Serum pattern of circulating soluble receptor of leptin throughout the menstrual cycle.

AIM: The aim of the study was to investigate the pattern of circulating soluble receptor of leptin (sLeptinR) during the menstrual cycle and the association of sLeptinR to leptin, sex hormones and gonadotropins. METHODS: Fasting blood samples were collected on alternate days throughout a full cycle from fifteen healthy volunteers. Immunoenzymatic assays were employed to record the relevant levels. RESULTS: sLeptinR concentrations throughout the cycle were found to vary negligibly. No significant correlations between sLeptinR and leptin, gonadotropins or progesterone, were established. During the follicular phase, subjects presenting with higher estradiol levels tended to have higher sLeptinR concentrations. CONCLUSION: The little variation of sLeptinR concentrations during the menstrual cycle indicates that the rise of leptin during the luteal phase implies an increase of its bioactivity.

Chromatin decondensation of human sperm in vitro and its relation to fertilization rate after ICSI.

The inability of sperm chromatin to decondense has been implicated in the failure of fertilization, This study was undertaken to identify the relationship between sperm chromatin decondensation in vitro after incubation with follicular fluid at various points in time and fertilization or pregnancy rates after intracytoplasmic sperm injection. Moreover, an attempt was made to determine whether this test could be used as a predictive test for the outcome of ICSI. Thirty-two infertile couples undergoing ICSI therapy were included in this prospective study. One milliter of semen from each sample was mixed with 1 mL of follicular fluid obtained from ICSI patients at the time of oocyte retrieval and incubated for 24 h. Many smears were made directly after semen liquefaction at the following time intervals: 30, 60, and 120 min and 24 h. Chromatin decondensation was evaluated with acridine orange staining. The mean percentage of uncondensed chromatin of spermatozoa in the native semen samples was 25 +/- 18.3%, which increased within 24 h to 91 +/- 9.5%. On the other hand, the fertilization and ongoing pregnancy rates were 64 +/- 21.7% and 20%, respectively. However, no correlations were found between chromatin decondensation at various point of time (30, 60, and 120 min and 24 h) and fertilization rate. No correlation was shown between the chromatin decondensation and sperm counts in the ejaculate. morphology, or the percentage of condensed chromatin. In light of this study, chromatin decondensation in vitro cannot be recommended for predicting the fertilization potential of spermatozoa and pregnancy rates in the ICSI program. Further research is necessary, especially in cases where ICSI is being considered as a therapeutic option.

Evaluation of cryoinjury of spermatozoa after slow (programmed biological freezer) or rapid (liquid nitrogen vapour) freeze-thawing techniques.

PURPOSE: This study was initiated to determine the negative effect (cryodamage) on human spermatozoa after freeze-thawing and to find out whether freezing of spermatozoa with a computerized biological freezer is more advantageous than freezing above static liquid nitrogen vapour with regard to spermatozoa vitality, chromatin normality, morphology, and membrane integrity. METHODS: Forty-four semen samples were obtained from patients attending andrology laboratory, and each sample was divided into two aliquots. One aliquot was frozen using static liquid nitrogen vapour (G.II) and the second with a computerized biological freezer (G.III). Acridine orange was used for assessment of chromatin cryoinjury, whereas the morphology was evaluated according to WHO criteria. Hypoosmotic swelling test was used to identify membrane integrity and eosin-nigrosin staining was used to determine the vitality of spermatozoa. RESULTS: The mean percentage of normally condensed chromatin in the native semen sample (G.I) decreased significantly (p < .001) after freeze-thawing by using either liquid nitrogen vapour (G.II), or a biological freezer (G.III), which was significantly higher (p < .001) after freezing with liquid nitrogen vapour than after freezing with the biological programmed freezer. Morphologically normal spermatozoa decreased significantly (p < .001) in both freezing methods in comparison to the native semen samples. In addition, membrane integrity of spermatozoa (HOS-test positive) was significantly lower (p < .001) after the freeze-thawing procedure in G.II and G.III compared to G.I. In both these parameters the deterioration was similar among the two freezing procedures. Finally the mean percentage of live spermatozoa decreased significantly (p < .001) in both freezing techniques in relation to the mean value in the neat semen samples. CONCLUSIONS: Freeze-thawing procedure has a detrimental effect on chromatin, morphology, membrane integrity, and vitality of human spermatozoa not only by freezing above static liquid nitrogen vapour but even by using a computerized biological freezer. However, the chromatin deterioration rates are significantly higher by freezing above static liquid nitrogen vapour in comparison to freezing with a programmed biological freezer. Therefore, we recommend the use of this technique for freezing semen especially when ICSI technique is considered as the main therapeutic procedure.

Can maturation arrest occur at the stage of spermiogenesis?

This article attempts to clarify the pathological condition during which the maturation of the germinal epithelium is unable to evolve beyond a certain stage and is characterized as maturation arrest. Emphasis is given to the histological entity named spermiogenic arrest.

Comparison between computerized slow-stage and static liquid nitrogen vapour freezing methods with respect to the deleterious effect on chromatin and morphology of spermatozoa from fertile and subfertile men.

The purpose of this study was to determine the negative effects (cryodamage) on human spermatozoa after freeze-thawing and to determine whether freeze-thawing of spermatozoa with a programmed slow freezer is better than freezing with liquid nitrogen vapour (rapid freezing) with regard to alterations in sperm chromatin and morphology in semen from fertile (donor) and subfertile, IVF/ICSI, patients. Ninety-five semen samples were obtained either from patients attending our IVF unit for treatment (n=34) or from donors (n=25) with proven fertility and normal sperm quality according to WHO guidelines. Each semen sample was divided into two parts after liquefaction and addition of the cryoprotectant. The first part was frozen using a programmed biological freezer and the second part was frozen by means of liquid nitrogen vapour. Smears were made before the freezing and after the thawing procedure to assess morphology (strict criteria) and chromatin condensation (Acridine Orange test). The mean percentage of chromatin condensed spermatozoa in the samples from donors (control group) was 92.4 +/- 8.4% before freezing and this decreased significantly (p < 0.0001) to 88.7 +/- 11.2% after freeze-thawing with the computerized slow-stage freezer and to 87.2 +/- 12.3% after using static liquid nitrogen vapour (p < 0.001). The corresponding values for semen obtained from patients was 78.9 +/- 10.3% before freezing which decreased to 70.7 +/- 10.8 and 68.5 +/- 14.8%, respectively (p < 0.001). On the other hand, the mean percentage of normal sperm morphology in the control group decreased from 26.3 +/- 7.5% before freezing to 22.1 +/- 6.4% (p < 0.0001) after thawing with the computerized slow-stage freezer and to 22.2 +/- 6.6% (p < 0.0001) after the use of static liquid nitrogen vapour. In the patient group, the mean percentage of normal morphology decreased from 11.7 +/- 6.1% after freezing with the biological freezer to 9.3 +/- 5.6% and to 8.0 +/- 4.9% after freezing with static liquid nitrogen vapour. This study demonstrates that chromatin packaging and morphology of human spermatozoa decrease significantly after the freeze-thawing procedure, not only after the use of static liquid nitrogen vapour but also after the use of a computerized slow-stage freezer. However, the chromatin of semen samples with normal semen parameters (donor sperm) withstand the freeze-thaw injury better than those with low quality semen samples. Therefore, the computerized slow stage freezer could be recommended for freezing of human spermatozoa, especially for subnormal semen samples, for example, ICSI and ICSI/TESE candidates and from patients with testicular tumours or Hodgkin's disease, in order to avoid further damage to the sperm chromatin structure.

Acrosome reaction: methods for detection and clinical significance.

The present article reviews the methods for detection and the clinical significance of the acrosome reaction. The best method for the detection of the acrosome reaction is electron microscopy, but it is expensive and labour-intensive and therefore cannot be used routinely. The most widely used methods utilize optical microscopy where spermatozoa are stained for the visualization of their acrosomal status. Different dyes are used for this purpose as well as lectins and antibodies labelled with fluorescence. The acrosome reaction following ionophore challenge (ARIC) can separate spermatozoa that undergo spontaneous acrosome reaction from those that are induced, making the result of the inducible acrosome reaction more meaningful. Many different stimuli have been used for the induction of the acrosome reaction with different results. The ARIC test can provide information on the fertilizing capability of a sample. The ARIC test was also used to evaluate patients undergoing in vitro fertilization since a low percentage of induced acrosome reaction was found to be associated with lower rates of fertilization. The cut-off value that could be used to identify infertile patients is under debate. Therapeutic decisions can also be made on the basis of the value of the ARIC test.

Sex hormone binding globulin inhibits strongly the uptake of estradiol by human breast carcinoma cells via a deprivative mechanism.

A controversy exists for many years about the role of sex hormone binding globulin (SHBG) in the uptake of estradiol by the cells. Using the estradiol-sensitive human breast carcinoma cell line MCF-7 and SHBG isolated from human serum by a new method, we observed a strong inhibition of estradiol uptake. The inhibition was higher when the concentration of the hormone was low. On the other hand, there seemed to be a lag period in inhibition when the concentrations of SHBG were very low, followed by an exponential increase, when the concentration exceeded a critical value. The inhibitory activity was higher when SHBG was added before or along with estradiol in the cell culture, as well as when the incubation period was elongated, while was dramatically minimized by the presence of dihydrotestosterone. Despite the inhibition of estradiol uptake caused by SHBG, the distribution of the hormone in various cell components remained practically the same. In conclusion, all indications from experimental data seem to suggest a simple deprivative mechanism being responsible for the inhibitory activity of SHBG on estradiol uptake by MCF-7 cells in culture.

Nonlinear binding of sex steroids to albumin and sex hormone binding globulin.

We have studied the binding of sex steroids to albumin and sex hormone binding globulin (SHBG) using gel filtration chromatography for the separation of the bound from the free fraction of the steroid. It was found that estradiol binds to the globulin and albumin in a nonlinear manner: a lag period of binding was observed at low concentrations of the proteins, followed by an exponential increase of the bound hormone as the protein concentration increased. The same was observed with dihydrotestosterone (DHT) and albumin but not with globulin. In the presence of a constant concentration of albumin, the increase of SHBG concentrations resulted in a rapid transfer of estradiol from albumin to globulin while the transfer of DHT was moderate. When whole serum was used, the increase of its amount again resulted in the transfer of estradiol from albumin to globulin. Our study showed that a substantial increase of globulin-bound hormone can occur, following small variations of the protein. This offers obvious advantages to the organism, by saving energy, material and time and plays a basic role in estradiol transfer from albumin to the much more biologically active globulin.