Diaa M. El-Mowafi - Zagagig University, Egypt

Follicular Fluid MMP-2 and MMP-9 in Stimulated IVF Patients

Diaa M. El-Mowafi*, Umnia A. El-Hendy, Melvina Araman, Paul Bischof
* To whom correspondence should be addressed: 4 Ghazza St., El-Hossania, El-Mansoura 35111, Egypt

Abstract

Objectives: To detect the level of both MMP-2 and MMP-9 and their gelatinolytic activity in the follicular fluid of stimulated IVF patients and to correlate all the other hormonal and clinical criteria in those patients to each other.
Design: Prospective randomized clinical study.
Setting: Assisted Reproduction Unit, Benha University Hospitals, Egypt.
Patients: Twenty patients complaining of infertility for more than one year and indicated for IVF-ET or ICSI procedures.
Intervention: Patients were stimulated according to the long protocol. Follicular fluid samples were collected during oocyte retrieval.
Main outcome measures: The different clinical, ultrasonographic and hormonal parameters of the patients were correlated together and to the MMP-2 and MMP-9 detected by zymography and ELISA, as well as their gelatinolytic activity.
Results: Our results showed that MMP-9 in the follicular fluids from patients treated with Metrodin was significantly higher (p< 0.05) than in patients treated with Metrodin HP. There was a positive correlation between the number of gonadotrophin ampoules given to the patient and MMP gelatinolytic activity. The activity of MMP-9 is significantly correlated (p< 0.05) with the log of serum progesterone level at time of embryo transfer.
Serum E2 at pick-up was strongly correlated (p< 0.0001) with serum E2 at time of hCG injection.
Conclusion: Level of MMP-9 which may be one of the triggers of ovulation increases in the follicular fluid of stimulated IVF patients particularly with Metrodin than Metrodin HP and with the increase number of given gonadotrophin ampoules. This increase is still positively correlated with the level of serum progesterone at time of embryo transfer. None of the investigated parameters was related to the pregnancy outcome in our studied group.
Key words: MMP-2, MMP-9, Follicular fluid, IVF-ET.

Introduction

The follicular fluid is that fluid filling the antrum of the Graafian follicle. In the mature Graafian follicle, the antrum occupies most of the space within the follicle. During the development of the follicle, fluid is secreted by granulosa cells and accumulates between cells to form fluid-filled spaces. The follicular fluid within the antrum contains proteins, steroids, peptide growth factors, mucopolysaccharides, and electrolytes. In addition to serving as a reservoir for metabolites and medium for the exchange of hormones and growth factors, follicular fluid may act to increase the pressure within the follicular wall and aid in the extrusion of the oocyte at ovulation (1).
Unfertilized oocytes secrete low, stable amounts of metalloproteinase-9 (MMP-9) activity in vitro and follicular granulosa cells secrete high amounts of MMP-9 in culture (2). It was also found that follicular granulosa cells secrete high amounts of MMP-9 activity in follicular fluid, and it can be assumed that the appearance of this enzyme in the follicular fluid is probably connected to follicular rupture (3).
Gelatin zymography of follicular fluid revealed bands of gelatinase of 72/67 kilodaltons, consistent with latent and active MMP-2. The area digested by both latent and active MMP-2 increased with approaching time of ovulation and corpus luteum formation in the ewe (Russell et al., 1995). The role of MMP-2 and MMP-9 in implantation has been proved (4).
Our study aimed at detecting the MMP-2 and MMP-9 levels and MMPs gelatinolytic activity in the follicular fluid of stimulated IVF cycles. Also, to correlate the different parameters of the stimulated cycles to each other as well as to the MMPs levels.

Materials and Methods

Patients and Samples

Twenty patients complaining of infertility for more than one year attending the Assisted Reproduction Clinic, Benha University Hospitals were included in this study. The mean age of the patients was 34.5 years (range, 28-42). Patients were stimulated according to the long protocol. Down regulation of the pituitary was achieved by daily IM injection of either Decapeptyl 3.75 mg or Lucrin 0.1 mg starting from day 21 of the menstrual cycle preceding the treatment cycle. Pituitary down regulation was confirmed when there were no ovarian follicles > 10 mm in diameter on scanning by vaginal ultrasound and serum oestradiol was below 60 pg/ml. Follicular growth was stimulated by daily injection of 75-225 IU of human menopausal gonadotrophin (hMG) (Pergonal, Sorono, Switzerland or Humegon, Organon, Switzerland). Follicular growth was stimulated in some randomly selected patients by follicle stimulating hormone (Metrodin or Metrodin-HP). The daily injected ampoules ranged from 1-3 according to the patient's response as detected by serum oestradiol and follicular growth. Monitoring by serum oestradiol level was started after 5 days of hMG or Metrodin injection and repeated every 2-3 days, and gonadotropin dosage was titrated accordingly. Human chorionic gonadotrophin (hCG) 5000-10.000 IU ( Profasi, Sorono, Switzerland) was injected when the leading follicle was ³ 18 mm in diameter and there were at least three follicles >15 mm in diameter. Follicular fluid samples were collected during oocyte retrieval under vaginal ultrasound guidance 38 hours after hCG administration. The follicular fluid samples that were taken after flushing with a heparinized medium were discard. The sterile tubes containing the follicular fluid were centrifuged at 1500 rpm to separate the granulosa cells. The samples were divided into aliquots and stored at -70oC till time of assay.

Gelatin Zymograghy

Gelatinolytic zymography was performed as described by Fischer et al., (1989)(5). Briefly, 10% polyacrylamide gel in 0.1% sodium dodecyl sulphate containing 1 mg/ml gelatin (Merk, Darmstadt, Germany) were cast at a final dimension of 80X85X0.8 mm. After polymerization of the gel, 2% polyacrylamide staking gel was cast above the previous one. The culture supernatants were diluted 6:1 with sample buffer 7X (17.4% sodium dodecyl sulphate, 7% sucrose and phenol red in tris HCl ph 6.8). Twenty ml of samples was added to the wells under non-reducing and non-denaturing conditions. A lane of molecular weight standards (Pharmacia, Uppsala) was added. The electorphoresis was run at 200 V, 50 mA for 45 min at 4oC in the cold room. After electrophoresis, the gel was put through six (5 minutes) washes in 2.5% tritonX100 (2.5% in water) and 3 times (10 min) in phosphate-buffered saline solution. The gel was then placed in phosphate-buffered saline solution, pH 7.4 containing 0.9 mmol calcium chloride and magnesium chloride and incubated overnight at room temperature on a moving platform. The next morning, gel was stained with Coomassie Brilliant Blue G250 (0.1% in 25% methanol and 10% acetic acid in water) and destained in the same solution in absence of the dye.
Zymograms were scanned in an "apple one scanner" and the surface of the digestion bands measured by NIH image 1.60 program on Power Macintosh 7/00/66 computer. All zymograms were evaluated using the same pre-set standards.

MMP-2 ELISA

Microplates (96 wells) were coated overnight at 4oC with 100 of sheep anti-human MMP 2 (30 mg/ml in Na Carbonate buffer, 50 mM pH 9.6). Unbounded sites were blocked for 2 hours at room temperature with 250 ml of 10% Blotto in PBS containing 0.02% NaN3. Plates were then washed twice with PBS containing 0.1% Tween 20 (PBST, 250 ml/ well) and once with PBST+ 10% Blotto (PBSTB).
Samples and standards were diluted in PBS containing 10% Blotto applied in duplicates (100ml/ well) and incubated overnight at room temperature. After incubation, the plates were washed and incubated with biotinylated anti-MMP 2 (100ml/well) for 2 hours at room temperature on a rotating platform. Plates were then washed three times with PBST and once with PBSTB and re-incubated for 30 min at 20oC with avidin-peroxidase (1/4000 in PBSTB, 100 ml/ well).
 After washing (4 times) with PBST, the plates were incubated in the dark for 10 min with OPD and H2O2 30% (10 mg and 10 ml/25 ml respectively in citrate-phosphate buffer 0.05 M, pH 5.0, 200 ml/well). The reaction was stopped by the addition of sulphuric acid (3M, 50 ml/well) and the absorbance measured at 492 nm in an ELISA plate reader (Labsystem Multiscan; Bioconcept, Allschwill, Switzerland).

MMP-9 ELISA

Washing and incubation procedures are essentially the same as for the MMP-2 ELISA. Our rabbit anti-human MMP-9 IgG preparation was used for coating the plates (48 mg/ml). The second antibody was a commercially available sheep anti-MMP-9, it was diluted 1/2000 in PBSTB. Peroxidase-labelled rabbit anti-sheep antibodies (100 ml/well) were incubated for 1 h at room temperature. Detection was the same as for MMP-2 ELISA.
The concentration of MMP-2 and MMP-9 were calculated by comparison to the respective standard curves expressed as log OD versus the log concentration of MMPs. These calculations were performed on a Power Macintosh 7100/66 computer using a regression analysis from state view program.

Gelatinolytic activity assay

Quantitative estimation of gelatinolytic activity in the follicular fluids was performed by measuring the degradation of heat-denatured radiolaballed collagen type IV, using a method modified from Emonard et al., (1990)(6). Briefly, tritiated human type IV collagen (N-[2,3-3H] propionate) sp. Act. 0.0067 GBq/mg; du Pont de Nemours, Geneva, Switzerland) was neutralized at pH 7 with tris-Hcl buffer (50 mM) containing 0.15 mm NaCl, 4 mm CaCl2 and incubated in a water bath at 60oC for 30 min to form gelatin. After cooling, N-ethylmaleimide (NEM, 0.5 mM; Sigma) and phenylmethyl-sulphonyl fluoride (PMSF, 0.1 M; Sigma) were added to inhibit thiol and serine proteases respectively. This solution contained 10 000 cpm of radiolabeled gelatin per 100 ml. Collagenase (EC 3.4.24.3) from clostridium histolyticum (330 U/mg; Sigma) was used as standard. The standard curve ranged from 0.8 to 100 ng/ml.
Alliquots of 100 ml of sample or standards were pipetted in duplicates into assay tubes. In order to activate the gelatinase to be measured, all tubes received 15 ml of 2.4 mM 4-amino- phenyl mercuric acetate (APMA; Sigma) in tris buffer (0.05 Mtris, 0.05% triton X-100, 5 mM CaCl2, 0.02% Na N2, pH 7.0) and the tubes were incubated at 25oC for 15 min. Substrate (100ml, 10 000 cpm) was added and the tubes incubated at 37oC overnight. After incubation, the tubes were put on ice and undigested gelatin was precipitated by addition of 100 ml of 36% trichloroacetic acid (TCA; Sigma) and incubated for 1 h at 4oC. The incubation mixture was finally centrifuged at 6000 g for 25 min at 4oC and an aliquot of the supernatant (100 ml) was added to 3 ml of Luma Gel (Lumac, LSc. B. V. Groningen, Netherlands) and the samples counted in a liquid scintillation counter (Packard) at an efficiency of 35%.
ANOVA and T test were used for statistical analysis of the results and P < 0.05 was considered as significant.

Results

The following parameters were correlated to each other: patient's age, type and dose of gonadotophin releasing hormone analogues (GnRH), type and number of given ampoules of gonadotrophins, dose of hCG, level of oestradiol (E2) at time of hCG injection, level of E2 and progesterone (P) at time of oocyte pick up, E2, P, and endometrial thickness at time of embryo transfer (ET), number and mean size of ovarian follicles, MMP-2 and MMP-9 by zymography, MMP-2 and MMP-9 by ELISA, and their gelatinolytic activity.
Different parameters of the patients' hormonal profile during stimulated cycles are shown in table I. The ultrasonograghic criteria of the stimulated patients are shown in table II.
It was found that age of the patient (28-42 years, mean 34.5 ±3.5) does not affect any of the parameters: MMP-2 (zymography), MMP-9 (zymography), MMP-2 (ELISA), MMP-9 (ELISA), and MMPs gelatinolytic activity.
Table III shows the results of MMPs and their gelatynolytic activity. MMP-9 in the follicular fluids from patients treated with Metrodin (4 patients) was significantly higher (p=0.033) than it in patients treated with Metrodin HP(10 patients) (table IV). None of the other parameters was changed.
There was a positive correlation between the number of gonadotrophin ampoules given to the patient and MMP gelatinolytic activity. No significant difference was observed when the number of the ampoules was correlated to the other different measured parameters.
The activity of MMP-9 is significantly correlated (p< 0.05) with the log of serum progesterone level at time of embryo transfer. None of the other parameters were correlated with log of progesterone at time of ET.
None of the parameters was dependent on log of progesterone at time of oocytes pick-up.
The number of gonadotrophin ampoules that were given to the patient was not statistically different among the different gonadotrophin regimen.
None of the parameters was correlated with the log of E2 at time of oocytes pick-up, but E2 at pick-up was strongly correlated (p< 0.0001) with serum E2 at time of hCG injection. Apart from that, none of the parameters was correlated with the log of E2 at time of hCG injection.
None of the parameters was significantly correlated with the ratio of E2 at oocyte pick-up/number of ovarian follicles.
None of the parameters was significantly correlated with either the number of follicles, size of the follicles or the total follicular mass (diameter X number of follicles). There was a positive correlation between MMP-2 and 9 shown in Fig. 1.
The only patient who got pregnant in our study had no significant difference in her parameters including MMPs.
The results obtained by the two different methods of detection (zymography and ELIZA) for both MMP-2 and 9 were matched together. The correlation coefficients were -0.185 and -0.528 respectively.

Discussion

In most organs, remodeling of tissues after morphogenesis is minimal; however, normal ovarian function depends upon cyclic remodeling of extracellular matrix (ECM). ECM has a profound effect on cellular functions and probably plays an important role in the processes of follicular development and atresia, ovulation and development, maintenance and regression of corpora lutea (12).
The significance of proteolytic enzymes in the follicle maturation has been widely studied in non-stimulated animal models and humans (7,8,9,3). ECM surrounding ovarian follicles consists of the basement membrane under the germinal epithelium, the tunica albuginea, the theca externa, the theca interna, and the basement membrane adjacent to the granulosa cells. During follicular growth in normal spontaneously ovulatory cycles, this layer of connective tissue becomes thinner, but it remains intact until ovulation (3). Various proteinases have been reported to be involved in the destruction of the follicle apex (10). MMP-2 (11) and MMP-9 (3) are two of these proteinases that had been investigated in the follicular fluid of spontaneously ovulated cycles. In such cycles, the follicular fluid escapes into the peritoneal cavity particularly in the Douglas pouch. By this way, the follicular fluid can pass into the fallopian tubes, where natural fertilization and early embryo development take place.
Our study showed no correlation between patient's age and Follicular fluid MMP-2, MMP-9, or MMPs gelatinolytic activity. So the difference of implantation rate according to the age is probably depending on other factors rather than the level of MMP-2, 9 or its gelatinolytic activity. The significantly higher level of MMP-9 with the Metrodin compared to highly purified Metrodin may reflect abundant secretion of MMP-9 by the granulosa cells. Our results also showed that the more the number of given gonadotrophin ampoules the more the MMPs gelatinolytic activity. These last two observations may lead to easily ruptured follicles in spontaneously ovulated cycles stimulated by Metrodin than Metrodin HP and with more number of gonadotrophin ampoules.
Puistola et al., (1986) (3) stimulated ovulation with clomiphene citrate and hMG. Their results of MMP-9 in the follicular fluid were similar to ours. They reported that during normal ovulation, the two basement membrane layers of the ovarian follicle are degraded locally. The main component of basement membrane is type IV collagen, which is specifically cleaved by type IV collagenase (MMP-9). They showed that MMP collagenolytic activity is present within the follicular fluid and increases towards ovulation, decreasing rapidly as the follicle rupture. Shalev et al., (2001) (13) reported higher levels of MMP-9 and MMP-2 in the follicular fluid from women with polycystic ovarian disease (PCOD) stimulated for IVF than the normal ovulatory women stimulated for IVF. A time-dependent increase in the production of MMP-9 was observed in cells from both normal and PCOS women, although the increase was more pronounced in the later.
The association between the MMPs gelatinolytic activity and serum progesterone level at time of transfer is logic as both are factors that enhance embryo implantation. Development and luteolysis of the corpus luteum are accompanied by extensive remodeling of the ECM. Differentiation and regression of luteal cells are associated with construction and degradation of ECM results in luteal cell death (12). HCG was found to suppress MMP-2 and MMP-9 production by human luteinized granulosa cells. This in turn leads to rescue of the corpus luteum and hence continuous progesterone production to maintain early pregnancy (14).
We can conclude that the level of MMP-9 which may be one of the triggers of ovulation increases in the follicular fluid of stimulated IVF patients particularly with Metrodin than Metrodin HP and with the increase number of given gonadotrophin ampoules. This increase is still positively correlated with the level of serum progesterone at time of embryo transfer. None of the investigated parameters was related to the pregnancy outcome in our studied group.

References

1. Oliver RH, Chen GD, Yeh J. Graafian Follicle. In: Encyclopedia of Reproduction, Knobil, E. and Neill, J.D. (eds.), Academic Press, San Diego, CA, USA, 1998; vol. 2 p 567.
2. Puistola U, Ponnberg L, Martikainen H, Turpeenniemi-Hujanen T. The human embryo produces basment membrane collagen (type IV collagen)-degrading protease activity. Hum Reprod 1989; 4(3):304-11.
3. Puistola U, Salo T, Martikainen H, Ponnberg L. Type IV collagenolytic activity in human preovulatory follicular fluid. Fertil Steril 1986; 45: 578-80.
4. Shimonoviz S, Huriwitz A, Dushnik M. Developmental regulation of the expression of 72 and 92 kD type IV collagenase in the human trophoblasts: a possible mechanism for control of trophoblast invasion. Am J Obstet Gynecol 1994; 171: 832-8.
5. Fisher SJ, Lizhang TC, Hartmann L. Adhesion and degradative properties of human placental cytotrophoblast. J Cell Biol 1989; 109:891-902.
6. Emonard H, Christiane Y, Smet M, Grimaund JA, Foidart JM. Type IV and interstitial collagenolytic activity in normal and malignant trophoblast cells are specifically regulated by extracellular matrix. Invasion Metast 1990; 10: 170-7.
7. Morales TI, Woessner JF Jr, Howell DS, Marsh JM, Le Maire WJ. A microassay for the direct demonstration of collagenolytic activity in Graafian follicles of the rat. Biochem Biophys Acta 1978; 524: 428-31.
8. Fukumoto M, Yajima Y, Okamura H, Midorikawa O. Collagenolytic enzyme activity in human ovary: an ovulatory enzyme system. Fertil Steril 1981; 36:746-9.
9. Reich R, Miskin R, Tsafriri A, Follicular plasminogen activator: involvement in ovulation. Endocrinology 1985; 116: 516-20.
10. Woessner J F, Uterus, cervix and ovary. In Collagen in Health and Disease, Edited by J.B. Weiss, M.I.V. Jayson. Edinburgh, Churchill Livingstone, 1982; p 506.
11. Russell DL, Salamonsen LA, Findlay JK. Immunization against the N-terminal peptide of the inhibin alpha 43-subunite (alpha N) disrupts tissue remodeling and the increase in matrix metalloproteinase-2 during ovulation. Endocrinology 1995; 136 (8): 3657-64.
12. Smith MF, McIntush EW, Ricke WA, Kojima FN, Smith GW. Regulation of ovarian extracellular matrix remodelling by metalloproteinases and their tissue inhibitors: effects on follicular development, ovulation and luteal function. J Reprod Fertil Suppl 1999;54:367-81.
13. Shalev E, Goldman S, Ben-Shlomo I. The balance between MMP-9 and MMP-2 and their tissue inhibitor (TIM‎P)-1 in luteinized granulosa cells: comparison between women with PCOS and normal ovulatory women. Mol Hum Reprod 2001; 7(4): 325-31.
14. Stamouli A, O'Sullivan MG, Frankel S, Thomas EJ, Richardson MC. Suppression of matrix metalloproteinase production by hCG in cultures of human luteinized granulosa cells as a model for gonadotrophin-induced luteal rescue. J Reprod Fertil 1996; 107(2): 235-9.

Table I. Hormonal Profile of the patients during stimulated cycles

	No.of  gonadotro-phins Amp.	E2 at hCG injection (pg)	E2 at pick up (pg)	P at pick up (ng)	E2 at ET (pg)	P at ET (ng)
Range	20-44	726-5959	495-3935	2.3-23.5	450-4066	29.6-210
Mean	30	2391.5	1347.5	12.7	1316.9	109
SD	7.1	1639.2	949.2	6.1	948.9	56.6
SEM	1.7	397.6	230.2	1.5	263.18	15.7

E2 = estradiol
P= Progesterone
ET= Embryo transfer

Table II. Ultrasonographic criteria of the patients

	Endometrial Thickness at ET (mm)	No. of Follicles	Size of Follicles (mm)
Range	6.5-18.1	2-23	12.1-25
Mean	11.7	13	15.6
SD	2.8	5.2	3.1
SEM	0.7	1.3	0.7

Table III. Results of MMPs

Table IV. MMP-9 with Metrodin and Metrodin HP

	MMP-9 with Metrodin		MMP-9 with Metrodin HP
	Zymography	ELISA	Zymography	ELISA
Range	3532.5-7741.0	87.8-216.5	1703.1-4999.3	46.1-117.4
Mean	5163.3	185.6	3091.3	75.5
SD	1806.2	56.9	964.0	28.1
SEM	903.1	28.5	304.9	8.3