Separation of sperm cells by sedimentation technique is not suitable for in vitro fertilization purposes.

Two methods of sperm preparation for in vitro fertilization were compared: the swim-up technique vs. the migration-sedimentation technique. The study comprised fresh semen samples obtained from 25 couples treated in the In Vitro Fertilization Unit. Oocytes aspirated in a single cycle were divided into two groups, each inseminated by sperm prepared by one of these techniques. Motility, degree of motility, and normal morphology were improved by both methods. The improvement was greater when the migration-sedimentation technique was applied. However, fertilization rate was significantly higher after the swim-up technique. In order to clarify this contradiction, an additional group of 26 semen samples was divided and then prepared by the swim-up or migration-sedimentation techniques. Sperm quality was examined up to 72 h after separation. Compared with the swim-up technique, sperm characteristics were better after separation by the migration-sedimentation technique. However, this difference abated after 24 h. The better results of the swim-up technique in the "survival experiment' may explain its improved performance in in vitro fertilization, despite lower separation capacity. Thus, the migration-sedimentation technique is not recommended for sperm preparation in in vitro fertilization.

Pentoxifylline improves sperm binding to the zona pellucida in the hemizona assay.

OBJECTIVE: To evaluate the effect of pentoxifylline on sperm binding capacity to zona pellucida (ZP) using the hemizona assay (HZA). DESIGN: The fertility potential of 82 men was evaluated by routine semen analysis. Each ejaculate was incubated with or without pentoxifylline (3 mM) in Ham's F-10 medium (Flow Laboratories, Irvine, Scotland) before the HZA. The effect of the pentoxifylline treatment on sperm-binding capacity to ZP was assessed by the hemizona index. RESULTS: The mean hemizona indexes with medium or pentoxifylline treatment were 23% +/- 2.1% (mean +/- SE) and 41% +/- 3.4%, respectively. Taking into consideration a significant change of the hemizona index on rising above the intra-assay coefficient of variation (+/- 8%) after pentoxifylline treatment, 73.1% of specimens improved, 19.5% deteriorated, and 7.4% remained unchanged. Using a threshold hemizona index of 23% as a discriminator between fertile and infertile specimens, 43.5% of the "pentoxifylline-improved" samples ascended to the fertile zone (> 23%). No correlations were found between sperm variables in the raw semen and the effect of pentoxifylline on sperm binding capacity. CONCLUSION: Pentoxifylline may improve the binding capacity of human spermatozoa. However, this effect is confined to a selected group of patients and cannot be predicted by the basic sperm variables. Thus, to avoid uncertain or damaging effects of pentoxifylline while preparing sperm suspension for assisted reproductive techniques, it is recommended that its effect be tested by the HZA system before its use.

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

The objective of the study was to evaluate the benefit of different sperm preparation methods by using the hemizona assay. A total of 58 men admitted to the male infertility clinic for evaluation were tested by routine semen analysis and hemizona assay. Five different techniques (swim-up, TEST-yolk buffer, Percoll, pentoxifylline and progesterone) were used for preparation of sperm suspensions. The effect of these treatments on the sperm-binding capacity using the hemizona assay was assessed. The routine swim-up preparation was used as the reference method. Of the four preparation methods, only the TEST-yolk buffer and pentoxifylline exhibited an overall statistically significant improvement in sperm-binding capacity in comparison with the swim-up preparation method (P = 0.01 and 0.001 respectively). Following preparation with Percoll and progesterone there was no change in the mean value of binding capacity, compared with swim-up. However, examination of the effect of the four treatments on each specimen individually yielded a diversity in the response, e.g. having the capability to enhance, damage or be ineffective in sperm binding capacity. The results support the conclusion that in-vitro sperm preparation methods can affect sperm binding to the zona pellucida. Since there is a diversity in the response of sperm samples to different treatments, the hemizona assay can be used in selecting the optimal sperm preparation method prior to its use for assisted reproductive techniques. This is advocated mainly for the 'male factor' group.

Advanced methods for evaluation of sperm quality.

Routine semen analysis includes measurements of sperm concentration, motility, and morphology. In our study, three additional tests were evaluated in relation to fertilization rate in an in vitro fertilization program: the freezing and hypo-osmotic swelling tests that evaluate the functional integrity and stability of the sperm membrane under extreme osmotic conditions, and migration sedimentation test that isolates high-quality motile sperm cells. The study was performed on semen delivered by men of couples treated at the In Vitro Fertilization Unit, and men who served as semen donors (fresh or cryopreserved semen). No correlation was found between the sperm fertilization rate, and the decrease in motility percent following the freezing-thawing process. Thus, the freezing test cannot be used to predict semen fertilization capacity. The hypo-osmotic swelling test was applied on semen given by a similar population of men. While the hypo-osmotic swelling test values carried out with fresh semen was found to have good correlation with fertilization, no correlation was found when frozen thawed semen was used. Post-migration sedimentation test sperm characteristics, and especially the recovery rate of the motile sperm, were significantly better in in vitro fertilization cycles with fertilizations. Both the hypo-osmotic swelling test and migration sedimentation test can assist in evaluating semen quality, judged by the fertilization rate in an in vitro fertilization program.

The effect of egg yolk on the binding capacity of human spermatozoa to zona pellucida.

OBJECTIVE: To evaluate the effects of Tes and Tris (TEST)-yolk buffer (TYB) incubation on sperm function. DESIGN: Sperm samples obtained from 61 patients attending the infertility clinic were incubated in TYB and control medium. Binding to the zona pellucida (ZP), evaluated by the hemizona assay, membrane functional integrity evaluated by the hypoosmotic swelling test, and acrosome reaction and penetration through cervical mucus were assessed to ascertain the effect of TYB treatment. RESULTS: The only substantial effect of TYB treatment was enhanced sperm binding to the ZP. The mean number of sperm cells attached to the hemizonae with TYB or medium treatment, was 42 +/- 6.1 (+/- SE) and 31 +/- 5.8, respectively (n = 61). However, this improvement was most obvious in 12 patients whose sperm samples exhibited a marked increase in sperm binding capacity following TYB treatment compared to medium without TYB, 71 +/- 5.6 and 12 +/- 2.7, respectively. In the other sperm samples (n = 49), sperm binding capacity was similar following TYB and medium treatment (both 35 +/- 6.0). CONCLUSIONS: A 2-hour incubation of sperm samples with TYB-containing medium (at room temperature) enhances the binding capacity of human spermatozoa in approximately 20% of patients considered to have male factor infertility. The basis of this selective effect has yet to be studied further.

The use of a seminal vesicle specific protein (MHS-5 antigen) for diagnosis of agenesis of vas deferens and seminal vesicles in azoospermic men.

Azoospermia is the cause of infertility in 8% of infertile male patients. Ten percent of those patients suffer from agenesis of the seminal vesicle (SV) and vas deferens (VD) agenesis. Currently, the diagnosis of SV and VD agenesis is based on low semen volume, low pH, and low fructose content of the seminal fluid of azoospermic men who have normal serum gonadotropins. In this study, an SV-specific sperm-coating antigen, the MHS-5 antigen, was used as a marker for the presence of SVs. The SV-specific protein (SVSP), MHS-5, was present in the control group but was not found in any of the seven samples from azoospermic men with proven agenesis of SV and VD. Another semen component, the prostate-specific antigen (PSA), whose presence in the semen is not influenced by the SV and VD agenesis, was found in both the study and the control groups. Its presence ruled out the possibility of azoospermia due to ejaculatory duct obstruction. The absence of MHS-5 antigen in seminal fluid can be used as a tool for a reliable diagnosis of agenesis of SV and VD in azoospermic men.

The hemizona assay is of good prognostic value for the ability of sperm to fertilize oocytes in vitro.

OBJECTIVES: To assess the prognostic value of hemizona assay (HZA) in predicting the success of IVF. DESIGN: Samples from 133 patients, who were referred for semen evaluation, were tested by HZA. Thirty samples were tested twice to assess interassay variation. Seventy couples were also referred for IVF. Results of HZA were compared with standard parameters of sperm quality, fertilization rates, and pregnancies. RESULTS: The intra-assay and interassay coefficient of variation were 8% and 14%, respectively. Hemizona assay results had the highest correlation with sperm morphology (r = 0.60). Of all parameters evaluated, fertilization rates were best predicted by hemizona index (HZI) (r = 0.75). The assay was found to have high sensitivity and specificity rates, at a threshold HZI of 23%. CONCLUSIONS: The HZA is a valuable prognostic test for IVF. With a threshold HZI of 23%, it has a good predictive value for fertilization rates in IVF, and may thus be used for patient preselection before IVF.

Management of bilateral nonpalpable testes: laparoscopic diagnosis and orchidectomy.

Bilateral nonpalpable testes in the adult human are associated with testicular malignancy, infertility and other abnormalities. Investigation for localization of the testes is mandatory, and either orchidopexy or orchidectomy is indicated. Laparoscopy was performed in an azoospermic male with bilateral unpalpable testes. A diagnosis of intra-abdominal testes was made and bilateral orchidectomy was performed. Laparoscopy is recommended for diagnosis and precise localization, as well as for orchidectomy, thus avoiding open abdominal operation in cases of intra-abdominal testes.

Retrograde ejaculation.

Retrograde ejaculation is an uncommon cause of male infertility. It should be suspected in any case of azoospermia, and might be congenital, acquired or idiopathic in origin. When pharmacological attempts to restore anterograde ejaculation fail, it is suggested that spermatozoa should be recovered from post-ejaculation urine to be applied in one of the modern techniques of assisted reproduction. The successful recovery of viable spermatozoa from the urine is dependent upon careful regulation of pH and osmolarity of the urine at the time of ejaculation. Careful handling of the retrieved spermatozoa enables isolation of sperm cells with good quality for insemination of ovulated oocytes (in vivo) or retrieved oocytes (in vitro).

Serum prolactin response to ether stress in diabetic rats: opiate system contribution.

Diabetes in streptozotocin-treated rats is associated with alterations in various neuroendocrine systems, including endogenous opioids. These changes are suggested to be responsible for the significant reduction in serum prolactin (PRL) response to a brief restraint stress in diabetic male rats, as compared with normoglycemic controls. The present study examines serum PRL response to ether exposure in diabetic male rats. The animals' response to ether stress, which is known to be related to the opioid system, was examined twice in each rat: shortly after cannula insertion (Day 1), and seven days later. In order to evaluate the opiate system involvement, the experiment was repeated on Day 1 and 7 after surgery in a group of rats which were pretreated with naltrexone (Nalt), an opioid receptor antagonist. Opioid receptor sensitization was also performed by prior acute morphine administration on Day 7 after cannulation surgery. Following adaptation to the cannulation, no difference in serum PRL response to ether stress was found between diabetic and normoglycemic rats. However, on Day 1 after surgery, a significant difference was found between the diabetic and control groups: the normoglycemic (control) group exposed to ether responded to the surgical stress by augmented serum PRL levels. This response was not recorded in the diabetic rats. Opioid receptor blockade by Nalt administration 30 min before ether exposure eliminated this difference. Opioid receptor sensitization by morphine pretreatment facilitated PRL secretion in normoglycemic rats exposed to either, while no effect could be distinguished in the diabetic group. It is therefore concluded that the streptozotocin-induced diabetic rats do not differ from normoglycemic ones in their ability to respond to acute ether stress by itself. However, enhanced PRL secretion induced by ether exposure under additional surgical stress, or by presensitization of the opioid receptors by morphine, is prevented in diabetic rats, probably due to diminished opioid receptor response.

Blood luteinizing hormone and prolactin concentrations in response to naltrexone challenge: studies on rats with diabetes induced by different doses of streptozotocin.

Opiate system involvement in diabetes induced by three different doses of streptozotocin (STZ; 40, 50, and 60 mg/kg body weight [BW]) was studied by monitoring luteinizing hormone (LH) and prolactin (PRL) levels as a response to naltrexone (Nalt) administration. After four weeks of diabetes a marked decrease in BW, as well as severe hyperglycemia and increased blood urea nitrogen (BUN) levels were found. The rats, whose diabetes was induced by 50 mg/kg of STZ, exhibited the highest amount of blood glucose (P < 0.05, compared with the 40 mg/kg induced group) and BUN levels (P < 0.004 compared with the other two groups) and BW loss. The normal response to Nalt, which is expressed by elevation of plasma LH and decreased plasma PRL levels was observed only in the low-dose STZ (40 mg/kg BW) diabetes-induced group, while in the other two diabetic groups (50 and 60 mg/kg BW) there was no significant change in plasma LH and PRL as a result of the Nalt challenge. Presensitization of the endogenous opioid receptors by morphine in normoglycemic (control) and "low-dose diabetic" rats (40 mg/kg BW of STZ), presented a clear difference between the two. Morphine pretreatment inhibited LH response to Nalt in the low-dose, STZ-induced diabetic rats, while no effect of morphine pretreatment on LH response to Nalt could be recorded in the normoglycemic group. Thus it can be concluded that in STZ-induced diabetes, plasma glucose and BUN levels do not reflect the neuroendocrine injury observed when monitored by LH and PRL secretion in response to Nalt challenge. Supersensitization of the opioid receptors before the Nalt challenge may increase the ability to reveal neuroendocrine system impairment.

[Hemizona assay for evaluating fertilizing capacity of human spermatozoa].

The hemizona assay (HZA), which tests the binding capacity of human spermatozoa to the zona pellucida, is described. This assay has very high sensitivity (86-100%) in predicting fertilizing capacity in the in vitro fertilization (IVF) system. It is recommended that it be carried out on semen samples before entering the IVF program. In addition, it can also help evaluate unexplained infertility and assess the quality of ova. The necessity for a control fertilized sample in each HZA might complicate the assay. To simplify the test, the use of sperm from the sperm bank was investigated. It was shown that fresh and frozen-thawed sperm have the same capacity to bind to the hemizonae. Thus, frozen donor semen preserved in the sperm bank can be used for controlling the HZA.

Experimental uremia in male rats: effect on the reproductive tract and fertility.

Chronic renal failure was induced in 3-month-old male rats by 5/6 nephrectomy. Potency and fertility studies were performed after 3 months of chronic uremia. The mean serum testosterone at the end of the experiments was significantly lower: 0.96 +/- 0.14 ng/mL compared to a control group of sham-operated male rats, 2.86 +/- 0.59 ng/mL, p < .001. All the uremic male rats had normal accessory gland weights at the end of the study. Fertility and, in most animals, sexual behavior and mating were not different from the normal control group. It is concluded that in 5/6-nephrectomized uremic male rats, in spite of low testosterone level, fertility and reproductive system are maintained similar to normal control male rats.

Migration sedimentation technique as a predictive test for the fertilizing capacity of spermatozoa in an in-vitro fertilization programme.

The migration-sedimentation technique (MST) has been proposed as a means of separating high quality motile spermatozoa. The present study was conducted in order to evaluate whether sperm performance following separation by MST predicts their fertilizing capacity in an in-vitro fertilization (IVF) programme. Ninety semen specimens were analysed for use in an IVF-embryo transfer (ET) programme. Each specimens was divided into two parts: one was processed in the IVF programme and was used after sperm swim-up separation for insemination of human ova. The other aliquot (0.2 ml) was separated by MST, and the sperm then characterized by their concentration, motility, degree of motility and morphology. Sperm characteristics after separation by MST were then correlated with the results of the IVF-fertilization rates. In 79 of 90 IVF-ET cycles, at least one oocyte was fertilized. All post-MST sperm characteristics were significantly higher in cycles with fertilizations compared to IVF cycles without fertilization. A larger percentage of the total motile spermatozoa were recovered after MST in semen specimens with fertilization, compared to semen specimens without fertilization (39.9 +/- 3.6 and 20.6 +/- 6.6%, respectively; P < 0.05). This value was correlated with the percentage of fertilized oocytes (r = 0.24; P < 0.02). More IVF cycles with fertilizations were recorded in cases in which the recovery of motile sperm was > 25% (P < 0.005), or when more than 1.5 x 10(6) motile spermatozoa were recovered after MST (P < 0.0001). As sperm characteristics after MST correlated significantly with their fertilizing capacity, the MST test could be used in evaluation of the fertilizing capacity of spermatozoa.

Fresh and frozen-thawed human sperm bind in a similar pattern to the zona pellucida in the hemizona assay.

This study evaluated the impact of sperm cryopreservation on sperm quality. The HZA was used to test the binding capacity of fresh as opposed to frozen-thawed sperm from 12 donors. Fresh and frozen-thawed sperm motility was 47% +/- 1.5% and 24% +/- 3.8% (mean +/- SE), respectively. However, the number of sperm cells attached to the hemizonae was 75 +/- 12.0 and 74 +/- 11.9, respectively. We conclude that cryopreservation results in a reduced number of motile sperm cells but does not adversely affect the ability of rescued sperm cells to bind to the ZP. The study also supports the use of frozen-thawed rather than fresh donor sperm for control in the HZA procedure.

Ca(2+)-independent induction of acrosome reaction by protein kinase C in human sperm.

We report that activated protein kinase C (PKC) can induce acrosome reaction independently of elevated Ca2+. Addition of 12-O-tetradecanoyl phorbol-13-acetate or the membrane-permeable diacylglycerol analog 1-oleoyl-2-acetylglycerol to ejaculated human sperm resulted in stimulation of acrosomal reaction (2- to 3-fold), provided the sperm underwent capacitation. Induction of acrosome reaction by 12-O-tetradecanoyl phorbol-13-acetate was blocked by the PKC inhibitor staurosporine or by down-regulation of endogenous PKC, but not by removal of extracellular Ca2+. Acrosome reaction was also enhanced by the Ca2+ ionophore ionomycin in a Ca(2+)-dependent, PKC-independent fashion. Immunohistochemical analysis with type-specific PKC antibodies revealed the presence of PKC alpha and PKC beta II in the equatorial segment, whereas PKC beta I and PKC epsilon staining was found in the principal piece of the tail. Acrosome reaction, thus far believed to be induced only by elevated Ca2+, can therefore be triggered by activated PKC in a Ca(2+)-independent fashion. The PKC subtypes potentially involved in acrosome reaction are most likely alpha and beta II, whereas the beta I- and epsilon-subspecies might be involved in regulation of flagellar motility of human sperm.

Cryptorchidism: incidence and sperm quality in infertile men.

In a population of 8500 men attending the andrology outpatient clinic, 200 men (2.35%) were recorded as having some disturbances with the descent of the testes into the scrotum. Medical history of the patients revealed that 51 underwent unilateral orchidopexy; 40 bilateral orchidopexy; and 24 were treated with human chorionic gonadotropin in order to induce descent of their testes. In addition, 6 patients reported spontaneous descent of the testes, and 13 others were found to be unilaterally cryptorchid upon physical examination. Results of semen analysis, hormonal profile, testes position, and testicular volume were compared to those of 105 proven fertile men. The major finding of this study shows that post-partum undescended testes suffer from primary Sertoli cell malfunction as reflected by elevated serum follicle stimulating hormone levels. Serum luteinizing hormone and testosterone levels were within the normal range. Surgical descent of the testes did not improve sperm production, proved by low sperm quality of all the study groups, compared to the cryptorchid group. Among the patients who were operated on, no correlation was found between age at operation and semen variables. All groups showed poor sperm quality which can be defined as oligoteratoasthenozoospermia. The degree of spermatogenic damage was in the following order of diagnosis or treatment: bilateral orchidopexy greater than cryptorchid testes greater than hormonal treatment greater than unilateral orchidopexy greater than late spontaneous descent of the testes. Thus, it is advisable to postpone surgical treatment of cryptorchidism and apply this only after a waiting period, and if the hormonal approach has failed to descend the testis.

Efficacy of varicocele embolization versus ligation of the left internal spermatic vein for improvement of sperm quality.

Efficacy of surgical varicocelectomy versus embolization of the spermatic vein was studied in 137 men diagnosed as suffering from left varicocele. The men were divided randomly into three groups according to the methods of treatment: A--embolization of the internal spermatic vein (51 men); B--Ivanissevich technique of high ligation of the spermatic veins (43 men); and C--Bernardi technique of high ligation (43 men). The groups were similar in terms of age, duration of infertility and possessed semen characterized as oligoteratoasthenozoospermia. The fertility of the female partners was evaluated carefully and they were found to be potentially fertile. Varicocele was diagnosed by at least two of the following methods: physical palpation during valsalva manoeuvre, venography, or scrotal scanning using the technetium pertechnetate radioactive method. Semen quality was assessed before treatment and at 3, 6 and 9 months post-treatment. Fecundity was followed-up for 18 months. The major results were: (i) Shrinkage of the varicocele was found in all three groups studied. The same rate of recurrence was recorded in the three groups (24%, 37% and 35% in groups A, B and C, respectively). (ii) Improvement of sperm quality was significant in groups A and B, with better results in group B. (iii) The pregnancy rate was significantly higher in group B, compared with A (38.2% vs. 20.6%; P less than 0.05). Thus, high ligation of the internal spermatic vein yields better results than low ligation or embolization as far as semen quality and pregnancy is concerned.

Improved fertilization rate in an in vitro fertilization program by egg yolk-treated sperm.

High fertilization and PRs have been achieved by using thawed donor sperm cryopreserved with medium containing EY. The aim of our study was to examine the possible effect of EY on the fertilizing capacity of fresh sperm in an IVF program. Preincubation of spermatozoa in EY for 2 hours at room temperature significantly improved fertilization (P less than 0.001) in couples who had low fertilization rates in previous cycles, whereas no effect was found concerning couples with high fertilization rates.

The predictive fertilization value of the hypoosmotic swelling test (HOST) for fresh and cryopreserved sperm.

BACKGROUND: The hypoosmotic swelling test (HOST) was recommended as a predictive test for in vitro fertilization (IVF) outcome. These results, however, were controversial and the results for thawed semen were insufficient. The present study was conducted in order to clarify the predictive value of the HOST for IVF in fresh and thawed sperm. METHODS: The hypoosmotic swelling test was performed in three groups: husband's fresh semen "subfertile" group, donor's thawed semen group, and donor's fresh semen "fertile" group. RESULTS: No correlation was found between HOST values and sperm characteristics in fresh or thawed sperm. Fresh sperm HOST values correlated with IVF fertilizations. No such correlations were found when thawed sperm was used. HOST values were significantly higher in the fresh fertile donor group than in the fresh subfertile group (P less than 0.001). Following the freezing and thawing process, HOST values decreased dramatically. Nevertheless, the fertilization rate was still higher compared to that of the fresh subfertile group (P less than 0.001). There were significantly more IVF cycles with no fertilizations when HOST values were below 45%. CONCLUSION: The HOST has a predictive value for fertilization of oocytes in IVF cycles when fresh semen, but not thawed sperm, is used. The freezing-thawing process affects the outer membrane of the spermatozoon and changes its characteristics, leading to a decrease in HOST values. Sperm characteristics that play a role in the fertilization process are not expressed directly by HOST values.