Successful implantation after reducing matrix metalloproteinase activity in the uterine cavity

Abstract

Background: Recently, the concept of recurrent implantation failure (RIF) in assisted reproductive technology has been enlarged. Chronic uterine inflammation is a known cause of implantation failure and is associated with high matrix metalloproteinase (MMP) activity in uterine cavity flushing. MMP activity of women with RIF has been reported to be higher than that of fertile women. In the present retrospective study we evaluated the efficacy of treatment for high MMP activity in the uterine cavity of patients with RIF.

Methods: Of the 597 patients recruited to the study, 360 patients underwent MMP measurements and 237 patients did not (control group). All patients had failed to become pregnant, despite at least two transfers of good-quality embryos. Gelatinase MMP-2 and MMP-9 activity in uterine flushing fluid was detected by enzymology (MMP test). All samples were classified into two groups (positive or negative) based on the intensity of the bands on the enzyme zymogram, which represents the degree of MMP activity. Patients who tested positive on the initial test were treated for 2 weeks with a quinolone antibiotic and a corticosteroid, and subsequently underwent a second MMP test. Negative results on the second MMP tests after treatment and subsequent rates of pregnancy and miscarriage were used to evaluate the efficacy of treatment. Data were analyzed by the Mann-Whitney U-test and the chi-square test.

Results: Of the patients who underwent the MMP test, 15.6% had positive results (high MMP activity). After treatment, 89.3% of patients had negative results on the second MMP test. These patients had a significantly better pregnancy rate (42.0%) than the control group (26.6%), as well as a lower miscarriage rate (28.5% vs 36.5%, respectively).

Conclusions: A 2-week course of antibiotics and corticosteroids effectively improves the uterine environment underlying RIF by reducing MMP activity.

Figure 1

Flow chart of the study design. In all, 597 patients with RIF were recruited and divided into two groups based on whether or not they agreed to undergo the matrix metalloproteinase (MMP) test. Patients with MMP scores ≥5 were deemed to have a positive response (Group P) and were treated with a corticosteroid and an antibiotic for 2 weeks. Patients with low MMP scores (<5) were determined as negative (Group N). Patients in Group P were given a second MMP test after the treatment and were divided into two subgroups: Group P_r (patients who had again positive results and were considered as resistant to the treatment) and Group P_e (patients in whom the treatment was effective). Patients who did not undergo an MMP test comprised the control group (Group C). Patients in all groups underwent another embryo transfer procedure and the outcomes were compared among the four groups.

Figure 2

A schedule of the MMP tests, the medication and the next embryo transfer. According to the results of the first MMP test, all the patients were divided into two groups, Group N (Panel a) and Group P (Panel b). Group P patients who had shown a positive result had a two-week treatment composed of antibiotics and corticosteroids at the next proliferative phase (panel b). After the treatment, Group P patients had the 2nd MMP test at the mid luteal phase of the same cycle, whereas Group N patients did not have the 2nd test (Panel a). In the subsequent cycle (the next cycle after the completion of the treatment), Group P patients had the next embryo transfers.

Figure 3

A representative image of a gelatin zymogram used for MMP scoring in a MMP test. The lanes represent uterine fluid samples from individual patients with recurrent implantation failure (RIF). Lanes 1, 2, 3, 4 and 6 were scored 4, 2, 4, 8 and 0, respectively. Lane 5 shows a result from the standard used in the present study (supernatant of BHK9 breast carcinoma cell culture). Details of the scoring system are described in the Methods section.

Figure 4

The MMP test results before and after the treatment of patients with RIF. Prior to the treatment, 15.6% of patients showed a positive test result (Group P). These patients were then treated with a corticosteroid and an antibiotic for 2 weeks and subjected to another MMP test. Most patients (89.3%) became negative (Group P_e) after the treatment, whereas 10.7% of patients remained positive (Group P_r).

Figure 5

Clinical pregnancy rate after the next embryo transfer for the four groups of RIF patients. Group P_e, a positive initial MMP test, but a negative second MMP test after the treatment; Group P_r, a positive initial MMP test, and a positive second MMP test despite the treatment; Group N, a negative response to the MMP test; Group C, the control group who did not undergo the MMP test. Patients in Group P_e had a significantly greater pregnancy rate than patients in Groups N and C (*P < 0.05).