PREDICTIVE VALUE OF SPERM CHROMATIN CONDENSATION (ANILINE BLUE STAINING) IN THE ASSESSMENT OF MALE FERTILITY

Little is known about the physiological regulation of spermatogenesis of the African catfish (Clarias gariepinus), a species used as an experimental model for two decades. After a brief introduction to the cystic type of spermatogenesis in fish, the role of androgens and estrogens will be discussed, leading to a conclusion that androgens are required for spermatogenesis while their mode of action is poorly understood. In the cystic mode of spermatogenesis in fish, the Sertoli cells are formed from cysts, in the somniferous lobules enclosing a single germ cell clone, providing suitable experimental models to address questions of relevance for vertebrate spermatogenesis in general.     

INCREASING SPERM CONCENTRATION TO ADJUST FOR SUBNORMAL SPERM MORPHOLOGY DID NOT ADVERSELY AFFECT IMPLANTATION AFTER EMBRYO TRANSFER

In 10 men with idiopathic infertility, the authors evaluate hypophyso-gonadal axis by measuring the intratesticular concentrations of several steroids in relation with the serum hormonal status. The data are compared with those from men ( n = 20) after cerebral death (stage IV coma), taken as a control group. There was no difference between the two groups. The histological studies of the testes revealed no significant differences between control and infertile men both in the development of the interstitial tissue and in the spermatogenic score. Conversely, the testicular concentrations of pregnenolone, dehydroepiandrosterone, progesterone, androstenedione, testosterone, and estradiol were higher in infertile men compared to control. Therefore, it is difficult to evoke a specific parameter involved in that peculiar pathology, but the large amounts of endogenous estradiol may account for the impairment of fertility.     

SPERM CHROMATIN

The effect of amezinium, a new type of antihypotensive agent, on retrograde ejaculation was evaluated in 3 patients with retrograde ejaculation. The patients received 10 mg amezinium orally once a day. All patients achieved antegrade ejaculation. Semen analyses revealed 6-50 &;#50 10 6 /mL (mean 28.7 &;#50 10 6 /mL) sperm with a motility of 20-50% (mean 36.7%). The wives of 2 patients became pregnant within 6 months of the initial treatment. None of the patients had any side effects. It would appear that amezinium is a useful treatment for retrograde ejaculation.     

PYRIFORM HEAD: A FREQUENT BUT LITTLE-STUDIED MORPHOLOGICAL ABNORMALITY OF SPERM

This study was conducted to investigate the frequency of sperm with a pyriform head in semen samples, to determine the percentage of the occurrence of this abnormal sperm form, and to assess its possible correlation with other semen parameters. The study was designed as a retrospective data analysis in the setting of an andrology laboratory at a tertiary-care academic hospital. Semen quality data were analyzed from 114 subfertile men and 60 fertile men. The Student's t test, the Mann-Whitney nonparametric test, and the Pearson correlation coefficient were used for statistical analysis. Sperm with a pyriform head were present in the semen samples of 98% of the subfertile men and 100% of the fertile men; the percentage of this abnormal sperm form was 22 &#45 14.9% in subfertile and 13% &#45 7.8 in fertile men ( p <.001); 16% of the subfertile men presented a higher percentage of these abnormal sperm than the normal upper limit. In some subfertile men with a high percentage of sperm with a pyriform head, their subfertility could be attributed to the cause that produces this morphological abnormality. Moreover, morphological abnormalities in the neck and the tail, as also a cytoplasmic droplet, are significantly more frequent in sperm with a pyriform head than in sperm with a normal head.     

ASSESSMENT OF HUMAN SPERM MORPHOLOGY BY STRICT CRITERIA: COMPARISON OF WET PREPARATION VERSUS STAINED WITH THE MODIFIED DIFF-QUIK METHOD

Routine semen examination remains an important tool for the diagnosis and treatment in human subfertility. Of all semen parameters, sperm morphology seems to be one of the most powerful indicators of a man's fertilizing potential in vitro and in vivo. Lack of standardization of sperm morphology assessments remains the main reason for the usefulness of this parameter. The aim of this study was to analyze the agreement between the wet-stained preparations versus those stained with modified Diff-Quik for sperm morphology. A total of 100 unselected semen samples from infertile couples were analyzed. Sperm morphology was evaluated with unstained specimens and following modified Diff-Quik staining according to the strict (Kruger classification) criteria by two different examiners (intralaboratory blind assessment). Mean percentages of morphologically normal spermatozoa were identical on wet and stained preparation slides (4.79 vs. 4.61, p >.05). Wide divergence of results was found with respect to the percentage of sperm with head and midpiece defects with the two different preparations ( p >.001). The percentage of sperm tail defects was similar in both methods ( p >.05). Simple linear regression analysis between the two methods revealed very good correlation for the morphologically normal spermatozoa ( r =. 83), but poor correlation for the sperm head, midpiece, and tail defects ( r =. 25,. 25, and. 28, respectively). Wet preparation is suitable only for the morphologically normal spermatozoa, but to determine the percentage of the defective spermatozoa, staining the smear is recommended.     

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 &#45 18.3%, which increased within 24 h to 91 &#45 9.5%. On the other hand, the fertilization and ongoing pregnancy rates were 64 &#45 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.     

SEMEN ANALYSIS AT THE TURN OF THE CENTURY: AN EVALUATION OF POTENTIAL USES OF NEW SPERM FUNCTION ASSAYS

Semen analysis is a critical assay in the evaluation of infertility and may yield critical information regarding the etiology and prognosis of many types of reduced male fertility. However, basic semen analysis does not directly measure sperm fertilizing capacity, or many of the biochemical events both prior to and subsequent to fertilization. In the last two decades numerous assays of sperm function have been developed. These assays can be classified as: 1)Assays of general biochemistry and ultrastructure, 2) Assays of zona binding and oocyte penetration, and 3) Assays of postpenetration events. Sperm function assays not only allow an accurate diagnosis of many infertilities not diagnosed by the semen analysis, but can also lead to improved treatment modalities. In this review, basic semen analysis and many sperm function assays are briefly reviewed. Novel uses of sperm function are demonstrated in brief case studies.     

PARTICIPATION OF DNA STRUCTURE ON SPERM CHROMATIN ORGANIZATION

The in vitro interaction between purified bovine liver and sperm DNA with somatic histones, to form nucleosomes, and with bovine and salmon protamines were studied. DNAse or microccocal nuclease digestion of liver DNA-histone reassociated chromatin produced the expected polynucleosome type of fragments. Electrophoretic patterns of digested sperm-DNA nucleosomes were different. Micrococcal nuclease digestion produced mainly fragments smaller than 100 bp and some nucleosome-type particles. Under DNAse activity most of the products were smaller than 100 bp, indicating an increased susceptibility of the sperm DNA-histone complexes to the hydrolytic activity of both nucleases, particularly toward DNAse I. This differential susceptibility was confirmed by sucrose gradient spectrophotometric analysis. Acridine orange (AO) staining of histone-DNA reassociated nucleosomes showed significant differences in fluorescence intensity, sperm DNA-histone complexes being almost twice as fluorescent as liver DNA-histone complexes. On the contrary, liver DNA/protamine complexes stained with AO were consistently more fluorescent than sperm DNA-protamine complexes. Finally, no differences in either fluorescence intensity or spectra were observed when liver and sperm DNA were stained with AO after interaction with salmon protamines. The data suggest that sperm DNA has important structural characteristics that differentiates it from somatic DNA. These differences seem to be species specific and must surely play an important role on the determination of the dramatic sequence of that participates sperm chromatin organization.     

AUTOSTAGE SPERM TRACING SYSTEM FOR SEMEN EVALUATION

To overcome the limitation of the microscope field, the study proposed an autostage sperm tracing system (ASTS), which could trace a particular sperm for a long time and distance. The ASTS was constructed by assembling a commercial microscope, an image frame grabber, a personal computer, and a motorized stage. Its performance was tested by evaluating 6 semen samples and by comparing the evaluation with those of other semen evaluations. The ASTS broke through the limitation of the microscope field and traced a particular sperm as long as possible. It analyzed the sperm track and calculated the motility parameters, such as curvilinear velocity (V_cl), straight-line velocity (V_sl), and linearity (L_in). The sperm quality was then evaluated in real time, and the user could decide to capture or abandon a particular sperm in the IVF. The ASTS enables users to evaluate sperm progression for a long time and to have the global quality of a particular sperm in real time. Its open structure has the flexibility for micromanipulating a semen sample, and has the potential application associated with a modern IVF technique.     

吸烟、饮酒与精子形态的相关性及双因素对精子形态影响

[目的]探讨吸烟深度、饮酒量与精子形态的相关性及吸烟饮酒双因素对精子形态影响。[方法]659例男性不育患者,分为有吸烟史（263）、经常饮酒（137）和吸烟饮酒（259）3组,生育组（329）为已有生育的健康男性。患者自愿填写问卷,对吸烟、饮酒患者资料分析,采用精子形态检测系统下人工修正方法进行精子形态分析。[结果]吸烟年数与正常形态精子百分率之间有显著负相关关系（r=-0.155,P﹤0.001）;每天饮酒的量（g/d）与正常形态精子百分率之间有显著负相关关系（r=-0.146,P﹤0.05）;吸烟饮酒组正常形态精子百分率显著低于吸烟组（P﹤0.005）,亦显著低于饮酒组（P﹤0.01）。[结论]长期吸烟、大量饮酒影响精子形态,吸烟饮酒双因素对精子形态影响大于单一因素的影响。     

TEMPORAL TRENDS IN HUMAN SEMEN PARAMETERS IN NEW ENGLAND IN THE UNITED STATES, 1989-2000

The current study explores trends in semen parameters in New England in the United States. A retrospective review was performed of 551 semen analysis records from 1989 to 2000 at Vincent Memorial Andrology Laboratory of Massachusetts General Hospital. After age adjustment, semen pH and motility significantly increased 0.05 units/year and 2.33%/year, respectively, while sperm with normal morphology decreased 0.33%/year. Sperm concentration showed a small upward trend. The year of birth in the present study ranged from 1932 to 1981; 2% were born between 1932 and 1941, 13% between 1942 and 1951, 48% between 1952 and 1961, 36% between 1962 and 1971, and 1% were born between 1972 and 1981. There were significant positive relationships between year of birth and semen volume (0.04 mL/1-year interval increase in year of birth) and motility (0.61 percent/1-year interval increase in year of birth), as well as with sperm concentration and morphology. Overall, there were temporal and year of birth trends in several human semen parameters.     

EVALUATION OF SPERM NUCLEAR MORPHOLOGY IN SPECIMENS WITH ABNORMAL SPERM CHROMATIN STRUCTURAL ASSAYS

Two recent tests have claimed to identify the subfertile male even when other semen parameters were normal: the sperm chromatin structure assay (SCSA) and abnormal sperm nuclear morphology using much higher magnification. The present study attempted to determine if having a high (> 30%) DNA fragmentation index (DFI), thus resulting in an abnormal SCSA test, is associated with a greater likelihood of sperm with abnormal nuclei. Four males with high DFI scores (57.6%, 65.4%, 31.0%, and 35.3%) had their nuclei evaluated by a complex microscope set-up that magnifies the sperm at least 6000 × . The corresponding % of normal nuclei was 0%, 20.0%, 23.7% and 40.0%. The mean and median % of normal nuclei was 20.9±16.43 and 21.8, respectively. More studies of similarly matched refractory in vitro fertilization cases, where males have normal DFI scores, are needed to determine if having a high DFI index is associated with a lower percentage of normal nuclei.     

DETERMINATION OF LABORATORY SPECIFIC PERCENT NORMAL SPERM MORPHOLOGY VALUE BASED ON PREVASECTOMY (FERTILE) EJACULATES

Semen assessments were performed on ejaculates from 25 men with proven fertility requesting vasectomy. Multiple cytological slides were made simultaneously from each ejaculate and analyzed by a single technician. The morphology data obtained were analyzed for repeatability. The mean +/- SD for normal sperm morphology was 30.6 +/- 7.3, and the within-subject repeatability was 1 SD of the mean for 17 of 25 ejaculates. Technician and laboratory specific criteria for the percent normal sperm morphology component of a semen analysis report (that are clinically meaningful) can be developed using the approach described herein.     

FERTILIZATION RATES OF SMALL-HEAD SPERM IN CONVENTIONAL IVF AND ICSI

Five men produced 23 ejaculates in 23 assisted reproductive technology cycles for semen analysis. In 11 of the 14 ICSI cycles and in 5 of the 9 IVF cycles, small-head sperm were found in more than 70% of the cells, having a length of <3.5 µm and a width of <2 µm. In 6 of the 14 ICSI cycles, the embryologist who performed the ICSI was not alerted to the presence of small-head sperm. Subsequent fertilization rate was significantly lower than a) the fertilization rate of the remaining 5 ICSI cycles that acted as a control, in which the embryologist was alerted to the presence of small-head sperm, and b) was lower than the 3 ICSI cycles with normal sperm head ( p <. 05). The 8 cell embryo and blastocyst rates in the former group were also decreased, although not significantly, compared to the latter groups. Likewise, in the 5 IVF cycles with small-head sperm, the fertilization rate was significantly lower than in the 4 IVF with normal sperm head ( p <. 05). Embryologists should be alerted when a high percentage of small-head sperm are detected during routine semen analysis because they may be associated with reduced fertilization and embryo outcome.     

SPERM PROCESSING: GLASS WOOL COLUMN FILTRATION

The cervical mucus selectively allows only progressively motile sperm of normal shape and size to penetrate and migrate through the cervix. The glass wool column filtration (GWCF) method for sperm processing, proposed in 1977 and later modified and standardized in 1986, selects such a sperm population in vitro. Sperm recovered from GWCF is significantly higher in quality than the original ejaculate. GWCF processing is reliable and highly repeatable, and when compared to other sperm processing techniques, GWCF yields significantly higher sperm quality. Sperm penetration into denuded hamster oocytes improves significantly with GWCF, while binding to human zona pellucida also occurs in higher numbers. IVF outcome also increases as compared to other processing techniques. GWCF, based on proven clinical success, is therefore invaluable for sperm processing.     

解脲脲原体对人精子形态结构及顶体酶活性的影响

[目的]探讨解脲脲原体(Uu)对人精子形态、超微结构及顶体酶活性的影响。[方法]Uu临床标本和标准菌株分别与精子按照25︰1比例混合,通过改良巴氏染色、透射电镜、比色法观察精子形态、超微结构及顶体酶活性的改变。[结果]精子出现头部、颈中段和尾部的缺陷;透射电镜观察可见Uu吸附于精子表面,精子顶体、膜结构、线粒体、中心粒、外周致密纤维等超微结构改变;临床株组和标准株组顶体酶活性均低于对照组;顶体酶活性与形态正常精子百分率呈明显正相关关系。[结论]Uu感染可改变精子形态、超微结构,降低精子顶体酶活性。     

COMPUTERIZED SEMEN ANALYSIS (CASA): EFFECT OF SEMEN CONCENTRATION AND CHAMBER DEPTH ON MEASUREMENTS

The gender of the offspring is determined by the fertilizing sperm. Previous gender studies were based on washed sperm, but not on sperm in seminal plasma. The objective was to correlate motility parameters assessed during semen analyses with the offspring gender. For comparison, fixed sperm head DNA quantitated by Hoechst 33342 fluorescence microscopy was also analyzed. Forty-six patients undergoing assisted reproduction procedures resulted in livebirth deliveries with either male or female-predominant offsprings. Sperm head fluorescence was weakly correlated to the gender in 61% of the cases. Sperm of patients with male offsprings had slower curvilinear (44.2?±?1.8 mean?±?SEM, versus, 49.9?±?2.7?µ/sec) and slower average path velocities (32.4?±?1.2 versus 36.3±1.7?µ/sec). Using cut-off values for the curvilinear (<49?µ/sec) and average path (<36?µ/sec) velocities of sperm swimming in seminal plasma, the two parameters predicted 75 and 68% of the male offspring births, respectively. The data suggest that sperm movement in seminal plasma is a marker for factors that skew the ratio of the X- to Y-sperm populations.     

PHOSPHOLIPID COMPOSITION OF HUMAN SPERM AND SEMINAL PLASMA IN RELATION TO SPERM FERTILITY

The phospholipid and fatty acid composition of sperm was studied in 8 healthy and 16 infertile men. Infertile men randomly formed from the patients with normal semen parameters according to WHO criterion. Therefore, all semen parameters of infertile patients were similar to the same characteristics of the semen of healthy men, except the abnormal forms. The amount of abnormal forms in infertile men was significantly higher than in healthy men. Sperm from infertile men show a drastic loss of phosphatidyl ethanolamine. At the same time, the significant increase of phosphatidyl serine in the sperm and seminal plasma of sterile patients was found. Lysophosphatidyl serine in the sperm of the infertile men was detected. Fatty acid composition of the semen of infertile men was altered. The levels of stearic and n-3 polyunsaturated fatty acids (eicosopentaenoic and docosahexaenoic acids) was dramatically lowered, but the values of some n-6 polyunsaturated fatty acids (linolenic and docosatetraenoic) acids increased. There was significant positive correlation between docosahexaenoic acid and sperm motility (r =. 82, p &lt;. 001) and negative correlation between linolenic acid and spermatozoa motility (r=-0.58, p&lt;.05). Infertility of men with normal semen quality can originate from the disorder of sperm lipid metabolism. The drastic loss of phosphatidyl ethanolamine and n-3 polyunsaturated fatty acids with simultaneous enhancement of phosphatidyl serine and some n - 6 polyunsaturated fatty acids in sperm could be an important cause of male infertility.