Ultramorphological sperm characteristics in the risk assessment of health effects after radiation exposure among salvage workers in Chernobyl.

We present a pilot study of individuals (liquidators) who were engaged in clean-up operations after the disaster at the nuclear power plant at Chernobyl in Ukraine. In the 10 years since the disaster, adverse health effects among exposed individuals have not been clearly defined. There is widespread fear of damage to the reproductive system, with implications for fertility problems and adverse effects on offspring. Bearing this in mind, methods to evaluate the potential for production of fertile semen have been applied using quantitative ultramorphological (QUM) analysis. QUM analysis examines the organization and integrity of sperm organelles by electron microscopy, using both transmission electron microscopy and scanning electron microscopy. Significant differences were observed between clean-up workers and controls of similar age regarding certain ultramorphological parameters of the sperm head. The results of this pilot study suggest that QUM analysis of human sperm is a feasible approach for evaluating the fertility potential of individuals who were exposed to ionizing radiation from the Chernobyl nuclear power plant accident.

Reduced semen quality caused by chronic abacterial prostatitis: an enigma or reality?

OBJECTIVE: To investigate whether there is an inter-relationship between chronic abacterial prostatitis and potential infertility. DESIGN: As part of the eligibility studies for hyperthermia treatment for chronic abacterial prostatitis patients, a large number of chronic prostatitis patients were referred to us from peripheral outpatient clinics. Sperm analysis was a routine portion of the eligibility studies. To exclude bacterial prostatitis, urine cultures, expressed prostatic secretion, and semen cultures were performed. The patient population was not differentiated on the basis of those suffering from either nonbacterial prostatitis or prostatodynia according to the commonly accepted classification. The control group was the laboratory normal standard group. SETTING: Normal human volunteers in an academic and clinical research environment. PATIENTS: The first group includes 86 patients suffering from long-standing (1 to 20 years) chronic abacterial prostatitis, according to the commonly accepted classification, with a mean age of 39.9 +/- 9.5 years. The second group includes 101 normal fertile men with a mean age of 31.4 +/- 5.5 years. INTERVENTIONS: The routine semen analysis performed included biochemical tests of seminal plasma, bacteriology, and light microscopy. MAIN OUTCOME MEASURE: The original hypothesis was based on a reduction in semen quality in these patients caused by chronic abacterial prostatitis. Measurements for sperm motility parameters, morphology characteristics, prostate markers, and white blood cells (WBC) were designed accordingly. RESULTS: Statistical comparisons of the two groups showed that several sperm motility parameters, morphology characteristics, prostate markers, and WBC are outside of the normal value ranges in the chronic abacterial prostatitis group. In addition, there is a correlation between the duration of the disease and two important sperm analysis variables: increased prostatic markers and appearance of sperm morphological defects. CONCLUSION: From the results obtained, the high incidence of secondary infertility in these patients may be explained.

The relationship between sperm ultrastructural features and fertilizing capacity in vitro.

OBJECTIVE: To evaluate the relationship between ultramorphological features of the human sperm and its fertilizing capacity in vitro. DESIGN: The study was performed retrospectively. Ultrastructural features were assessed using scanning and transmission electron microscopes in sperm samples of individuals who underwent an in vitro fertilization (IVF) treatment cycle no more than 6 months before the study. SETTING: Institutional clinical care. PATIENTS: Fifty-six infertile couples in whom mechanical infertility was diagnosed in the female partner. Patients were categorized as fertilizing when fertilization of at least 30% of the oocytes occurred (n = 27) and nonfertilizing when none of the oocytes fertilized in at least two consecutive IVF treatment cycles (n = 29). RESULTS: The two groups differed significantly only in ultramorphological parameters of the sperm head and acrosome (head, F(8,36) = 2.8, P less than 0.02; acrosome, F(4,40) = 2.8, P less than 0.04), and especially in the following malformation patterns: hyperelongated head, acrosome deficiency, and acrosome damage. The suggested score based on these findings was able to predict 90% and 76% of the cases with and without fertilizing potential, respectively. CONCLUSION: The ultrastructural morphology of the sperm head components is a key parameter for assessing the sperm fertilizing capacity in vitro.

Sperm quality of subfertile males before and after treatment with human follicle-stimulating hormone.

OBJECTIVE: To assess the potential of short-term systemic administration of FSH for improving sperm quality, including ultrastructure, in teratozoospermic patients having normal endocrine profiles. DESIGN: Semen parameters were assessed prospectively using light microscopy (LM), biochemical analysis, and quantitative ultramorphological analyses within 2 months before FSH administration and within 5 days after the end of treatment. SETTING: Samples were collected from patients who were referred to the male fertility clinic at Bar-Ilan University. PATIENTS: Thirty-one patients with teratozoospermia who exhibited normal hormonal profiles and who failed to fertilize their wives in at least two previous IVF attempts (n = 17) or who had wives with apparent normal fertility unable to conceive for > or = 5 years (n = 14) were classified as subfertile. One hundred one males with no previous history of infertility, whose wives conceived after < or = 12 months of pregnancy expectation, served as the control group. INTERVENTION: Treatment was 75 IU FSH administered daily for 30 days. MAIN OUTCOME MEASURES: Pretreatment and post-treatment sperm evaluation of basic and quantitative ultramorphological analyses parameters. The hypothesis was FSH treatment may improve spermatid morphogenesis by its multiple actions on the Sertoli-gamete cell compartment without interfering with the testicular hormonogenic function. RESULTS: A significant improvement in agenesis of the acrosome and in the amorphous heads was observed, reaching normal values after treatment with FSH. The axonema deteriorated. No significant changes were observed in basic semen analysis parameters. CONCLUSIONS: Because malformations of the fine structure of the sperm head subcellular organelles seem to be prerequisites for the success of FSH treatment, ultramorphological examination of the sperm may serve as an indication for the probability of success of this treatment.

Ultrastructural changes in sperm with improved fertilizability after treatment with egg yolk.

OBJECTIVES: To determine the existence of differences in the ultrastructural parameters between the medium-washed sperm samples of the affected and nonaffected egg yolk (EY) groups and to verify whether ultrastructural changes occur in the EY-affected spermatozoa after EY preincubation. SETTING: The study was performed in the Laboratory of the IVF Unit, Serlin Maternity Hospital, and the Laboratory of Male Fertility, Bar-Ilan University, Ramat Gan, Israel. PATIENTS: The positive group included 12 males who underwent 1.9 IVF cycles with 0% fertilization rate that increased to 68% after EY treatment. The negative group included 11 males with 1.2% fertilization rate in 1.1 IVF cycles with no improvement after preincubation of spermatozoa in EY. RESULTS: Compared with the laboratory standard, patients of both groups exhibited a lower normalcy of the head sperm cell subcellular organelles. With EY treatment, the positive group exhibited a decrease in the frequency of some sperm head organelle specific malformations. CONCLUSIONS: Fertilization capacity of mature spermatozoa might be reduced because of an excess of acrosome malformations, postacrosomal lamina, and chromatin caused by in vitro sperm manipulations. The manipulation effect may be avoided by EY treatment.

Follicle-stimulating hormone treatment for men with idiopathic oligoteratoasthenozoospermia before in vitro fertilization: the impact on sperm microstructure and fertilization potential.

OBJECTIVE: To assess the effect of FSH on sperm fertilization potential and sperm intracellular structure in men with oligoteratoasthenozoospermia and a proven low fertilization rate in IVF. DESIGN: Prospective, randomized, partial crossover study. SETTING: IVF Unit, Golda Campus, Rabin Medical Center, Petah Tikva, Israel. PATIENT(S): Forty normogonadotropic, normogonadal men with oligoteratoasthenozoospermia and at least one previous IVF attempt in which fertilization failed or the fertilization rate was <30%. INTERVENTION(S): The men were randomly assigned to treatment with daily injections of 75 IU of FSH or 150 IU of FSH for at least 60 days before IVF treatment. A control group of men underwent an IVF cycle without treatment and then were randomly assigned tojoin group 1A or 1B for an additional IVF cycle with treatment. MAIN OUTCOME MEASURE(S): LH, FSH, and testosterone levels during FSH treatment, evaluation of ultramorphologic changes in sperm by electron microscopy, and comparison of fertilization rates in the control and study groups. RESULT(S): After treatment with 75 IU or 150 IU of FSH, the mean fertilization rates were 19.7% and 20.5%, respectively, compared with a 5.8% fertilization rate in the study control cycles. CONCLUSION(S): Prolonged treatment with FSH results in a significant increase in fertilization rates. This effect may be related to improvements in subcellular components of the sperm.

How to improve IVF-ICSI outcome by sperm selection.

In previous studies, a new IVF method of intracytoplasmic morphologically selected sperm injection (IMSI) was introduced, based on motile sperm organellar morphology examination (MSOME). It was concluded that microinjection of morphologically selected sperm cells with strictly normal nucleus, defined by MSOME, improves IVF-ICSI outcome. The aim of the present study was to confirm this conclusion in new, enlarged study groups. Comparison between 80 couples, who underwent an IVF-IMSI trial, with matched couples, who underwent a standard IVF-ICSI procedure, confirmed that pregnancy rate following IVF-IMSI was significantly higher, and abortion rate significantly lower than in the routine IVF-ICSI (60.0 versus 25.0%, and 14 versus 40% respectively, P

Transmission electron microscope morphogram of progressive motile sperm cell fraction separated from teratozoospermic human semen.

Teratozoospermic semen specimens were separated, with the aid of a specially designed separation chamber, into an upper progressive motile sperm fraction and a lower fraction containing a high percentage of sperm cells of poor or no motility, and immature germinal cells. Analysis of the TEM morphograms of progressive motile cell fractions and original teratozoospermic semen revealed that 15 various malformations were improved significantly in the motile cell population due to the cell separation technique.

Morphological characterization of abnormal human spermatozoa using transmission electron microscopy.

A morphological analysis of ejaculated human abnormal spermatozoa is presented. Eighteen different malformations are characterized, based on (a) the cell's organelle where they appeared, (b) the possible developmental stage when they occurred, and (c) the possible mechanism responsible for a specific appearance. The forces that govern the shaping of the spermatozoan head are described in detail. The possible mechanism responsible for chromatin subcondensation, acrosome malformation, kinked tail forms, and mitochondrial malformations are discussed.

Interaction between membrane properties and proteins synthesis in reticulocytes - a two step inhibition of protein synthesis by valinomycin.

In this work we consider the differential effect of Valinomycin used at different concentrations both on the protein synthesis of reticulocytes and on 42K exchange. We demonstrate that there is a two step action of this antibiotic. At 10(-6)M and below the drug has no effect on the 42K exchange, but it stops, however reversibly, protein synthesis. At 10(-5)M the drug has a very sharp action on the 42K exchange and stops protein synthesis in an irreversible way. Ribosomal population checked by two ways, sucrose gradient and direct counting on E.M. sections shows that at low concentrations of Valinomycin (10(-8)M to 10(-6)M) there is no breakdown of the polysomes which can be detected by either one of these methods. On the contrary, after short incubation with 10(-5)M of Valinomycin the breakdown of ribosomes is very clear, as evidenced by sucrose gradient analysis. By direct ribosomes clusters counting on E.M. sections this breakdown is seen only after long incubation.

Effect of acupuncture on sperm parameters of males suffering from subfertility related to low sperm quality.

The aim of this prospective controlled study was to assess the effect of acupuncture on the sperm quality of males suffering from subfertility related to sperm impairment. Semen samples of 16 acupuncture-treated subfertile patients were analyzed before and 1 month after treatment (twice a week for 5 weeks). In parallel, semen samples of 16 control untreated subfertile males were examined. Two specimens were taken from the control group at an interval of 2-8 months. The expanded semen analysis included routine and ultramorphological observations. The fertility index increased significantly (p < or = .05) following improvement in total functional sperm fraction, percentage of viability, total motile spermatozoa per ejaculate, and integrity of the axonema (p < or = .05), which occurred upon treatment. The intactness of axonema and sperm motility were highly correlated (corr. = .50, p < or = .05). Thus, patients exhibiting a low fertility potential due to reduced sperm activity may benefit from acupuncture treatment.

Quantitative ultramorphological (QUM) analysis of human sperm: diagnosis and management of male infertility.

The advantages of quantitative ultramorphological (QUM) sperm analysis in the diagnosis and treatment of male infertility are presented. QUM methodology is based on three elements: (1) complementary SEM and TEM observations of 7 sperm cell subcellular organelles: acrosome, postacrosomal lamina, nucleus, neck, axoneme, mitochondrial sheath, and outer dense fibers; (2) systematic classification of the specific ultramorphological malformations into 4 pathological and the normal categories, which indicate the morphological state of each subcellular organelle; and (3) comparison between well-defined reference groups with opposite fertility status or treatment conditions. QUM analysis has enabled the establishment of two indices that optimally express the in vivo and in vitro male fertility potential: The Natural Fertility Index (NFI), which allowed an accurate prediction (97% sensitivity and 90% specificity) of 80% of the naturally fertile and suspected infertile male patients, and the in vitro fertilization (IVF) score, which enabled prediction of 76% of the nonfertilizing and 90% of the fertilizing IVF groups. Validation tests confirmed these data. QUM also enabled assessment of ultramorphological indications for varicocele and radiation exposure: Both male factor etiologies indicated a persistent effect on the natural fertility potential, as expressed by structural changes in the nucleus. Varicocele was found to cause defects in the sperm head organelles related to early spermatid development, whereas ionizing radiation resulted in amorphous head shape. Criteria for specific non-in vitro therapeutic interventions such as varicocelectomy, follicle-stimulating hormone (FSH) administration, and acupuncture treatment were established. A varicocele index, which enabled the correct classification of 79 and 89% of the patients pre- and post-high ligation, respectively, was suggested to be a good indicator for varicocele which affects the fertility potential. Males exhibiting idiopathic impairment of sperm acrosome and nucleus were found to be potential responders to FSH treatment, whereas patients exhibiting low sperm activity proved to be good candidates for acupuncture treatment. Indications for selecting the optimal appropriate assisted reproduction technique (ART) procedure were found: Patients with a low Natural Fertility Index should be recommended for ART. A first choice ART selection should be performed according to an ART index based on the ultramorphological examination of the tail axoneme. The above index enabled correct prediction of 78% of the patients who achieved pregnancy following conventional ART (intrauterine insemination or IVF) and 74% of those whose wives conceived only following intracytoplasmic sperm injection. QUM sperm analysis is clinically informative, nontraumatic, and in the long run also cost-effective. This analysis should be performed when the male infertility factor cannot be clearly diagnosed by routine tests and prior to the first ART trial.

Sperm ultramorphology as a pathophysiological indicator of spermatogenesis in males suffering from varicocele.

Varicocele of spermatic veins is considered to be one of the major causes of male infertility associated with reduction of sperm quality. The pathophysiology of this condition is not yet completely understood. The aim of this study was to shed light on the pathophysiology of varicocele by identifying semen parameters, especially sperm ultramorphology, which improve following high ligation of the spermatic vein. Seventy-five males with diagnosed varicocele were included in this study. Semen parameters were assessed prospectively using light microscopy, semen biochemistry and sperm quantitative ultramorphological analysis, before high ligation and 3-9 months after high ligation. The control group consisted of twenty-five untreated varicocele patients who underwent two semen examinations within 3-9 months. No statistical difference in any of the examined variables was found between the two examinations in the control group. The treated patients exhibited a significant improvement in sperm density, progressive motility, percentage of normally formed spermatozoa, agenesis of sperm acrosome, chromatin condensation and incidence of amorphous heads compared with the pretreatment condition (P < or = 0.01). In contradiction, no significant improvement was observed following treatment in any of the sperm tail subcellular organelles. It is concluded that varicocele may cause deleterious alterations in early spermatid head differentiation during spermiogenesis and that varicocele patients with a high incidence of sperm acrosome and nucleus malformations are appropriate candidates for varicocele correction.

Relation of human sperm acrosin activity and fertilization in vitro.

The aim of the study described here was to determine the possible contribution of the acrosin activity test to routine semen analysis in enhancing the precision of the prognosis of IVF success in a group of patients in which the contribution of the egg factor to infertility was ruled out (20 cases) compared to a control IVF group (39 cases). Semen analysis, acrosin activity and acrosome ultrastructure were determined for all semen samples. The group with high fertilization rates was comprised of normozoospermic patients while the group with low fertilization rates was comprised of astheno-teratozoospermic patients. The mean acrosin level of the positive IVF group was significantly higher than that of the negative group (51.7 +/- 33.2 and 28.6 +/- 13.7, respectively). Two parameters: per cent motile spermatozoa and acrosin level, were found to have a significant positive correlation with subsequent successful IVF (r = 0.36, P < 0.006; r = 0.37, P < 0.004, respectively); and agenesis of the acrosome was found to have a significant negative correlation (r = -0.33, P < 0.01). The ability of these parameters to correctly predict fertilization success was 59%, with 5% false positive, among which 15.4% was predicted solely by the acrosin level (above 54 microIU 10(6) cells-1) and 23% solely by per cent motile spermatozoa (above 50%). Abnormalities of the acrosome ultrastructure did not contribute further to the correct classification. The apparent clinical benefit of the acrosin level test is discussed.

Quantitative ultramorphological analysis of human sperm: fifteen years of experience in the diagnosis and management of male factor infertility.

The advantages of quantitative ultramorphological (QUM) sperm analysis in the diagnosis and treatment of male infertility are presented. The QUM methodology is based on three elements: complementary scanning electron microscopy and transmission electron microscopy observations of 7 sperm cell subcellular organelles (acrosome, postacrosomal lamina, nucleus, neck, axoneme, mitochondrial sheath, and outer dense fibers); systematic classification of the specific ultramorphological malformations into 4 pathological and the normal categories, indicating the morphological state of each subcellular organelle; and comparison between well-defined reference groups with opposite fertility status or treatment conditions. QUM has established 2 indices for the in vivo and in vitro male fertility potential: (1) Natural Fertility Index (NFI), with accurate prediction (97% sensitivity and 90% specificity) of 80% of the male patients; and (2) IVF score, with prediction of 76% of the nonfertilizing and 90% of fertilizing IVF groups. QUM has enabled assessment of ultramorphological indications for varicocele and radiation exposure. Varicocele causes defects in sperm head organelles related to early spermatid development, whereas ionizing radiation causes amorphous head shape. QUM established criteria for specific non-in-vitro therapeutic interventions, including varicocelectomy, follicle-stimulating hormone (FSH) administration, and acupuncture. The varicocele index enabled correct classification of 79 and 89% of patients with and without varicocele. Males with idiopathic impairment of sperm acrosome and nucleus are potential responders to FSH treatment, whereas patients exhibiting low sperm activity are candidates for acupuncture treatment. Patients with a low Natural Fertility Index are recommended for an assisted reproduction technique (ART). based on the ultramorphology of the tail axoneme. Patients who achieved pregnancy following intrauterine insemination or in vitro fertilization and those whose wives conceived only following intracytoplasmic sperm injection were classified with accuracy of 78 and 74%, respectively. QUM sperm analysis is clinically informative, nontraumatic, and cost-effective, and is recommended when the male infertility factor cannot be clearly diagnosed by routine tests prior to first ART trial.

Estimating fertility potential via semen analysis data.

The aim of this study was to evaluate diagnostic profiles for the assessment of semen analysis data with respect to male fertility potential. Semen samples taken from 208 patients of known fertility and suspected infertility were studied by conventional semen analysis methods. The data throw doubt upon the validity of an approach based on the number of deviations from the normal standard values defined by the World Health Organization. The alternative approach of a specific semen characteristic (particularly morphology) as the major predictor of fertility produced no beneficial results. However, the semen analysis index based on semen volume, sperm count, percentage motility and normal forms resulted in a high accuracy of classification but for only 44% of the cases, with 3% false negatives and 10% false positives using cut-off indices of > or = 0.6 and < or = -1.0 for defining 'fertile' and 'infertile' zones, respectively. In conclusion, it is emphasized that there are a number of specific semen analysis variables, each expressing a different aspect of male fertility potential which, when combined in correct proportion, do provide the optimal evaluation of the male fertility status. However, in order to increase the prognostic potential of the semen sample, new and meaningful parameters must be discovered.

Improved diagnosis of male fertility potential via a combination of quantitative ultramorphology and routine semen analyses.

The aim of this study was to develop a new male fertility diagnostic profile based on quantitative ultramorphology parameters and to determine the contribution of this profile to the enhancement of the routine semen analysis index reported previously. Semen samples from 208 males of known fertility and suspected infertility were evaluated for the ultrafine structure of the following sperm cell organelles: acrosome, post-acrosomal lamina, nucleus, neck, axonema, mitochondrial and fibrous sheaths. For each of these organelles, four pathological states (agenesis, incomplete genesis, malformation and degradation) and an intact state were defined. A quantitative ultramorphology index based on the incidence of intact nucleus, acrosome and fibrous sheath malformations enabled high accuracy in the classification (97% sensitivity and 90% specificity) of 74% of the cases. A combined semen quality index based on a proportional combination of the semen analysis and quantitative ultramorphology indices was found to increase the percentage of cases classified correctly to 80%. It was proposed that semen specimens of males whose fertility status cannot be predicted clearly using routine semen analysis should be fixed and sent for quantitative ultramorphology analysis to specialized laboratories so that their fertility potential can be determined more accurately using the semen quality index.

Does acupuncture treatment affect sperm density in males with very low sperm count? A pilot study.

Classic therapies are usually ineffective in the treatment of patients with very poor sperm density. The aim of this study was to determine the effect of acupuncture on these males. Semen samples of 20 patients with a history of azoospermia were examined by light microscope (LM) and scanning electron microscope (SEM), with which a microsearch for spermatozoa was carried out. These examinations were performed before and 1 month after acupuncture treatment and revealed that the study group originally contained three severely oligoteratoasthenozoospermic (OTA), two pseudoazoospermic and 15 azoospermic patients. The control group was comprised of 20 untreated males who underwent two semen examinations within a period of 2-4 months and had initial andrological profiles similar to those of the experimental group. No changes in any of the parameters examined were observed in the control group. There was a marked but not significant improvement in the sperm counts of severely OTA males following acupuncture treatment (average = 0.7 +/- 1.1 x 10(6) spermatozoa per ejaculate before treatment vs. 4.3 +/- 3.2 x 10(6) spermatozoa per ejaculate after treatment). A definite increase in sperm count was detected in the ejaculates of 10 (67%) of the 15 azoospermic patients. Seven of these males exhibited post-treatment spermatozoa that were detected even by LM. The sperm production of these seven males increased significantly, from 0 to an average of 1.5 +/- 2.4 x 10(6) spermatozoa per ejaculate (Z = -2.8, P < or = 0.01). Males with genital tract inflammation exhibited the most remarkable improvement in sperm density (on average from 0.3 +/- 0.6 x 10(6) spermatozoa per ejaculate to 3.3 +/- 3.2 x 10(6) spermatozoa per ejaculate; Z = -2.4, P < or = 0.02). Two pregnancies were achieved by the IVF-ICSI procedure. It is concluded that acupuncture may be a useful, nontraumatic treatment for males with very poor sperm density, especially those with a history of genital tract inflammation.

Separation of sub-populations of sperm with higher fertility potential from normal and pathological semen by peanut agglutinin.

Incubation of human sperm from semen with counts below 20 x 10(6)/ml with peanut agglutinin (PNA) resulted in agglutination of about 70% of sperm. An inverse correlation was found between non-agglutinating (PNA) sperm and sperm density (r = 0.48, p less than 0.01) and a direct correlation with acrosome-damaged sperm (r = 0.83, p less than 0.001). Binding of 125I-PNA to sperm revealed high, and possibly also low affinity binding sites on sperm from both normozoospermic and oligozoospermic origins. Quantitative transmission electron microscopy analysis revealed that agglutinated, PNA+, sperm had lower frequency of acrosomeless spermatozoa than PNA(-)-sperm (44% versus 77%) and higher fertility score (+0.18 +/- 0.1 and -1.5 +/- 0.87 respectively, p less than 0.01). Removal of PNA- sperm from oligozoospermic semen may increase the fertility score of the remaining sperm.