen, spontaneous miscarriage, therapeutic abortion).

All patients in anamnesis had one or few foci of chronic inflammation of the pelvic organs. With almost equal frequency inflammatory diseases of the uterus were registered (21 - 26%), appendages (32-34%) and cervix (16-21%), 16% of patients with tubal-peritoneal genesis of infertility had pelvic peritonitis. Almost every woman had STD - 1,2 - 1,3 diseases per patient in both groups in anamnesis. 5% of patients in 2nd group were diagnosed with uterine fibroids, in 21% of cases - endometriosis, 26% of woman were registered with endometrial hyperplastic processes in different years. Those patients during a long time underwent treatment and rehabilitation programs and hormonal correction for the restoration of reproductive function. 70% of women for therapeutic and diagnostic purposes, there were performed surgical interventions on the pelvic organs, in many cases single patient could have several surgeries. So, patients from the first group mainly had several surgeries on the fallopian tubes - tubectomy for ectopic pregnancies (32%), tubectomy for hydrosalpinx and sactosalpinx (16%), stomatoplasty was performed to 45% of women. Anamnesis showed that patients from 2nd group prevailed surgery on the ovaries and uterus – ovarian resection for cysts (26%), conservative myomectomy (5,3%), 42% of women had intrauterine surgery for endometrial polyps and endometrial hyperplastic processes. As a result of carrying out the ECO and PE programs, 26% of patients got pregnant. Stimulation of folliculogenesis carried out according to an individually selected scheme. With​ «long scheme» usage, 20 patients used daily subcutaneous injections GRHA(drug diferelin, BeafourIspen, France) in the luteal phase of the previous menstrual cycle. With «short protocol», 18 patients had anti-GRHA injections (cetrotide, SeronoAres, Switzerland) on the first day of treatment cycle and daily until start of ovulation induction. Choosing a treatment regimen were selected due to the anamnesis patient had, also results of previous attempts at ovarian stimulation. Standard starting dose of human menopausal gonadotropins (HMG) (drug menopur, Ferring Germany) and RFSA (drug gonal - F, MerkSerono, Switzerland) 150 - 225 ME depending on the age of the patient, case histories. Ovulatory dose of HCG (10000 ME) were created by the choragon, Ferring, Germany. After 36 hours after injecting ovulatory dose of HCG under ultrasound control a transvaginal follicle puncture was performed and aspiration of their contents. The resulting biological material ( LF ) was subjected to freezing and storage at temperature - 20 ºС.

Contents of MMP-1 and TIMP-1 in LF were determined by methods of quantitative enzyme-linked immunosorbent assay of the type "sandwich". Concentration of MMP-1 was set using Matrixmetalloproteinase-1 kit ELISA AbFrontier at measurement range 0,08 – 10 ng/ml. TIMP-1 was determined using kits eBioscience ( BenderMedSystems,USA ) at measurement range 0,06 – 10 ng/ml. Linked immunosorbent assay carried out on an automatic tablet photometer MultiskanFC ( 2002 ) in MEI laboratory and FC ULSU.

 Statistical analysis of the results was performed using the software package: Statistica 6.0 forWindows ( StatSoftInc., USA). Continuous variables are represented as M±m, where M – average value, m – medium error. We calculated the Student​ criterion while​ comparing averages. Differences were considered statistically significant with an error probability p < 0,05. 

 Research results.​ MMP-1​ level in LF is more than 500 times lower than average TIMP-1. Large scale of individual indicators MMP-1 takes place – from 1,02 up to 24,5 ng/ml, which indicates the dependence of concentration of MMP-1 from factors, characterizing the pathogenesis of infertility (table -1). 

Table 1 Average levels MMP-1 and TIMP-1 in LF

Indicator	MMP-1 (n=38)	TIMP-1 (n=38)
Average, ng/mL	4,9 ± 1,0	2462,2 ± 64,3
Range, ng/mL	1,02 – 24,5	1700 – 3000
Median (Me)	1,7	2500

 

Contents of MMP-1 women had in the 1st group are reliably higher, than in the 2nd group. Contents of TIMP-1, on​ the contrary,​ patients had with tubo-endocrine genesis of infertility from 2nd group, have statistically significant predominance (table -2). 

Table – 2.  Levels of MMP-1, TIMP-1 in LF depending on the genesis of infertility

Indicator	1st group – tubal-peritoneal infertility (n = 20)	2nd group – tubal endocrine sterility (n = 18 )	p
MMP-1, ng/ml	7,9 ± 1,7 (1,26 – 24,5)	1,5 ± 0,1 (1,02 – 1,96)	t = 3,76 р=0,000624
TIMP-1, ng/ml	2365,8 ± 94,9 (1700 – 3000)	2696,4±87,8 (2000 – 3000)	t = 2,56 р 0,015046

 

Significant difference of contents of MMP-1 depending on the duration of infertility is not detected. Patients’ ( n=25 ), which had infertility for the first four years, level of MMP-1 amounted to 2,79 ± 0,52 ng/ml, over four years ( n=13 ) amounted to 6,9 ± 2,3 ng/ml ( t=1,74;  p = 0,090120 ). The scale of individual indicators of TIMP-1 turned out to be small, the differences amounted to 1300 ng/ml ( table 1 ). We obtained significant differences of average levels of TIMP-1 women 25 – 30 years old had ( n=18 ) ( 2137,5 ± 139,7 ng/ml ) and older patients 31 - 36 years old ( n = 20 ) ( 2535,0 ± 125,7 ng/ml ) – ( t=2,12 , p = 0,041605 ),​ also​ significant differences depending on the duration of infertility. So, level of TIMP-1 patients had with infertility for the first four years is 2276,8 ± 70,7 ng/ml; level of TIMP-1 patients had with infertility for more than 4 years is 2611,5 ± 126,7 ng/ml; accordingly, ( t=2,31; p = 0,027102

). ​  Effectiveness of ART amounted to 26%, moreover, the positive outcomes were in both groups: with tubo-peritoneal genesis of infertility ( n=6 ) and with tubo-endocrine genesis of infertility ( n = 4 ). From table 3 we can see contents of TIMP-1 women with different outcomes of ART had, did not differ in subgroups and average indicator. Individual values of TIMP-1 are associated with its deep influence on ovarian structure, folliculogenesis processes and depend on a number of pathological factors.  Patients with different genesis of infertility with​ positive ECO and PE programs results, level of MMP-1 in LF amounted to 1,4 ± 0,1 ng/ml, which is 3,5 times lower than average in the group. Level of MMP-1 - 6,1±1,3 ng/ml with negative results, it is 4 times higher, than with positive outcomes of ART. Average age of women, which had positive outcome of ART program, amounted to 30,0 ± 0,96 years, negative outcome – 31,0 ± 0,64 years. Therefore, age didn’t affect the result of ECO and PE. Infertility duration also didn’t significantly affect the outcomes of ART program.

Table 3. Levels of MMP-1 and TIMP-1 in LF patients had with different outcomes of ART

Indicator	Average,  M ± m	Positive outcome of ART (n = 10)	Negative outcome of ART (n = 28)	p
MMP-1, ng/ml	4,9 ± 1,0 (1,02 – 24,5)	1,4 ± 0,1 (1,02 – 1,84)	6,1 ± 1,3 (1,1 – 24,5)		t = 3,60
					p=0,001247
TIMP-1, ng/ml	2462,2 ± 64,3 (1700 – 3000)	2510,0 ± 135,8 (2000 – 3000)	2445,2 ± 78,1 (1700 – 3000)		t = 0,41
					p=0,682402

 

            In​ this way, levels of MMP-1 in LF statistically reliably depends on patients’ age, and severity of residual inflammatory process. Level of MMP-1 might reflects the morphological changes in the reproductive system after inflammatory and endocrine disorders [1 - 4]. We assume, that MMP-1 can be considered as a marker for the level of degradation of extracellular matrix, condition of ovarian tissue architectonics, folliculogenesis process, MMP-1 value might be predictive outcome factor of ART [ 1]. Certainly, examination of MMP-1 preferably must be carried out before ovarian stimulation by puncturing follicles in the previous cycle and estimating the forecast of the planned technology.

Older patients’ with long-term infertility level of TIMP-1 statically turned out to be significantly higher. Most likely, TIMP-1 has an impact on change in ovarian morphostructure, causing persistent irreversible changes in stroma and folliculogenesis suppression. With prolonged infertility these impacts of TIMP-1 get worse, which, apparently, reduces the chances of success by treatment and usage of ART technology. 

            In conclusion, clinical and laboratory findings allowed to obtain convincing data of biological significance of the MMP-1/TIMP-1 system in the development of infertility. Understanding key factors of pathogenesis of proliferative diseases contributes to the formation of the main criteria for creating treatment regimens, exerting a purposeful impact on diseased cells - targets, which are one of the key points in preparing for ART. In clinical practice this is achieved by using therapy, which is aimed at the correlation of the formation of collagen. According to the treatment of chronic inflammatory diseases protocol ( №16 dated November 20, 2015 ) usage of hormonal drugs, containing estrogens is recommended, also prescription of vitamins A, E, D, C, B ( orotic acid ), magnesium preparations, enzymes. As stated, most women when preparing to ART received several courses of similar treatment and were conditionally ready for using ART technology. However the state of the follicular apparatus by contents of MMP-1 and TIMP-1 in LF of stimulated follicles, previously performed anti-inflammatory and immunomodulating therapies didn’t lead to alignment of the adverse effects of metalloproteinases on the development of pathological hardening. These factors explain the low efficiency of ART, examined female groups went through. We assume, that metalloproteinase determination during patients examination in preparation for ART is promising in terms of development of prognostic performance criteria of ART and timing definitions, most favorable for carrying ART. The planned technology should be postponed with unfavorable performance of MMP-1 and TIMP-1, additional training for patients should be planned as well, so they could achieve normal levels of metalloproteinases.

 

 

 

 

About the authors

Olga A. Marinova

Ulyanovsk State University

Email: mari5604@mail.ru
ORCID iD: 0000-0003-3690-8881

Senior Lecturer

Russian Federation, Ulyanovsk

Larisa I. Trubnikova

Ulyanovsk State University

Author for correspondence.
Email: trubnicova-li@yandex.ru
ORCID iD: 0000-0002-0720-0369
SPIN-code: 5343-4958
Scopus Author ID: 454565

D. Sci. (Med.), Prof.

Russian Federation, Ulyanovsk

Marina L. Albutova

Ulyanovsk State University

Email: albutova.m@yandex.ru
ORCID iD: 0000-0001-8510-4930
Scopus Author ID: 22051969

Cand. Sci. (Med.)

Russian Federation, Ulyanovsk

References

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