International Journal of Reproductive BioMedicine
Volume:3 Issue: 1, May 2005

Infertility is one of the most stressful conditions amongst married couples. Male factor infertilityis implicated in almost half of these cases. Recent advances in the field of reproductive medicinehave focused the attention of many researchers to consider reactive oxygen species (ROS) as one ofthe mediators of infertility causing sperm dysfunction. Although, ROS is involved in manyphysiological functions of human spermatozoa, their excess production results in oxidative stress.Mitochondria and sperm plasma membranes are the two locations of ROS production that involvescomplex enzyme systems such as creatine kinase and diaphorase. ROS causes damage to thespermatozoa DNA, resulting in increased apoptosis of these cells. The production of ROS is greatlyenhanced under the influence of various environmental and life style factors such as pollution andsmoking. An effective scavenging system is essential to counteract the effects of ROS. Variousendogenous antioxidants belonging to both enzymatic and non-enzymatic groups can remove theexcess ROS and prevent oxidative stress. Since, ROS is essential for the normal sperm physiology,rationale use of antioxidants is advocated.

For screening sequence variations in genes, rapid turnover time is of fundamentalimportance. While, many of the current methods are unfortunately time consuming and technicallydifficult to implement. Denaturing high-performance liquid chromatography (DHPLC) method hadbeen shown to be a high-throughput, time saving, and economical tool for mutation screening.

In the present study DHPLC method was used to explore the potential associationbetween estrogen receptor β gene (ESR2) variants and male infertility.

DNA from 96 men with infertility and 96 normal male as control werescreened for mutation in the nine exons of the ESR2 gene, using WAVE® DHPLC device equippedwith a DNA separation column and automated sequence analysis on the ABI Prism 310.

DHPLC evaluation of ESR2 gene in 96 infertile patients, revealed one heterozygoussequence variation (IVS 8–4G>A) near the 5’ splicing region of intron 8 in 5 patients. No variationwas identified in control population.

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’vefound might increase susceptibility to infertility; but without cDNA screening, the consequences ofthese genetic alterations cannot be predicted.

Polycystic ovarian syndrome (PCOS) patients are prone to premature LH surge andovarian hyperstimulation syndrome (OHSS). Long GnRH analogue protocol and GnRH antagonistprotocol are two methods utilized for induction ovulation in patients undergoing IVF/ICSI.

The aim of this study was to compare the effects of GnRH agonists and antagonists inPCOS patients.

A total of 60 PCOS patients under 35 years old were enrolled in thisstudy. The patients have no history of thyroid disorder and hyperprolactinemia. All patientsreceived 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) wasadministered 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.

There were no significant differences in age, duration of infertility, BMI, number of HMGampules, number of follicles≥18mm, serum estradiol level on 6th day of HMG initiation and HCGinjection time, fertilization and pregnancy rate between two groups. However there weresignificant differences regarding duration of treatment, duration of HMG usage, LH level at theinitiation of HMG, OHSS rate and number of Metaphase II oocytes between two groups (p<0.05).

Usage of the GnRH antagonist may have more advantages such as the shorter durationof treatment and less gonadotrophin requirement. Furthermore, the incidence of OHSS can bereduced in GnRH antagonist comparing to agonist. For decreasing the risk of OHSS and abortionrate, we recommend long term use of OCP before starting the treatment.

The cryopreservation of human oocyte would make a significant contribution toinfertility treatment, such as using it for oocyte donation and for patients a bout to lose ovarianfunction due to surgery or chemotherapy. Despite of using standard freezing straws and cryovials oreven open pulled straws, only a few successful pregnancies have been arisen from cryopreservedhuman oocytes. This situation has been primarily attributed to poor survival, fertilization anddevelopment of cryopreserved oocytes.

The aim of this study was to evaluate the novel cryoloop vitrification method forcryopreservation of human oocytes.

Nine infertile couples participated in this study. In all women properregulation and desensitization was done using GnRH agonist during luteal phase. Mature oocytesallocated 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 theoocytes were thawed, incubated for 2 hours and subsequently the ICSI was done on survivedoocytes. To verify normal fertilization of vitrified oocytes the number of pronuclei in the cytoplasmwas counted 16-18 hours after ICSI and good morphological quality embryos were transferred onday 2 or 3 after sperm injection. Pregnancy was identified by the serum ß HCG level, checked 14days after embryo transfer.

The present study shows that the rate of survival of vitrified human oocytes in two groupshas no significant difference (52.94% in group I versus 63.63% in group II) but the fertilization rateof 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 resultedin the delivery of healthy baby.

Vitrification by using cryoloop can improved the fertilization rate and developmentalcapacity of vitrified thawed oocyte.

Pentoxifylline (PX) is a methyxanthin derivative that influences the sperm motioncharacteristics. In general, PX has been reportedly effective in preserving sperm motility in vitro,also when administered orally to the asthenozoospermic patients. The main objective of this prospective clinical trial study was to rule out the effect oforal administration of PX on sperm progressive motility of asthenozoospermic ejaculates obtainedfrom men with or without mild testicular varicoceles. In addition, the role of patient’s age on spermmotility following PX administration was investigated. A total of 68 infertile men with asthenozoospermia were allocated to thisstudy. Following physical examination, 20 cases were found with mild varicocele of testis. Adosage of 400 mg PX/ twice daily for duration of 3 months was administered to each patient. Twosemen 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 asthenozoospermicpatients with testicular varicocele were compared with cases lacking varicocele. The subjects weredivided into two age groups of <30 and ≥30 years old. 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 alsoeffective in improving the fast progressive motility of sperm of samples from cases with or withoutmild testicular varicocele (p<0.01). Fast progressive motility was also significantly enhanced inejaculates of men from both age groups. Our results demonstrate that low dose of oral therapy of PX is significantly useful inenhancing fast progressive motility of sperms from infertile men with asthenozoospermia. Also,testicular varicocele did not interfere with enhancing effect of PX on sperm motility.

Auto antibodies to zona-pellucida (AZA) seem to be important autoantibodiesimplicated in reproduction, with substantial role in both endocrine and reproductive functions of thehuman ovary. There are some debates on the relation of AZA with infertility, repeated In VitroFertilization (IVF) attempts, and outcome of it.

In this study, we assessed the presence of AZA in the follicular fluids (FFs) of womenwho underwent intra cytoplasmic sperm injection (ICSI), in relation to etiology of infertility andmultiple puncture of ovaries.

In this prospective study, follicular fluids were evaluated from 96 infertilewomen, (19-40 years old, 31.5±5.1), who were candidates for ICSI based on the etiology ofinfertility. From these 80 women had explained infertility whereas 16 had unexplained infertility.All FFs were evaluated for presence of AZA by ELISA test.

Twenty patients (20.8%) were positive for AZA in follicular fluid. In patients withunexplained infertility, AZA antibody in follicular fluid, was significantly higher than the groupwith proven etiology of infertility (p=0.001). In addition, 20.4 % of patients who had beenpunctured previously showed AZA in their FFs which is statistically similar to the patients whowere punctured for the first time.

The high incidence of AZA in infertile women, especially women with unexplainedinfertility has to be considered. Relation of the presence AZA and repeated puncture of ovaries isstill debatable. Determinations of AZA are highly recommended in evaluation of infertile couplesespecially in patient with unexplained infertility.

Addition of amino acids to the culture medium is beneficial for embryonicdevelopment in many species.

The objective of this study was to investigate the effects of amino acids on the in vitromaturation and embryonic development of the bovine oocyte.

Bovine ovaries were collected from a local abattoir and brought intolaboratory. Cumulus-oocyte complexes (COCs; n=1212) were aspirated from follicles (2-8 mm indiameter) and randomly assigned to four groups for maturation in culture: (1) Basic medium aloneas control; (2) Basic medium supplemented with 2% MEM essential amino acids solution; (3) Basicmedium supplemented with 1% MEM non-essential amino acids solution; and (4) Basic mediumsupplemented with 2% MEM essential amino acids solution + 1% MEM non-essential amino acidssolution. COCs were incubated in 1 ml maturation medium in an Organ culture dish at 38.5°C in anatmosphere of 5% CO2 with high humidity. After 24 h of culture, 372 oocytes were fixed todetermine 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 403oocytes were further cultured.

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 the8-cell stage and blastocyst were significantly higher (p<0.05) in Group (2) and (4) than in theControl and Group (3).

These results indicate that the presence of amino acids, especially essential aminoacids in the maturation medium is beneficial to oocyte cytoplasmic maturation and subsequent earlyembryo development in vitro.

Recently, embryonic stem (ES) cells have become very important resources in basicmedical researches. These cells can differetiate into derivatives of all primary germ layers.

In order to isolate embryonic stem cells in vitro, the blastocyst were cultured and themorphological aspects, population doubling time, alkalin phosphatse and differentiation properties ofthe cells were investigated.

The balstocysts from NMRI mice were cultured for 3 days up to time thatinner cell mass (ICM) reach to the outgrowth stage. The cells were disaggregated and trypsinizedevery 3 days until the appearance of the colonies of ES cells. The colony positive cells were fixed andstained for alkaline phosphatase. The ES cells were cultured in suspension state for 5 days, at thesame time Leukaemia Inhibitory Factor (LIF) was removed from media to form embryoidbodies(EBs). The EBs were cultured for 8 - 20 days on collagen coated dish to induce thespontaneouse differentiation.

During the 6-9 days after the disaggregation of ICM in the expansion stage, the colony ofES cells appeared as a flat monolayer mass with strike boundaries and nondistinguish cytoplasmincluding a few nuclei. In colony formation stage, the morphology changed from flat monolayer toround multilayer with strike define boundaries. Undifferentiated cells were seen as intensely smallcells attached together compactly with high nucleus/cytoplasm (N/C) ratio. The cells of coloniestend to differetiate by separation from each other and became larger and diffused on substrate byattaching to dish. The positive alkaline phosphatase cells were seen in typical morphology of EScolonies. The EBs cells were seen in culture after 5 days in suspension and began to spontaneouslydifferentiate into various types of cells such as nerve and hematopoitic lineages.

Despite strike morphology of ES colonies, it is difficult to distinguish thedifferentiated from undifferentiated cell colonies in the colony formation stage. New ES cells arecapable to give rise into EBs and are susceptible of spontaneously differentiation in various type ofcells.

Leydig cell tumor is a rare form of testicular neoplasm which comprises 1-3% of alltesticular tumors and only about 3% of these tumors are bilateral. A few Leydig all tumor have beendescribed 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 ofinfertility for one year. Physical examination, semen analysis, testes sonography and hormonalassay were done for him. Right side simple orchiectomy was performed for patient.

This tumor is always benign in children and approximately 90% are benign in adults.Clinical presentation is testicular enlargement, gynecomastia, sexual activity disturbances such asdecreased libido, infertility and azoospermia. We recommend complete exam and karyotype inpatients with infertility and azoospermia.