Activity of matrix metalloproteinase 2 and 9 in follicular fluid and seminal plasma and its relation to embryo quality and fertilization rate

Background: the purpose of this study was to evaluate the activity of matrix metalloproteinase 2 [MMP-2] and 9 [MMP-9] in follicular fluid and seminal plasma and the correlation of their activities with parameters that are important in successful intracytoplasmic sperm injection [ICSI]

Methods: seventy-four infertile couples admitted to the Research Center for Endometrium and Endometriosis to carry out ICSI method were enrolled in this study. Follicular fluid was collected after retrieving the oocyte. In addition, semen samples were collected and seminal plasma was used for determination of MMP2 and MMP- 9 activity. Gelatin zymography electrophoresis was applied to measure MMPs activities in follicular fluid and seminal plasma

Results: in follicular fluid, there was a positive correlation between MMP-2 activity with oocyte [r=0.27, p=0.021] or embryo quality [r=0.30, p=0.014], but no correlation was observed between MMP-2 activity and oocyte count or fertilization. Activity of MMP-9 showed positive correlation with oocyte morphology [r=0.29, p= 0.014]. In addition, MMP-2 activity of seminal plasma had positive correlation with sperm count [r=0.28, p=0.015], fertilization [r=0.28, p=0.02], and embryo quality [r=0.28, r=0.026]

Conclusion: MMP2 and MMP9 activities in seminal plasma have a positive effect on sperm count and motility. MMP-2 and MMP-9 activity in follicular fluid and seminal plasma could be important factors in embryo quality in patients undergoing ICSI and may affect the outcome of ICSI

Gene polymorphism of matrix metalloproteinase 9 in asthenozoospermic male subjects

Background: Matrix metalloproteinase [MMPs] play important roles in the structural and functional properties of reproductive organs. The aim of this study is to determine the prevalence of C-1562T MMP-9 [rs3918242] gene polymorphism in fertile and infertile men. In addition, we aim to determine the association between C-1562T MMP-9 and G-1575A MMP-2 gene polymorphisms

Materials and Methods: A total of 400 subjects, including 200 fertile and 200 infertile men, were recruited for this case control study. The allele frequencies and genotype distributions of single nucleotide polymorphism in the promoter regions of MMP-9 [C-1562T] were determined using polymerase chain reaction-restriction fragment length polymorphism [PCR-RFLP] analysis. The chi-square [?2] test was used to assess the distribution of genotype frequencies

Results: There were no significant differences found in the genotype distributions or allele frequencies between fertile and infertile men for the C-1562T MMP-9 gene polymorphism. The percent of immotile sperm in infertile men with the CC and CT genotypes of C-1562T MMP-9 gene polymorphism significantly differed compared with that of subjects with the TT genotype. The frequency of CC/GA-combined genotypes of C-1562T MMP-9 and G-1575A MMP-2 gene polymorphisms significantly differed in fertile and infertile men [P=0.031]

Conclusion: Our results suggest that genetic polymorphisms in MMP may impact male fertility

Association between seminal plasma neopterin and oxidative stress in male infertility: a case-control study

Background: Neopterin is a significant and sensitive marker in estimating the activity of cellular immune system. Oxidative stress plays a role in the etiology of male infertility. Increased reactive oxygen species is accompanied with increase in neopterin level. Hence neopterin may be involved in male infertility

Objective: The objective of this case-control study was to determine neopterin level in idiopathic infertile and normospermic men; furthermore, to identify its relationship with oxidative stress markers including total oxidant, malondialdehyde, sperm DNA fragmentation, and total antioxidant capacity of seminal plasma

Materials and Methods: Forty seven infertile and forty three normospermic males were selected according to WHO criteria. Their semen and blood samples were taken; subsequently, the levels of neopterin, total oxidant, total antioxidant, malondialdehyde, and sperm DNA fragmentation were measured

Results: The levels of neopterin, total oxidant, and malondialdehyde in seminal plasma of infertile males were significantly higher than those of normospermic group [p=0.038, 0.018, and 0.028, respectively]. Furthermore, sperm DNA fragmentation in infertile men was higher than that of control group [p<0.001]. Moreover, total antioxidant capacity of seminal plasma in infertile males was significantly lower than that of normospermic subjects [p=0.002]. No significant difference was observed in serum neopterin, total oxidant, and malondialdehyde between the infertile and normospermic groups

Conclusion: The significant inverse correlation between seminal plasma neopterin and total antioxidant in the infertile males supports a possible role of neopterin in male infertility. Neopterin can be suggested as a marker in monitoring and diagnosis of idiopathic male infertility

effect of 1alpha,25[OH]2D3 on osteogenic differentiation of stem cells from dental pulp of exfoliated deciduous teeth

Statement of the Problem: Stem cells from human exfoliated deciduous teeth [SHEDs] are a population of highly proliferative cells, being capable of differentiating into osteogenic, odontogenic, adipocytes, and neural cells. Vitamin D3 metabolites such as 1 alpha, 25-dihydroxyvitamin D3 are key factors in the regulation of bone metabolism

Purpose: The aim of this study was to investigate the effect of 1 alpha, 25-dihydroxyvitamin D3 on osteogenic differentiation [alkaline phosphatase activity and alizarin red staining] of stem cells of exfoliated deciduous teeth

Materials and Method: Dental pulp was removed from freshly extracted primary teeth and immersed in a digestive solution. Then, the dental pulp cells were immersed in alpha-MEM [minimum essential medium] to which 10% fetal bovine serum was added. After the third passage, the cells were isolated from the culture plate and were used for osteogenic differentiation. As a control group, the cells were cultured in osteogenic cell culture medium. As the case group, the cells were cultured in osteogenic culture medium supplemented with 100 nM 1 alpha, 25 [OH]2D3. The alkaline phosphatase [ALP] activity and alizarin red staining were analyzed to evaluate the osteogenic differentiation at day 21. The results were analyzed by using t-test

Results: Compared with the control group, significant increase was observed in ALP activity of SHEDs after being treated with 1 alpha, 25[OH]2D3 [p= 0.002]. Alizarin red staining demonstrated that the cells exposed to 1 alpha, 25[OH]2D3 induced higher mineralized nodules [p< 0.001]

Conclusion: Osteoblast differentiation in SHEDs was stimulated by 1 alpha,25[OH] 2D3. It can be concluded that 1 alpha,25[OH]2D3 can improve osteoblastic differentiation

Serum levels of lycopene, beta-carotene, and retinol and their correlation with sperm DNA damage in normospermic and infertile men

Background: Oxidative stress in reproductive system leads to sperm DNA damage and sperm membrane lipid peroxidation and may play an important role in the pathogenesis of male infertility, especially in idiopathic cases. Antioxidants such as carotenoids function against free radical damages

Objective: The aim of this study was to determine the levels of lycopene, beta-carotene and retinol in serum and their relationship with sperm DNA damage and lipid peroxidation in infertile and normospermic males

Materials and Methods: Sixty two infertile men and 71 normospermic men participated in this study. Blood and semen samples were collected from all subjects. Sperm DNA damage was measured using TUNEL method. Carotenoids, retinol, and malonedildehyde in serum were also determined

Results: DNA fragmentation was higher in infertile group comparing to control group. Serum levels of lycopene, beta-carotene and, vitamin A in infertile men were significantly lower than normospermic men [p< 0.001, =0.005, and =0.003 respectively]. While serum MDA was not significantly different between two groups, MDA in seminal plasma of infertile men was significantly higher than control group [p< 0.001]

Conclusion: We concluded that lycopene, beta-carotene, and retinol can reduce sperm DNA fragmentation and lipid peroxidation through their antioxidant effect. Therefore the DNA fragmentation assay and determination of antioxidants factors such as lycopene, beta-carotene and retinol, along with sperm analysis can be useful in diagnosis and treatment of men with idiopathic infertility

Comparisons between two antioxidant butylated hydroxytoluene and glutathione supplemented cryopreservation medium on human sperm DNA integrity

Introduction: Cryopreservation of semen is routinely used in a variety of circumstances including before assisted reproduction treatments, pre- radiation or chemotherapy treatment and etc. The aim of this study was to compare the effect of Butylated hydroxytoluene [BHT] and Glutathione supplemented cryopreservation medium on sperm parameters and amount of DNA fragmentation during the freeze-thaw process

Methods: Semen samples were obtained from 60 donors. After the determination of basic parameters, groups of three sample with similar parameters were pooled and processed by Pure Sperm gradient centrifugation. The semen samples were then diluted with normal freezing medium [control] or a medium containing 5mM glutathione [test] and 0.5 mM BHT [test] stored in liquid nitrogen. Frozen cryovials were thawed individually for 20 seconds in a water bath [37 degree C] for evaluation

Results: Significant differences were observed in motility, viability and DNA fragmentation. Motility and viability were significantly higher in treated groups with 0.5 Mm in 5 min BHT than the control group and Glutathione 5mM [P<0.001]

Conclusion: Significant differences were observed in motility, viability and DNA fragmentation. Motility and viability were significantly higher in treated groups with 0.5 Mm in 5 min BHT than the control group and Glutathione 5mM [P<0.001]

Effect of chitosan on osteogenic properties of mesenchymal stem cell of exfoliated deciduous teeth

The exfoliated human deciduous tooth contains multipotent stem cells [Stem Cell from Human Exfoliated Deciduous tooth [SHED]] that identified to be a population of highly proliferative and clonogenic. These cells are capable of differentiating into a variety of cell types including osteoblast/osteocyte, adiopcyte, chondrocyte and neural cell. The aim of this study was to evaluate the differentiation of SHED to osteoblast in standard osteogenic medium and comparing the results with medium which supplemented with glucosamine in form of chitosan. Dental pulp cells were isolated from freshly extracted primary teeth, digested with 4 mg/ml collogenase/dispase, and grown in Dulbecco's modified Eagle's medium with 10 percent fetal bovine serum. The clonogenic potential of cells was performed after 3 weeks of culture. Flowcytometric analysis, performed at day 21 of culture to identify surface markers of mesenchymal stem cells. The cells from 3rd passage were used for osteogenic differentiation in routine osteoinductive medium. Chitosan [10 microg/ml] was added to the culture medium of case group. Alizarin Red Staining and Alkaline Phosphatase [ALP] activity were done to evaluate osteogenic differentiation in the developing adherent layer on the third passage. The results were analyzed using T-test. For the analysis of normal distribution of data, non-parametric Kolmogrov-Smirnov test was used. The colonogenic efficiency was more than 80%. Flowcytometric analysis showed that the expression of mesenchymal stem cell marker CD90, CD 105 and CD146 were positive in SHED, while hematopoietic cell marker CD34, CD45 and endothelial cell marker CD31 were negative. Quantitative analysis of Alizarin Red Staining demonstrated that: mineralized nodule formation was higher in the group supplemented with glucosamine [chitosan]. Results from Alkaline Phosphatase activity test, on day 21, demonstrated a significantly higher ALP activity in the group supplemented chitosan [P<0.001]. Stem cells isolated and cultured from exfoliated deciduous teeth pulp can be differentiated to osteoblast. Addition of chitosan can be beneficial to promote osteogenic differentiation of these cells

Metformin therapy decreases hyperandrogenism and ovarian volume in women with polycystic ovary syndrome

It is well known that there is a close relationship between elevated androgen plasma levels and the ultrasound findings of stromal hypertrophy in polycystic ovary syndrome [PCOS]. The objective of this study was to investigate the effects metformin on the hyperandrogenism and ovarian volume in PCOS. The study is an unrandomized clinical trial with before-after design. Twenty eight patients with infertility [male or female factor] meeting the Rotterdam ESHRE/ASRM criteria for PCOS were studied during the 2008-2009. The anthropometric characteristics of the patients, mean bilateral ovarian volume, and morphology by trans vaginal sonography as well as the plasma levels of leutinizing hormone, follicle stimulating hormone, estradiol, testosterone, 17- alpha -hydroxyprogesterone, and dehydroepianderosterone sulfate were obtained before and after treatment with metformin [500 mg three times a day] for three months. Paired t, Pearson's Correlation Coefficient, or Partial Correlation test was used to analyze the findings. The patients had a mean age of 25.67 years. A significant reduction in mean ovarian volume [11.70 +/- 4.31 ml vs 8.27 +/- 3.71 ml P=0.001], body mass index [BMI, 28.11 +/- 4.55 kg/m[2] vs 26.84 +/- 4.55 kg/m[2] P=0.000] and serum androgen levels was seen after three months of treatment with metformin. There was positive correlations between the ovarian volume and serum testosterone level [r=0.589, P=0.001] or BMI [r=0.663, P=0.000]. Metformin therapy may lead to a reduction in ovarian volume. It is likely that the reduction of ovarian volume reflect a decrease in the mass of androgen producing tissues

effect of retinoic acid on in vitro maturation and fertilization rate of mouse germinal vesicle stage oocytes

Retinoids are recognized as important regulators of cell differention and tissue function, Previous studies, performed both in vivo and in vitro, indicate that retin-oids influence several reproductive events. In this study, we investigated the effect of all-trans retinoic acid [t-RA] on maturation and fertilization rate of immature oocytes [germinal vesicle]. Germinal vesicle [GV] oocytes were recovered from 4-6 week old female mice 48 hours after injection of 10 IU pregnant mare serum gonadotropin [PMSG]. Collected oocytes were divided into seven groups: control, sham and five experimental groups. t-RA at concentrations of 1, 2, 4, 6, 8 jjM were added to oocyte maturation medium in the experimental groups. The maturation rate was recorded after 24 hours of culture in a humidified atmosphere of 5% CO[2] at 37°C. Fertilization and developmental rates of matured oocytes were recorded after in vitro fertilization [IVF] and 24 hour culture. The rate of oocytes that developed to the metaphase II stage of maturation significantly increased with 2 and 4 microM t-RA compared to the control and sham groups [p<0.05]. In addition, the number of fertilized oocytes was significantly higher in 4 microM retinoic acid compared to the control [p<0.05], but the difference between the number of fertilized oocytes which developed to the 2-cell stage was not significant between the two groups. The results show that t-RA enhanced mouse oocyte maturation in vitro and improved fertilization and development rates in a dose dependent manner

The outcome of pregnancy rate after the embryo donation in natural cycle at Hamadan Infertility Center

Today, the donation of embryos, produced in vitro, is a common way to help the infertile cases who cannot produce embryo themselves by any of assisted reproductive procedures. The quality of endometrium and the time of embryo transfer are the most important factors in achieveing high pregnancy rate in these cases. The aim of this study was the assessment of pregnancy rate after embryo donation in natural cycle. This descriptive study was carried out in the year 2005 at Hamadan Infetility Center on 24 couples. A total of 24 couples went through the treatment cycles with embryos donated by 17 couples. Embryo donation was done on days of 14-17 of natural cycle and 2-3 days after the progesterone administration to the recipients. A pregnancy test was performed 2 weeks after ET, and positive results were confirmed by sonographic evaluation of the embryo. Individual, hormone administration results and embryo donation data were collected in a questionnaire, analyzed by descriptive statistics and frequency distribution tables. The mean age of the recipient and donor women were 32.2 and 26.2 years respectively.An average of 2.3 embryos were transferred on each occasion. The clinical pregnancy rate in the recipients was 30% [8.24] per embryo transfer. Transfer of donated embryos in natural cycle, results in a high pregnancy rate and is an easy, acceptable and cost-effective way of treatment for infertility

Nitric oxide level in seminal plasma and its relation with sperm DNA damages

Evidence supports the involvement of nitric oxide [NO] in a Varity of male reproductive processes such as spermatogenesis, spermiogenesis, sperm motion, sperm metabolism and sperm capacitation. However, Low concentration of NO is essential in biology and physiology of spermatozoa, but high amounts of NO is toxic and has negative effects on sperm functions. On the other hand, it is established that high amounts of NO have detrimental effects on DNA. The integrity of sperm DNA is an important factor in successful fertility and embryo development. It is hypothesized that supra physiological concentrations of NO in seminal plasma cause sperm DNA damage. The aim of this study was to determine sperm DNA damage by comet assay and its correlation with NO level in seminal plasma of fertile and infertile men. Semen samples were collected from 45 patients and 70 healthy donors. The stable metabolites of NO [nitrite and nitrate] in seminal plasma were measured by Griess assay and DNA damage was determined using single cell gel electrophoresis [comet] assay method. The NO concentration in the seminal plasma of infertile males was significantly higher than fertile males [5.74 +/- 1.01 microM/L vs. 3.88 +/- 0.53 microM/L]. There was a significant positive correlation between the NO concentration and sperm DNA comet value in infertile males [P<0.01, R = 0.598]. These results indicate that the overproduction of NO in genital tract of infertile males has a potential pathogenetic role in the reduction of sperm DNA integrity

Day 3 serum inhibin-B level is not predictive of ovarian assisted reproductive technologies outcome

The ability of the ovary to respond to exogenous gonadotrophin stimulation and development of several follicles is essential in assisted reproductive technology. Neither age and regularity of menses nor follicular phase FSH and estradiol concentrations are reliable predictors of ovarian response. Day 3 serum inhibin-B level, during induction ovulation, has been proposed as a predictor of ovarian response. To determine day 3 serum inhibin-B as a predictor of ovarian response to induction ovulation in IVF/ ICSI cycles. Seventy one infertile patients under 40 years old were enrolled in this study. All women have both ovaries, basal FSH level under 15 mIU/ml, and no evidence of endocrine disorders. Day 3 FSH, estradiol, inbibin-B concentrations and ovarian volume were measured before treatment. All patients underwent standard long GnRH agonist protocol. The number of oocytes retrieved, fertilization rate, clinical pregnancy rate, days of stimulation and number of HMG ampoules were determined. The patients were divided into two groups, normal responders and poor responders [number of oocytes retrived <4]. The mean inhibin-B level in normal responders was 166.9 +/- 141 pg/ ml versus 115.8 +/- 87 pg/ml in poor responders, which the difference was not statistically significant [p=0.24]. We could not find a cut off between normal and poor responders. The use of day 3 inhibin-B level as a predictive marker of ovarian response in IVF/ICSI cycles is not reliable

effect of nucleus tractus solitarius nitric oxidergic neurons on blood pressure in diabetic rats

It has been shown that nitric oxide is synthesized in the central nervous system as well as in vascular endothelial cells. Recently, it was reported that nitric oxide was involved in central cardiovascular regulation, baroreflex modulation, and involved in a reciprocal release with excitatory amino acids in the nucleus tractus solitarii of rats. The purpose of the present study was to investigate the possible interaction of nitric oxide and glucose in the nucleus tractus solitarii on blood pressure regulation. Male Wistar stereptozotocin induced diabetic rats were anesthetized with urethane. A cannula was inserted above the nucleus tractus solitarii and blood pressure was monitored intra-arterially. Unilateral microinjection of L-glutamate [2.3 nmol/60 nL] into the nucleus produced a decrease in blood pressure in diabetic rats. Microinjection of lidocaine [0.5 Mul%2] increased blood pressure. Unilateral microinjection of sodium nitroprusside [100 mmoV6O nL] into the nucleus increased blood pressure in diabetic rats. After microinjection of sodium nitroprusside, the depressive responses to glutamate were significantly attenuated. These results demonstrated the probable role of glucose on blood pressure regulation in diabetic animals affecting on nitric oxidergic neurons and so it implicates an interaction between nitric oxide and glucose in central cardiovascular regulation

Nitric oxide - induced apoptosis in human granulosa cells

Recent evidence suggests that nitric oxide [NO] acts as an important factor in a variety of physiological and pathological processes, including reproductive function. The purpose of the present study was to investigate whether NO might significantly induce any apoptotic changes in cultured human granulosa cells. The granulosa cells [GC] were obtained from women taking part in an in vitro fertilization [IVF] program. After 48h culture, 1mM DETA/NO was added to the culture medium and then the apoptosis of granulosa cells was evaluated by in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling [TUNEL] immediately and after one hour culture. Nitric oxide significantly increased apoptotic index after one hour in human granulosa cell culture [p<0.024], but it did not significantly affect the controls and the group which apoptotic index was calculated immediately after NO donor addition. These results suggest that, apoptosis of human granulosa cells is mediated by DETA/NO, and this effect is directly proportional to the duration of the exposure

effects of cumulus cells on in vitro maturation of mouse germinal vesicle stage oocytes

In vitro maturation [IVM] of oocytes is a promising technique to reduce the costs and avert the side-effects of gonadotropin stimulation for in vitro fertilization [IVF]. The pregnancy rates from oocytes matured in vitro are much lower than those of in vivo stimulation cycles, indicating that optimization of IVM remains a challenge. In this study, we investigated the effect of cumulus cells on maturation and fertilization rate of immature oocytes [Germinal vesicle]. Germinal vesicle [GV] oocytes were recovered from 6-8 weeks old Balb C female mice 48hr after injection of 10 IU pregnant mare serum gonadotropin [PMSG]. Collected oocytes were divided into two groups. Group A: GV oocytes without cumulus [denuded oocyte]. Group B: GV oocytes with cumulus cells [cumulus-oocyte complex]. The oocytes in both groups were cultured in TCM-199 medium in a humidified atmosphere of 5% CO2 in air at 37?C. The maturation, fertilization and developmental rates were recorded after 24hr. Maturation, fertilization and developmental rates in denuded oocytes [DO] were 65.1%, 68.02%, 78.63% respectively, and in cumulus-oocyte complex [COC] were 78.20%, 85.57% and 85.05%, respectively. The maturation, fertilization and developmental rates of COC were significantly higher than those of DO [p<0.05]. The results show that cumulus cells have beneficial effects on maturation, fertilization and cleavage rates of mice oocytes

[ effect of hyperglycmia on nitric oxidergic neurons in nucleus tractus solitarius and blood pressure regulation in rats with induced diabetes]

The role of nucleus tractus solitarius in cardiovascular system regulation is controversial. On the other hand, study on the problem of hypertension in diabetic animals is the subject of many research programs. The aim of the present study was to determine wheather inactivation of nucleus can affect blood pressure in diabetic rats. To this end, stereptosotocin-induced diabetic rats were anesthetized with Urethane and a cannula was inserted above nucleus. Blood pressure and heart rate were monitored using an intraarterial cannula. The cannulas were filled with L-glutamate [78 pmol/60 nL, to functionally identify the NTS; see below], L-NAME[1nmol, to inhibit the nitric oxidergic neurons] and sodium nitroprusside [100mmol,as a NO-donor]. The results indicated that inactivation of nucleus in diabetic rats, had no effect on systolic and mean arterial pressure but enhanced diastolic blood pressure [P<0.05]. There was no significant difference in heart rate between control and test groups. Glucose affect on increasing blood pressure in rats with induced diabetes, in part, is caused by nitric oxidergic neurons resided in neucleus tractus solitarius

role of L-arginine in control of apoptosis in preimplantation mouse embryos cultured in high glucose media

Maternal hyperglycemia causes delay in early stages of embryonic growth and development, higher incidence of congenital malformations and spontaneous miscarriage compared with those of non-diabetic conditions. High glucosis tratogenicity seems to be related to reduction of Nitric Oxide production [NO] in hyperglycemic condition. In order to test this hypothesis, 2-cell stage embryos of normal mice were cultured with high concentration of glucose [30mM] and different concentrations of L-arginine [5,10,20 mM] or L-NAME, an NO syntase [NOS] inhibitor. In the end of culture, blastocysts were stained by by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling [TUNEL] technique and apoptotic cells were detected by using a Fluorescence microscope. Finally the amount of nitrite in the cultured media was assayed by Griess method. The results indicated that high glucose reduces Nitric Oxide production by preimplantation embryos and increases apoptosis of embryonic cells, but 5-20mM of L-arginine significantly increases Nitric Oxide production and decreases apoptosis. On the contrary L-NAME significantly inhibits the development of pre-implantation embryos. In conclusion, this study indicated that reduced nitric oxide production in high glucosis condition is a main factor for embryonic damage, and supplementation of high glucose media with L-arginine has an important role in prevention of high glucosis embryotoxicity

effect of nitric oxide on mouse Pre-implantation embryo development and apoptosis

Clinical studies have shown that in pathological conditions such as endometriosis and reproductive tract infection [male and female] there is an activation status of macrophages that produce large quantities of nitric oxide [NO] in addition to other effector molecules. Large amounts of NO may have embryotoxic roles and produce infertility. This study was designed to evaluate the effect of different concentrations of NO on mouse pre-implantation embryo development in vitro. Mouse embryos [2-cell stage] were cultured in media containing different concentrations of sodium nitroprusside [SNP], an NO donor, or L-arginine methyl ester [L-NAME], an NO syntase [NOS] inhibitor. At the end of culture, cell apoptosis was studied by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling [TUNEL] technique. The results showed that development of preimplantation embryos were inhibited by high concentration of SNP [1 and 10