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.     

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.     

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.     

Simultaneous detection of viability and sex of bovine spermatozoa

The viability and sex of bovine spermatozoa were simultaneously evaluated. After viability and acrosome staining with trypan blue/Giemsa, only live spermatozoa became decondensed by a modified papain-dithiothreitol method. Owing to this specific effect, live sperm heads were easily distinguished by their enlarged size and dark violet colour from small, light blue dead sperm heads. In the same sperm sample, X- and Y-chromosome-bearing sperm were distinguished by their fluorescent signal, using fluorescence in situ hybridization (FISH) with an XY paint set and 4,6-diamino-2-phenylindole counterstaining. The combined staining provides a method for morphological and viability evaluation before FISH and permits identification of the proportions of X- and Y-chromosome-containing live spermatozoa in a semen sample. However, only 25% of the undecondensed dead sperm express signals allowing detection of the sex of the chromosome. The method may be an effective tool in evaluating sex-oriented semen samples.     

Segregation of sperm subpopulations in normozoospermic infertile men

Sperm function is essential for fertilization and embryogenesis yet semen contain a heterogeneous population of sperm. This study was designed to evaluate two different sperm populations separated by the density gradient method. Semen from 25 idiopathic normozoospermic infertile men was processed by double density gradient centrifugation and evaluated for sperm present in the 50% (upper) layer and the 90% (lower) layer for reactive oxygen species (ROS), sperm chromatin integrity, and morphology. The population of sperm in the 90% layer showed significantly lower ROS levels (22.90 (0.92, 85.32) vs. 382.03 (158.30, 1409.51) and lower DNA fragmentation index (DFI) (24.26 (22.54, 25.50) vs. 29.93 (28.48, 31.25) and higher number of sperm with normal morphology (55 (45.0, 60.0) vs. 32.5 (20, 40) compared to sperm in the 50% layer. However, in the original raw semen, sperm DFI (27.02 (26.19, 27.76)) and percentage high DNA stainability (% HDS) (3.1 (2.40, 3.78)) cells were significantly higher compared to the 90% layer population. Density gradient separation of the sperm subpopulation from the original semen favors the selection of sperm with genome integrity, low levels of ROS, and normal morphology. Therefore presence of pathological sperm in the semen may disrupt the function of normal spermatozoa, and hence the selection of the normal sperm subpopulation may be a better candidate for assisted conception. Further studies are required to evaluate the gradient separated sperm population in assisted reproductive techniques (ART).     

DHR123: an alternative probe for assessment of ROS in human spermatozoa

The aim of this study was to assess the potential of dihydrorhodamine 123 (DHR123) to measure oxidative stress produced by human spermatozoa. The results were compared with 2', 7'-dichlorodihydrofluorescein diacetate (DCFH-DA) that is routinely used for assessment of H₂O₂ produced by spermatozoa. Fluorescence intensity and percentage R123 and DCF positive sperm were measured by flow cytometry. The optimal condition for assessment of reactive oxygen species (ROS) produced by sperm with DHR123 was 0.05 µM for 1 million sperm per ml for 20 minutes. The results of ROS measurement by DHR123 showed a significant correlation (r?=?+0.818, P?<?0.001) with DCFH-DA staining. Immunofluorescence of sperm stained with DHR123 revealed ROS production in the sperm mid-piece. In addition a significant correlation was observed between oxidant production assessed by DHR123 and semen parameters. Therefore, DHR123 may be considered a suitable probe for estimating oxidants produced by human spermatozoa, and can present heterogeneity in oxidant production between different samples.     

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。     

DHR123: an alternative probe for assessment of ROS in human spermatozoa

The aim of this study was to assess the potential of dihydrorhodamine 123 (DHR123) to measure oxidative stress produced by human spermatozoa. The results were compared with 2', 7'-dichlorodihydrofluorescein diacetate (DCFH-DA) that is routinely used for assessment of H(2)O(2) produced by spermatozoa. Fluorescence intensity and percentage R123 and DCF positive sperm were measured by flow cytometry. The optimal condition for assessment of reactive oxygen species (ROS) produced by sperm with DHR123 was 0.05 μM for 1 million sperm per ml for 20 minutes. The results of ROS measurement by DHR123 showed a significant correlation (r=?+0.818, P<0.001) with DCFH-DA staining. Immunofluorescence of sperm stained with DHR123 revealed ROS production in the sperm mid-piece. In addition a significant correlation was observed between oxidant production assessed by DHR123 and semen parameters. Therefore, DHR123 may be considered a suitable probe for estimating oxidants produced by human spermatozoa, and can present heterogeneity in oxidant production between different samples.     

Seasonal variation of human sperm cells among 4,422 semen samples: A retrospective study in Turkey

We aimed to assess the possible presence of a seasonal pattern in three parameters of semen analysis: sperm concentration, morphology, and motility as a function of the time of ejaculation and sperm production (spermatogenesis) in normal and oligozoospermic men. This retrospective study included a consecutive series of 4,422 semen samples that were collected from patients as a part of the basic evaluation of the infertile couples attending the Reproductive Endocrine Outpatient Clinic of a tertiary women’s hospital in Ankara, Turkey, between January 1, 2012 and December 31, 2013. The samples were classified according to sperm concentration: ≥15 x10⁶/mL as normozoospermic samples and 4 -14.99 x10⁶/mL as oligozoospermic samples and seasonal analysis of the semen samples were carried out separately. When the data was analyzed according to the season of semen production, there was no seasonal effect on the sperm concentration. A gradual and consistent decrease in the rate of sperm with fast forward motility was observed from spring to fall with a recovery noticed during the winter. The percentage of sperms with normal morphology was found to be statistically significantly higher in the spring samples compared with the summer samples (p=0.001). Both normozoospermic and oligozoospermic semen samples appeared to have better sperm parameters in spring and winter. The circannual variation of semen parameters may be important in diagnosis and treatment desicions.

Abbreviations: WHO: World Health Organization; mRNA:messenger ribonucleic acid     

Contraindication of ART following a sperm FISH analysis,even though only 12% of the spermatozoa had enlarged heads

We report on a couple with a five-year history of idiopathic primary infertility. Two early miscarriages had followed intrauterine insemination (IUI). The man's fertility was then re-evaluated, in order to establish whether or not IUI was the best treatment option. Although the semen parameters were normal (sperm concentration: 89 million/ml; progressive motility: 40%; percentage of typical forms: 20%), a computer-assisted sperm morphology analysis with strict criteria found that 12% of the spermatozoa had enlarged heads. All of the latter had a normal form and none had multiple flagella. Using fluorescence in situ hybridization (FISH) analysis, we found that the proportion of aneuploid and diploid spermatozoa was 78% for the sample as a whole and 68% for normally-shaped spermatozoa with a normal-sized head. Although treatment options are well documented for men with macrocephalic sperm head syndrom, there is no consensus on individuals with a low but non-negligible proportion of spermatozoa with enlarged heads. Here, our FISH results contraindicated the use of assisted reproductive technology with the man's sperm. The couple decided to resort to donor sperm.     

Predicting the clinical outcome of ICSI by sperm head vacuole examination

To assess whether high magnification sperm head vacuole examination (SHVE) and/or standard sperm morphology assessment can predict ICSI outcomes in terms of fertilization, embryo quality, and delivery rates, a prospective observational bicentric study was conducted in two publicly funded assisted reproductive technology (ART) units in France between January and July of 2012. A total of 111 ICSI cycles for exclusively male infertility factors were included. A Spearman’s correlation test was performed to validate SHVE reproducibility between the ART units. The normal morphology rate and SHVE performed on selected spermatozoa were respectively determined according to David’s and Vanderzwalmen’s classifications used for motile sperm organelle morphology examination (MSOME) on the day of the ICSI. Receiver Operating Characteristic (ROC) curve analysis was performed to determine thresholds associated with the occurrence of a delivery. There was an excellent correlation between the two operators (r=0.98), thus validating the study’s SHVE data. Percentages of normal morphology grade spermatozoa using the standard classification and first-best morphology grade spermatozoa determined by SHVE were not significantly associated with (i) delivery (p=0.58; 0.90 /area under curve (AUC) =0.532; 0.507), (ii) fertilization (p=0.88; 0.90), (iii) top-quality embryos (p=0.27; 0.98), and (iv) good quality embryo rates (p=0.73; 0.98), respectively. In conclusion, high magnification SHVE and standard sperm morphology assessment cannot predict clinical or biological ICSI outcomes.

Abbreviations: ART: assisted reproductive technology; HBV: hepatitis B virus; HCV: hepatitis C virus; HIV: human immunodeficiency virus; ICSI: intra-cytoplasmic sperm injection; IVF: in vitro fertilization; LNVs: large nuclear vacuoles; MSOME: motile sperm organelle morphology examination; SHVE: sperm head vacuole examination; WHO: World Health Organization     

精子DNA碎片与精液各参数的相关性分析

目的探讨男性不育患者精子DNA碎片与精液各参数的相关性,研究精子DNA完整性在评估男性生育力方面的应用价值。方法回顾性分析2014年1月至2015年11月就诊于邢台不孕不育专科医院生殖中心男科的不育患者265例,采集精液标本,通过计算机辅助行常规精液分析,采用瑞-吉染色镜检观察精子形态及通过精子染色体扩散试验(SCD法)行精子DNA碎片率检测,对精子DNA碎片率与年龄、精液各参数(精子浓度、前向运动精子、正常精子形态)进行分组比较及相关分析。结果不同年龄段精子DNA碎片组间比较差异有统计学意义(P0.05);不同精子DNA碎片化指数(DNA fragmentation index,DFI)、精子浓度组间比较差异无统计学意义(P0.05),前向活动精子、正常形态精子组间比较差异有统计学意义(P0.05);年龄与精子DNA碎片存在正相关关系,前向运动精子率、正常形态精子率均与精子DNA碎片存在负相关关系。结论年龄是影响精子DNA损伤的因素,精子DNA损伤可能是导致精子活力和正常形态精子的影响因素。     

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。     

广西地区正常生育力男性精液质量的调查分析

目的了解广西地区正常生育力男性的精液质量状况。方法选择广西地区有生育力的男性进行精液检测,对精液的各主要参数进行分析,比较壮族和汉族不同居住地间的差别。结果 687例正常生育力男性的精子密度、存活率、精液量和pH值与世界卫生组织(World Health Organization,WHO)——第4版的正常参考值符合率较高,液化时间、正常精子形态率和活动力的符合率偏低。其中有321例(46.72%)达到世界卫生组织的精液分析正常值标准。汉族正常精子形态率则低于壮族,差异具有统计学意义(P<0.05)。居住在农村的男性和城市的相比较,除pH值无明显差异(P>0.05)外,其他参数均明显优于城市,差异有统计学意义(P<0.05)。结论广西地区正常生育力男性的精液参数与国内其他调查分析结果相似;广西汉族、壮族间精液质量差别不大,农村男性的精液质量优于城市男性。     

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.     

男性不育患者精液分析和精子形态学分析

目的：回顾性分析助孕门诊男性不育患者精液分析与精子形态学检测，为防止临床对女方过度治疗提供依据。方法：选择2007年10月1日～2008年2月29日在本院助孕门诊就诊的不孕夫妇，男方至少行精液分析2次，在第一次精液分析即进行精子形态学分析。精液分析采用清华同方精液分析系统，精子形态学分析采用改良巴氏法染色，应用Microsoft Excel进行分析。结果：进行精液分析共1206人次，进行精子形态学分析共517人次。精子密度0-180^★10^9／L，平均55．3±32．2^★10^9／L，75．1％的病例精液密度为20-100^★10^9／L，仅13．3％为少精症，无精症占1．5％；a级精子为14．0％±9．9％，b级精子为10．6％±7．8％，a级＋b级〉50％者仅占0．6％，超过半数的为30％-50％（51．1％），精子形态学分析，仅有20．5％病例是正常的（〉14％）。结论：除无精症显然与不育相关外，精液密度可能不是评估男性不育的一个很好的指标，精子活动力低下或精子畸形率过高更能预示不孕因素，精液分析和精子形态学分析可以作为助孕门诊男性不育的首要筛查手段，防止对女方的过度治疗。     

RELATIONSHIP BETWEEN SOME REPRODUCTIVE PARAMETERS AND BIOCHEMICAL PROPERTIES OF BLOOD SERUM IN RAMS

The objective of the present study was to describe the relationship between some reproductive parameters, hormonal levels, and some biochemical properties of blood serum in rams under the Afyon province conditions. The total protein and globulin were positively (P?<?0.01) correlated with sperm motility and sperm concentration in all of rams. Total lipid was negatively (P?<?0.05) correlated with sperm motility and concentration in Daglic, and it was positively (P?<?0.01) correlated with percentage of abnormal spermatozoa in Chios rams. Total lipid and cholesterol was positively (P?<?0.01) correlated with triiodothyronine in all of rams and was negatively (P?<?0.05) correlated with testosterone in Daglic rams. Percentage of abnormal spermatozoa were negatively (P?<?0.01) correlated with alanine amino transaminase (ALT) level, but were positively (P?<?0.01) associated with aspartate amino transaminase (AST) level and AST/ALT ratio in all of rams.     

Characterization of viability,mitochondrial activity,acrosomal integrity and capacitation status in boar sperm during in vitro storage at different ambient temperatures

Extended storage of unfrozen boar semen becomes an alternative because the use of frozen-thawed boar sperm results in low fertility. Sperm viability, mitochondrial activity, capacitation and acrosome integrity of freshly ejaculated boar semen stored in vitro for up to 48 h at 4 degrees C, 15 degrees C, 20 degrees C and 39 degrees C was characterized during the study. The viability of boar sperm was assessed by both Hoechst 33258 and SYBR-14/PI staining. Mitochondrial function was assessed by JC-1 staining. Capacitation status was determined by chlortetracycline (CTC)/Hoechst 33258 staining. The acrosome integrity was analysed with Coomassie blue staining. These data were derived from three ejaculates each from three crossbred boars. The viabilities assessed with SYBR-14/PI, Hoechst 33258 and JC-1 staining correlated highly (r > 0.980). In freshly ejaculated boar semen, 96 +/- 1% of the sperm did not take up the Hoechst 33258, whereas 95 +/- 2% were stained by SYBR-14 and 96 +/- 2% of the sperm had mitochondria exhibiting positive JC-1 staining. Staining with CTC/Hoechst 33258 suggested that a high percentage of sperm became capacitated after 24 h storage at 15 degrees C and 20 degrees C. There were 62 +/- 2% (15 degrees C) and 89 +/- 2% (20 degrees C) capacitated sperm by 48 h. Moreover, most of the capacitated sperm were acrosome intact. These results suggest that SYBR-14/PI, Hoechst 33258 or JC-1 staining can be used to effectively evaluate the quality of boar sperm during in vitro storage.     

Natural Sperm Birefringence Can be Used to Estimate Sperm Viability and Morphology

Semen parameters were evaluated by a variety of tests. A polarizing microscope (PM) which allows, in a single step, for the evaluation of the number, motility, viability of sperm using the phenomenon of birefringence was used. This approach avoids a Papanicolaou staining procedure modified for sperm (PAP) performed in fixed material. The aim of this study was to examine the birefringence of sperm structures, in live cells, for the direct analysis of morphology on fresh samples. Semen samples from 15 men of recently proven fertility and from 66 male patients who attended our center for semen analysis were examined by polarization microscope (PM) analysis in order to find an index representing a pool of sperm with normal morphology and progressive motility. The receiver operating characteristics (ROC) curves were used to determine the performance of the proposed diagnostic method. The difference between the two areas under the ROC curves (PAP=0.76 and PM=0.82) was quantitatively not significant (P=0.308); however, the curve of the PM method was always higher than the curve of PAP, revealing that, qualitatively, PM was more sensitive than PAP. The PM index can represent the percentage of motile sperm with normal morphology, which is the actual pool of sperm that can reach and fertilize the oocyte. We suggest a PM diagnostic cut-off value of 20%, since this value was the lowest found for individuals of proven fertility.     

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。