Amnon Makler

A New Multiple Exposure Photography Method for Objective Human Spermatozoal Motility Determination

A new method for objective spermatozoal motility determination is described. In this method, spermatozoa from undiluted specimens are always examined under the same standard conditions by inserting them into a new counting chamber 10xa0μm thick. This constant thickness allows free, frictionless, horizontal movement of the spermatozoa to be seen clearly in one focal plane when microscopically observed. Using a phase-contrast microscope, these samples are photographed with a still camera and a multiple exposure technique, where six short light pulses are given during 1xa0second with the aid of a mechanical stroboscope. As a result, nonmotile spermatozoa appear 6 times brighter than do motile spermatozoa. The latter are seen as six-ringed chains; their length and shape describe the distance traveled by the spermatozoa during 1xa0second. From these pictures it is possible to obtain the exact percentage of motility, individual and average speed (expressed as microns per second), frequency of distribution of spermatozoal speed, and spermatozoal concentration. By statistical evaluation it was shown that a high degree of accuracy can be obtained from analyzing about four to six pictures, or 300 to 400 spermatozoa in each sample. It was also found that, when this objective motility determination was compared with subjective determination, human judgment tended to overestimate motility–about 20% more than did the objective method. This inexpensive, easy to perform, and highly informative technique can be of great value in the routine clinical work-up and in research studies. The average sperm speed may become a new index in any routine semen analysis.

Factors affecting sperm motility. vii. sperm viability as affected by change of ph and osmolarity of semen and urine specimens

The effects of pH and osmolarity of semen and urine specimens on motility and velocity of human spermatozoa were studied objectively with the aid of the multiple exposure photography (MEP) method. The pH of fresh ejaculates ranged from 7.2 to 8.2 and specimens were slightly hyperosmotic ranging between 300 to 380 mOsm/kg. Gradually changing the pH and osmolarity to either side of normal values led to progressive loss of sperm motility. However, sperm velocity was slightly increased by mild alkalinization and hyperosmolarity. Spermatozoa that became immobilized by acidification regained their motility shortly after pH was restored to normal values. In the majority of instances spermatozoa lost their motility when mixed with fresh urine specimens. Neutralization of urinary pH could not protect them from this effect unless urine osmolarity was also isotonically adjusted. It is suggested that patients with retrograde ejaculation should adequately increase their fluid intake before recovery of sperm from their bladder for artificial insemination.

A New Chamber for Rapid Sperm Count and Motility Estimation

A new chamber for sperm count and motility estimation is described. This chamber, which is only 10 micron deep, enables free horizontal movement of spermatozoa in one focal plane and provides conditions for the examination of undiluted samples. Therefore, with the aid of this instrument it is possible to compare sperm motility in various samples from the same person or in different samples at different times. This can be done either by simple estimation or with any other method of motility evaluation chosen by the examiner. The sperm count can be made rapidly and directly from an undiluted, preheated sample by counting spermatozoa in the area of a grid located within the eyepiece; the count is expressed in millions per milliliter. Thirty-seven specimens were analyzed with this chamber. Statistical evaluation of the results revealed high precision, accuracy, and reliability of sperm counts when compared with the hemocytometric method. Better results were obtained when motility estimation was compared with the ordinary slide technique. Easy performance, rapid sperm counts, and improvement of motility estimation make this chamber a useful tool where sperm analysis is carried out.

Use of the Elaborated Multiple Exposure Photography (Mep) Method in Routine Sperm Motility Analysis and for Research Purposes

Elaboration of the three basic components incorporated in the recently developed multiple exposure photography (MEP) method for objective sperm motility determination is described. This includes an improved version of the 10-micrometers counting chamber, modified procedures of photography, and a system for easier calculation of the results. During the last 2 years several thousands of sperm motility analyses were performed with the elaborated technique. In our institute this method has totally replaced motility evaluation by subjective estimation and has become the sole method for semen assessment performed during routine clinical work-up and research studies. This method is compared with other still-camera photographic techniques, and data about duration of performance, details of the relatively low cost of analysis, and specifications of some of its components are presented as well.

Premature ovarian failure—the prognostic application of autoimmunity on conception after ovulation induction

OBJECTIVEnTo assess whether the presence of autoimmune activity in patients with premature ovarian failure (POF) can predict the response to ovulation induction and conception.nnnDESIGNnAssessment of autoimmune activity in patients with POF, correlating the response to ovulation induction with this autoreactivity.nnnSETTINGnTertiary care academic center.nnnPATIENTSnForty women with POF, 15 of them treated by ovulation induction because of infertility.nnnINTERVENTIONSnAll patients were tested for the presence of autoimmune activity, antibodies against various tissues, and 15 of them were treated with combinations of hMG/hCG, glucocorticosteroids as immunosuppressant, and some of them also with a long-acting GnRH agonist. Those patients not interested in infertility were put on hormone replacement therapy (HRT).nnnMAIN OUTCOME MEASURESnSerum E2 and P were measured during ovulation induction as well as follicular diameter monitoring by transvaginal sonography. Achievement of gestations and their outcome were monitored in the group in which ovulation induction was accomplished.nnnRESULTSnAntibodies against thyroglobulin, nuclear antigens, heart, tissue gluten, or increased levels of immunoglobulin (Ig)M, or decreased levels of complement C3 and C4 were significantly different in the patients with POF than in the control population. Autoreactivity of at least one class of the tested antibodies was found in 31 of 40 patients (77%). In 15 patients with autoimmune activity who have undergone ovulation induction using hMG/hCG, 14 pregnancies were achieved in 8 patients. Two of the pregnancies were spontaneous, and 12 were generated by hMG/hCG and fluocortolone, with or without pretreatment with GnRH-a. Twelve healthy babies were generated by 10 gestations, 3 ended in spontaneous abortions (23%), and 1 is ongoing. All the nonspontaneous pregnancies were achieved in the first three cycles of ovulation induction.nnnCONCLUSIONSnPatients with POF and autoimmune activity, suggesting an autoimmune etiology to the ovarian failure, may respond to ovulation induction and have a conception rate of approximately 40% in three cycles. Those who do not conceive in three treatment cycles have a very low probability to conceive; therefore, further attempts of ovulation induction should be discouraged. However, some patients may spontaneously conceive in association with HRT.

Evaluating the accuracy of different sperm counting chambers by performing strict counts of photographed beads

PurposeTo suggest a method for evaluating the accuracy of different kinds of sperm counting chambers by eliminating errors concerned with human skills or semen properties. In this method, various concentrations were prepared from stocks of commercially available latex beads (Accu-beads, Hamilton-Thorn Research, Beverly, MA). Samples from identical preparations were loaded into different types of chambers, namely, hemocytometer (Neubauer, improved double, Superior Ltd, Germany), Makler (Sefi Medical Instruments, Haifa, Israel) and Horwell (Horwell Ltd, London, UK). Beads were counted by both direct microscopic observation and by strict scanning of their photographed images.ResultsIn all cases, counts by direct observation were about 5% higher than strict counts of the same photographed beads. Counting photographed beads showed high reproducibility (average CV of 5.1%) between samples in the two wells of the hemocytometer. Counts of photographed beads, sampled from identical stocks, were on average slightly lower in the Makler chamber (20.7 × 106/ml) and much higher in the Horwell chamber (47.4 106/ml) than counts in the hemocytometer (21.5 × 106/ml). Samples from three different batches of Accubeads revealed slight variation in counts between the batches and an average concentration of 11% above the number indicated on the commercial product.ConclusionsA technique that combined loading latex beads from identical stock into various chambers, proper covering of the tested samples and strict counting of photographed beads provided precise and reproducible results. By eliminating most errors related to human skills and semen properties, this method is suitable for evaluating the accuracy of counting chambers.

A new model for investigating in real-time the existence of Chemotaxis in human spermatozoa

OBJECTIVEnTo suggest a new approach to the research of chemotaxis between various media and human spermatozoa to solve the riddle of its existence.nnnDESIGNnLaboratory experiments in which chemotaxis between human spermatozoa and follicular fluid (FF) as well as N-formyl was investigated.nnnSETTINGnMale physiology laboratory and in vitro fertilization program unit.nnnPATIENTSnFollicular fluid was collected from 15 patients and used either fresh or after storage at -5 degrees C for less than 2 weeks. High-quality semen specimens were obtained from normal donors and underwent one-step washing with Hams F-10 solution (Biological Industries, Kibbutz Beth Haemek, Israel).nnnTECHNIQUESnAssays were performed in real time by direct microscopical observation and quantitative determination of the disturbance in random movements of human spermatozoa caused by the tested media. Spermatozoa and tested media, including FF and N-formyl-Met-Leu-Phe were placed in a sealed minichamber and photographed with the aid of the multiple exposure photography technique.nnnRESULTSnBy analyzing photographed tracks of more than 80,000 moving spermatozoa, a physiologically insignificant deviation from randomness, corresponding to a 95% confidence interval of -0.57 +/- 0.46%, was found when spermatozoa swam along the concentration gradient of the tested media. The average velocity of spermatozoa swimming toward the test media was slightly lower (less than 5%) than that for spermatozoa swimming away, indicating, if anything, negative chemoattraction.nnnCONCLUSIONSnDespite the high sensitivity of the model, neither natural FF nor synthetic N-formyl exerted any chemotaxis on human sperm. The advantages and potential use of this model for investigating chemotaxis by other biological and synthetic media are discussed.

Factors affecting sperm motility. VI. sperm viability under the influence of bacterial growth in human ejaculates

The influence of bacterial growth on human sperm motility and viability was evaluated objectively with the multiple-exposure photography method. Experimental semen specimens, obtained from normal donors bh nonaseptic means of masturbation, were incubated with antibiotics at room temperature or body temperature for 24 hours. Although bacteria, grew in control specimens, were totally eradicated in all antibiotic-treated specimens, no significant difference was found between these groups with regard to sperm motility throughout the time of incubation. Sperm survival was not inhibited, nor was it extended as a result of suppression of bacterial growth. In both groups, survival time was much shorter in specimens incubated at body temperature than in those kept at room temperature. Sperm motility was not affected after 2 hours of incubation of fresh specimens with concentrations of various pathogenic bacteria similar to those found in severe prostatitis. The question of whether the use of antibiotics in the treatment of asthenospermia per se has a prognostic value is discussed.

Use of a Sealed Mini-Chamber to Investigate Human Sperm Motility in Real Time Under Aerobic and Anaerobic Conditions

To study the correlation between metabolism and motility, ejaculated human spermatozoa were washed in media containing glucose, pyruvate, and deoxyglucose in various combinations. Spermatozoa suspended in these media were incubated in sealed mini-chambers and subjected to aerobic or anaerobic conditions at 37 degrees C. The effect on the patterns of sperm motility was investigated in real time by direct observation and objective determination with the multiple exposure photography (MEP) method. The motility of spermatozoa incubated in media containing excess of glucose showed similar changes of motility quality with time, whether exposed to aerobic or anaerobic conditions, and in both cases motility lasted about 13 h. Motility of sperm incubated with pyruvate only was of a much lower quality, especially under anaerobic conditions, and in both circumstances lasted about 7 h. When glycolysis of fructose remnants was totally inhibited by deoxyglucose and sperm were incubated with pyruvate only, motility lasted for 2 h under aerobic conditions and only for about 1 h under anaerobic conditions. It is concluded that the main metabolic process that supplies energy for sperm motility is glycolysis, under both aerobic and anaerobic conditions. Oxidative respiration was less efficient as a source of energy for sperm motility. When glycolysis was inhibited and oxidative respiration was eliminated under anaerobic conditions, sperm motility lasted only for about 1 h, probably by using intracellular energy reserves.

Factors affecting sperm motility. V. Washing and resuspension of human spermatozoa in various artificial media.

Fresh semen specimens from fertile donors were subjected to one-step and two-step washings in six various commonly used artificial media. It was found that washing procedures per se, in most cases, had an immediate and extended harmful effect on sperm motility which was much more prominent after the second washing. However, human albumin, when added, could usually protect spermatozoa from this deleterious effect. None of the tested media showed any stimulatory or revitalization effect, and the increase in sperm velocity after one washing in some of these media was attributed to a simple decrease in viscosity of the original seminal fluid. The possible mechanism of the deleterious effect of sperm washings as related to the property of the medium and some implications for practical clinical and research studies are discussed.