jalali 2004-11-1 gregorian 2005 5 1 3 1 online 1 fulltext

Mutation detection in human estrogen receptor ? gene in infertile male patients by denaturing high-performance liquid chromatography - Background: For screening sequence variations in genes, rapid turnover time is of fundamental importance. While, many of the current methods are unfortunately time consuming and technically difficult to implement. Denaturing high-performance liquid chromatography (DHPLC) method had been shown to be a high-throughput, time saving, and economical tool for mutation screening. Objective: In the present study DHPLC method was used to explore the potential association between estrogen receptor ? gene (ESR2) variants and male infertility. Materials and Methods: DNA from 96 men with infertility and 96 normal male as control were screened for mutation in the nine exons of the ESR2 gene, using WAVE® DHPLC device equipped with a DNA separation column and automated sequence analysis on the ABI Prism 310. Results: DHPLC evaluation of ESR2 gene in 96 infertile patients, revealed one heterozygous sequence variation (IVS 8–4G>A) near the 5’ splicing region of intron 8 in 5 patients. No variation was identified in control population. Conclusion: Mutation detection by DHPLC, as it is presented in this context, is a high-throughput, quick, and economical tool for mutation screening. The gene alterations in ESR2 gene that we’ve found might increase susceptibility to infertility; but without cDNA screening, the consequences of these genetic alterations cannot be predicted - DHPLC, Male infertility, ESR2 gene, Mutation 9 13 Mir Davood Omrani davood_omrani@umsu.ac.ir No Mir Davood Omrani davood_omrani@umsu.ac.ir No Agneta Nordenskhold - No

A comparative study of GnRH antagonist and GnRH agonist in PCO patients undergoing IVF/ICSI cycles - Background: Polycystic ovarian syndrome (PCOS) patients are prone to premature LH surge and ovarian hyperstimulation syndrome (OHSS). Long GnRH analogue protocol and GnRH antagonist protocol are two methods utilized for induction ovulation in patients undergoing IVF/ICSI. Objective: The aim of this study was to compare the effects of GnRH agonists and antagonists in PCOS patients. Materials and Methods: A total of 60 PCOS patients under 35 years old were enrolled in this study. The patients have no history of thyroid disorder and hyperprolactinemia. All patients received OCP (LD) before starting the treatment. Then patients randomly divided into two groups. The agonist group underwent standard long GnRH analogue protocol. In antagonist group, HMG (150 IU/day) was started from third day of cycle. Then GnRH antagonist (0.25mg) was administered from 6th day after HMG initiation (LH?5 IU/ml) to the day of HCG injection. Follicular development monitored by vaginal ultra sonography and serum estradiol measurement. Results: There were no significant differences in age, duration of infertility, BMI, number of HMG ampules, number of follicles?18mm, serum estradiol level on 6th day of HMG initiation and HCG injection time, fertilization and pregnancy rate between two groups. However there were significant differences regarding duration of treatment, duration of HMG usage, LH level at the initiation of HMG, OHSS rate and number of Metaphase II oocytes between two groups (p<0.05). Conclusion: Usage of the GnRH antagonist may have more advantages such as the shorter duration of treatment and less gonadotrophin requirement. Furthermore, the incidence of OHSS can be reduced in GnRH antagonist comparing to agonist. For decreasing the risk of OHSS and abortion rate, we recommend long term use of OCP before starting the treatment - PCOS , GnRH agonist, GnRH antagonist , OHSS, IVF, ICSI 14 18 Mahnaz Ashrafi info@royaninstitute.org No Mahnaz Ashrafi info@royaninstitute.org No Ashraf Moini - No Afsaneh Mohammadzadeh - No Zahra Ezabadi - No Fatemeh Zafarani - No Ahmad Reza Baghestani - No

Vitrification of human oocyte using cryoloop - Background: The cryopreservation of human oocyte would make a significant contribution to infertility treatment, such as using it for oocyte donation and for patients a bout to lose ovarian function due to surgery or chemotherapy. Despite of using standard freezing straws and cryovials or even open pulled straws, only a few successful pregnancies have been arisen from cryopreserved human oocytes. This situation has been primarily attributed to poor survival, fertilization and development of cryopreserved oocytes. Objective: The aim of this study was to evaluate the novel cryoloop vitrification method for cryopreservation of human oocytes. Materials and Methods: Nine infertile couples participated in this study. In all women proper regulation and desensitization was done using GnRH agonist during luteal phase. Mature oocytes allocated into two groups randomly. In group I, 34 oocytes were vitrified in conventional straws, while in group II, 33 oocytes were vitrified in cryoloop. After a store time of 1-6 months the oocytes were thawed, incubated for 2 hours and subsequently the ICSI was done on survived oocytes. To verify normal fertilization of vitrified oocytes the number of pronuclei in the cytoplasm was counted 16-18 hours after ICSI and good morphological quality embryos were transferred on day 2 or 3 after sperm injection. Pregnancy was identified by the serum ß HCG level, checked 14 days after embryo transfer. Results: The present study shows that the rate of survival of vitrified human oocytes in two groups has no significant difference (52.94% in group I versus 63.63% in group II) but the fertilization rate of vitrified oocytes by cryoloop was greater than vitrified oocytes by conventional straws (73.7% versus 55.55% respectively). One of the embryo transfers achieved clinical pregnancy and resulted in the delivery of healthy baby. Conclusion: Vitrification by using cryoloop can improved the fertilization rate and developmental capacity of vitrified thawed oocyte. - Vitrification, Human oocyte, Survival rate, Fertilization 19 24 Ghasem Saki ghasemsaki@yahoo.com No Ghasem Saki ghasemsaki@yahoo.com No Fatemeh Ghalambor Dezfuly - No

Anti-zona pellucida antibodies in infertile patients in relation to multiple puncture of ovaries and unexplained infertility - Background: Auto antibodies to zona-pellucida (AZA) seem to be important autoantibodies implicated in reproduction, with substantial role in both endocrine and reproductive functions of the human ovary. There are some debates on the relation of AZA with infertility, repeated In Vitro Fertilization (IVF) attempts, and outcome of it. Objective: In this study, we assessed the presence of AZA in the follicular fluids (FFs) of women who underwent intra cytoplasmic sperm injection (ICSI), in relation to etiology of infertility and multiple puncture of ovaries. Materials and Methods: In this prospective study, follicular fluids were evaluated from 96 infertile women, (19-40 years old, 31.5±5.1), who were candidates for ICSI based on the etiology of infertility. From these 80 women had explained infertility whereas 16 had unexplained infertility. All FFs were evaluated for presence of AZA by ELISA test. Results: Twenty patients (20.8%) were positive for AZA in follicular fluid. In patients with unexplained infertility, AZA antibody in follicular fluid, was significantly higher than the group with proven etiology of infertility (p=0.001). In addition, 20.4 % of patients who had been punctured previously showed AZA in their FFs which is statistically similar to the patients who were punctured for the first time. Conclusions: The high incidence of AZA in infertile women, especially women with unexplained infertility has to be considered. Relation of the presence AZA and repeated puncture of ovaries is still debatable. Determinations of AZA are highly recommended in evaluation of infertile couples especially in patient with unexplained infertility - Anti Zona Antibody, Multiple puncture of ovaries,Unexplained infertility 30 35 Soheila Arefi soheilaarefi@yahoo.com No Soheila Arefi soheilaarefi@yahoo.com No Mahmoud Jeddi Tehrani - No Mohammad Mehdi Akhondi - No Ali Reza Mousavi - No Mahnaz Heidari - No Ahmad Ali Bayat - No

Effects of essential and non-essential amino acids on in-vitro maturation, fertilization and development of immature bovine oocytes - Background: Addition of amino acids to the culture medium is beneficial for embryonic development in many species. Objective: The objective of this study was to investigate the effects of amino acids on the in vitro maturation and embryonic development of the bovine oocyte. Materials and Methods: Bovine ovaries were collected from a local abattoir and brought into laboratory. Cumulus-oocyte complexes (COCs; n=1212) were aspirated from follicles (2-8 mm in diameter) and randomly assigned to four groups for maturation in culture: (1) Basic medium alone as control; (2) Basic medium supplemented with 2% MEM essential amino acids solution; (3) Basic medium supplemented with 1% MEM non-essential amino acids solution; and (4) Basic medium supplemented with 2% MEM essential amino acids solution + 1% MEM non-essential amino acids solution. COCs were incubated in 1 ml maturation medium in an Organ culture dish at 38.5°C in an atmosphere of 5% CO2 with high humidity. After 24 h of culture, 372 oocytes were fixed to determine maturation rate and the remaining oocytes were used for in vitro fertilization (IVF). Following 18 h of insemination, 437 oocytes were fixed and examined for fertilization and 403 oocytes were further cultured. Results: There were no differences in maturation rates and penetration rates among the four groups. Although oocyte cleavage rates were not different in the four groups, embryo development up to the 8-cell stage and blastocyst were significantly higher (p<0.05) in Group (2) and (4) than in the Control and Group (3). Conclusion: These results indicate that the presence of amino acids, especially essential amino acids in the maturation medium is beneficial to oocyte cytoplasmic maturation and subsequent early embryo development in vitro. - In vitro maturation, Bovine oocyte, Amino acids 36 41 Nourollah Rezaei noor454@yahoo.com No Nourollah Rezaei noor454@yahoo.com No Ri-Cheng Chian - No

Isolation and differentiation of mouse embryonic stem cells - Background: Recently, embryonic stem (ES) cells have become very important resources in basic medical researches. These cells can differetiate into derivatives of all primary germ layers. Objectives: In order to isolate embryonic stem cells in vitro, the blastocyst were cultured and the morphological aspects, population doubling time, alkalin phosphatse and differentiation properties of the cells were investigated. Materials and Methods: The balstocysts from NMRI mice were cultured for 3 days up to time that inner cell mass (ICM) reach to the outgrowth stage. The cells were disaggregated and trypsinized every 3 days until the appearance of the colonies of ES cells. The colony positive cells were fixed and stained for alkaline phosphatase. The ES cells were cultured in suspension state for 5 days, at the same time Leukaemia Inhibitory Factor (LIF) was removed from media to form embryoid bodies(EBs). The EBs were cultured for 8 - 20 days on collagen coated dish to induce the spontaneouse differentiation. Results: During the 6-9 days after the disaggregation of ICM in the expansion stage, the colony of ES cells appeared as a flat monolayer mass with strike boundaries and nondistinguish cytoplasm including a few nuclei. In colony formation stage, the morphology changed from flat monolayer to round multilayer with strike define boundaries. Undifferentiated cells were seen as intensely small cells attached together compactly with high nucleus/cytoplasm (N/C) ratio. The cells of colonies tend to differetiate by separation from each other and became larger and diffused on substrate by attaching to dish. The positive alkaline phosphatase cells were seen in typical morphology of ES colonies. The EBs cells were seen in culture after 5 days in suspension and began to spontaneously differentiate into various types of cells such as nerve and hematopoitic lineages. Conclusion: Despite strike morphology of ES colonies, it is difficult to distinguish the differentiated from undifferentiated cell colonies in the colony formation stage. New ES cells are capable to give rise into EBs and are susceptible of spontaneously differentiation in various type of cells. - Embryonic stem cells, Embryoid bodies, Differentiation, Mice 42 46 Mahmoud Hashemi-Tabar Hashemi_tabar@Hotmail.com No Mahmoud Hashemi-Tabar Hashemi_tabar@Hotmail.com No Fatemeh Javadnia - No Mahmoud Orazizadeh - No Maryam Baazm - No

Bilateral Leydig cell tumor and male infertility: A case report - Background: Leydig cell tumor is a rare form of testicular neoplasm which comprises 1-3% of all testicular tumors and only about 3% of these tumors are bilateral. A few Leydig all tumor have been described in patients with klinefelter’s syndrome so far. Case: The patient described in this case report was a 24 year-old man with chief complaint of infertility for one year. Physical examination, semen analysis, testes sonography and hormonal assay were done for him. Right side simple orchiectomy was performed for patient. Conclusion: This tumor is always benign in children and approximately 90% are benign in adults. Clinical presentation is testicular enlargement, gynecomastia, sexual activity disturbances such as decreased libido, infertility and azoospermia. We recommend complete exam and karyotype in patients with infertility and azoospermia. - Leydig cell tumor, Infertility, Azoospermia 47 49 Behrouz Ilkhanizadeh ilkhani_b@yahoo.com No Behrouz Ilkhanizadeh ilkhani_b@yahoo.com No Mohammad Taghizadieh - No Mehrzad Mahzad-Sadaghiani - No Farahnaz Noroozinia - No Bahman Jahandideh - No

Oxidative stress and antioxidants in male infertility: a difficult balance - Infertility is one of the most stressful conditions amongst married couples. Male factor infertility is implicated in almost half of these cases. Recent advances in the field of reproductive medicine have focused the attention of many researchers to consider reactive oxygen species (ROS) as one of the mediators of infertility causing sperm dysfunction. Although, ROS is involved in many physiological functions of human spermatozoa, their excess production results in oxidative stress. Mitochondria and sperm plasma membranes are the two locations of ROS production that involves complex enzyme systems such as creatine kinase and diaphorase. ROS causes damage to the spermatozoa DNA, resulting in increased apoptosis of these cells. The production of ROS is greatly enhanced under the influence of various environmental and life style factors such as pollution and smoking. An effective scavenging system is essential to counteract the effects of ROS. Various endogenous antioxidants belonging to both enzymatic and non-enzymatic groups can remove the excess ROS and prevent oxidative stress. Since, ROS is essential for the normal sperm physiology, rationale use of antioxidants is advocated. - Reactive oxygen species, Male infertility, Oxidative stress 1 8 Ashok Agarwal Agarwaa@ccf.org No Ashok Agarwal Agarwaa@ccf.org No Sushil Anandh Prabakaran - No

The effect of oral administration of Pentoxifylline on sperm motility of asthenozoospermic ejaculates from men with or without testicular varicoceles - Background: Pentoxifylline (PX) is a methyxanthin derivative that influences the sperm motion characteristics. In general, PX has been reportedly effective in preserving sperm motility in vitro, also when administered orally to the asthenozoospermic patients. Objective: The main objective of this prospective clinical trial study was to rule out the effect of oral administration of PX on sperm progressive motility of asthenozoospermic ejaculates obtained from men with or without mild testicular varicoceles. In addition, the role of patient’s age on sperm motility following PX administration was investigated. Materials and Methods: A total of 68 infertile men with asthenozoospermia were allocated to this study. Following physical examination, 20 cases were found with mild varicocele of testis. A dosage of 400 mg PX/ twice daily for duration of 3 months was administered to each patient. Two semen samples (one before and one after the PX therapy) were evaluated under blind condition. Semen parameters of sperm concentration, total and fast progressive motility (%) and morphology (%) were analyzed for each sample. Also, the sperm motion characteristics of asthenozoospermic patients with testicular varicocele were compared with cases lacking varicocele. The subjects were divided into two age groups of <30 and ?30 years old. Results: PX was significantly effective on the fast progressive motility of sperm (p<0.01). Also, total progressive motility was enhanced from 26.82±16.8 to 29.60±22.2 with PX therapy. However, PX did not have any negative effect on other semen parameters. Oral therapy of PX was also effective in improving the fast progressive motility of sperm of samples from cases with or without mild testicular varicocele (p<0.01). Fast progressive motility was also significantly enhanced in ejaculates of men from both age groups. Conclusion: Our results demonstrate that low dose of oral therapy of PX is significantly useful in enhancing fast progressive motility of sperms from infertile men with asthenozoospermia. Also, testicular varicocele did not interfere with enhancing effect of PX on sperm motility. - Sperm Motility, Pentoxifylline, Varicocele, Asthenozoospermia 25 29 Mohammad Reza Moein - No Mohammad Ali Khalili khalili59@hotmail.com No Mohammad Ali Khalili khalili59@hotmail.com No Arash Davoudi - No