Violeta S. Rilcheva, Nina P. Ayvazova, Danail I. Martinov, Cvetomir I. Ivanov and Emiliana I. Konova
The aim of the study was to explore the impact of oxidative stress on frozen seminal plasma in fertile and infertile men by examining the total antioxidant capacity. Patients: Infertile patients from male infertility clinic with various diagnoses and fertile men. Design: Seminal plasma from proven fertile men [n=50] and infertile patients [n=50] were examined for total antioxidant capacity (TAC) level, semen parameters such as morphology, motility and concentration, and DNA integrity test. Interventions: Seminal plasma TAC measurement by luminometric assay using the TAC assay kit, semen analysis parameters, DNA integrity test. Fertile men showed higher TAC values (median and SD): 1201µM (SD±548), as compared with the infertile patients: 831μM (SD±343). The result from sperm morphology of fertile patients showed a mean percentage of 4.8 % (SD±1.68) whereas the percentage in the infertile group was 2.68% (SD ±1.68). The same group of samples, analyzed for DNA damage showed a mean of DFI 10.38% (SD±5.17%) in fertile men and a mean of DFI 17.22% (SD±7.22%) in infertile men. Total antioxidant capacity of the seminal plasma as measured by the luminоmetric assay is a reliable and simple test for diagnosing and management of male infertility.
Nina P. Ayvazova, Lyubomira O. Ilieva, Emiliana I. Konova and Milena A. Atanasova
Recently, the important role of matrix metalloproteinases (MMPs) has been identified in follicular development and subsequent ovulation. Although the role of MMP in ovarian tissue remodeling during folliculogenesis has been well studied, the relationship between matrix protease activity and their inhibitors - Tisue inhibitors of matrix metalloproteinases (TIMP) and aging of the oocytes is still unclear. The present study aimed to establish the probable relationship between the expression levels of MMP-2 and TMP-1 and TIMP-2 in follicular fluid with the degree of oocyte maturity and quality. Follicular fluids from 20 women collected on the day of follicular puncture were tested for the presence of MMP-2, TIMP-1, and TIMP-2 using enzyme-linked immunosorbent assay (ELISA). The oocytes obtained were described in terms of maturity, morphology, and fertilization, as well as the embryo’s quality and rate of development. MMP-2 was significantly higher in follicular aspirates in the first prophase of meiosis - germinal vesicle (GV), compared to aspirates with first metaphase (MI) (p=0.011) and second metaphase (MII) of mature oocytes (p=0.010). The MMP-2/TIMP-1 ratio was significantly higher for GV compared to M1 (p=0.011), M2 (p=0.006) and atretic oocytes (p=0.032); (F(3, 71)=2.909, p=0.040). Based on our results, we can conclude that MMP-2 concentration in follicular fluids during the IVF / ICSI procedure had a significant relationship to oocyte maturation levels. It was significantly higher in the case of immature oocytes. On the other hand, oocytes with normal morphology were associated with a significantly higher MMP-2 concentration in follicular fluids.
Nina P. Ayvazova, Violeta S. Rilcheva, Emiliana I. Konova, Roumen G. Roussev and Pavel I. Rashev
Matrix metalloproteinases (MMPs) areagroup of proteases containing Zn ions asacofactor, which are involved in degrading ofalarge number of extracellular matrix proteins, and bioactive molecules. They also playamajor role in processes such as cell proliferation, cell migration, differentiation and apoptosis. Very little is known about the expression and function of MMPs in the male reproductive tract. Occurrence of MMP-2 and MMP-9 activity in human seminal plasma has been previously reported but their origin and function are still not fully understood. The aim of this study was to examine the presence of MMP-2 and MMP-9 in normal and abnormal human sperm samples and find if any correlation existed between the levels of expression of MMPs and fertilization potential of the spermatozoa. Human spermsamples were examined for the presence of MMP-2 and MMP-9 by gel zymography and western blot analysis. A DNAfragmentation test was performed. The samples were divided into two groups - samples with normozoospermia and teratozoospermia. The gelatin zymography showed gelatinolytic bands with molecular weight 64 and 72 k Da corresponding to active and inactive form of MMP-2. MMP-9 was not detected. The MMP-2 enzymatic activity appeared to be much higher in samples with compromised sperm morphology as compared to the normozoospermic samples. The mean DNAfragmentation index (DFI) of the group with teratozoospermia was relatively higher (22.16%) and over the upper reference limits, compared to the normozoospermic group, in which it was within the normal range (17.26%).
Violeta S. Rilcheva, Nina P. Ayvazova, Lyubomira O. Ilieva, Svetlana P. Ivanova and Emiliana I. Konova
The objective of the study was to investigate the influence of sperm DNA fragmentation index (DFI) by DNA integrity test on pregnancy outcome and pregnancy loss after assisted reproductive technology (ART) procedure: autologous intracytoplasmic sperm injection (ICSI), donation eggs ICSI, and intrauterine insemination (IUI). We investigated men from 531 couples undergoing autologous ICSI procedure (n=416), from couples undergoing donation eggs procedure (n=39) and IUI (n=76). We performed the following interventions: semen analysis, DNA integrity test, embryo scoring by Gardner and Schoolcraft grading system (1999). The study showed no statistically significant differences between the group regarding pregnancy rate (χ2=0.55; p>0.05; OR=1.25, 95% Cl 1.23-1.46; p>0.05). However, with increased levels of DFI, the number of pregnancy losses became higher (including biochemical pregnancies and spontaneous abortions) at OR=5.65 (95% Cl 4.32-7.11; p=0.05). We examined the percentage of grade I blastocysts (by Gardner and Schoolcraft, 1999) before donation eggs embryo transfer and found a statistically significant correlation with both the DFI (χ2=7.80; p<0.05) and sperm morphology (χ2=6.14; p<0.05). Analysis of the relationship between DFI and IUI output (clinical pregnancy, miscarriage) revealed significant correlations in both directions: between DFI and pregnancy rate after IUI (χ2=6.29; p<0.05) and between the DFI and pregnancy development after IUI (χ2=6.87; p<0.05). The three group categories (autologous, heterologous ICSI procedures and IUI) studied showed that sperm samples with DFI>27% were associated with increased riskofearlypregnancyloss. Men with infertility should undergo DNA fragmentation assay in addition to the standard semen analysis. When DFI exceeds 27%, ICSI should be a method of choice, even in cases where the conventional parameters of semen analysis tests are normal.