The usefulness of Real Time Morphology software in semen assessment of teratozoospermic boars

The aim of the present study was to compare two different techniques of sperm cell morphology evaluation in teratozoospermic boars: computer assisted semen morphology analysis and conventional assessment of stained semen smears. The semen samples were collected manually from 30 boars with reduced semen quality. In all samples the percentage of morphologically normal spermatozoa was below 70%. Computer assisted semen morphology assessment was performed using the Real Time Morphology (RTM) software (IVOS ver. 12.2, Hamilton Thorne Bioscience). The assessment was made by phase contrast optics, with the magnification of 20 x 3.8 and without staining. Conventional morphology assessment was performed by bright field microscopy with 1,000 x magnification after staining with Giemsa. At least 200 spermatozoa were evaluated per slide in both methods. The Bland-Altman plot indicated a general agreement between both methods of sperm morphology evaluation. The plots revealed the widest limits of agreement (mean?±?1.96 SD) for the percentage of midpiece anomalies (from ?16 to 13.2), and the narrowest for the percentage of looped tail (from ?1.49 to 1.09). The Bland Altman plot indicates general agreement between RTM and Giemsa staining in the percentage of major and minor defects. However, it was not possible to evaluate acrosomes using RTM. Otherwise, RTM proved to be a valuable tool in sperm morphology assessment, with accuracy equal to typical conventional methods.     

精索静脉曲张对精子形态的影响

目的:探讨精索静脉曲张对精子形态的影响。方法:分析71例已有生育男性和417例男性不育患者精液样本,其中不育患者分为精索静脉曲张组Ⅰ°(130例)、Ⅱ°(64例)、Ⅲ°(88例)和无精索静脉曲张组(135例)4组。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:精索静脉曲张Ⅱ°和Ⅲ°组正常形态精子百分率均显著低于生育组(P<0.05,P<0.001),但两组之间差异没有显著性(P>0.05)。精索静脉曲张Ⅰ°组正常形态精子百分率与生育组之间差异无显著性(P>0.05)。精索静脉曲张不育组正常形态精子百分率显著低于生育组和无精索静脉曲张组(P<0.005,P<0.05),梨形头精子、尾部/中部缺陷精子百分率显著高于生育组(P<0.05,P<0.005),大头精子、颈部/中段缺陷精子和尾部缺陷精子百分率显著高于无精索静脉曲张组(P<0.05),而其它畸形精子百分率显著低于无精索静脉曲张组(P<0.005)。结论:精索静脉曲张能够影响精子形态,Ⅱ°和Ⅲ°能导致正常形态精子百分率下降。     

男性不育患者精子形态及质量与精液白细胞的关系

目的探讨男性不育患者精子形态及质量与精液白细胞的关系。方法随机选取202例男性不育患者的精液,严格按照WHO人类精液实验手册要求检测精液质量、形态及精液中的白细胞。结果 202例不育患者中有58例（占28.7%）精液中的白细胞〉1×106个/ml,设为白细胞精子组,其余144例（占71.3%）患者精液中白细胞≤1×106个/ml,设为非白细胞精子组。白细胞精子症组患者精液与非白细胞精子症组患者比较在精子密度、精子活动率方面明显降低,液化时间明显增加（P〈0.05）;白细胞精子症组形态正常精子所占比例明显低于非白细胞精子症组（P〈0.05）,并且精液白细胞与正常形态精子率呈显著负相关（P〈0.05）。结论男性不育患者的精液白细胞增多影响精子质量和精子形态的正常。     

SPERM MORPHOLOGY AND CHROMATIN CONDENSATION BEFORE AND AFTER SEMEN PROCESSING

Semen analysis constitutes the most important investigation of male infertility. However, the true anomalies present in defective sperm cells have been only partially characterized. The integrity of the sperm chromatin may play the most important role, particularly in ICSI, where most of the natural selection mechanisms are bypassed. This study was carried out to characterize sperm morphology (strict criteria), to evaluate chromatin condensation and sperm count in native semen as well as after semen preparation by the swim-up technique, and to eventually evaluate any correlation between these parameters. Semen from 90 men was analyzed for the above parameters in both the fresh and processed semen. Whereas the sperm count decreased after sperm preparation by the swim-up technique in comparison to the value in the fresh semen (p&lt;.001), there was an increase in the percentage of morphologically normal (p&lt;.001) and chromatin-condensed sperm (p=.99). However, there was no correlation between sperm morphology, chromatin condensation, and sperm count either in the fresh or in the processed semen samples. These results suggest that sperm morphology, sperm count, and chromatin condensation are independent parameters that should be evaluated separately in the assessment of male fertility in an assisted reproduction program.     

输精管吻合术后精子超微结构观察

目的:观察输精管吻合术后精子超微结构的变化。方法:采用光学显微镜、扫描和透射电子显微镜对20例接受输精管吻合术者和10例正常生育男性精子的形态和超微结构改变进行观察、统计、分析。结果:①光镜和扫描电镜分析显示吻合术后精液中正常形态精子显著减低;②两组精子尾畸在光镜下有明显差异,在扫描电镜下无差异;③两组精子头畸在扫描电镜下有显著性差异;④在透射电镜下吻合术后精子多数均属于复合型精子结构异常,大部分均有多个部位和多个细胞器结构异常。结论:吻合术后精子有多种类型和复合型的异常改变,这些改变与不育男子精子的改变相似。提示:输精管结扎术后精子形态学的改变,是使吻合术后生育能力降低的主要原因之一。     

The importance of transmission electron microscopy analysis of spermatozoa: Diagnostic applications and basic research

This review is aimed at discussing the role of ultrastructural studies on human spermatozoa and evaluating transmission electron microscopy as a diagnostic tool that can complete andrology protocols. It is clear that morphological sperm defects may explain decreased fertilizing potential and acquire particular value in the field of male infertility. Electron microscopy is the best method to identify systematic or monomorphic and non-systematic or polymorphic sperm defects. The systematic defects are characterized by a particular anomaly that affects the vast majority of spermatozoa in a semen sample, whereas a heterogeneous combination of head and tail defects found in variable percentages are typically non-systematic or polymorphic sperm defects. A correct diagnosis of these specific sperm alterations is important for choosing the male infertility’s therapy and for deciding to turn to assisted reproduction techniques. Transmission electron microscopy (TEM) also represents a valuable method to explore the in vitro effects of different compounds (for example drugs with potential spermicidal activity) on the morphology of human spermatozoa. Finally, TEM used in combination with immunohistochemical techniques, integrates structural and functional aspects that provide a wide horizon in the understanding of sperm physiology and pathology.Abbreviations: transmission electron microscopy: TEM; World Health Organization: WHO; light microscopy: LM; motile sperm organelle morphology examination: MSOME; intracytoplasmic morphologically selected sperm injection: IMSI; intracytoplasmic sperm injection: ICSI; dysplasia of fibrous sheath: DFS; primary ciliary dyskinesia: PCD; outer dense fibers: ODF; assisted reproduction technologies: ART; scanning electron microscopy: SEM; polyvinylpirrolidone: PVP; tert-butylhydroperoxide: TBHP     

精子核成熟度与精子形态的关系

目的:研究精子核成熟度与精子形态关系,探讨精子形态异常机制。方法:收集55例精液样本,其中生育组19例,畸形精子症组36例。应用CASA分析精子密度、活力,伊红染色检测精子活率,联苯胺染色分析精液白细胞浓度,改良巴氏染色分析精子形态,苯胺蓝染色法评价精子核成熟度。结果:畸形精子症组苯胺蓝染色阳性率显著高于生育组(P<0.05)。头部异常、颈部异常、尾部异常、无定型、其它畸形精子组苯胺蓝染色阳性率显著高于形态正常精子组苯胺蓝染色阳性率(P<0.05)。结论:精子核成熟异常可导致精子形态异常。     

Inhibition of spermatozoa maturation in rhesus monkey by cyproterone acetate

The morphology of spermatozoa from the initial segment, caput, corpus and cauda epididymides of normal and cyproterone acetate-treated animals was studied using light and scanning electron microscopy to understand the changes taking place during spermatozoa maturation. A progressive and significant decrease in the percentage of spermatozoa that retained the cytoplasmic droplet and a shift in its position from proximal end of the midpiece to its distal end were seen during epididymal transit; these events were inhibited in cyproterone acetate-treated animals. A large percentage of spermatozoa from the initial segment and the caput epididymides showed coiling of the spermatozoa tail which involved the midpiece, principal piece and the endpiece. The percentage of spermatozoa that showed the coiled tail decreased; a gradual straightening of the spermatozoa tail with less complex types of coiling was also seen during epididymal transit. Cyproterone acetate reversed these changes occurring during maturation. These results indicate that spermatozoa maturation in the rhesus monkey, occurring between the corpus and cauda epididymides, is an androgen-dependent event.     

High Resolution Light Microscopic Evaluation of Boar Semen Quality Sperm Cytoplasmic Droplet Retention in Relationship with Boar Fertility Parameters

The purpose of this study was to investigate the relationship between fertility and quantitative measures of boar semen quality, including various patterns of sperm cytoplasmic droplet (CD) retention, as determined by high power differential interference contrast (DIC) microscopy. A total of 116 ejaculates were collected from a nucleus herd of 18 Large White boars over an eight month period. Semen quality parameters were analyzed for each ejaculate by calculating the percentage of normal spermatozoa, spermatozoa possessing a CD in the proximal, distal, or distal midpiece reflex position, total spermatozoa with an attached cytoplasmic droplet, spermatozoa with non-CD related aberrations and total spermatozoa with abnormalities. Of the 116 ejaculates received, 71 ejaculates from 13 boars had corresponding fertility data from single-sire inseminations of multiparous sows. The fertility data included farrowing rate (FR) and total number born (TNB). The monthly FR encompassed one month before and one month after the date of semen collection. Detection of differences for fertility and semen quality parameters was performed by separating the boars into either an above-average or below-average group based on the mean FR (74.01 ± 1.43%) or TNB (12.34 ± 0.17) for the study. For FR, the boars in the below-average group had a significantly lower percentage of normal spermatozoa and significantly higher percentage of spermatozoa possessing distal CDs, total attached CDs and total abnormalities compared to the boars in the above-average group. Conversely, for TNB there were no significant differences between the above- and below-average groups for the semen quality parameters. These data suggest that the attached CD may negatively affect FR, but not TNB. The detection of relationships between the boar fertility parameters and the retention of the sperm CD after ejaculation, document the advantage of high power DIC microscopy in conventional semen evaluation.     

REVISION OF HYPO-OSMOTIC SWELLING (HOS) TEST WITH A PROPOSITION OF CLASSIFIED GRADING SYSTEM: iTS COMPARISONS WITH FOUR DIFFERENT TYPES OF HUMAN SPERM SEPARATION TECHNIQUE

In this study, we arbitrarily classified the morphological changes of sperm under hypo-osmotic condition and compared the results with four different sperm separation techniques. The morphology of classification ranged from a good swelling (SG 3 > SG 2 > SG 1), to non-swelling but reactive (SG 0-R), and completely non-swelling (SG 0). Thirty fresh semen from patients were divided into 4 groups and each processed by washing, swim up, 2-layer column and real time micro-separation system. The prevalent patterns of the swollen sperm in sequence after treatment was SG 0 > SG 1 > SG 3 > SG2 > SG 0-R in fresh semen, and the proportion of SG 0 was almost half of the total % HOS test results. However, SG 3 became the most common swollen form with a significant increase in number with any method of sperm preparation after treatment. The real time technique yielded the highest % rate of SG 3 type of swollen spermatozoa and was the richest in concentration (53±3.9%, P < 0.05) as compared with that of swim up (26±4.6%), 2-layer (20±4.4%), sperm washing (23±3.9%), and fresh semen (17± 2.8%), respectively. By contrast, 2-layer collected more number of SG0-R (5.3±1.2%, P < 0.05) pattern of spermatozoa when compared to real time and swim up. A positive correlation (r = 0.81, P < 0.002) was shown between the % total HOS spermatozoa and total motility of fresh semen but not with morphology. Collectively, an abnormally functional sperm may exist in almost half of a total sperm count from men. It would appear that a better potential fertilization capacity may reside in the more swollen sperm from HOS test than the less swollen sperm and that may be more related with the motility rather than the morphology. The HOS classification and grading system appears valuable in further evaluating sperm quality.     

男性不育患者精浆锌含量与精液主要参数相关性研究

目的:探讨不育男性精浆的锌含量与精液质量的关系。方法:回顾性分析2011年8月～2012年1月在广西壮族自治区妇幼保健院生殖中心就诊的343例男性不育患者的相关资料,依据精浆锌含量分为正常A组(n=274例)和异常B组(n=69例),比较两组间精液参数的差异;同时根据精液黏稠度分为黏稠C组(n=54例)与非黏稠D组(n=289例),比较两组间精浆锌含量及其他精液参数的差异。结果:A组与B组患者年龄比较差异无统计学意义(P>0.05)。A组的精液量、每次射精精子总数、前向运动精子总数显著高于B组,而黏稠精液的比例明显低于B组,差异均有统计学意义(P<0.05)。其他精液各指标比较差异无统计学意义(P>0.05);C组与D组精液圆细胞浓度、精液量、精子浓度、精子总数比较差异无统计学意义(P>0.05);D组的前向运动精子百分率、前向运动精子总数、活动率、精浆锌明显高于C组,差异有统计学意义(P<0.05)。精浆锌含量与精液量、每次射精精子总数、前向运动精子总数显著正相关,与其他参数无显著相关性。结论:精浆锌含量直接影响精液量、精子总数、前向运动精子总数和精液黏稠度,精浆锌含量是男性生殖力的重要评估指标。     

Seasonal patterns of LH, testosterone and semen quality in the Northern pintail duck (Anas acuta)

This study characterized seasonal changes in circulating LH and testosterone and in semen production and quality in the Northern pintail duck. Plasma LH and testosterone were measured in blood samples collected weekly throughout the year from eight males exposed to natural fluctuations in day length and temperature. Semen quality was evaluated weekly in these same males from April-June, the months when spermatozoa were produced. Semen quality (based on sperm concentration and normal morphology) peaked 0-2 weeks after sperm production onset and decreased sharply before sperm production cessation in late June. Nadir LH concentrations were measured in July and August with peak LH observed in May and November. There were clear seasonal patterns in circulating testosterone with July-September values being less (P<0.05) than October-December which, in turn, were less (P<0.05) than January-March. Maximal circulating testosterone (P<0.05) occurred during April-June, coincident with semen production. Weekly circulating LH during the breeding season was directly related to testosterone concentrations (P<0.01), but was not correlated to any specific semen or sperm trait (P>0.05). Testosterone concentrations throughout the breeding season were correlated (P<0.05) to total numbers of spermatozoa produced (volume x cell concentration) and percent normal sperm morphology. In summary, the Northern pintail experiences seasonal hormone fluctuations, with maximum circulating testosterone coinciding with peak ejaculate quality reflected by the production of high numbers of morphologically normal spermatozoa.     

Relationship between phospholipase C-zeta,semen parameters,and chromatin status

The need for additional tests to complement basic sperm analysis in clinics is well appreciated. In this regard, a number of tests such as sperm DNA integrity test as a tool in diagnosis and treatment of infertility are suggested. But recent studies have focused on main sperm factors involved in oocyte activation such as phospholipase C-zeta (PLCζ) that initiate intracellular Ca²⁺ signaling and embryogenesis. Therefore, this study aimed to investigate the relationship between PLCζ, basic semen parameters, sperm DNA fragmentation (SDF), and protamine deficiency in men with normal (n=32) and abnormal (n=23) semen parameters. Unlike SDF and protamine deficiency, as negative factors related to fertility, the mean value of PLCζ as positive factor related to infertility was significantly lower in men with abnormal semen parameters compared to men with normal semen parameters. Significant correlations were also observed between sperm concentration, motility, and abnormal morphology with the percentage of PLCζ positive spermatozoa. In addition, logistic regression analysis revealed that sperm morphology is more predictive than sperm motility and concentration for PLCζ presence. In addition, a statistically significant negative relationship was observed between the percentage of PLCζ positive spermatozoa and SDF. These findings suggested during ICSI, selection of sperm based on morphology has a profound effect on its ability to induce oocyte activation based on the likelihood of PLCζ expression. Therefore, assessment of PLCζ as an index for fertilization potential of a semen sample in men with severe teratozoospermia may define individuals who are candidates for artificial oocyte activation (AOA) and may avoid failed fertilization post ICSI.     

EFFECT OF SEMINAL PLASMA CALCITONIN LEVELS ON SPERM MOTILITY

This study investigated the effect of the seminal and blood plasma calcitonin levels on the sperm motility in idiopathic infertile patients. The number of sperm cells and their motility were evaluated in the spermiograms of 52 idiopathic infertile patients. The levels of seminal plasma calcitonin were studied with double antibody technique using a DPC kit. Fifty-two patients were divided into 2 groups according to the motility rates of sperm and 20 healthy volunteers were assigned to a control group. The difference between the groups was evaluated by using Kruskall-Wallis and Mann-Whitney U tests, and the correlation of seminal and blood calcitonin levels with sperm motility were determined. The difference in motility rates between the 3 groups was statistically significant ( p =. 000, p <. 05). Blood plasma calcitonin levels were in normal ranges in all cases and no significant difference was found among the 3 groups ( &#104 2 = 2.7219, p =. 2589, p >. 05). While sperm motility was correlated with seminal calcitonin levels ( r =. 8581), blood calcitonin levels did not show a correlation with sperm motility rate ( r = -.0265). Moreover, there was no correlation between seminal and blood plasma levels of calcitonin ( r = -.0010). Motility rates decreased in the patients with low seminal calcitonin levels and seminal calcitonin levels had a significant effect on sperm motility.     

Immunolocalization of aquaporin 7 in human sperm and its relationship with semen parameters

Aquaporins (AQPs) are a family of 13 small hydrophobic trans-membrane proteins expressed in numerous tissues and cells. Some AQPs work as strict water channels, others are permeable to a range of substances, including glycerol. In the male reproductive system their localization in testis, efferent ducts, epididymis, and spermatozoa has been described. We studied the distribution of AQP7 in ejaculated human sperm and the relationship between AQP7 labeling and sperm characteristics. Semen samples from 33 men were examined by light and transmission electron microscopy (TEM). TEM data were quantified using a mathematical formula that calculates a fertility index (FI) and the percentages of sperm apoptosis, immaturity, and necrosis. Immunocytochemistry with a polyclonal antibody anti-AQP7 was performed on the sperm samples. Normal sperm were labeled in the pericentriolar area, midpiece, equatorial segment, and weakly in the tail (grade 1). Abnormal sperm showed a diffuse low intensity of fluorescence evident in the cytoplasmic residues, coiled tails, in the entire head, and acrosome (grade 2). A high number of motile sperm obtained by swim up were labeled in a dotted manner in the mitochondria. A significant positive correlation was found between the spermatozoa with AQP7 grade 1 labeling and the percentage of normal form (P<0.008), progressive motility and FI (P<0.005); a negative correlation was noted with the percentages of cytoplasmic residues (P<0.010) and immaturity (P<0.006) and coiled tails (P<0.012). The link between AQP7 distribution and sperm morphology and the particular dotted labeling in swim up selected motile sperm are novel and deserve additional studies.     

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.     

Variation of sperm head shape and tail length in a species of Australian hydromyine rodent: the spinifex hopping mouse, Notomys alexis

In Australia, there are around 60 species of murid rodents that occur in the subfamily Hydromyinae, most of which produce highly complex, monomorphic, spermatozoa in which the head has an apical hook together with two ventral processes containing filamentous actin and a long tail of species-specific length. One of the few exceptions to this is the spinifex hopping mouse, Notomys alexis, whose spermatozoa have previously been shown to have pleiomorphic heads. In this study, the structural organisation of the sperm head has been investigated in more detail and the variability in length of the midpiece and total length of the sperm tail has been determined for this species. The findings confirm that pleiomorphic sperm heads are invariably present in these animals and that this variability is associated with that of the nucleus, although nuclear vacuoles were not evident. The total length of the sperm tail, as well as that of the midpiece, was also highly variable both within, as well as between, individual animals. The reason(s) for this high degree of variability in sperm morphology is not known but it may relate to a relaxation of the genetic control of sperm form owing to depressed levels of inter-male sperm competition.     

输精管吻合术后精液参数恢复的研究

为探讨输精管吻合术后精液参数恢复情况,我们对46例接受输精管吻合术者进行术后18个月追踪分析,共检查精液85份。结果提示:输精管吻合术后18个月内,精液中射出的精子总数和精子密度(几何均值)减少;正常形态学的精子减少;精子膜完整性的百分率减少;活动良好的精子减少(a级)和活动不良的精子(c级)增高,这6个异常参数在吻合术后18个月时间内较为恒定。精子总活动率和精子低渗膨胀试验有良好的正相关关系,这两个参数在吻合12个月后有逐步恢复至正常水平的趋势。     

吸烟对精液的影响

目的 探讨吸烟对精液的影响。 方法 选择门诊265例男性不育症患者精液标本,其中165例为吸烟者、100例为非吸烟者,观察比较2组精液标本的各项参数。 结果 吸烟者和非吸烟者的精子形态正常率、精子头尾畸形率、精子体畸形率2组间差异有显著性(P<0.001,P<0.05),其它精液参数差异无显著性。结论 吸烟与精子畸形关系密切。香烟烟雾中含有大量的氧化剂和诱变剂,可能是造成精细胞的DNA损伤断裂导致畸形精子增多的重要原因。     

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.