Understanding the molecular mechanisms of bisphenol A action in spermatozoa / 대한생식의학회지

Bisphenol A (BPA) is an endocrine-disrupting chemical that is capable of interfering with the normal function of the endocrine system in the body. Exposure to this chemical from BPA-containing materials and the environment is associated with deleterious health effects, including male reproductive abnormalities. A search of the literature demonstrated that BPA, as a toxicant, directly affects the cellular oxidative stress response machinery. Because of its hormone-like properties, it can also bind with specific receptors in target cells. Therefore, the tissue-specific effects of BPA mostly depend on its endocrine-disrupting capabilities and the expression of those particular receptors in target cells. Although studies have shown the possible mechanisms of BPA action in various cell types, a clear consensus has yet to be established. In this review, we summarize the mechanisms of BPA action in spermatozoa by compiling existing information in the literature.

Clinical assessment of the male fertility

The evaluation of infertility in males consists of physical examination and semen analyses. Standardized semen analyses depend on the descriptive analysis of sperm motility, morphology, and concentration, with a threshold level that must be surpassed to be considered a fertile spermatozoon. Nonetheless, these conventional parameters are not satisfactory for clinicians since 25% of infertility cases worldwide remain unexplained. Therefore, newer tests methods have been established to investigate sperm physiology and functions by monitoring characteristics such as motility, capacitation, the acrosome reaction, reactive oxygen species, sperm DNA damage, chromatin structure, zona pellucida binding, and sperm-oocyte fusion. After the introduction of intracytoplasmic sperm injection technique, sperm maturity, morphology, and aneuploidy conditions have gotten more attention for investigating unexplained male infertility. In the present article, recent advancements in research regarding the utilization of male fertility prediction tests and their role and accuracy are reviewed.

Comparision in the yield of fetal nucleated red blood cell between the first-and second-trimester using double density gradient centrifugation / 대한산부인과학회지

OBJECTIVE: The aim of our study was to make a practical comparative evaluation of the first and second trimesters in order to determine the period during which a higher yield of fetal nucleated red blood cells (FNRBCs) can be obtained. METHODS: NRBCs were isolated from maternal blood during the first and second trimesters of pregnancy using double Percoll gradients with different osmolarities. Magnetic activated cell sorting was performed with Kleihauer-Betke stain. We isolated fetal NRBCs from 10 mL of samples of maternal blood and determined fetal sex and fetal aneuploidy by fluorescence in situ hybridization (FISH). RESULTS: The average number of NRBCs was 9.85 in samples obtained during the first trimester and 14.88 in samples obtained during the second trimester (P=0.07). The average number of NRBCs with Y chromosome signals was 5.73 in the first trimester and 8.22 in second trimester (P=0.56). However, the percentage of NRBCs with Y chromosome signals in the first trimester (70.6%) was significantly higher than in the second trimester (59.8%) (P=0.049). We diagnosed the blood samples from 7 pregnant women having fetal aneuploidy using this method and the number of NRBCs was 18.4. CONCLUSION: The method using Percoll osmolarity and a double density gradient system may be a very useful method for separation of NRBCs in the first trimester of pregnancy and also in the second trimester.

Prenatal diagnosis by isolation of fetal nucleated RBCs in maternal peripheral blood / 대한산부인과학회지

OBJECTIVE: To identify prenatal fetal sex and chromosomal aneuploidies by FISH using isolation of fetal nucleated RBCs. METHODS: peripheral blood samples was collected from 37 women between 11 and 24 weeks of gestation. we tried to enrich nucleated RBCs morphologically by Kleihaur-Betke staining after double gradient centrifugation and magnetic activating cell sorting (MACS) from maternal blood. Fluorescence in situ hybridization (FISH) analyses with CEP X and CEP Y probes for K-B positive nucleated RBCs were performed to detect whether fetal cells were existed among nucleated RBCs by observation of sex chromosomes. RESULTS: The average number of K-B positive nucleated RBCs separated from 10ml of maternal blood was 17.3 (+/-17.2) and the maximum number of nucleated RBCs was 54. We observed FISH signals in nucleated RBCs separated from 18 pregnant women, and Y probe signals were observed in 67.3% of nucleated RBCs separated from 10 pregnant women. CONCLUSION: We confirmed that separated nucleated fetal RBCs can be used to identify fetal sex and chromosomal aneuploidies by FISH. Since nucleated RBCs from maternal origin were not excluded, further studies are needed to overcome this limitation.

The Study of 5,10-Methylenetetrahydrofolate Reductase Polymorphism and Its Effect on Pregnancy Outcomes among the Korean Population / 대한주산의학회잡지

OBJECTIVE: The purpose of this study was to analyze MTHFR polymorphism among the Korean population and to evaluate the relationship between serum levels of homocysteine and MTHFR polymorphism and also to investigate the effect on pregnancy outcomes. METHODS: DNA was extracted from whole blood of 600 pregnant women. All samples were genotyped for the C677T and A1298C polymorphisms in MTHFR gene by PCR-RELP assay. Serum levels of homocysteine and folate were measured by high performance liquid chromatography for homocysteine and radioassay for folate. Pregnancy outcomes were estimated by gestational weeks and birth weights of newborns. RESULTS: Serum homocysteine was higher in women with the T/T genotype than those with the C/T or C/C genotype of the MTHFR C677T polymorphism (p<0.05). And also serum homocysteine was higher in women with the A/A genotype than those with the A/C or C/C genotype of the MTHFR A1298C polymorphism (p<0.05). Serum homocysteine was negatively correlated with serum folate in all MTHFR genotypes, especially prominent in T/T genotype of MTHFR C677T polymorphism and A/A genotype of MTHFR A1298C polymorphism. Gestational age and the birth weight of infant from hyperhomocysteinemic mothers whose homocysteine levels higher than 15 micromol/L were 36.1 weeks, 3053.8g, respectively, which were significant lower than those from normohomocysteinemic mothers (38.3 weeks, 3,215.3g) (p<0.05). CONCLUSION: Serum homocysteine was influenced significantly by MTHFR C677T polymorphism and MTHFR A1298C polymorphism. MTHFR C677T and A1298C polymorphism and serum homocysteine levels affect pregnancy outcomes, although not mainly by serum folate level.

The Total Serum Homocysteine and Folate by C677T Methylene-tetrahydrofolate Reductase Mutation in Korean Preeclamptic Pregnant Women / 대한산부인과학회잡지

OBJECTIVE: The purpose of this study was to evaluate whether the C677T Methylene-TetraHydroFolate Reductase (MTHFR) polymorphism affects the total maternal serum homocysteine and folate concentration in preeclamptic pregnant women. METHODS: Patients admitted to the hospital for the delivery during 2000-2002. 126 controls without the pregnancy complications and 34 patients with severe preeclampsia were enrolled. The serum homocysteine analysis was conducted using the high performance liquid chromatography methods. The serum folate and vitamin B12 concentration were determined using a radioimmunoassay kit. The C677T MTHFR gene mutation was examined by the polymerase chain reaction of the genomic DNA fragments. RESULTS: The total maternal serum homocysteine concentration and the serum vitamin B12 concentration were not significantly different between controls and the preeclampsia patients (p=0.44 for homocysteine, p=0.06 for vitamin B12). However, the maternal serum folate concentration was significantly higher in the preeclampsia patients than in controls (27.00 +/- 9.54 nmol/L versus 18.03 +/- 12.97 nmol/L, respectively, p=0.01). The total maternal serum homocysteine concentration, the serum folate concentration, and serum vitamin B12 in the C677T MTHFR CC type and TT type were not significantly different (p=0.21 for homocysteine, p=0.22 for folate, p=0.14 for vitamin B12). CONCLUSION: The C677T MTHFR mutation does not significantly affect the maternal homocysteine and folate concentration in both the controls without pregnancy complication and the preeclampsia patients.

Increased Oxidative Stress Affects the Neonatal Birth Weight in Preeclamptic Patients / 대한산부인과학회잡지

OBJECTIVE: To assess the maternal circulating levels of homocysteine, the thiobarbituric acid reactive substances (TBARS) and the glutathione peroxidase (GPX) activities, and to determine whether or not these markers have any effect on the neonatal birth weight in preeclamptic patients. METHODS: A case control study of 74 normal pregnant women and 47 preeclamptic patients who were hospitalized for delivery and had singleton live births between 2000 and 2002 was conducted. The birth weight, gestational age, and maternal circulating level of homocysteine, the TBARS, the GPX activities, and the antioxidant capacity (GPX/TBARS) were measured. GPX genotyping was done with the maternal DNA samples. The results were analyzed with a Chi-square test, student T-test, and logistic regression analyses. RESULTS: The homocysteine and TBARS levels were significantly higher in the preeclamptic patients than in the pregnant controls (p=0.0098 for homocysteine, p0.05). After controlling for the covariates, homocysteine reduced the gestational age (p=0.0035) and reduced the birth weight by 27 g (p=0.0259). CONCLUSION: The levels of the oxidative stress markers (homocysteine and TBARS) are higher and the antioxidant capacity (GPX/TBARS) is lower in preeclampsia patients. Homocysteine significantly reduces the fetal growth rate.

Influence of Sperm Fertilizing Capacity on Embryonic Development and Pregnancy in in Vitro Fertilization / 대한불임학회지

OBJECTIVES: To assess the fertilizing capacity using sperm penetration assay (SPA) to predict the outcome of the in vitro fertilization-embryo transfer (iVF-ET) outcome. MATERIALS AND METHODS: Semen samples were provided by 129 patients undergoing iVF. We attempted to correlate the extent of sperm penetration under enhanced SPA protocol with the results of fertilization, cleavage, preimplantation embryo development, and pregnancy. RESULTS: Univariate analysis demonstrated a statistically significant correlation between fertilizing capacity and motility, kinetics, fertilization, cleavage and embryo development, and pregnancy rate. By logistic regression analysis, fertilizing capacity was found to be the only variable that was statistically significant with respect to pregnancy rate. Fertilizing capacity, cleavage rate and pregnant rate were significantly higher in pregnant group. However, the fertilization rates was comparable with both group. CONCLUSIONS: Lower fertilizing capacity could denote a poorer prognosis for establishing a pregnancy, even after satisfactory fertilization rate is achieved.

Effects of Reactive Oxygen Species on Sperm Function, Lipid Peroxidation and DNA Fragmentation in Bovine Spermatozoa / 대한불임학회지

OBJECTIVE: To evaluate the effects of the reactive oxygen species (ROS) generated with a xanthine(X) and xanthine oxidase (XO) system on sperm function, the change of sperm characteristics, lipid peroxidation, and DNA fragmentation in bovine spermatozoa. MATERIALS AND METHODS: ROS were produced using a combination of 100 micrometer X and 50 mU/ml XO. The ROS scavengers: superoxide dismutase (SOD)(200mu/ml) and catalase (500mu/ml) were also tested. Spermatozoa were incubated for 2 hours in BWW medium with a combination of X-XO supplemented with or without ROS scavengers at 37degrees C under 5% CO2 incubator. Sperm movement characteristics by CASA (computer-aided sperm analysis), HOST (hypoosmotic swelling test), Ca-ionophore induced acrosome reaction, malondialdehyde formation for the analysis of lipid peroxidation, the percentage of DNA fragmentation using the method of TdT-mediated nick end labelling (TUNEL) by flow cytometry were determined after 2 hours incubation. RESULTS: The action of ROS on bovine spermatozoa resulted in a decreased in capacity for sperm motility, Ca-ionophore induced acrosome reaction and membrane integrity, an increased in malondialdehyde formation and the percentage of sperm with DNA fragmentation. In the effects of antioxidant, catalase completely alleviated the toxic effects induced by the ROS in terms of sperm function and characteristics, however SOD exhibited no capacity to reduce the toxic effects. CONCLUSION: The ROS can induce significant damages to sperm functions and characteristics. The useful ROS scavengers can minimized the defects of sperm function and various damages of spermatozoa.

Comparison of Sperm Morphology Evaluation Using Strict Criteria, Acrosome Reaction Following Ionophore Challenge and Zona-free Hamster Ova Sperm Penetration Assay as Prognostic Factors in Diagnosis of Male Infertility and In Vitro Fertilization / 대한불임학회지

OBJECTIVE: This study was designed to investigate the interrelationship and clinical usefulness of sperm morphology by strict criteria (SM), acrosome reaction following ionophore challenge test (ARIC) and sperm penetration assay (SPA) using zona-free hamster ova as prognostic factors in in vitro fertilization. MATERIALS AND METHODS: Semen samples were provided by 83 patients undergoing IVF. We first evaluated the differences between normal fertilization group and poor fertilization group on three andrologic tests. Secondly, we analyzed the relationship between the three andrologic tests and in vitro fertilization on IVF settings. Finally, we evaluated the effectiveness of the three andrologic tests as the prognostic indicators for fertilizing ability. RESULTS: The fertilization rate of all men in the poor fertilization group was less than 30%; but there was no evidence that this poor fertilization was due to oocyte defects. The results of three andrologic tests were significatly higher in normal fertilization group. Fertilization rate (%) in vitro was highly correlated (p<0.001) with % normal sperm by SM, ARIC value (%), and SPA result. By using Receiver-Operator-Characteristic curve (ROC), we evaluated the effectiveness of these three tests. The sensitivity and specificity of SM, ARIC test and SPA in predicting fertilization potential in IVF setting were 76% and 75%, 84% and 90%, and 76% and 95%, respectively. CONCLUSION: Our data suggest that the three andrologic tests can be reliable tools as prognostic factors of sperm fertilizing ability. Among these test, ARIC test and SPA gave more accurate information on fertilizing capacity. ARIC test was shown to have a predictive value for fertilizing ability comparable to that of SPA that appears to be a simple and cost-effective addition to current andrology laboratory. Combined application of these three tests may give more information on predicting sperm fertilizing capacity.

A study on the association between angiotensinogen gene and angiotensin-converting-enzyme gene and pregnancy-induced hypertension in Korean women / 대한산부인과학회잡지

OBJECTIVES: Previous studies have suggested an association of pregnancy-induced hypertension(PIH) with several genes involved in cardiovascular control. The objectives of this study were to evaluate the association between PIH and angiotensinogen(AGT) M235T gene and also to study the association between PIH and angiotensin-converting- enzyme(ACE). METHODS: DNA was extracted from whole blood, cheek swabs, and blood spot cards of 39 PIH patients and 54 controls. Controls consisted of women who had undergone at least two term pregnancies unaffected by PIH. All samples were genotyped for all the polymorphism using PCR of known alleic variants. Results were ananlyzed with a kappa2 contingency table. RESULTS: Four of 13 women with mild PIH(30.8%) and thirteen of 26 women with severe PIH(50.0%) were heterozygous for AGT M235T mutation compared with 26 of 54 controls(48.1%). Two of 13 women with mild PIH(15.4%) and two of 26 women with severe PIH(7.7%) were homozygous for AGT M235T mutation compared with 10 of 54 controls(18.6%). Six of 7 women with mild PIH(85.7%) and ten of 21 women with severe PIH(47.6%) were ID type for ACE gene compared with 31 of 56 controls(55.4%). One of 7 women with mild PIH(14.3%) and seven of 21 women with severe PIH(33.4%) were DD type for ACE gene compared with 15 of 56 controls(26.7%). There was no significance between mild, severe PIH patients and controls for AGT M235T mutation and ACE gene polymorphism. CONCLUSION: In Korean population, AGT M235T mutation and ACE gene are not associated with an increased risk for PIH.

Preimplantation Genetic Diagnosis of Chromosomal Abnormality with Clinical Application of Multicolor FISH in Embryos Developed after ICSI in Male Factor Infertile Patients / 대한산부인과학회잡지

No abstract available.

Intracyplasmic Sperm Injection in Patients with Past History of Failed or Poor Fertilization in Previous IVF - ET Cycles : Comparison with Patients with Severe Male Factor / 대한산부인과학회잡지

OBJECTIVE: The purpose of this study was to determine whether intracytoplasmic sperm injection(ICSI) could overcome the defects of oocytes in IVF-ET patients with previous fertilization failure by conventional fertilization technique. Design: Retrospective study Materials and METHODS: A total of 119 ICSI cycles in 57 IVF-ET patients performed from May, 1995 to December, 1997 was enrolled. Subjects were divided into two groups: FR group included 66 ICSI cycles in 35 patients with normal sperm who underwent ICSI due to past history of failed or poor fertilization in the previous IVF-ET cycles, and OAT group included 53 ICSI cycles in 22 patients with severe oligoasthenoterato- zoospermia(OAT) which was defined as sperm concentration < 20 million/ml, mo#dlity < 30% and normal morphology < 4% by strict morphologic criteria. The outcomes of ICSI were analyzed and compared in both groups. RESULTS: The age of female patients, basal serum FSH level, and the numbers of oocytes retrieved and metaphase II oocytes were all comparable in both groups. The fertilization rate after ICSI was similar in both groups(68.7+/-25.3% vs. 67.7+/-24.5%), as were the cleavage rate of normally fertilized oocytes(93.1+/-21.4% vs. 89.3+/-21.6%), the number of embryos transferred(4,00+/-1.98 vs. 4.64+/-2.10), and cumulative embryo score(CES) indicating the quality of embryos(47.3+/-33.2 vs. 54.1+/-33.2). The implantation rate(4.3+/-10.5% vs. 3.8+/-11.0%) and the clinical pregnancy rate per cycle(15.2% vs. 13.2%) were also comparable in both groups. CONCLUSIONS: Although it has been shown that there is a higher risk of chromosomal abnormalities in oocytes from IVF-ET patients with pevious failed or poor fertilization, higher implantation and clinical pregnancy rates wer#e not observed in patients with OAT following ICSL Therefore, the functional defect of sperm such as loss of capacitation, defect of aaasome reaction, and abnormality of nucleus decondensation should be also considered in patients with previous failed or poor fertilization.

Analysis of Strict Morphology of Human Spermatozoa / 대한산부인과학회잡지

The proportion of male factor infertility due to quantitative and qualitative sperm disorders is approximately 50-60% in infertile couples. In IVF-ET, lower or failed fertilization of oocytes usually results from subnormal count of total motile sperms, but this may occur in infertile couples even with normal sperm count. It has been suggested that some functional defects in sperms are responsible for lower or failed fertilization. Routine semen analysis based on numerical background has limits for the assessment of fertilization capacity of sperm in infertile males, and the andrologic test for the prediction of fertilization capacity must be objective, repeatable, quick, economic, and easily applicable for the clinical settings. The purposes of this study were to develop the analysis method of strict morphology of sperm using the strict criteria as a simple, inexpensive and useful test of sperm fertilization capacity, to establish the normal fertile range and the cut-off value of strict morphology, and to evaluate the validity of strict morphology as a prognostic indicator of fertilization capacity in IVF-ET. In establishing the effectiveness of strict morphology of sperm, ROC curve was used. Among the various thresholds for the prediction of fertilizing ability, normal morphologic value 10.0 corresponding to the value with higher sensitivity and lesser false positive rates was determined as a cut-off value. Using this cut-off point, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of strict morphology for the prediction of fertilization capacity was 73.9%, 81.0%, 80.6%, and 72.7%, respectively. To evaluate the clinical validity of strict morphology as a prognostic indicator of fertilization capacity, this cut-off point was applied to 133 patients undergoing IVF-ET. For the prediction of fertilization rate >30% in IVF-ET, the sensitivity, specificity, PPV, and NPV was 77.3%, 77.8%, 87.2%, and 63.6%, respectively. In conclusion, the strict morphology of sperm is one of the most simple and useful test for the assessment of fertilization capacity of sperm and the prediction of IVF-ET outcomes in infertile couples.

The Effects of Isotypes and Regional Distribution of Antisperm Antibodies on Semen Parameters and Fertilizing Ability / 대한불임학회지

SUMMARY: To investigate the influences on semen parameters and fertilizing capacity of immuno- globulin(Ig) isotypes and regional distribution of antisperm antibody (ASA) on the human sperm surface. Sixty-seven ASA-positive patients were compared with 96 ASA-negative donors. ASAs in semen showed significant negative effects on both semen parameters and fertilizing capacity; in those with ASAs in the sperm head and/or tail, the reductions were significant. In the head as well as the tail, there was close correlation between fertilizing capacity and both IgG and IgA. Both semen parameters and fertilizing capacity are significantly affected by the presence of ASA in semen. In particular, antibodies IgG to sperm head and/or tail, and antibodies IgA to sperm tail appeared to have a highly detrimental effect on fertilizing capacity.

Chromosome Analysis in Clinical Samples by Chromosome Diagnostic System Using Fluorescence in Situ Hybridization / 대한불임학회지

Fluorescence in situ hybridization (FISH) techniques allow the enumeration of chromosome abnormalities and from a great potential for many clinical applications. In order to produce quantitative and reproducible results, expensive tools such as a cooled CCD camera and a computer software are required. We have developed a Chromosome Image Processing System (Chips) using FISH that allows the detection and mapping of the genetic aberrations. The aim of our study, therefore, is to evaluate the capabilities of our original system using a black-and-white video camera. As a model system, three repetitive DNA probes (D18Zl, DXZI, and DYZ3) were hybridized to variety different clinical samples such as human metaphase spreads and interphase nuclei obtained from uncultured peripheral blood lymphocytes, uncultured amniocytes, and germ cells. The visualization of the FISH signals was performed using our system for image acquisition and pseudocoloring. FISH images were obtained by combining images from each of probes and DAPI counterstain captured separately. Using our original system, the aberrations of single or multiple chromosomes in a single hybridization experiment using chromosomes and interphase nuclei from a variety of cell types, including lymphocytes, amniocytes, sperm, and biopsied blastomeres, were enabled to evaluate. There were no differences in the image quality in accordance with FISH method, fluorochrome types, or different clinical samples. Always bright signals were detected using our system. Our system also yielded constant results. Our Chips would permit a level of performance of FISH analysis on metaphase chromosomes and interphase nuclei with unparalleled capabilities. Thus, it would be useful for clinical purposes.

The Optimization of Human Sperm Decondensation Procedure for Fluorescence in Situ Hybridization / 대한불임학회지

Studies were conducted to determine the efficiency of decondensation protocols. Sperm obtained from seven normal donors was immediately washed after liquefaction and then decondensed using the method of West or al. (1989) and my original protocol. My optimized protocol entailed mixing 1 ml aliquots of semen with 4 ml phosphate buffered saline (PBS) Following centrifugation, pellets were resuspended in 1 ml PBS containing 6 mM EDTA. After centrifugation, pellets were resuspended in 1 ml PBS containing 2 mM dithiothreitol at 37degrees C for 45 min. Following mixing with 2 ml PBS and centrifugation, pellets were resuspended by vortexing. While vortexing, 5 ml of fixative were gently added. Slide preparation was accomplished using the smear method and it was stored at 4degrees C. When comparing these protocols, the degree of sperm decondensation and head swelling was monitored by measuring nuclear length, area, perimeter, and degree of roundness using FISH analysis software. Apparent copy number for chromosome 1 and, separately, for the sex chromosomes was determined by FISH using satellite DNA probes for loci DIZ1, DXZ1 and DYZ3. Sperm treated by my decondensation protocol showed significant increases (p<0.05) in length, area, perimeter, and degree of roundness. There was a significant decrease (p<0.05) in the frequency of nuclei displaying no signal but no change in the frequency of nuclei with two signals in samples decondensed by my protocol.. My data suggested that decondensation using my original protocol may lower the frequency of cells with spurious 'nullisomy' due to hybridization failure without inducing spurious 'disomy' resulting from increased distances between split signals.

Study on the clinical validity of sperm penetration assay / 대한불임학회지

No abstract available.

Correlation of human sperm chromatin heterogeneity with sperm penetration assay / 대한산부인과학회잡지

No abstract available.

Comparison among the sperm preparation methods on the human spermatozoa / 대한불임학회지

No abstract available.