Cell Journal (Yakhteh)
Volume:9 Issue: 2, 2007

Over-diagnosis and under treatment of varicocele may be responsible for poor outcome of varicocelectomy. In this study, we used Color Doppler ultrasound for accurate diagnosis and grading of varicocele and to predict the outcome of microsurgical subinguinal varicocelectomy. A total of 104 patients undergoing microsurgical subinguinal varicocelectomy for treatment of infertility were included in this study. Patients were evaluated with routine history, physical examination, semen analysis, hormonal assessment and scrotal ultrasound and Doppler. After varicocelectomy, improvement index, in sperm concentration, was calculated by dividing the difference between the postoperative and preoperative sperm concentration by the preoperative sperm concentration. Improvement Index greater than 0.5 is considered a good outcome. Statistical analysis was done to study the correlation between microsurgical varicocelectomy outcome and testicular vein diameter at the inferior pole of the testis and the degree of reflux measured by color Doppler ultrasound. Improvement index in sperm concentration, motility and morphology more than 0.5 was achieved in 58.8%, 27.3% and 17.6% of cases respectively. We found that patients with testicular vein diameter, at the inferior pole of the testis, more than 2.5 have significantly higher improvement index in sperm concentration, motility and morphology than in patients with testicular vein diameter less than 2.5mm (p=0.006, 0.016, 0.041 respectively). We also found that patients with clear reflux detected by color Doppler ultrasound at the inferior pole of the testis have a significantly higher improvement index in sperm concentration, motility and morphology than patients with reflux detected only in the supratesticular venous channels (p=0.013, 0.015 and 0.045 respectively). Color Doppler ultrasound is a useful tool for accurate diagnosis and grading of varicocele and to predict the outcome of varicocelectomy. We recommend doing varicocelectomy in cases of testicular vein diameter more than 2.5mm and in cases of reflux detected at veins at the lower pole of the testis.

Gynecology and Obstetrics Deparemnt, University of Schleswig-Holstein, Section of Andrology, Campus of Luebeck, Ratzeburger Allee 160, 23560 Luebeck, Germany Email: sf_alhasani@hotmail.com Functions of seminal vesicle, prostate and bilateral testes can generally be determined by parameters of sperm analysis such like concentration, motility, morphology, semen volume, and pH. Infertile men frequently have lower sperm parameters than their fertile counterparts. However the results of sperm analyses among these groups are sometimes overlapping. As well traditional spermiogram and classical morphological assessment even based on strict morphological criteria sometimes fail in certain prediction of reproductive outcome of infertile men who undergone artificial reproductive techniques with their partners. Thus new and more sensitive methods, especially for detecting sperm function and viability, were being needed by many experts. Human sperm DNA is one of the most attractive targets for majority of experts during advances. Therefore various clinicians assumed that sperm DNA might comprise many useful clinical parameters and predictors for reproductive outcome. Conversely to loose structure of chromatin (DNA and nuclear proteins) in somatic cells, chromatin is tightly compacted due to unique associations between the DNA and sperm nuclear proteins. OraOl rParl ePsreensteanttioatnison Abstract of the 8th Royan International Twin Congress, Tehran, Iran, 5-7 September 2007 8 Yakhteh Medical Journal, Vol 9, Sup 1, Summer 2007 It has been shown that in cases of DNA damage nuclear protein ratios determined as protamine and histone ratio altered favoring histone resulting in loose package of sperm DNA. Different from nuclear DNA small and circular mitochondrial DNA is also structured in sperm mainly reflecting maternal inheritance and linked with sperm motility. Sperm DNA causes, much like cause of male infertility, have many factors such as reactive oxygen species and their damage, febrile disease, testicular hyperthermia, varicoceles, drugs, chemotherapy, radiation, genital tract infections, environmental causes, genetic deficiencies and hormonal factors. Nevertheless DNA damage of sperm mainly related with male infertility. There are several tests such as TUNEL, COMET assays, sperm chromatin structure assay and nuclear protein composition with protein separation. All of these tests verify sperm DNA damage and its ratio or its link with male infertility and sperm function. Majority of their mechanism are showing DNA breaks and others examine either ratio of protamine / histone or the susceptibility of sperm DNA to denaturation. Increased sperm DNA damage has been shown precisely to be related with poor reproductive outcome in natural cycles. As well it has also been reported that naturally sperm with damaged DNA are unlikely to attached fallopian tube epithelium resulting in natural selection. However IVF and ICSI bypass this selection leading oocyte fertilization by sperm with mild and moderate DNA damage. Therefore some of the studies those included IVF and ICSI patients indicated only fertilisation failure whereas some of them reported poor embryo quality and lower ongoing pregnancy rates. More or less the studies met some topics that till four cell stage embryonic genome is not activated and resulting in unaffected embryonic morphology beyond this stage. As well today there are some attitudes on recovery of sperm DNA damage by oocyte after fertilisation forming healthy embryonic genome up to several damage degrees. However majority of these studies indicated that embryonic quality beyond 4 cell stage, ongoing pregnancy and fertilisation rates less or more linked with sperm DNA damage. Therefore sperm DNA damage is closely related with reproduction either naturally or with ART. Conclusively sperm DNA damage without any doubt should be included in the current and advanced management of infertile couples. In future sperm DNA damage seemed to have a severe role in assisted reproduction especially in selection of treatment choice.

WHO has made a significant contribution by producing successive revisions of the semen analysis manual. The 5th edition to be published soon has a number of improvements with clearer and simpler instructions for standard analysis particularly for sperm concentration and motility. Morphology continues to be done by the strict method. There are tables for comparing duplicates. New reference values (weighted 5th percentiles) from about 1600 fertile men (recent fathers) from different countries are included. The section on sperm preparation has been expanded to cover common methods of isolation of sperm for assisted reproductive technology. A chapter on sperm cryopreservation has been added. The quality control chapter has been rewritten. There is less material in appendices but there is a new appendix on the basics of microscopy. The aim of the manual is to standardise semen analysis but experience with external quality assurance indicates this is difficult to achieve. This is particularly the case with sperm morphology. Clinicians will continue need to know the reference ranges for their own laboratories. We have experience with routine use of computer assisted semen analysis (CASA) for sperm concentration, percent progressively motile sperm and sperm kinematics: straight line velocity (VSL), using the Hamilton Thorn CASA with IDENT fluorescent staining of the sperm. This requires dilution of samples with high sperm Abstract of the 8th Royan International Twin Congress, Tehran, Iran, 5-7 September 2007 Yakhteh Medical Journal, Vol 9, Sup 1, Summer 2007 9 concentration with seminal plasma and selection of fields across the counting chamber to achieve accuracy. For sperm concentrations above 2x106/mL the quality control is very good. We have a sperm morphometry system based on automated focusing and slide movement that provides 32 morphometric measurements of the sperm head and upper neck regions including features related to density of staining that allow orientation of the sperm head and assessment of the acrosomal region. An index (%Z) derived from features selected by the sperm-ZP binding process was related to natural conception rates in about 1200 subfertile couples. VSL and female age were also significant in regression analysis models. We believe these and other advances in CASA will greatly improve semen analysis.

During natural conception and standard in vitro fertilisation (IVF), motile capacitated sperm with intact acrosomes bind to the surface of the zona pellucida (ZP) and this binding triggers the acrosome reaction (AR). The sperm then passes through the ZP and binds to the oolemma via the plasma membrane that persists over the equatorial segment. The sperm is then engulfed into the ooplasm where decondensation of the nucleus occurs to form the male pronucleus. We have developed tests for human sperm- ZP binding, the ZP-induced AR and spermoolemma binding using oocytes which failed to fertilise in the clinical IVF program. The patients consent to the use of this material for testing or research. Usually the oocytes maintain their ability to bind sperm and stimulate the AR. The ZP can be preserved in concentrated salt solution at 4oC for months. Using these assays we have found defective sperm-ZP binding and disordered ZP-induced AR are common causes of failure of IVF when there are sperm defects, but can also occur with normal semen analysis. These defects of sperm-oocyte interaction could account for about 25% of patients with idiopathic infertility and if diagnosed before IVF is attempted would allow the patients to be treated by ICSI and avoid an IVF cycle with low or zero fertilisation. In contrast, oolemma binding defects appear to be rare. Using experimental conditions in which the amount of ZP was not limiting, we showed that only a small proportion

Males suspected to be infertile should have a detailed medical history and physical examination. Semen analysis is the main investigation. Unless the clinical picture is clear, several semen analyses need to be done because of the day-to-day variability. Measurement of gonadotrophin and Abstract of the 8th Royan International Twin Congress, Tehran, Iran, 5-7 September 2007 10 Yakhteh Medical Journal, Vol 9, Sup 1, Summer 2007 testosterone levels is helpful in distinguishing primary from secondary testicular failure. Testis biopsies are useful for confirming obstructive azoospermia and determining the type of spermatogenic defect with primary seminiferous tubule disorders. Other investigations: karyotype, genetic tests for Yq microdeletions, cystic fibrosis, imaging for pituitary tumour or ejaculatory duct obstruction, are performed where indicated. A number of conditions are untreatable and cause sterility, in particular primary spermatogenic disorders where no live sperm are produced. These patients need to consider other alternatives for having a family by donor insemination or adoption. Over the last 15 years it has become clear that sperm or elongated spermatids that can be used for intracytoplasmic sperm injection (ICSI) may be found in a proportion of patients with severe testicular disorders such as Klinefelter syndrome and Sertoli cell only syndrome, either in the semen or in testis biopsy specimens. Conditions that may be treatable to increase the chances of natural conception include: gonadotropin deficiency or suppression, sperm autoimmunity, genital tract obstruction and reversible toxin exposures or illness effects and some coital disorders. However ICSI is often a better alternative for conditions such as sperm autoimmunity and genital tract obstruction. The remaining patients have less severe abnormalities of the semen, ranging from oligospermia to normal standard semen analyses but defective sperm function. These patients may have varicoceles, features of low grade genital tract inflammation, increased abnormal DNA in the sperm and increased production of reactive oxygen species by their sperm. There are no good controlled trials which prove treatment of these problems will increase natural conception rates. These patients are subfertile rather than sterile and pregnancies may occur but at lower than normal rates. Infrequent or mistimed coitus and female factors such as ovulatory disorders may be contributing. Thus the male and female partners should be treated as a couple and reversible factors treated where possible. The estimation of prognosis for natural conception is also important. If this is low, ICSI is usually effective.

This article represents a step forward in our study on ram sperm functioning and the relationship with fertility. Artificial insemination in sheep has not been widely adopted, probably due to the low fertility rate obtained with frozen-thawed semen, possibly due to premature capacitation-like changes. Therefore, a good knowledge of the sperm capacitation process could help in the formulation of better diluents that prevent these changes during sperm freezing or storage, and, therefore, improving the sperm fertilizing capacity. In previous studies we reported that in vitro capacitation and acrosome reaction induced a decrease in the content and the redistribution of P14 and P20, two ram seminal plasma proteins that protect spermatozoa against coldshock. Our results suggested that the protective effect of these proteins could be related to their decapacitating role (JAndrol2005). Likewise, we showed that membrane protein tyrosine phosphorylation is related to the capacitation state of ram spermatozoa (MRD2001). However, there had not been any report about the molecular regulation mechanism of this process in ram. Therefore, in this study we investigated basic aspects of the signal transduction pathways that are activated during capacitation. Our results demonstrated that in ram sperm, capacitation and the associated protein tyrosine phosphorylation is not absolutely dependent on the presence of BSA and calcium, and that the PKA/cAMP pathway is, at least, partially implicated in the tyrosine phosphorylation of some proteins. Our data indicate that the signal transduction mechanisms of capacitation in ram sperm differ from those in other mammals, which suggests that species specificities might exist Abstract of the 8th Royan International Twin Congress, Tehran, Iran, 5-7 September 2007 Yakhteh Medical Journal, Vol 9, Sup 1, Summer 2007 11 with respect to this process. Our findings might benefit our understanding of the biochemical mechanisms involved in mammalian sperm capacitation and ultimately, fertility. Furthermore, in this article we validate the chlortetracycline (CTC) technique for the assessment of ram sperm capacitation state, performing a specific determination in viable cells exclusively.

The lack of effective diagnosis of testicular disorders leading to infertility is a major problem in reproductive biomedicine. A better understanding of male infertility related to abnormal spermatogenesis associated with differential gene expression is of interest. We are working on a novel hyaluronan binding protein (HABP1) that interacts specifically with HA and facilitates HA mediated processes including sperm-oocyte interaction1 and sperm motility2. Sequence analysis of HABP1 from human fibroblasts reveal its identity with SF2/p32, the protein copurified with alternate splicing factor3 and globular head of C1Q thus represented in human Chromosome 17p13.34 as synonym HABP1/p32/C1QBP. HABP1 is synthesized as a proprotein which forms mature protein by cleavage of initial 73 amino acids. This proprotein form is extremely labile and detected only in pachytene spermatocytes and round spermatids in germ cells in adult testis5. Further analysis demonstrates that though mature form of HABP1 is present in the testis, its precursor form was not found in testis of 7, 14, 21 and 28 day old rat, but is present only in pachytene spermatocytes and round spermatids of testis of 21 day and 28 day old rats. With spermatogenic arrest, HABP1 is lost from pachytene and round spermatids suggesting that the expression of HABP1 proprotein may be crucial for spermatogenesis6. Earlier we reported on the reduction in the level of HABP1 on sperm surface in asthenozoospermic and oligospermic patient7. In continuation, we have also demonstrated the interaction of HABP1 with zona pellucida of buffalo and shown that the supplementation of IVF medium with rHABP1 can promote the capacity of sperm binding to oocytes under invitro fertilization conditions even in presence of D-mannosylated albumin (DMA), known to inhibit sperm oocyte interaction8. Thus we propose to use HABP1: 1. as a diagnostic marker for male infertility and spermatogenic arrest in testicular biopsy. 2. in IVF medium to promote sperm oocyte interaction.