Combined supravital staining and hypoosmotic swelling test.

The parameter of sperm viability in hypoosmotic solution (VHOS) provides information concerning the membrane integrity of the sperm head. The objective of this study was to determine the association between the VHOS parameter and the sperm penetration assay. The VHOS parameter correctly predicted 70.0-71.4% of the failed sperm penetration assay samples in the short duration preincubation groups. A combination of both the VHOS parameter and the hypoosomotic sperm swelling (HOS) test significantly reduced the number of false negative results. In general, a sperm sample with an abnormal VHOS result and an abnormal HOS test result would be associated with a negative sperm penetration assay. Washing by centrifugation appeared to weaken the sperm head membranes while the swim-up method selected for sperm with strong head and tail membranes. After the various processing methods unique changes in the integrity of the sperm head and tail membranes for each sperm sample may help to identify the optimal method of preparation for individual patients undergoing the newer assisted reproductive technologies such as sperm microinjection.     

Sperm-associated oocyte-activating factor is released from the spermatozoon within 30 minutes after injection as a result of the sperm- oocyte interaction

We have previously shown that sperm plasma membrane damage makes the sperm plasma membrane permeable and the sperm nucleus accessible for low molecular weight molecules such as eosin and dithiothreitol. In the present study, we investigated whether this damage is associated with a passive release of the sperm-associated oocyte activating factor (SAOAF) from the spermatozoon and, if so, its time sequence. In a first study, human oocytes remaining unfertilized after conventional in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) and freshly ovulated mouse oocytes were injected with a whole spermatozoon or a sperm head respectively. They were randomly allocated to one of three groups: oocytes in group 1 were injected with a spermatozoon immobilized or sperm head detached immediately prior to the injection; oocytes in group 2 were injected with a spermatozoon immobilized or sperm head detached 2-4 h before injection; oocytes in group 3 were injected with a spermatozoon or sperm head that had been subjected to heat treatment. The activation rate of oocytes injected with a spermatozoon or sperm head was the same for groups 1 and 2, and significantly higher than in group 3 (P < 0.001). In a second series of experiments, human oocytes remaining unfertilized after IVF or ICSI were injected with a sperm head that was subsequently removed from the ooplasm 20-30 min after injection. The activation rates were compared to that of oocytes injected with heat-treated spermatozoa which subsequently were removed from the ooplasm. We found that the removal of the spermatozoon 30 min after injection did not prevent oocyte activation. Our data indicate that the initial damage to the sperm plasma membrane induced at immobilization, although essential for the onset of sperm nuclear swelling after ICSI, does not by itself lead to the release of SAOAF from the spermatozoon. We postulate, however, that SAOAF is released during the sperm nuclear swelling phase, which is induced by the so-called sperm nucleus decondensing factor (SNDF) of the oocyte.      

Determination of optimal view angles for quantitative facial image analysis

In quantitative evaluation of facial skin chromophore content using color imaging, several factors such as view angle and facial curvature affect the accuracy of measured values. To determine the influence of view angle and facial curvature on the accuracy of quantitative image analysis, we acquire cross-polarized diffuse reflectance color images of a white-patched mannequin head model and human subjects while varying the angular position of the head with respect to the image acquisition system. With the mannequin head model, the coefficient of variance (CV) is determined to specify an optimal view angle resulting in a relatively uniform light distribution on the region of interest (ROI). Our results indicate that view angle and facial curvature influence the accuracy of the recorded color information and quantitative image analysis. Moreover, there exists an optimal view angle that minimizes the artifacts in color determination resulting from facial curvature. In a specific ROI, the CV is less in smaller regions than in larger regions, and in relatively flat regions. In clinical application, our results suggest that view angle affects the quantitative assessment of port wine stain (PWS) skin erythema, emphasizing the importance of using the optimal view angle to minimize artifacts caused by nonuniform light distribution on the ROI. From these results, we propose that optimal view angles can be identified using the mannequin head model to image specific regions of interest on the face of human subjects.     

用双色荧光原位杂交检测人精子染色体非整倍体率

目的检测人精子染色体非整倍体率。方法采用双色荧光原位杂交（ＦＩＳＨ）方法,取少量精标本经洗后制片,用二硫苏糖醇（ＤＴＴ）和二碘水杨酸锂（ＬＩＳ）处理,使精子头部染色质去凝集。然后,与生物素标记的α卫星Ｘ染色体特异ＤＮＡ探针（ＤＸＺ１）和地高辛标记的α卫星Ｙ染色体特异ＤＮＡ探针（ＤＹＺ３）进行原位杂交。用ＣＹ３－链亲和素、山羊抗链亲和素检测Ｘ染色体探针杂交信号;用鼠抗地高辛抗体、与荧光素结合的兔抗鼠抗体检测Ｙ染色体探针杂交信号。结果在Ｎｉｋｏｎ荧光显微镜下可以清楚看到精子头部的杂交信号,头部有１个红色荧光杂交信号的精子为Ｘ染色体精子（Ｘ精子）,有１个绿色荧光杂交信号的精子为Ｙ染色体精子（Ｙ精子）。精子头部有２个荧光杂交信号的精子为染色体数目异常精子。若用１条常染色体探针和１条性染色体探针进行ＦＩＳＨ,可以区别头部有２个相同颜色荧光杂交信号的精子属非整倍体精子或二倍体精子。结论双色荧光原位杂交（ＦＩＳＨ）方法,可以用于测定接触致突变剂和非整倍体诱导剂后,人精子染色体非整倍体率的变化。     

In vitro sperm capacitation to treat antisperm antibodies bound to the sperm surface.

Our objective was to study antisperm antibody bound to the acrosome region during in vitro capacitation and to determine whether acrosome-antibody free sperm can be obtained from previously acrosome-antibody-coated sperm. The spermatozoa from a selected series of 14 patients were tested for sperm antibodies bound to the sperm surface using d-IBT and focusing on the acrosome positivity. The tests were carried out before the incubation and after 3, 6, 9, and 12 h of incubation in Tyrode's solution with 0.5% human serum albumin as the capacitation medium. Tests to evaluate acrosome region, sperm motion parameters, and zonae binding ability were carried out. In this way we were able to evaluate sperm function during capacitation protocol. The patients were 14 subjects selected according to good seminal characteristics, good post-rise sperm parameters, and high percentage of ASA bound to the sperm surface. In all cases the results showed that antisperm antibodies bound to the acrosome region were shed prior to the acrosome reaction. During sperm capacitation in human a modification, migration, or shedding of plasma membrane molecules takes place. The presence of antibodies in such an important area of the sperm head could certainly interfere in the fertilization process. Our data indicate that in vitro capacitation could provide an in vitro therapy capable of eluting antibodies from the acrosome region.     

Fertilization potential of spermatozoa with abnormal morphology

One of the best discriminators for the fertilization potential of human spermatozoa is sperm morphology. The problem in the assessment of the sperm morphological characteristics is their pleiomorphism. Examination of spermatozoa with the light microscope can provide only limited information on their internal structure. More detailed examination of sperm structure using electron microscopy can reveal major, often unsuspected ultrastructural abnormalities. Results and cut-off values for sperm analysis depend on the criteria for normal morphology. World Health Organization recommendations provide a classification suitable for clinical practice. Clinically reliable cut-off limits for normal sperm morphology according to strict Tygerberg criteria were suggested to be 4% in in-vitro fertilization procedures. Patients with severe sperm head abnormalities have a lower chance of establishing successful pregnancies, even though fertilization may be achieved. The outcome of intracytoplasmic sperm injection is not related to any of the standard semen parameters or to sperm morphology. Sperm decondensation defects and DNA anomalies may be underlying factors for the unrecognized derangements of the fertilizing capacity of spermatozoa, regardless of sperm morphology. Centrosome dysfunction may also represent a class of sperm defects that cannot be overcome simply by the insertion of a spermatozoon into the ooplasm. In this article an overview on the composition and ultrastructure of spermatozoa is presented, while emphasizing sperm ultrastructural and sperm DNA anomalies and their effects on fertilization.     

Aetiology of failed and abnormal fertilization after intracytoplasmic sperm injection1

The aim of this study was to determine why oocytes remain unfertilizedor develop three pronuclei after intracytoplasmic sperm injection(ICSI). Unfertilized and abnormally fertilized oocytes werefixed in glutaraldehyde,stained with Hoechst 33342 and examinedby fluorescence microscopy to identify oocyte, sperm and polarbody DNA.One-pronuclear oocytes were considered to be unfertilized.Atotal of 285 unfertilized oocytes were examined (104 ICSI cycles).Overall, 83% of these oocytes were not activated (still at metaphaseII) while 17% had activated and formed a single (female) pronucleus.About 66% of the unfertilized, metaphase II oocytes containeda swollen sperm head, indicating that the oocyte was correctlyinjected but had failed to activate and complete the secondmeiotic division. Premature chromosome condensation of the spermDNA was evident in 6% of these metaphase II oocytes (4% of theunfertilized oocytes). The swollen sperm head was located amongthe oocyte chromosomes in 5%of the metaphase II oocytes. Othercauses of failed fertilization in the metaphase II oocytes werethe failure of sperm head decondensation (11%) and ejectionof the spermatozoon from the oocyte (23%). A similar patternwas observed in one-pronuclear oocytes (52%, swollen sperm head;28%, intact, undecondensed sperm head; 20%, ejection of thespermatozoon), which indicates that asynchronous pronucleardevelopment does not explain the presence of one-pronuclearoocytes. A total of 41 threepronuclear oocytes were examinedand all had a single polar body, which indicates that the retentionof the second polar body leads to the formation of the thirdpronucleus.In conclusion, this study demonstrates that: (i)the major cause of fertilization failure after ICSI is failureof oocyte activation; (ii) ejection of the spermatozoon intothe perivitelline space is not a major cause of fertilizationfailure;and (iii) sperm head decondensation and oocyte activationafter ICSI can occur independently.     

Formation and organization of the mammalian sperm head

The formation and organization of a mammalian sperm head occurs through diverse cellular and molecular processes during spermiogenesis. Such cellular events include sequential changes in the nucleus and the acrosome-which is derived from the Golgi apparatus-in concert with prominent bundles of microtubules, the manchette. However, these complex processes are readily impaired by a variety of intrinsic and extrinsic factors, eventually causing various types of male infertility--such as teratozoospermia--which include the deformation of the acrosome and nucleus. In order to comprehend such idiopathic male infertility syndromes, it is important to clarify the mechanism involved in sperm head formation and organization. In addition to the manchette, two key structures in these events are the acroplaxome and the perinuclear theca. The acroplaxome forms the acrosome plate with periodic intermediate filament bundles of the marginal ring at the leading edge of the acrosome, and its nature has recently been characterized. The perinuclear theca, which is located in the perinuclear region in the sperm head, contains not only a cytoskeletal element to maintain the shape of the sperm head but also functional molecules leading to oocyte activation during fertilization. This review discusses recent developments regarding the formation and organization of the mammalian sperm head in relation to its relevant functions.     

Binding of ethylene oxide in spermiogenic germ cell stages of the mouse after low-level inhalation exposure

Mice received inhalation exposures of 3H-labeled ethylene oxide (EtO) gas at levels from 0.65 to 3.2 parts per million-hours (ppm-hr), which are below the exposure limits currently allowed for humans. Subsequently, spermatozoa were recovered from the reproductive tracts of the animals over a two-week period and assayed for the amount of bound EtO. A strong increase in the level of EtO binding occurred in late spermatid stages; these stages are also genetically sensitive to the action of EtO. The maximum binding of EtO in late spermatids amounted to 6 X 10(3) alkylations/sperm head/ppm-hr of exposure. Alkylation of the DNA within the sperm accounted for a very small fraction of the total sperm head alkylation, averaging about 20 DNA alkylations per sperm per ppm-hr of exposure over the two-week period. However, alkylation of protamine, a protein unique to sperm cells, was found to be correlated with total sperm head alkylation and accounted for nearly all of the EtO binding. Protamine alkylation appears to be a significant cause of EtO-induced genetic damage in spermiogenic cells of the mammal.     

Human Sperm Anatomy: Different Expression and Localization of Phosphatidylinositol 3-Kinase in Normal and Varicocele Human Spermatozoa

Abstract

Recent reports support the possible role of PI3K in sperm capacitation and acrosome reaction, although studies regarding PI3K identity in human sperm, under certain disease states such as varicocele, are still lacking. The authors, therefore, examined the expression profile and ultrastructural localization of PI3K in human semen samples, comparing healthy donors and patients with varicocele. The results obtained performing western blotting assay showed decreased PI3K expression in varicocele with respect to the “healthy” sperm. Immunogold labeling revealed human sperm cellular compartments containing PI3K, evidencing it in the head at both the membrane and nucleus and the entire tail, from the middle to the end piece of normal sperm. In varicocele PI3K label was confined to the head, with a strong reduction of specific reaction in the neck, middle piece, and tail. In conclusion, the data suggest that PI3K may play a role in the maintenance of male factor infertility associated with varicocele, and it may be further exploited as an additional molecular marker for the diagnosis of male infertility disorders.     

Embryo quality and IVF treatment outcomes may correlate with different sperm comet assay parameters

BACKGROUND: Standard semen parameters have proven poor at predicting the outcomes of IVF treatment cycles. As recent studies suggest that the male genome may play an important role in early embryogenesis, this study attempts to correlate the level of sperm DNA damage in fresh semen and prepared sperm with the outcomes of conventional IVF treatment cycles. METHODS: Forty patients embarking on IVF treatment were recruited into this prospective observational study. Both fresh semen and PureSperm-prepared sperm were processed using a modified comet assay 3-6 months prior to the patients' IVF treatment cycles. Comet head DNA (mean and integrated head density) and tail DNA parameters (length and moment) were measured separately. RESULTS: Significant correlations between total sperm concentration and between comet length, moment, mean head density with embryo quality were detected in fresh semen and prepared sperm. Surprisingly, no significant correlations between head and tail parameters were detected. CONCLUSIONS: Comet head and tail DNA parameters appear to be potentially useful as predictors of embryo quality and IVF outcomes, especially in couples with unexplained subfertility. The lack of correlation between head and tail parameters may be due to a different mechanism of DNA damage within these two compartments.     

A multiresolution diffused expectation-maximization algorithm for medical image segmentation

In this paper a new method for segmenting medical images is presented, the multiresolution diffused expectation-maximization (MDEM) algorithm. The algorithm operates within a multiscale framework, thus taking advantage of the fact that objects/regions to be segmented usually reside at different scales. At each scale segmentation is carried out via the expectation-maximization algorithm, coupled with anisotropic diffusion on classes, in order to account for the spatial dependencies among pixels. This new approach is validated via experiments on a variety of medical images and its performance is compared with more standard methods.     

Exposure of actin on the surface of the human sperm head during in vitro culture relates to sperm morphology, capacitation and zona binding

BACKGROUND: The aim of this study was to determine the relationship between the proportion of motile sperm with actin exposed on the surface of the head and sperm function. METHODS: Semen samples were obtained from normozoospermic men and sperm function tests were performed. Motile sperm selected by swim-up were incubated with actin monoclonal antibody (A-mAb, 1:100) for 2 h, then anti-mouse IgG Dynabeads were used to detect sperm-bound A-mAb. Sperm capacitation was increased by phorbol myristate acetate (PMA) and decreased by bicarbonate-free medium. RESULTS: The proportion of sperm with exposed actin increased with time for up to 2 h incubation. Bicarbonate-free medium significantly decreased the proportion of sperm with exposed actin. PMA significantly enhanced this phenomenon. Sperm bound to zona pellucida (ZP) had a significantly higher proportion with exposed actin than did sperm remaining in medium. Of the 79 samples studied, an average of 9.4% (range 1-27%) of motile sperm had exposed actin after 2 h incubation and this was significantly correlated with sperm normal morphology and ZP binding. CONCLUSION: Exposure of actin on the surface of the sperm head during in vitro culture may be related to membrane modification during sperm capacitation and hence may be a useful marker for this subpopulation of sperm.     

The effects of coital lubricants on sperm motility in vitro

Infertility affects approximately 15% of couples, and in about one- third the primary cause is a male factor. Patients undergoing infertility investigations frequently experience sexual dysfunction, which often is due to inadequate vaginal lubrication. This can lead to increased use of coital lubricants. The effects of such lubricants on sperm motility have not been widely studied, although sperm motility is one of the best prognostic indicators of fertilization. Using a prospective longitudinal control-based study, we analysed the effect of adding four lubricants: KY jelly, baby oil, olive oil and saliva on sperm motion in 16 samples from patients undergoing infertility investigations. Sperm samples were prepared by density gradient centrifugation prior to mixing with lubricants. Motility parameters were determined using computer-assisted semen analysis after 5, 15 and 30 min. All lubricants except baby oil significantly decreased percentage progressive motility, progressive velocity, curvilinear velocity and lateral head displacement at 12.5% concentration. At a lower concentration of 6.25%, both olive oil and saliva still significantly reduced progressive motility parameters, while KY jelly diminished head movement parameters. Hence, even at these very low concentrations, coital lubricants impair sperm motility and thus may adversely affect fertility.      

Glycosylation related actions of glycodelin: gamete, cumulus cell, immune cell and clinical associations

Glycodelin is an example of a glycoprotein whose complex-type glycans mediate biological actions in human reproduction and immune reactions. Being attached to an identical protein backbone, glycodelin oligosaccharides vary significantly from one reproductive tissue to another and have an effect on its own secretion and role in cell communication. For instance, uterine glycodelin-A inhibits sperm-oocyte interaction by binding on the sperm head. This is a glycosylation-dependent phenomenon, in which fucosyltransferase-5 plays a key role. Glycodelin-S from seminal plasma binds evenly around the sperm head and maintains an uncapacitated state in the spermatozoa, until the isoform is detached during sperm passage through the cervix. Glycodelin-F from follicular fluid and Fallopian tube binds to the acrosomal region of the sperm head, thereby inhibiting both the sperm-oocyte binding and premature progesterone-induced acrosome reaction. The cumulus cells surrounding the oocyte can capture glycodelin-A and -F from the surrounding environment and convert these isoforms to a cumulus cell isoform, glycodelin-C. It differs by glycosylation from the other isoforms, and it too attaches on the sperm head, with the highest density in the equatorial region. Glycodelin-C is capable of detaching the sperm-bound inhibitory isoforms so that the sperm-oocyte binding is enhanced. Glycodelin-A also has immunosuppressive actions directed to cellular, humoral and innate immunity. Although these actions depend mainly on the protein backbone, glycosylation also plays a part. Glycosylated glycodelin may be involved in the protection of spermatozoa against maternal immune reactions, and glycodelin also has apoptogenic activity. Some glycosylation patterns of glycodelin may mask its apoptogenic domain. This review updates the recent research and clinical associations of glycodelin, highlighting the role of glycosylation.     

An electron microscopic study of sperm penetration into the rabbit egg after natural mating

The ultrastructure of fertilization has been studied in rabbit eggs recovered 11 to 15 hours after natural mating. Many sperm passing between the granulosa cells had undergone the acrosomal reaction, but this was not invariable, and, occasionally, intact sperm were present close to the zona pellucida. The cells of the corona radiata sometimes develop pseudopodial processes at the abovular surface and can ingest sperm after natural mating. The bulk of the content of the acrosome and the vesiculated elements formed during the acrosomal reaction, are lost before the sperm penetrates the zona pellucida, at which point the naked inner membrane of the acrosome is brought into intimate apposition with the zona. As the sperm cleaves a path through the zona pellucida, the posterior equatorial segment of the acrosome remains intact, and later persists as such in perivitelline sperm and quite possibly after incorporation of the sperm head into the vitellus. Sperm head entry into the vitellus is a two-fold process. The fertilizing sperm invariably fuses first with the vitelline membrane over the midposterior region of the head, whereas the rostral or acrosomal portion is drawn into the vitellus while encased by a flattened vesicle; this vesicle is comprised by the persistent inner membrane of the acrosome and externally by vitelline membrane sequestered from the egg surface. Soon after exposure to ooplasm, the sperm nucleus begins to decondense at a variable rate into a web of electron-dense strands; this process begins in the midposterior region, and then extends rostrally and caudally. At the same time the encasing membranes are reflected away from the anterior region of the nucleus, exposing subacrosomal material to the ooplasm. At this point the perforatorium remains, but this and the associated membranes are presumed to disintegrate eventually within the egg. After decondensation of the nucleus is complete, the faintly staining chromatin becomes enveloped by a series of compressed vesicles which together will form the porous limiting membrane of the male pronucleus. The last region to be incorporated is the sperm tail, the plasma membrane of which is lost as organelles of the tail pass into the ooplasm. During its incorporation, the midpiece engenders some reaction at the egg surface, and the mainpiece sometimes becomes fused with surface processes before it enters the body of the egg. The midpiece then commonly disintegrates, with dispersion of the mitochondrial sheath, whereas the mainpiece usually remains essentially intact until the time of syngamy or beyond.     

Sperm actin and calmodulin during fertilization in the hamster: an immune electron microscopic study.

The distribution of actin and CaM in hamster spermatozoa was examined during the early events of fertilization using postembedding immunogold procedures. Actin was immunolocalized with a polyclonal antibody and two monoclonal antibodies. CaM was immunodetected with a polyclonal antibody. In epididymal sperm, actin labeling was found solely in the principal piece of the flagellum. CaM labeling was observed in the postacrosomal lamina, subacrosomal ring, and tip of the perforatorium. These distributions were not modified after capacitation and acrosome reaction. During the successive steps of sperm-egg fusion actin remained undetected in the sperm head whereas its location did not change in the flagellum. CaM distribution remained unmodified until the sperm head begins to decondense. At later stages of sperm head decondensation the postacrosomal lamina and its CaM labeling disappeared, whereas gold particles were still detected in the subacrosomal layer. The predominant location of actin into the egg cortex, particularly the microvillus-free area was confirmed. Except for the CaM labeling of the meiotic spindle, no special CaM location could be found throughout the egg. Thus, in hamster, a role for sperm actin in sperm-egg fusion appears unlikely. In contrast the CaM present in the Ca(2+)-rich postacrosomal lamina could be involved in the regulation of egg activation.     

Automatic classification of the acrosome status of boar spermatozoa using digital image processing and LVQ

We consider images of boar spermatozoa obtained with an optical phase-contrast microscope. Our goal is to automatically classify single sperm cells as acrosome-intact (class 1) or acrosome-damaged (class 2). Such classification is important for the estimation of the fertilization potential of a sperm sample for artificial insemination. We segment the sperm heads and compute a feature vector for each head. As a feature vector we use the gradient magnitude along the contour of the sperm head. We apply learning vector quantization (LVQ) to the feature vectors obtained for 320 heads that were labelled as intact or damaged using stains. A LVQ system with four prototypes (two for each class) allows us to classify cells with an overall test error of 6.8%. This is considered to be sufficient for semen quality control in an artificial insemination center.     

Removal of bone in CT angiography by multiscale matched mask bone elimination

For clear visualization of vessels in CT angiography (CTA) images of the head and neck using maximum intensity projection (MIP) or volume rendering (VR) bone has to be removed. In the past we presented a fully automatic method to mask the bone [matched mask bone elimination (MMBE)] for this purpose. A drawback is that vessels adjacent to bone may be partly masked as well. We propose a modification, multiscale MMBE, which reduces this problem by using images at two scales: a higher resolution than usual for image processing and a lower resolution to which the processed images are transformed for use in the diagnostic process. A higher in-plane resolution is obtained by the use of a sharper reconstruction kernel. The out-of-plane resolution is improved by deconvolution or by scanning with narrower collimation. The quality of the mask that is used to remove bone is improved by using images at both scales. After masking, the desired resolution for the normal clinical use of the images is obtained by blurring with Gaussian kernels of appropriate widths. Both methods (multiscale and original) were compared in a phantom study and with clinical CTA data sets. With the multiscale approach the width of the strip of soft tissue adjacent to the bone that is masked can be reduced from 1.0 to 0.2 mm without reducing the quality of the bone removal. The clinical examples show that vessels adjacent to bone are less affected and therefore better visible. Images processed with multiscale MMBE have a slightly higher noise level or slightly reduced resolution compared with images processed by the original method and the reconstruction and processing time is also somewhat increased. Nevertheless, multiscale MMBE offers a way to remove bone automatically from CT angiography images without affecting the integrity of the blood vessels. The overall image quality of MIP or VR images is substantially improved relative to images processed with the original MMBE method.