Upregulation of the RNF8 gene can predict the presence of sperm in azoospermic individuals / 대한생식의학회지

Objective@#In this study, specimens from testicular biopsies of men with nonobstructive azoospermia (NOA) were used to investigate whether RNF8 gene could serve as a biomarker to predict the presence of sperm in these patients. @*Methods@#Testicular biopsy specimens from 47 patients were classified according to the presence of sperm (positive vs. negative groups) and investigated for the expression of RNF8. The level of RNF8 gene expression in the testes was compared between these groups using reverse-transcription polymerase chain reaction. @*Results@#The expression level of RNF8 was significantly higher in testicular samples from the positive group than in those from the negative group. Moreover, the area under the curve of RNF8 expression for the entire study population was 0.84, showing the discriminatory power of RNF8 expression in differentiating between the positive and negative groups of men with NOA. A receiver operating characteristic curve analysis showed that RNF8 expression had a sensitivity of 81% and a specificity of 84%, with a cutoff level of 1.76. @*Conclusion@#This study points out a significant association between the expression of RNF8 and the presence of sperm in NOA patients, which suggests that quantified RNF8 expression in testicular biopsy samples may be a valuable biomarker for predicting the presence of spermatozoa in biopsy samples.

Variable localization of Toll-like receptors in human fallopian tube epithelial cells / 대한생식의학회지

OBJECTIVE: To determine the localization, expression, and function of Toll-like receptors (TLRs) in fallopian tube epithelial cells. METHODS: The localization of TLRs in fallopian tube epithelial cells was investigated by immunostaining. Surprisingly, the intensity of staining was not equal in the secretory and ciliated cells. After primary cell culture of fallopian tube epithelial cells, ring cloning was used to isolate colonies of ciliated epithelial cells, distinct from non-ciliated epithelial cells. The expression of TLRs 1–10 was examined by quantitative real-time polymerase chain reaction, and protein localization was confirmed by immunostaining. The function of the TLRs was determined by interleukin (IL)-6 and IL-8 production in response to TLR2, TLR3, TLR5, TLR7, and TLR9 ligands. RESULTS: Fallopian tube epithelial cells expressed TLRs 1–10 in a cell-type-specific manner. Exposing fallopian tube epithelial cells to TLR2, TLR3, TLR5, TLR7, and TLR9 agonists induced the secretion of proinflammatory cytokines such as IL-6 and IL-8. CONCLUSION: Our findings suggest that TLR expression in the fallopian tubes is cell-type-specific. According to our results, ciliated cells may play more effective role than non-ciliated cells in the innate immune defense of the fallopian tubes, and in interactions with gametes and embryos.

effect of vitamin e and selenium on sperm chromatin quality in couples with recurrent miscarriage

Background and Aims: Due to the paucity of studies, association between the morphology and function of sperm and recurrent miscarriage [RM] is not yet completely known. Increased reactive oxygen species and decreased antioxidant levels in men have been shown to be associated with RM. Recently it has been accepted that antioxidant therapy can approve sperm parameters. The goal of this study was to evaluate the effect of paternal factor and antioxidant therapy on sperm parameters in the couples with RM

Materials and Methods: Sixty ejaculate samples with RM patients were analyzed before and after 3 months of vitamin E and selenium therapy. Sperm chromatin assay was assessed by cytochemical tests including aniline blue, chromomycin A3, and toluidine blue. To measure DNA fragmentation index, terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL] test was used. Data were analyzed by SPSS software

Results: Patients had significantly higher percentage of sperm parameters [p<0.001] compared to the time before treatment. TUNEL positive spermatozoa were decreased in post treatment compared to pre-treatment phase [p<0.0001]

Conclusions: Our study demonstrates that antioxidants can improve sperm parameters and chromatin condensation in recurrent miscarriage male partner

Detection of 4977-bp deletion of mitochondrial DNA in in vitro fertilization failure women: a case-control study

Background: The quality of oocyte is often considered as a limiting factor for fertility, especially IVF. Some mitochondrial mutations, particularly the 4977-bp deletion increase with the age. Thus, this mutation can serve as a marker for cell aging, which indicates the reduced quality of the oocytes for fertilization. It has been suggested that this can also be investigated in the blood cells of women with IVF failure

Objective: 1-Determination of the frequency of 4977-bp deletion in women with IVF failure, 2-Investigation of the relationship between 4977-bp deletion and the age of patients

Materials and Methods: Polymerase chain reaction was used to detect the 4977-bp deletion in blood samples of 52 IVF failure women and 52 women who had at least one healthy child. After polymerase chain reaction with deleted and wild-type primers, the products were examined using agarose gel electrophoresis

Results: 48.07% of women with IVF failure and 34.62% of healthy women had a mitochondrial 4977-bp deletion, with p=0.163 and OR: 1.749. Also, in association with the age of these patients and the frequency of 4977-bp mutation, p and OR were obtained 0.163 and 1.749, respectively and frequency of this mutation was higher in patients over 35 yr old compared to other subgroups [Patients >/=35: 57.69]

Conclusion: According to the findings of this study, there is no a significant relationship between the frequency of mitochondrial 4977-bp mutation and failure in IVF

Frequency of CYP1A1 gene polymorphisms in infertile men with non-obstructive azoospermia

Background and Aims: The cytochrome P450 1A1 [CYP1A1] plays a curial role in phase I metabolism of polycyclic aromatic hydrocarbons to their ultimate biologically active intermediates that have potential reproductive toxicity in men. Reproductive functions in men may be impaired by many environmental, physiologic, and genetic factors. The majority of the environmental factors are xenobiotics. Metabolic active xenobiotics exert adverse effects via covalent interactions between intermediate metabolites and cellular macromolecules such as DNA or protein

Materials and Methods: Genotyping two polymorphisms, CYP1A1*2A and CYP1A1*2C, was done using polymerase chain reaction–restriction fragment length polymorphism [PCR-RFLP] assay in a case–control study including 105 infertile men and104 healthy fertile subjects

Results: The results showed that frequency of CYP1A1*2A was significantly different between the patients and the controls [p = 0.036]. Analysis indicated that CYP1A1*2A CC genotype was significantly associated with an increased risk of male infertility [OR = 5.4, CI=1.12-26.04; 95%] compared with the AA genotype. No significant association was detected between CYP1A1*2C polymorphism and male infertility

Conclusions: The CYP1A1*2A single nucleotide polymorphism can be considered as an effective agent in azoospermia

Association of -460C/T and +405 G/C polymorphisms of vascular endothelial growth factor gene and susceptibility to ovarian hyperstimulation syndrome

Background: Ovarian hyperstimulation syndrome [OHSS] is one of the most important complications of assisted reproduction treatment. Many substances are involved in the regulation of the vascular permeability, which have been concerned to cause OHSS. Vascular endothelial growth factor [VEGF] has emerged as one of the main angiogenic factors, which could be responsible for increased vascular permeability

Objective: In this study the association of vascular endothelial growth factor -460C/T and +405 G/C polymorphisms and susceptibility to ovarian hyperstimulation syndrome was evaluated

Materials and Methods: In this cross sectional study, VEGF gene polymorphisms were amplified by Polymerase chain reaction- Restriction Fragment Length Polymorphism in 75 women with established OHSS [case group] and 85 normoresponder [control group] which received conventional ovarian stimulation regimen

Results: There was no significant difference in the frequency of -460 C/T polymorphism between cases and controls [p=0.85]. The frequency of +405 G/C polymorphism was significantly higher in the OHSS women [p=0.03, OR=2.44; 95% CI=1.23-4.82]

Conclusion: In women who developed OHSS, VEGF gene polymorphism +405 could be effective. Two of the polymorphisms -460 C/T and +405 G/C were reported to be associated with increased VEGF basal promoter activity. However, only +405 G/C gene polymorphisms were more frequent in cases than controls

Frequency of TNFR1 36 A/G gene polymorphism in azoospermic infertile men: a case-control study

Background: tumor necrosis factor-alpha [TNF-alpha] is a multifunctional cytokine that regulates different cellular activities related to spermatogenesis. Tumor necrosis factor-alpha receptor 1 [TNFR1] mediates TNF-alpha activity and polymorphism in TNFR1 could lead to gene dysfunction and male infertility

Objective: the aim of this study is to determine the association of TNFR1 36 A/G polymorphism with the idiopathic azoospermia in Iranian population

Materials and Methods: this case-control study included 108 azoospermic and 119 fertile men. This research investigated the frequency of TNFR1 36 A/G polymorphism in cases who were idiopathic azoospermic men referred to Yazd Research and Clinical Center for Infertility, Iran in comparison with controls. polymerase chain reaction- restriction fragment length polymorphism [PCR-RFLP] method was used to investigate the polymorphism in both case and control groups. PCR fragments were digested by Mspa1I enzyme and products were appeared by gel electrophoresis. The abundance of A?G was calculated in the azoospermic and healthy men

Results: according to the present study, GG and AG genotypes frequency in the azoospermic men group were higher than the control group [OR= 2.298 [1.248-4.229], p=0.007], [OR=1.47 [0.869-2.498, p=0.149]. Our findings also showed that G allele frequency in azoospermic men had significant difference compared to the control group [OR=2.302 [1.580-3.355], p<0.001]

Conclusion: it seems that the GG genotype and G allele have an association with increased risk of non-obstructive azoospermia

Association between HLA-E gene polymorphism and unexplained recurrent spontaneous abortion [RSA] in Iranian women

Background: Human leukocyte antigen-E [HLA-E]is a non-classical major histocompatibility complex [MHC] class I antigens which expressed on extra villous cytotrophoblast, which interacts with NKG2A, is an inhibitory receptor on natural killer [NK] cells and leading to down regulation of immune response in the maternal-fetal interface and provides maternal immune tolerance of the fetus

Objective: This study was designated to investigate the gene frequencies of E0101 and E0103 in HLA-E gene in Iranian women with recurrent spontaneous abortion [RSA]

Materials and Methods: Amplification Refractory Mutation System [ARMS-PCR] technique was carried out to detect polymorphism in exon 3 of the HLA-E gene in women with RSA and controls [n=200]. Differences between groups were analyzed by SPSS19 software using X[2] test

Results: There was no significant difference in the allele frequencies of the HLA-E polymorphism between RSA and fertile controls but HLA-E 0101/0103 heterozygous genotype was found to be significantly higher in RSA group [p=0.006, OR=1.73], so this genotype might confer susceptibility to RSA

Conclusion: Our results suggest that HLA-E 0101/0103 heterozygous genotype leads to increase of RSA risk. It seems that by genotyping of HLA-E polymorphism, we can predict the risk of RSA in infertile women

association between single nucleotide polymorphism in interleukin-27 gene and recurrent pregnancy loss in Iranian women

Recurrent pregnancy loss [RPL] has been defined as two or more miscarriages before 20[th] week of gestation. It seems that IL-27 may reduce inflammatory responses and affect the survival of the embryo during human pregnancy. IL-27 polymorphisms may influence RPL by altering the levels or the activity of gene product. We studied for the first time the association of IL-27 -964 A>G single nucleotide polymorphism [SNP] with RPL in Iranian women. A case-controlled study was performed on two groups consisting of 150 healthy women with at least one delivery [control group] and 150 women with two or more primary RPLs history [RPL group]. The -964 A>G SNP in IL-27 gene was determined by PCR-RFLP technique. Genotype and allele frequencies were compared using Chi[2] tests between two groups. There was no difference between the two groups regarding age of women [29 +/- 4.4 [control] vs. 30.84 +/- 5.2 years [case]]. In the RPL group, the genotype frequencies of -964 A>G polymorphism were AG [49.3%], AA [40%], and GG [10.7%], and in the control group, they were AG [43.3%], AA [48.7%], and GG [8%]. There was no significant difference between the genotypes of AA, AG, and GG in two groups [p=0.23]. As the frequency of allele A was 64.7% in the RPL group and 70.3% in the control group, the difference in frequency of allele A in -964 A>G between two groups was not significant [p=0.19]. Our findings indicate that SNP of -964 A>G in IL-27 gene is not a risk factor for RPL in Iranian women

The imbalaace in expression of angigenic and anti- angiogenic factors as candidate predictive biomarker in preeclampsia

Preeclampsia is an importait pregnancy disorder with serious maternal and fetal complications uliich its etiology has not been conlpletely understood yet. Early diagxosis and management of disease could reduce its potential side effects. The vascular endothelial growth Factor [VEGF] family including VEGF-A is the most potent endothelial growth factor which induces angiogenesis and endotheha1 cell proliferation and has basic role in vasculogenesis. VEGF and its tyrosine kinase receptors [Fltl and 1U]R] are major factors for fetal and placental aigiogenic development. Finding mechanisms involved in expression of angiogenic factors may lead to new prognostic and therapeutic points in management of preeclampsia. Recent researches, has shown capability of some antiangiogenic factors as potential candidate to be used as early predictors for preeclampsia. Soluble fins-like tyrosin lcinase-1 [sFltl] is a truncated splice variant of the membrane-bound VEGF receptor Fltl, that is produced by the placenta and it can bind to angiogenic gsowth factors and neutraliz, their effects. It is also observed that the ratio of sFItl to placental growth factor is valuable as prognostic marker. In this review, VEGF family member's role in angiogenesis is evaluated as biomarkers to be used for prediction of preeclampsia.

Effectiveness of aspirin compare with heparin plus aspirin in recurrent pregnancy loss treatment: a quasi experimental study

Using aspirin, heparin, or both in women with unexplained recurrent miscarriage could be useful, because this problem might be initiated by thrombosis in decidual vessels. To investigate the association between thrombophilia and unexplained recurrent miscarriage and to evaluate the efficacy of anticoagulant treatment. In this quasi experimental, we enrolled 520 women, who had a history of recurrent miscarriage. Two hundred fifty two women with unexplained recurrent miscarriage were assigned to receive aspirin [80 mg daily] for two month before pregnancy and after confirmation of a viable pregnancy until 36 weeks of gestation or receive aspirin, as the same, plus heparin [5000 unit twice a day] subcutaneously after confirmation of viable pregnancy until 4 weeks after delivery. Type of medication was chosen for each woman according to number of abortion and age. Live-birth rates did not different significantly among the two study groups. The proportions of women who gave birth to a live normal infant were 74.5% in the group receiving aspirin plus heparin [combination-therapy group] and 79.8% in the aspirin group. Live-birth rates did not different significantly among the two study groups. So, using aspirin or aspirin plus heparin did not change pregnancy rate in these patients. Using aspirin is easier than injecting heparin which should be chosen case by case

Expression of Toll-like receptors 7-10 in human fallopian tubes

The human female reproductive tract [FRT] is constantly deal with the invading pathogens. Recognition of these pathogens is attributed to the family of Toll like receptors [TLR] as a major part of the innate immune system. We and others have previously revealed that TLRs1-6 express in the female reproductive tract. However, more studies should be done to detect TLRs 7-10 in the female reproductive tract, especially in the fallopian tubes. To examine the expression of TLRs7-10 in human fallopian tube tissue. Using immunostaining techniques, distribution of TLR7-10 was studied in surgical sections from the uterine tubes, obtained from patients undergoing tubal ligation and hysterectomy for benign gynecological conditions. RT-PCR was used to show the existence of TLR7-10 genes in fallopian tube tissue. TLR7-10 proteins were detected in the fallopian tube epithelium, although the intensity of staining was not equal in cases. TLR7-10 genes were expressed in human fallopian tube tissue. This study indicates that TLR7-10 is expressed in fallopian tubes tissues, and may play an important role in microbial recognition, and in host defense against ascending infection

cellular uptake of antisense oligonucleotid of E6 mRNA into cervical cancer cells by DOPE-modified hydroxyapatite nanoparticles

Although several chemical and physical methods for gene delivery have been introduced, their cytotoxicity, non-specific immune responses and the lack of biodegradability remain the main issues. In this study, hydroxyapatite nanoparticles [NPs] and 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine [DOPE]-modified hydroxyapatite NPs was coated with antisense oligonucleotide of E6 mRNA, and their uptakes into the cervical cancer cell line were evaluated. Calcium nitrate and diammonium phosphate were used for the synthesis of the hydroxyapatite nanoparticle. Thus, they were coated with polyethylene glycol [PEG], DOPE and antisense oligonucleotide of E6 mRNA using a cross-linker. Then, hydroxyapatite NPs and DOPE-modified hydroxyapatite NPs were incubated 48 hours with cervical cancer cells and their uptakes were evaluated by fluorescent microscopy. The hydroxyapatite NPs had different shapes and some agglomeration with average size of 100 nm. The results showed DOPE-modified hydroxyapatite NPs had higher uptake than hydroxyapatite NPs [P<0.05]. Hydroxyapatite NPs conjugated with DOPE are a good choice for gene delivery and silencing of viral genes in cervical cancer cells, but their efficacy should be addressed more in future studies

Analysis of MLH3 C2531T polymorphism in Iranian women with unexplained infertility

Background: meiotic genes are very important candidates for genes contributing to female and male infertility. Mammalian MutL homologues have dual roles in DNA mismatch repair [MMR] after replication errors and meiotic reciprocal recombination. The MutL homologs, MLH1 and MLH3, are crucial for meiotic reciprocal recombination and human fertility. In this study the functional polymorphisms of MLH3C2531T was investigated in Iranian women with unexplained infertility

Objective: investigating the association between a common SNP [single nucleotide polymorphism] C2531T in the MLH3 gene and female infertility

Materials and Methods: in total, 105 women with unexplained infertility as case group and 100 women with at least one child and no history of infertility or abortion as controls were recruited for this association study. The MLH3 C2531T polymorphism was tested by tetra-amplification refractory mutation system-PCR [4P-ARMS-PCR] method

Results: the MLH3 2531C and T alleles frequencies were 43.33% and 56.67% among infertile patients, and 61.5% and 38.5% among normal controls, respectively. In the patient and control subjects the CC [Pro 844 Pro] genotype frequency ofMLH3 C2531T was 4.76% and 25%, the CT [Pro 844 Leu] genotype was 77.15% and 73%, and the TT [Leu 844 Leu] genotype was 19% and 2%, respectively [p=0.0001]

Conclusion: the presence of the polymorphic allele T leads to an increased risk of 2.09 times [OR=2.09, 95% CI=1.38-3.16; p=0.0001] for developing infertility in relation to the control group. Therefore, our data suggest that the MLH3 C2531T polymorphism can be associated with the risk of unexplained infertility in Iranian women

Case control study of the factor V Leiden and factor II G20210A mutation frequency in women with recurrent pregnancy loss

Background: recurrent pregnancy loss [RPL] caused by various genetic and non-genetic factors. After chromosome abnormality, thrombophilia is one of the most important genetic factors that could cause RPL. Factor V Leiden and factor II G20210A mutation were the most common mutations cause thrombophilia in the world

Objective: the purpose of this study was to determine the frequency of factor V Leiden and prothrombin gene mutations in women with RPL compared with women who had uneventful pregnancies

Materials and Methods: this case control study evaluates the frequency of factor V-Leiden and factor II G20210 genotypes in 80 women with two or more pregnancy losses, compared with 80 women without adverse pregnancy outcome. The mutations were assessed by PCR-RFLP

Results: frequency of the factor V Leiden among cases was 2.5%, which was higher than controls [1.25%], but the difference was not significant. No factor II G20210 mutation was found among cases and controls

Conclusion: these data did not confirm that factor V Leiden and factor II G20210 mutation might play a role in recurrent pregnancy loss in Iranian women

Mental health status of infertile couples based on treatment outcome

Infertility is accompanied by numerous psychological and social problems. Infertile couples are more anxious and emotionally distressed than other fertile people. Previous studies suggested that infertility is more stressful for women than men. The purpose of this study was to determine the status of general health of infertile couples. This cross-sectional study evaluated general health of 150 infertile couples attending to Yazd Research and Clinical Center for Infertility that were selected consequently. The data were gathered by the researchers, based on face to face interview before and after three months of treatment by two questionnaires. The first questionnaire had questions on demographic information and the second one was the General Health Questionnaire-28 [GHQ-28]. This questionnaire has four sub- scales areas. All data were transferred directly to SPSS 15 and analyzed. The mean age of women was 28.3 and men were 32.4 years. The scores for all sub- scales of GHQ in women were more than men. There was significant difference between age and general health at physical symptoms scales [p=0.002], anxiety and sleep disorders [p=0.003]. The age group 25-29 years had higher scores [more than 7] than other age groups. There was significant difference between the scale of social dysfunction and results of treatment. Our results, similar to the previous studies have revealed negative social and mental effects of infertility on women is more than men, so there is need that they be educated specially

Treatment of recurrent pregnancy loss by Levothyroxine in women with high Anti-TPO antibody

Recurrent pregnancy loss [RPL] is defined as two or more consecutive pregnancy losses before twenty weeks of gestation. It is caused by a variety of genetics and non-genetics factors. Thyroid autoimmunity could associate with pregnancy loss. To investigate the effectiveness of Levothyroxine in treatment of RPL in women with high auto-thyroid antibodies. In this observational cross sectional study, 900 women who had a history of recurrent pregnancy loss were studied. All women with high anti-TPO antibody without any other problems entered in this study. Levothyroxine was given to them two months before pregnancy till the end of pregnancy. The doses of levothyroxine were depended on the anti-TPO levels, which were decided by endocrinologist. Women followed for the results of pregnancies. The success rate of pregnancy in women with abnormal anti-TPO with Levothyroxine therapy was 82.85%. Mean of anti-TPO in women with treatment before taking medication was 488.35 and after that it was 123.35 UI/ml. This difference was significant [p<0.05]. The mean of the antibodies was not significantly different in women without treatment. This study showed that Levothyroxine reduces the incidence of spontaneous abortions in women with high Anti-TPO antibody. It decreased anti-TPO antibody levels after 2-3 months treatment

Evaluation of the general health of the infertile couples

The prevalence of infertility is variable between 5-30% around the world. In Iran, more than 2 million couples suffer from infertility. Infertility causes depression, anxiety, social isolation and sexual dysfunction. This descriptive study was undertaken to determine general health in infertile couples. One hundred and fifty infertile couples attending Yazd Research and Clinical Center for Infertility were randomly selected during March till September 2009. The GHQ-28 questionnaires were completed by researchers, based on face to face interview. It contains 28 items, which have been divided to four sub- items. The results come out by scoring the patients answer from 0 to 84. All data were analyzed by Chi-square and t-test in SPSS software. GHQ scores of all sub-scales and total in women were more than men, which shows general health condition in women is worse than men. There was no relation between the duration of infertility and general health scores. This study suggests that the infertility has significant effect [p=0.001] on health situation of infertile couples, especially infertile women. They are at risk of somatic symptoms [p=0.0001], social dysfunction [p=0.0001] and severe depression [p=0.0001]. GHQ could provided help and support in order to improve the health situation of infertile couples

Frequency of polycystic ovary syndrome in patients with premenopausal breast cancer

Polycystic ovary syndrome [PCOS] is a heterogeneous, complex genetic disorder characterized by hyperandrogenemia, hyperinsulinemia, insulin resistance, and chronic anovulation. It is the most common endocrine disorder in women of reproductive age with an enigmatic pathophysiologic and molecular basis. Obesity, hyperandrogenism, and infertility occur frequently in PCOS, which mostly have a genetic predisposition, and are features known to be associated with the development of breast cancer risk. In present study, frequency of PCOS in patients with premenopausal breast cancer was compared with the frequency in women without breast cancer. This is a case-control study, which compared PCOS frequency in 166 patients with premenopausal breast cancer and 166 healthy controls with normal mammography in last 6 months. Eleven patients [6.62%] in case group and 16 patients [9.63%] in control group had polycystic ovary syndrome according to their questionnaire. The difference was not significant [p=0.645]. There was no relationship between frequency of polycystic ovary syndrome and breast cancer in this study. This might be due to the age of patients with breast cancer in this study, which was mostly over 40. It could be significant if the patients were chosen in lower age for showing more effect of genetic than environment. The adjustment or matching of other risk factors could help to find the better results