Andrology: The use of hemizona assay in the evaluation of the optimal sperm preparation technique

Semen preparation techniques for intrauterine insemination

Abstract: Background Semen preparation techniques for assisted reproduction, including intrauterine insemination (IUI), were developed to separate the motile morphological normal spermatozoa. Leucocytes, bacteria and dead spermatozoa produce oxygen radicals that negatively influence the ability to fertilize the egg. The yield of as many motile, morphologically normal spermatozoa as possible might influence treatment choices and therefore outcomes. Objectives To compare the effectiveness of gradient, swim-up, or wash and centrifugation semen preparation techniques on clinical outcome in subfertile couples undergoing intrauterine insemination (IUI). Search strategy We searched the Menstrual Disorders and Subfertility Group Trials Register (3 January 2007), Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library 2007, Issue 2), MEDLINE (1966 to January 2007), EMBASE (1980 to January 2007), Science Direct Database (1966 to January 2007), National Research Register (2000 to 2007), Biological Abstracts (2000 to January 2007), CINAHL (1982 to October 2006) and reference lists of relevant articles. We also contacted experts and authors in the field. Selection criteria Parallel randomized controlled trials (RCTs) comparing the efficacy of semen preparation techniques used for subfertile couples undergoing IUI in terms of clinical outcome were included. Data collection and analysis Two reviewer authors independently assessed trial quality and extracted data. Study authors were contacted for additional information. Main results Five RCTs, including 262 couples in total, were included in the meta-analysis (Dodson 1998; Grigoriou 2005; Posada 2005; Soliman 2005; Xu 2000). Xu compared the three techniques; Soliman compared a gradient technique versus a wash technique; Dodson and Posada compared a gradient technique versus a swim-up technique; whereas Grigoriou compared swim-up versus a wash technique. No trials reported the primary outcome of live birth. There was no evidence of a difference between pregnancy rates (PR) for swim-up versus a gradient or wash and centrifugation technique (Peto OR 1.57, 95% CI 0.74 to 3.32; Peto OR 0.41, 95% CI 0.15 to 1.10, respectively); nor in the two studies comparing a gradient technique versus wash and centrifugation (Peto OR 1.76, 95% CI 0.57 to 5.44). There was no evidence of a difference in them is carriage rate (MR) in two studies comparing swim- up versus a gradient technique (Peto OR 0.13, 95% CI 0.01 to 1.33). Authors' conclusions There is insufficient evidence to recommend any specific preparation technique. Large high quality randomised controlled trials, comparing the effectiveness of a gradient and/or a swim-up and/or wash and centrifugation technique on clinical outcome are lacking. Further randomised trials are warranted.

Binding of zona binding inhibitory factor-1 (ZIF-1) from human follicular fluid on spermatozoa.

Abstract: Previous studies showed that zona binding inhibitory factor-1 (ZIF-1) was the glycoprotein mainly responsible for the spermatozoa zona binding inhibitory activity of human follicular fluid. ZIF-1 has a number of properties similar to glycodelin-A. A binding kinetics experiment in the present study demonstrated the presence of two binding sites of ZIF-1 on human spermatozoa. These binding sites were saturable, reversible, and bound to (125)I-ZIF-1 in a time-, concentration-, and temperature-dependent manner. Glycodelin-A shared one common binding site with ZIF-1 on spermatozoa, and it could displace only 70% of the (125)I-ZIF-1 bound on human spermatozoa. ZIF-1 and glycodelin-A formed complexes with the soluble extract of human spermatozoa. Coincubation of solubilized zona pellucida proteins reduced the binding of ZIF-1 to two complexes of the extract, suggesting that the ZIF-1 binding sites and zona pellucida protein receptors on human spermatozoa were closely related. ZIF-1, but not glycodelin-A, significantly suppressed progesterone-induced acrosome reaction of human spermatozoa. The carbohydrate moieties derived from ZIF-1 reduced the binding of native ZIF-1 on human spermatozoa as well as the zona binding inhibitory activity of the glycoprotein, although the intensity of the effects are lower when compared with the native protein. These effects are not due to the action of the molecules on the motility, viability, and acrosomal status of the treated spermatozoa. Deglycosylated ZIF-1 had no inhibitory effect on both ZIF-1 binding and zona binding capacity of spermatozoa. We concluded that the carbohydrate part of ZIF-1 was critical for the functioning of the glycoprotein.

Sperm viability in the black-footed ferret (Mustela nigripes) is influenced by seminal and medium osmolality.

Abstract: Fundamental knowledge of spermatozoa cryobiology can assist with optimizing cryopreservation protocols needed for genetic management of the endangered black-footed ferret. Objectives were to characterize semen osmolality and assess the influence of two media at various osmolalities on sperm viability. We examined the influence of Ham’s F10 + Hepes medium (H) at 270, 400, 500 or 700 mOsm (adjusted with sucrose, a nonpermeating cryoprotectant) and TEST Yolk Buffer (TYB) with 0% (300 mOsm) versus 4% (900 mOsm) glycerol (a permeating cryoprotectant). Electroejaculates ( n = 16) were assessed for osmolality using a vapor pressure osmometer. For media comparison, semen ( n = 5) was collected in TYB 0%, split into six aliquots, and diluted in H270, H400, H500, H700, and TYB 0% or TYB 4%. Each sample was centrifuged (300 g , 8 min), resuspended in respective medium, and maintained at 37 °C for 3 h. Sperm motility and forward progression were monitored every 30 min for 3 h post-washing. Acrosomal integrity was monitored at 0 and 60 min post-washing. Results demonstrated that black-footed ferret semen has a comparatively high osmolality (mean ± SEM, 513.1 ± 32.6 mOsm; range, 366–791 mOsm). Ferret spermatozoa were sensitive to hyperosmotic stress. Specifically, sperm motility was more susceptible ( P P > 0.05) by hypotonic solutions. Exposure to TYB 4% glycerol retained more ( P

Preliminary results of hemizona assay (HZA) as a fertility test for canine spermatozoa

Abstract: The Hemizona assay (HZA) is considered to be an effective test for predicting the fertilizing potential of spermatozoa. It is a functional test that distinguishes the zona-binding capacity of spermatozoa from fertile and infertile males. The objective of this study was to validate the HZA for canine spermatozoa, as a test for diagnosing canine male fertility status. Various parameters that affect binding capacity were examined: the presence of an adequate number of capacitated and motile spermatozoa for an HZA, the influence of fertility status, sperm-binding variability within fertile dogs over 60 d, variability in sperm-binding capacity of different oocytes, the lower limit number of spermatozoa binding to a zona from the fertile control, and evaluation of HZI to determine the fertilizing capacity of spermatozoa. Hemizonae were obtained from frozen oocytes of spayed bitches. The oocytes were manually cut into nearly equal halves. Spermatozoa were capacitated by swim-up and 1 h incubation at 37°C in modified Ham's F10 medium. Spermatozoa and hemizonae were co-incubated in 100-μL drops at 37°C for 1 h. Spermatozoa from 7 fertile and 3 infertile dogs were used for this study. The optimal sperm concentration for hemizona insemination was 1x106/mL capacitated and motile spermatozoa. A significant difference (P

The factors affecting sperm binding to the zona pellucida in the hemizona binding assay

Abstract: 'To whom correspondence should be addressed Sperm binding to the zona pellucida is a prerequisite for fertilization. The hemizona binding assay (HZA) is commonly used to evaluate the zona-binding capacity of spermatozoa. The present study reports three factors that affect HZA. They were the base medium used, the protein source and the size of pipette used for removing loosely bound spermatozoa during HZA. The number of spermatozoa bound on the hemizona was compared between (1) Earle's balanced salt solution (EBSS) and Ham's F-10, and (2) between human serum and bovine serum albumin (BSA). Results indicated that EBSS and human serum both significantly increase the number of bound spermatozoa when compared to Ham's F-10 (P < 0.0001, paired Mest) and BSA (P < 0.05, paired Mest) respectively. Pipettes of different diameters were used to study the effect of size in removing loosely bound spermatozoa on hemizona. Data showed that the diameter of the pipette should be 2*200 mm, in order not to remove bound spermatozoa excessively. These results emphasize the importance of standardizati on of the protocol of the hemizona assay worldwide to be able to compare results between different laboratories.

Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa.

Abstract: Addition of the divalent cation ionophore, A23187, to washed populations of human spermatozoa resulted in a sudden burst of production of reactive oxygen species which peaked within 3-5 min. This activity was dependent upon the presence of calcium in the external medium and was unaffected by the mitochondrial inhibitors, oligomycin, antimycin and rotenone. Studies with scavengers of reactive oxygen species revealed that, while reagents directed against singlet oxygen and the hydroxyl radical were without effect, cytochrome C reduced the response to A23187 by about 50%, suggesting that the superoxide anion radical is a major product of the activated human spermatozoon. The clinical implications of these studies stem from the considerable variation observed between individuals in the levels of reactive oxygen species produced by the spermatozoa. This variability was shown to be inversely related to the ability of the spermatozoa to exhibit sperm-oocyte fusion on exposure to A23187; defective samples exhibited a basal level of reactive oxygen species production which was 40 times that observed with normal functional cells.

Significance of Reactive Oxygen Species and Antioxidants in Defining the Efficacy of Sperm Preparation Techniques

Abstract: The mechanisms responsible for mediating the influence of sperm preparation protocols on human sperm function have been investigated. Techniques that involved the separation of motile spermatozoa prior to centrifugation were found to yield sperm suspensions of highest quality. If the spermatozoa were centrifuged prior to isolation of the motile cells, sperm function was impaired. The detrimental effects of centrifugation were associated with a sudden burst of reactive oxygen species production by a discrete subpopulation of cells (characterized by significantly diminished motility and fertilizing capacity) that could be separated from normal functional spermatozoa on Percoll gradients. If unfractionated sperm suspensions were subjected to centrifugation, the reactive oxygen species generated by this subpopulation impaired the functional competence of normal spermatozoa in the same suspension. Assessment of the ability of the antioxidants, butylated hydroxytoluene, and vitamin E, to curtail the peroxidative damage inflicted by such cells in response to centrifugation revealed a significant improvement of sperm function in the presence of vitamin E.

The evaluation of morphological characteristics of human spermatozoa according to stricter criteria

Abstract: The evaluation of the morphology of human spermatozoa varies widely between and sometimes even within laboratories. The purpose of this study was to determine whether the method that has been developed in our laboratory and which resulted in the use of stricter criteria for the evaluation of sperm morphology is a practical, reliable and repeatable method and to establish the within and between observer variations. The criteria used for a 'normal' spermatozoon are based on the appearance of spermatozoa found in the mucus of the upper endocervical canal. The results of the morphological evaluations of 26 samples by four observers were statistically analysed by various methods. The method of Barnett showed a high degree of relative accuracy between observers with error variances of between 2.89 and 19.67 as well as high Spearman rank correlation coefficients of between 0.8675 and 0.6537 (P less than 0.0003). The Spearman correlation coefficient for 15 duplicate evaluations by one observer was 0.9650 (P less than 0.0001) while the coefficients of variation for repeated evaluations of single samples were also within acceptable limits. Based on these results, the method described in this article allows comparable and reliable results between and within observers to be obtained. From this and other studies it can be concluded that the method also has a good prognostic value for the prediction of expected IVF fertilization, the hamster test and hemizona assay.

The hemizona assay (HZA): development of a diagnostic test for the binding of human spermatozoa to the human hemizona pellucida to predict fertilization potential * † ‡

Abstract: The authors present their initial results with the hemizona assay (HZA), which was developed to predict the fertilizing potential of spermatozoa. The HZA uses the matching halves of a human zona pellucida from a nonfertilizable and nonliving oocyte, providing an internal control on zona-to-zona variability. Maximal binding of human sperm to the hemizona usually occurred after 4 to 5 hours of coincubation. Sperm from fertile men exhibited significantly higher binding capacity to hemizonae compared with sperm from men who had fertilization failure during in vitro fertilization (IVF) treatment. The HZA index is calculated as follows: (bound sperm from subfertile male) divided by (bound sperm from fertile male) X 100. These findings demonstrate that the HZA may be a useful diagnostic tool in male infertility evaluations.