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Excessive levels of free radicals diminish the functional integrity of spermatozoa. Levels of reactive oxygen species (ROS) produced by spermatozoa were negatively correlated with the quality of sperm in the original semen. “Intrinsic,” “extrinsic,” and “iatrogenic” sources of ROS production were identified within semen: intrinsic sources of ROS in semen are morphologically abnormal and arrested-maturity spermatozoa and leukocytes. Poor sperm quality showing attributes of arrested sperm maturation is linked to increased ROS generation as a consequence of excess residual cytoplasm related to the arrest of cytoplasmic extrusion in terminal spermiogenesis. In the past 30 years, the Huszar lab has studied several key events of sperm maturation, including cytoplasmic extrusion and expression of the HspA2 chaperone protein. In experiments related to sperm function and fertilizing potential, it has been established that, simultaneously with cytoplasmic extrusion during terminal spermiogenesis, there is a remodeling of the plasma membrane that facilitates the formation of the zona pellucida and hyaluronic acid (HA)-binding sites. The studies with HA immobilized to glass slides or Petri dishes showed that sperm firmly bind to HA. However, not all sperm exhibited HA-binding ability. These data supported the hypothesis that the ability of sperm-HA binding is related to sperm cellular maturity. Although, recent literature on the efficacy with HA selection of human sperm reported a lack of efficacy but with the caution that is either inadequately powered or of low-quality, HA-mediated sperm ICSI selection may increase fertilization rates and paternal contribution of sperm to the embryo even if the man to be treated is affected by seminal ROS production. Indeed, well-designed new studies are needed to evaluate this aspect in terms of potential evaluation and treatment of subfertile men with ROS-affected semen utilizing HA.

Our aim was the development of a safe sperm cryopreservation New Media (NM), composed of consistent and reproducible components devoid of any animal origin, and evaluation of NM in terms of its effect on sperm structure and function as compared to regularly used yolk media (TYM) (Irvine Scientific).We evaluated patient semen samples and cryopreserved them in duplicates in either NM or TYM. The samples were cryopreserved for either a short term of 1 week or long term of 1 month prior to thawing. The parameters investigated include sperm motility via computer-assisted semen analysis (CASA), sperm concentration, and sperm biomarkers that promote paternal contribution of spermatozoa to fertilization including hyaluronic acid binding, chromatin maturity, apoptotic markers, cytoplasmic retention, and sperm DNA integrity.As compared to TYM, NM was equally capable of sperm cryopreservation with both short-term and long-term storage in media, and after freeze-thaw and gradient processing of sperm. HA binding of sperm was comparable post thaw in both NM and yolk media. There are also no differences observed between the samples cryopreserved in NM or TYM in terms of their aniline blue staining, CK immunocytochemistry, caspase 3 immunostaining, or DNA nick translation.NM has the advantage of being xeno-free, yet in preservation of sperm motility and other sperm attributes, the NM is as effective as the TYM.

During human spermiogenesis, the elongated spermatids undergo a plasma membrane remodeling step that facilitates formation of the zona pellucida and hyaluronic acid (HA) binding sites. Various biochemical sperm markers indicated that human sperm bound to HA exhibit attributes similar to that of zona pellucida-bound sperm, including minimal DNA fragmentation, normal shape, and low frequency of chromosomal aneuploidies. In this work, we tested the hypothesis that HA-bound sperm would be enhanced in sperm of high DNA chain integrity and green acridine orange fluorescence (AOF) compared with the original sperm in semen. Sperm DNA integrity in semen and in their respective HA-bound sperm fractions was studied in 50 men tested for fertility. In the semen samples, the proportions of sperm with green AOF (high DNA integrity) and red AOF (DNA breaks) were 54.9% ± 2.0% and 45.0% ± 1.9%, whereas in the HA-bound sperm fraction, the respective proportions were 99% and 1.0%, respectively. The data indeed demonstrated that HA shows a high degree of selectivity for sperm with high DNA integrity. These findings are important from the points of view of human sperm DNA integrity, sperm function, and the potential efficacy of HA-mediated sperm selection for intracytoplasmic sperm injection.

Problem Interleukin (IL)-18 is a novel cytokine, previously known as interferon (IFN)-γ inducing factor. We evaluated the levels of IL-18 and IFN-γ in seminal plasma (SP) of fertile and infertile men. Method of study Semen samples were obtained by masturbation from 80 men, and were examined for the levels of IL-18 and IFN-γ by enzyme-linked immunosorbent assay. Seven groups were included: (i) fertile men (n = 18), (i) infertile men with genital tract infections (n = 17), (iii) with varicocele (n = 15), (iv) with Klinefelter syndrome (n = 6), (v) with cryptorchidism (n = 7), (vi) with mumps orchitis (n = 7), and (vii) with idiopathic testicular lesions (n = 10). Results Mean levels of IL-18 were higher in SP from infertile men with genital tract infections compared with SP from other groups except Klinefelter syndrome (P

Objective To explore the dimensional attributes of haploid and disomic X-bearing and Y-bearing spermatozoa. Design Morphometric evaluation of more than 2,000 X-bearing and Y-bearing spermatozoa after identification of the genotype with fluorescence in situ hybridization. Setting Academic clinical and research andrology laboratory. Main Outcome Measure(s) Sperm head area, perimeter, long axis, short axis, shape factor, elliptical form factor (long axis/short axis), and tail length. Result(s) We found no differences in dimensions or dimensional distributions between X-bearing and Y-bearing spermatozoa, whether in the native or the decondensed state, or in oligozoospermic or normozoospermic men. There were inconsistent differences and a 70% overlap in the dimensions of haploid and disomic spermatozoa. The other 30% of sperm with disomic nuclei were either smaller or larger compared to haploid spermatozoa. Conclusion(s) There are no differences, or distinguishing characteristics, in dimensions or dimensional distributions between X-bearing and Y-bearing spermatozoa. Dimensional attributes do not discriminate between dysomic and haploid spermatozoa.

Background We hypothesize that the potential relationship between abnormal sperm morphology and increased frequency of numerical chromosomal aberrations is based on two attributes of diminished sperm maturity: (i) cytoplasmic retention and consequential sperm shape abnormalities; and (ii) meiotic errors caused by low levels of the HspA2 chaperone, a component of the synaptonemal complex. Because sperm morphology and aneuploidies were assessed in semen, but not in the same spermatozoa, previous studies addressing this relationship were inconclusive. We recently demonstrated that sperm shape is preserved following fluorescence in situ hybridization (FISH). Thus, we examined the shape and chromosomal aberrations in the same sperm. Methods We performed phase contrast microscopy and FISH, using centromeric probes for chromosomes X, Y, 10, 11 and 17 in 15 men. The fluorescence and respective phase contrast images were digitized using the Metamorph program. We studied 1286 sperm (256 disomic, 130 diploid and 900 haploid sperm) by three criteria: head and tail dimensions, head shape and Kruger strict morphology. Furthermore, in each analysis, we considered whether disomic or diploid sperm may be distinguished from haploid sperm. Results There was an overall, but not discriminative, relationship between abnormal sperm dimensions or shape and increased frequencies of numerical chromosomal aberrations. However, approximately 68 of the 256 disomic, and four of 130 diploid sperm showed head and tail dimensions comparable with the most normal, lowest tertile of the 900 haploid spermatozoa. Considering all 1286 sperm, among those with the most regular, symmetrical shape (n = 367), there were 63 and five with disomic and diploid nuclei, respectively. In line with these findings, among the 256 disomic sperm, 10% were Kruger normal. Conclusions Sperm dimensions or shape are not reliable attributes in selection of haploid sperm for ICSI.

The relationship between human sperm maturity and apoptosis is of interest because of the persistence of immature sperm in ejaculates in spite of various apoptotic processes during spermatogenesis. We assessed sperm maturity by HspA2 chaperone levels, and plasma membrane maturity by sperm binding to immobilized hyaluronic acid (HA). We also utilized objective morphometry. Sperm were stained with three antibody combinations: active caspase-3/creatine kinase (CK, a marker of cytoplasmic retention), caspase-3/the antiapoptotic Bcl-(XL), and CK/Bcl-(XL). In semen, 13% of sperm stained with CK, caspase-3 or Bcl-(XL), and 28% had stained with two markers. In the mature HA-bound sperm fraction,4% were single- or double-stained. Regarding sperm regions, CK staining, whether alone or as double staining, occurred in the head and midpiece (15-20%), whereas caspase-3 and Bcl-(XL) were primarily (80% of sperm) in the midpiece. Morphometrical attributes of clear, single- and double-stained sperm, in line with their more pronounced maturation arrest, showed an incremental increase in head size (due to cytoplasmic retention) and shorter tail length. We hypothesize that during faulty sperm development, three alternatives may occur: (i) elimination of aberrant germ cells by apoptosis; (ii) in surviving immature cells, caspase-3 is activated, and in response the antiapoptotic Bcl-(XL), and perhaps HspA2, provide protection; (iii) in a third type of immature sperm, in addition to the CK, caspase-3 and Bcl-(XL) expression, there are related manifestations of increased head size and shorter tail length. Thus, immature sperm may vary in the type of developmental arrest and in protection mechanisms for apoptosis. These variations are likely to explain the persistence of immature sperm in the ejaculate.

BACKGROUND We have previously shown that after 80% Percoll centrifugation there is an overall 2.7-fold reduction of sperm with chromosomal disomies and diploidies (3.2-fold and 2.0-fold respectively), and of sperm with diminished maturity as detected by cytoplasmic retention. The relationship between disomies and immature sperm was r = 0.7, suggesting that disomy primarily originates in immature sperm. In the present work we studied the efficacy of the swim-up method in elimination of sperm with diminished maturity and with chromosomal aberrations in the swim-up sperm fractions of 10 patients (sperm concentration: 20 +/- 3.9 x 10(6)/ml, range 8.9-45.5; sperm motility: 45.2 +/- 2.4, all mean +/- SEM). METHODS The validity of the study was enhanced by assessing each sperm fraction with three-colour (X, Y and 17; 5000 sperm) and two-colour (10 and 11; 5000 sperm) chromosome probes using fluorescence in-situ hybridization (FISH). Thus, in each sample 10 000 sperm were evaluated. The incidence of diminished maturity sperm was assessed with creatine kinase immunocytochemistry. RESULTS In the swim-up fractions there was a reduction in the frequencies of disomic sperm, whether considering the sex chromosomes (1.4-fold) or the three autosomal chromosomes (1.5-fold based on the aggregate frequencies of disomy 10, 11 and 17). There was also a 1.5-fold reduction in diminished maturity sperm, indicating a relationship between the proportion of immature sperm and chromosomal aneuploidies (r = 0.46, P < 0.05, n = 20). Diploid sperm were reduced at a 2.7-fold rate, whether assessed with two- or three-colour FISH. There was a slight increase in the X/Y ratios. CONCLUSIONS Swim-up reduces the proportion of sperm with chromosomal aberrations and of sperm with diminished maturity. When compared with the results of the previous study with gradient centrifugation performed on semen samples with similar quality, the efficacy after swim-up is lower for disomies and higher for diploidies than that of gradient centrifugation.

Objective To test, both in semen and washed-sperm fractions, whether hyaluronic acid (HA) binding is restricted to sperm that have completed cellular maturation. Design Comparisons of sperm in semen and in HA-bound sperm fractions. Setting University-based diagnostic and research andrology laboratory. Patient(s) Semen samples originated in men being tested for infertility. Intervention(s) The attributes of sperm maturity were tested by immunocytochemistry with creatine kinase and HspA2 antisera (highlights cytoplasmic retention in diminished-maturity sperm), aniline blue chromatin staining (detects persistent histones), pisum sativum lectin staining (reveals acrosomal integrity), and the FertiLight viability kit (highlights viable and nonviable sperm). Result(s) All markers of sperm maturity and immaturity supported the hypothesis that HA-bound sperm are mature. Nonbinding sperm exhibited cytoplasmic and nuclear properties of diminished maturity. The acrosomal status of HA-bound sperm was either unreacted or slightly capacitated, but not acrosome reacted. Only viable sperm exhibited HA binding. Conclusion(s) Sperm that are able to bind to HA are mature and have completed the spermiogenetic processes of sperm plasma membrane remodeling, cytoplasmic extrusion, and nuclear histone–protamine replacement. Hyaluronic acid–bound sperm show unreacted acrosomes. These studies provide further insights into the relationship between spermiogenesis and sperm function.

The relationship between abnormal sperm morphology and chromosomal aberrations has been of interest. Thus far, however, studies have focused on frequencies of sperm with either abnormal morphology or aneuploidies in semen samples, not on detection of individual spermatozoa exhibiting both abnormal morphology and aneuploidy. To assess the feasibility of simultaneous evaluation of both attributes in an individual sperm cell, we investigated whether sperm shape is preserved after decondensation and denaturation, procedures that are required for fluorescent in situ hybridization (FISH). On 21 slides, 395 sperm were fixed, photographed, and then digitized by the computerassisted Metamorph morphometry program for individual evaluation before decondensation. To establish whether sperm of various shapes would behave in similar manners, the cells were also classified, according to their head shapes, into symmetrical (n 5 115), asymmetrical (n 5 115), irregular (n 5 115), and amorphous (n 5 50) categories. Following decondensation and subsequent denaturation, sperm that had been photographed initially were relocalized and digitized for morphometry. Head area, perimeter, long axis, short axis, shape factor, and tail length were evaluated in each of the 395 sperm in both the native and decondensed states. After the decondensation and denaturation protocol of the FISH procedure, the sperm exhibited a proportional increase in dimensions as compared to their original sizes. Their initial shapes were preserved with high fidelity whether the sperm were in the symmetrical, asymmetrical, irregular, or amorphous categories. Hybridization with the chromosome probes had no further effect on sperm shape or size. We provide images to demonstrate how these findings facilitate studies about the relationship between sperm shape and chromosomal content or aberrations in individual spermatozoa. sperm

To develop a method for delayed assessment of sperm motility, after shipment of semen to a remote laboratory. Sperm in semen were labeled with the MitoTracker(®) Red CM-H(2)XRos reagent, and fixed with 3.7 % formaldehyde by the laboratory technicians at the origin of the semen. This treatment reflected well sperm mitochondrial activity, and the MitoTracker(®) signal was related to sperm motility and velocity for 2-3 days following ejaculation.Sperm motility and velocity were evaluated manually and by computer assisted semen analysis (CASA), respectively. Fluorescence assessment of individual sperm was carried out with the computer assisted Metamorph v4.6.9 program. Emission levels of MitoTracker(®) spermatozoa were studied in room temperature and cooled semen, or in the respective room temperature swim-up sperm fractions following ejaculation, and on the second day (N = 103 samples, 89 men) and third day (N = 10 samples, 8 men).Sperm with optical density (O.D.) ≥0.7 showed close correlations with ejaculatory sperm motility and velocity even after second day (r = 0.92, p 0.001, N = 103 samples). Further, the multiple of sperm motility and velocity was also related to the proportion of high MitoTracker(®) reagent emission sperm (r = 0.83, p 0.001, N = 103 samples). MitoTracker(®) dye fluorescence on the second day accurately reflected the ejaculatory sperm motility (r = 0.90, p 0.001). Thus, a shipping delay would not adversely affect the results.The delayed assessment of sperm motility in samples treated with MitoTracker(®) Red CM-H(2)XRos reagent and shipped to remote laboratory truly reflects the level of sperm motility at the time of the ejaculation.

WOS:000337285300004 PubMed: 24077441 In mammalian species, acquisition of sperm fertilization competence is dependent on the phenomenon of sperm capacitation. One of the key elements of capacitation is protein tyrosine phosphorylation (TP) in various sperm membrane regions. In previous studies performed, the pattern of TP was examined in human sperm bound to zona pellucida of oocytes. In the present comparative study, TP patterns upon sperm binding to the zona pellucida or hyaluronic acid (HA) were investigated in spermatozoa arising from the same semen samples. Tyrosine phosphorylation, visualized by immunofluorescence, was localized within the acrosomal cap, equatorial head region, neck, and the principal piece. Tyrosine phosphorylation has increased in a time-related manner as capacitation progressed, and the phosphorylation pattern was identical within the principal piece and neck, regardless of the sperm bound to the zona pellucida or HA. Thus, the data demonstrated that the patterns of sperm activation-related TP were similar regardless of the spermatozoa bound to zona pellucida or HA. Further, sperm with incomplete development, as detected by excess cytoplasmic retention, failed to exhibit TP.

We previously described a putative creatine kinase M isoform in human sperm that is developmentally regulated and expressed during late spermiogenesis, simultaneous with cytoplasmic extrusion. We have now identified this protein as the testis-expressed 70-kDa heat shock protein chaperone known as HspA2 (the human homologue of mouse Hsp70-2). We have isolated and characterized HspA2 (formerly CK-M) by amino acid sequencing and have localized it by immunocytochemistry to spermatocytes at low levels, to spermatids, and in the tail of mature sperm. The specificity of the CK-M/HspA2 antiserum to HspA2 was demonstrated on immunoblots of one- and two-dimensional SDS-PAGE. In agreement with our earlier biochemical data, immunocytochemistry of testicular tissue indicated that HspA2 is selectively expressed in mature spermatids and in sperm about to be released in the seminiferous tubuli. The identity of HspA2 has been further confirmed by cross-absorption of the mouse HSP70-2 antibody by the HspA2/CK-M fraction, and by identical immunostaining patterns of human testicular tissue using either the anti-CK-M/HspA2 or an anti-mouse Hsp70-2 antisera. During spermiogenesis, both cytoplasmic extrusion and plasma membrane remodeling, which facilitate the formation of the zona pellucida binding site, involve major intrasperm protein transport, which may be chaperoned by HspA2. Accordingly, in immature human sperm, which fail to express HspA2, there is cytoplasmic retention and lack of zona pellucida binding. The present findings provide the biological rationale for the role of the human HspA2 as an objective biochemical marker of sperm function and male fertility, which we have established in earlier clinical studies.

Prosaposin is a well-characterized, approximately 68- kDa protein found in many tissues and as a normal component of human semen. A fragment of prosaposin apparently is involved in primary sperm-egg binding. We hypothesized that binding of sperm from some men to egg investments would be increased by in vitro exposure of their sperm to a synthetic fragment of human prosa- posin (FertPlus#{174} peptide). Hence, we evaluated samples of washed fresh or frozen-thawed human sperm after a 10-minute exposure to synthetic FertPlus#{174} peptide at 0 (control), 80, 160, 320, 640, or 1280 pM, followed by 1:50 dilution for evaluation of binding. The criterion of response was mean percentage of sperm bound to a substrate prepared from chicken egg membranes after sperm were incubated for 60 minutes at 37#{176}C in substrate-coated wells of a sperm-binding assay plate. For each seminal sample, data were normalized against the percentage of sperm bound for control aliquots, providing values for relative binding. With fresh sperm, relative binding was increased (P 30% of men, exposure of their sperm to this synthetic fragment of prosaposin at 1280 pM increased binding of sperm to an egg membrane substrate similar to that offered by the zona pellucida.

Annually, >1.3 million men are members of couples seeking help because of infertility. Semen from many of these men contains reasonable numbers of motile and normal sperm, but for a subset of individuals, many sperm are deficient in ability to bind to the zona pellucida during in vitro fertilization. Diagnosis of this defect has been hampered by lack of a low-cost test. Molecular similarity exists between the perivitelline membrane of a hen's egg and the mammalian zona pellucida. These facts and some preliminary data led to evaluation of binding of human sperm during incubation for 60 minutes at 37 degrees C to an extract of chicken perivitelline membrane coated in microwell assay plates. The sperm-binding assay had inter- and intraassay plate variations of 21 and 12%, respectively, using washed fresh sperm. All seminal samples were normal, except a few that had 36 to 50% motile sperm with a low rate of sperm movement (if there is a low rate of movement, World Health Organization [WHO] criterion for normalcy is >50% motile). Nevertheless, this sperm-binding assay detected differences among individuals in percentage of sperm bound. Based on data for two to four ejaculates from each of eight occasional sperm donors, the coefficient of variation for ejaculates within donor averaged 31%, and means for the donors differed (P < 0.02). Percentage of sperm bound ranged from

As part of our studies on sperm maturity and function, we examined the head, midpiece and tail of human spermatozoa using computerized morphometry in order to determine which regions reflect the differences between mature spermatozoa and spermatozoa of diminished cellular maturity. We studied 20 men, who were divided into two groups based on their lower (LCKM: 14.6 +/- 7.0%, n = 8) and higher sperm creatine kinase (CK-M) isoform ratios (HCKM: 48.0 +/- 4.3%, n = 12) in the initial semen. Using a sequential centrifugation method which relies on the lower density of immature spermatozoa with retained extra cytoplasm, we prepared three sperm fractions with progressively declining maturity, as confirmed with CK-M isoform ratio measurements. Following the sequential fractionation, we affixed the spermatozoa to glass slides, stained the midpiece and the sperm contour, and photographed 25 spermatozoa in each of the 60 fractions (1509 spermatozoa in all). The spermatozoa were then individually digitized on the Image-1 system, and the dimensions of the head, midpiece, and tail were determined. While the data showed significant differences in the midpiece and tail dimensions between the mature and diminished-maturity sperm fractions, the head dimensions were similar and did not reflect sperm maturity. We postulated that the relationship between the biochemical markers of sperm maturity and sperm morphology is based on common spermiogenic events. The data support this idea. In immature spermatozoa in which cytoplasmic extrusion, CK-M isoform expression, and tail sprouting are all diminished, the retained extra cytoplasm in the midpiece and shorter tail length contribute to the morphological variations that we identified by morphometry and considered in sperm morphology. These morphometric features, in association with fluorochrome-coupled biochemical probes, can facilitate the identification of mature spermatozoa in computer-assisted semen analysis.

Purpose:Our purpose was to measure sperm creatine phosphokinase (CK) activity, which reflects cytoplasmic retention in immature spermatozoa, in normospermic and oligozospermic Hungarian men.

Purpose:Our purpose was to evaluate sperm motility and viability and the maintenance of these parameters in already cryopreserved semen samples following repeated freezing/thawing cycles. Methods:Human spermatozoa were subjected to five cycles of cryopreservation/thawing. Recovery of sperm motility and viability and the proportion of viable nonmotile sperm were determined up to 6 hr after thaw. Results:Sperm motilities (prefreeze motility, 70.1%; n = 9 samples) after each of five freeze/thaw cycles were 24.4, 8.0, 3.5, 1.5, and 1.8%. The recovery of sperm viability was higher than that of motility after each cycle: 39.1, 25.3, 22.6, 17.8, and 16.5%. Recoveries of motility and viability were improved if the thawed samples were left in the original cryopreservation medium prior to refreezing vs. if a washing/resuspension step was included. The recovery of sperm motility in the first thawing cycle was indicative of the expected motile sperm recovery in the second thawing cycle. Conclusions:Cryopreserved semen that is intended to be reused in future assisted reproduction treatments should be thawed only once and aliquoted in the original freezing medium before refreezing. The recovery of sperm motility and viability in the second thawing cycle, thus the applicability of the sample in conventional in vitro fertilization or intracytoplasmic sperm injection may be anticipated in >90% of the samples. In view of intracytoplasmic sperm injection it is important that sperm viability is maintained better than motility; after the first, second, and third thawing cycles the ratios of motile:nonmotile viable sperm were 1:1, 1:4, and 1:7, respectively.

The general perception of how innovative assisted reproductive technologies (ART) are introduced is through a carefully controlled series of experiments in an animal model, such as the mouse. Only after the technique has been proven can one consider confirmatory studies on mammals closely related to humans, such as rhesus monkeys or other nonhuman primates. With this background of a peer-reviewed body of well-established published data, there is sufficient foundation and rationale to propose a clinical investigation to a responsible human subjects institutional review board (IRB). IRBs weigh the benefits and risks of the new methods to human subjects, and then consider the appropriate informed consent procedures for the particular case. Only after a large number of clinical studies are performed at multiple sites and are peer reviewed can the efficacy and safety of the innovative approach be clearly evaluated. At that time, the potential therapy can be responsibly offered to suitable beneficiaries.

We have demonstrated previously that hyaluronic acid (HA) improves the velocity and the retention of motility in freshly ejaculated human spermatozoa. In the present work, we examined the effect of HA on cryopreserved/ thawed spermatozoa in four paradigms: (i) effect of HA on sperm motility and velocity in semen; (ii) stabilizing effect of HA after 4 h of incubation when the decline of sperm motility is already detectable; (iii) the duration of improved motility after the separation of spermatozoa from HA by Percoll gradient centrifugation; and (iv) motility of sperm cryopreserved in the presence of HA. HA improved the retention of sperm motility in thawed spermatozoa. Indeed, the motility values after 30 h were approximately 100% higher in the HA compared with the control samples. This effect of HA was also evident in the stabilization of spermatozoa with already declining motility. After removal of the HA from the incubation medium, significantly increased motility in the HA-exposed spermatozoa was still detectable for at least 4 h. Cryopreservation of spermatozoa in the presence of HA did not improve the recovery of motility. The data indicate that HA improves the retention of motility of cryopreserved/thawed spermatozoa, even after the removal of HA from the incubation medium. The utilization of HA will probably prove beneficial in assisted reproduction: in intrauterine insemination and in in-vitro fertilization (IVF), the extended sperm motility and velocity will enhance the fertilizing efficiency; in intracytoplasmic sperm injection (ICSI), the improved motility will facilitate the identification of viable spermatozoa. Because HA is a physiological component of the cumulus and of the female and male reproductive tracts, administration of HA should not cause ethical concerns.

Exposures to environmental toxicants can have detrimental effects on several aspects of human male reproduction: fertility, sexual function, hormone status, and pregnancy/birth outcomes. However, no simple prescreening methods are available for reliably identifying potential hazards; questionnaires alone are relatively imprecise and inefficient in the absence of field data. Multidisciplinary field studies are required that include detailed exposure information, health and reproductive histories, physical examinations, semen analyses, and possibly, hormone analyses. Semen analysis is a critical component of field studies for evaluating two aspects of male reproduction: 1) changes in sperm or seminal content, which may be indicative of adverse effects on the male reproductive system with possible implications for fertility potential; and 2) defects in sperm DNA or chromosomes, which may be associated with subsequent changes in viability during embryonic development and health risks to the offspring. Semen analyses may be tiered: 1) initially, each semen study may include conventional semen assays (concentration, motility, and morphology) as well as specific biomarkers indicated by the health effect of concern in the study cohort: and 2) archived samples (i.e., frozen, videotaped, or smeared) may be utilized in later second-tier analyses to further characterize specific findings. Before initiating any field study, it is cost effective to critically evaluate the suitability of the cohort by confirming exposure and determining that there are adequate numbers of male participants in each exposure category. Such evaluations must be based on the statistical sensitivities of the specific tissue biomarkers and health endpoints for detecting changes. This article summarizes the components of the ideal field study and identifies research needs for improving field studies of male effects and for understanding the mechanisms of male reproductive toxicity. Several promising semen methods currently under development are also discussed.

Sperm creatine phosphokinase (CK) concentrations and the synthesis of the CK-M isoform reflect normal spermiogenesis and predict maturity and fertilizing potential of ejaculated human spermatozoa. Immature spermatozoa, characterized by cytoplasmic retention and low CK-M to CK-B isoform ratios, are deficient in zona binding and fail to cause pregnancies. Because these sperm lack zona-binding ability, we examined in this study whether beta 1,4-galactosyltransferase (GalTase), a key element of sperm-zona interactions in mice, is diminished in immature human sperm. Unexpectedly, GalTase was overexpressed in immature sperm relative to mature sperm: the levels of cytoplasmic CK and plasma membrane GalTase were positively correlated (r = 0.78, p < 0.001, n = 88). Sperm populations with various levels of cellular maturity, prepared by Percoll gradients, had different CK and GalTase concentrations, but within each subpopulation the relationship between CK and GalTase was maintained (p < 0.01-0.001). GalTase activities in intact and vortex-disrupted sperm fractions were similar, showing that GalTase is present on the surface membrane of human sperm--similar to the situation in all other species assayed. The changes previously reported by our laboratory in zona-binding ability and lipid peroxidation rates (which occur simultaneously with cytoplasmic extrusion), decline in CK activity, and increased expression of the CK-M isoform are suggestive of a remodeling of the sperm surface concomitant with cytoplasmic maturation. The changes reported here in GalTase expression on the surface of maturing spermatozoa prove this hypothesis.

In addition to their role as man-made membranes, vesicles continue to be investigated as carriers for drug delivery. While most research focuses on their injectable properties, here a new delivery strategy is proposed. It is shown that spermatozoa can transport vesicles of variable composition. For human spermatozoa, the vesicles started to show binding after 20mol% of the nonbinding vesicle backbone lipids were substituted with positive, negative, cerebroside or ganglioside lipids. Vesicle binding is a dynamic process with constant ‘on' and ‘off' binding. The physiological and motility attributes of the spermatozoa are not affected by the attached vesicles. Sperm swimming characteristics changed only marginally. Also, the activation status of the acrosomal membrane, tested with the fluorescent probe Pisum sativum agglutinin, was not affected by vesicle binding. Moreover, the hyaluronic acid-binding test showed that viable, fully developed spermatozoa will attach and remain bound to hyaluronic acid-coated slides regardless of vesicle binding. Therefore a new ‘hybrid' delivery system was created with human spermatozoa, and tested with a mouse IVF system. Large unilamellar vesicles physisorbed to mouse spermatozoa can not only penetrate the mouse oocytes in these proof-of-principle experiments, but also deliver the cargo placed within the vesicles. Vesicles are artificially prepared, small lipid membrane-enclosed sacks that can store substances. As vesicles encapsulate an aqueous solution inside a partly hydrophobic membrane, they can carry both hydrophobic and hydrophilic molecules. Due to their unique properties, liposomes are often used as model systems to create or study artificial cells, as well as drug delivery vehicles. In order to transport nucleic acids, proteins or other cargo, the vesicles should exhibit mobility. In the present paper we propose a new delivery strategy, using the motion of spermatozoa to transport attached vesicles into the oocyte during fertilization. To use spermatozoa as a vesicle-transport system, we have to accomplish three goals. One, the lipid composition of the vesicle membranes needs to be created such that they can bind to the sperm cells. Two, the spermatozoa should still be able to swim with the extra loaded vesicle cargo. And three, the bound vesicles should not inhibit the capability of sperm cells to fertilize. In this study, vesicles started to show binding after 20mol% neutral lipids were substituted with either positive, negative, cerebroside or ganglioside lipids. A new ‘hybrid' delivery system has been created in which vesicles are bound to spermatozoa in physiological conditions with no significant alterations in sperm motility and function. Moreover, with IVF experiments in the mouse model, it was confirmed that spermatozoa remained capable of fertilizing while delivering vesicles simultaneously.

This chapter summarizes the scientific and clinical utility of the nuclear and cytoplasmic sperm biomarkers in the diagnosis and treatment of male infertility. These biomarkers were developed and used by the Yale Sperm Physiology Laboratory in the past 15 years. These biomarkers tested and were proven to reflect the degree of sperm development and fertilizing potential, including cytoplasmic retention measured by sperm creatine kinase content, chromatin structure, the key factor of HSPA2 chaperon protein concentration, DNA chain integrity, chromosomal aneuploidies, sperm head and tail shape and length, and most important, spermiogenetic remodeling of the plasma membrane that is the basis of the sperm-hyaluronic acid-binding assay in the Andrology Laboratory, and of the hyaluronic acid-mediated ICSI sperm selection in the Embryology laboratory. Some aspects of ICSI sperm selection by sperm shape combined with high magnification are also discussed.

It is well established that the testis-expressed HspA2 chaperone is a measure of human sperm cellular maturity and function, including fertilizing potential. The presence of HspA2 in the synaptonemal complex also provides the link between low HspA2 expression and increased frequencies of chromosomal aneuploidies in arrested maturity, dysmature sperm. There is also a relationship between the levels of HspA2 expression in elongated spermatids and the related spermiogenetic events, such as cytoplasmic extrusion, formation of the normal sperm shape, the nuclear events of histone-transition protein-protamine replacement, and the associated changes in DNA packing, as well as the sperm plasma membrane remodeling. The membrane remodeling facilitates the formation of the zona pellucida (and the hyaluronic acid [HA]) binding sites, which enables the sperm to fertilize and provide paternal contribution to the embryo. In dysmature sperm, some or all of these developmental steps are arrested (there is a well-established relationship between nuclear and cytoplasmic dysmaturity). For this reason, the sperm selected by the zona pellucida or by hyaluronic acid are comparable via the common origin of plasma membrane remodeling and the formation of both receptors. With the advent of ICSI, the challenge to understand which sperm is empowered to fertilize the egg and the ongoing research focusing on biochemical markers of sperm function have taken higher prominence, as the pathology in male infertility patients, who require ICSI treatment, of higher complexity. The HA receptor of mature sperm, coupled with HA-coated slides or Petri dishes, allows the direct visual observation of sperm-HA binding, which is the basis for sperm maturity testing, and the assessment of sperm binding to HA which is related to sperm-zona pellucida binding. Sperm-Ha binding provides a major advancement in semen evaluation and it also facilitates; the selection of single mature sperm for ICSI [3]. The HA-binding step, similar to zona pellucida binding, eliminates dysmature sperm that exhibit cytoplasmic retention, persistent histones, and DNA chain breaks. Further, the frequencies of sperm with chromosomal disomy and diploidy are reduced by 4–6-fold in HA-bound sperm vs. semen sperm fractions [3]. This reduction is similar to the increase of chromosomal aberrations in ICSI children. Combined studies of sperm shape and chromosome probes demonstrated that sperm shape does not reliably aid selection of haploid sperm [34]. Thus, HA-mediated sperm selection is a novel and efficient technique that may alleviate potential future public health problems that are related to ICSI with visually selected sperm, such as chromosomal aneuploidies, shortened lifetime, and increased cancer rates.

In this chapter, the laboratory methods for detection of sperm biomarkers that are aimed at identifying arrested sperm development are summarized. These probes include sperm staining with aniline blue for persistent histones, representing a break in the histone-transition protein-protamine sequence, immunocytochemistry with cytoplasmic sperm proteins, highlighting cytoplasmic retention during spermiogenesis, DNA nick translation testing for DNA chain fragmentation due to various reasons, for instance low HspA2 chaperone protein levels, and consequential diminished DNA repair. Finally, we briefly provide references on our work on sperm hyaluronan binding, abnormal Tybergerg sperm morphology, and the increased levels of chromosomal aneuploidies in sperm with developmental arrest. A very interesting aspect of the biomarker field is the discovery (Sati et al, Reprod Biomed Online 16:570-579, 2008) that the various nuclear and cytoplasmic defects detected by the biomarkers are related, and may simultaneously occur within the same spermatozoa as evidenced by a combination of biomarkers, such as aniline blue staining (persistent histones) coupled with cytoplasmic retention, DNA fragmentation, Caspase-3, Tygerberg abnormal morphology, and increased levels of chromosomal aneuploidies. We show examples of this80% overlap in staining patterns within the same spermatozoa.

In this chapter, the laboratory methods for detection of sperm biomarkers that are aimed at identifying arrested sperm development are summarized. These probes include sperm staining with aniline blue for persistent histones, representing a break in the histone-transition protein-protamine sequence, immunocytochemistry with cytoplasmic sperm proteins, highlighting cytoplasmic retention during spermiogenesis, DNA nick translation testing for DNA chain fragmentation due to various reasons, for instance low HspA2 chaperone protein levels, and consequential diminished DNA repair. Finally, we briefly provide references on our work on sperm hyaluronan binding, abnormal Tybergerg sperm morphology, and the increased levels of chromosomal aneuploidies in sperm with developmental arrest. A very interesting aspect of the biomarker field is the discovery (Sati et al, Reprod Biomed Online 16:570-579, 2008) that the various nuclear and cytoplasmic defects detected by the biomarkers are related, and may simultaneously occur within the same spermatozoa as evidenced by a combination of biomarkers, such as aniline blue staining (persistent histones) coupled with cytoplasmic retention, DNA fragmentation, Caspase-3, Tygerberg abnormal morphology, and increased levels of chromosomal aneuploidies. We show examples of this >80% overlap in staining patterns within the same spermatozoa.

In this novel paper,1 the authors examined the role of a ubiquitously expressed testicular protein, Pumilio 1, which is a post-transcriptional regulator of spermatogenesis in the testis. The mechanism of action of this regulatory protein is based on its binding to the 3′ end of mRNAs that code for kinases that regulate the p53 protein (most prominently eight of these mRNAs), which causes a Pumilio 1 binding-mediated activation of p53, and the consequent suppression of apoptosis in spermatocytes. In Pumilio 1-mutant mice, apoptosis occurs primarily in spermatocytes, which interrupts sequential male gamete development and thus reduces the fertility of the affected mice. Additional support for this mechanism came from experiments in which p53 was inhibited in mice which subsequently showed a reduction in spermatocyte apoptosis. The data also provided further evidence regarding the mRNA regulation proposed by Keene2 which states that the multifunctional mRNAs are not physically linked, but that each mRNA cistron carries a common sequence motif that is recognized by a shared regulator, as exemplified by Pumilio 1, and its repression of the eight p53 activators. Overall, Pumilio 1 may be considered a master protein that directs spermatogenesis via germ cell production and apoptosis. This is not a direct effect, but occurs by suppressing the translation of the p53 regulator kinases, which modulate the activity of p53 necessary for optimal spermatogenetic homeostasis. In order to determine whether Pumilio 1 promotes elimination of the p53 target RNAs or inhibits their translation, the authors examined two possibilities: (i) that Pumilio 1 activates regulator kinases, when p53 activity would be low, apoptosis would not be accelerated, and spermatocyte and male germ cell numbers would remain normal; and (ii) that Pumilio 1 activates p53 kinases, spermatocyte apoptosis would increase and mature sperm numbers would be reduced, resulting in oligozoospermia. The evidence supported the first hypothesis: elevated synthesis of Pumilio 1 represses p53 activity, decreases the apoptotic process and maintains sperm homeostasis. The second hypothesis is true only when the pumilio 1 gene is deleted. These insights are important for our understanding of various testicular and reproductive events. For instance, is Pumilio 1 action direct or an upstream factor in the spermatogenetic and spermiogenetic phases of gamete development? In men with male factor infertility is daily sperm production subject to Pumilio 1 regulation? Is sperm development, plasma membrane remodelling in terminal spermiogenesis, as well as the formation and expression of the zona pellucida and hyaluronic acid receptors, and other sperm biomarkers,3 under Pumilio 1 regulation? In the case of accelerated apoptosis, due to a lower level of Pumilio 1 production, would any or all spermatozoa be subject to apoptosis, or spermatocytes have DNA defects or aneuploidy, or higher histone and lower transition protein and protamine levels? Would apoptosis under these conditions be a selective phenomenon that alters the proportion of fertile spermatozoa and rescue fertility in spite of lower sperm numbers? Also of interest is the relationship between Pumilio 1-mediated apoptosis and apoptosis protection involving other elements of the system, such as Bcl-2, Bcl-xL and BAK.4 Regarding physiological fluctuations in sperm production, it is well known in the horse and sheep that around breeding time, sperm production and testicular size increase substantially, measurable by both testicular weight and the ratio of developing male gametes to Sertoli cells. Is Pumilio 1 involved in this adaptation? Future research will have to address these issues.

Excessive levels of free radicals diminish the functional integrity of spermatozoa. Levels of reactive oxygen species (ROS) produced by spermatozoa were negatively correlated with the quality of sperm in the original semen. “Intrinsic,” “extrinsic,” and “iatrogenic” sources of ROS production were identified within semen: Intrinsic sources of ROS in semen are morphologically abnormal and arrested maturity spermatozoa and leukocytes. Poor sperm quality showing attributes of arrested sperm maturation is linked to increased ROS generation as a consequence of excess residual cytoplasm related to the arrest of cytoplasmic extrusion in terminal spermiogenesis. In the past 20 years, the Huszar lab has studied several key events of sperm maturation, including cytoplasmic extrusion and expression of the HspA2 chaperone protein. In experiments related to sperm function and fertilizing potential, Huszar et al. have established that, simultaneously with cytoplasmic extrusion during terminal spermiogenesis, there is a remodeling of the plasma membrane that facilitates the formation of the zona pellucida- and hyaluronic acid (HA)-binding sites. The studies with HA immobilized to glass slides or Petri dishes showed that sperm firmly bind to HA. However, not all sperm exhibited HA binding ability. These data supported the hypothesis that the ability of sperm–HA binding is related to sperm cellular maturity.

The lack of reliable methods to assess sperm fertilizing potential has been a long-standing problem for infertile couples and for their physicians. The most widely used tests, the measurements of sperm concentrations, motility, velocity and morphology in the ejaculate, are of limited utility. Indeed, following intrauterine insemination, a treatment that compensates for low motile sperm concentrations, there were no significant differences found in semen parameters among those who did or did not achieve pregnancies. Other available assays probing for selected sperm functions, such as membrane integrity, acrosome enzyme activity, bovine cervical mucus penetration test, zona-free hamster oocyte penetration test and sperm binding to various carbohydrates,10–13have all failed thus far to consistently predict male fertility. It became increasingly obvious that there was a need to identify cellular markers of sperm quality and fertilizing potential.

In the past two decades, the Huszar laboratory focused upon the objective biochemical markers of sperm cellular maturity and development, and the characteristics of sperm in which cellular development is arrested during spermatogenesis and spermiogenesis. In assays directed to sperm cellular maturity/development, several sperm attributes were studied, including (a) excess cytoplasm (arrested cytoplasmic extrusion), (b) low expression of the HspA2 chaperone protein, a protein of the synaptonemal complex that supports meiosis, as well as the delivery of essential components in developing sperm, such as DNA repair enzymes or chromatin elements, (c) sperm shape (affected by cytoplasmic extrusion and insertion of the tail) according to the criteria detected by the Metamorph computer-assisted program or by the Tygerberg strict morphology, and (d) the relationship between sperm nuclear events (such as presence of chromosomal aneuploidies and excess residual histones). The primary focus of this chapter is related to recent reports that studied human sperm chromatin complexity and sperm HA-binding capacity. The studies suggest that the numerical chromosomal aberrations and persistent histones, i.e., errors in the spermatogenetic and spermiogenetic phases of male germ cell development, are associated. This chromatin deficiency associated with high persistent histones and relative lack of protamines affects DNA folding and renders the sperm DNA more vulnerable. It appears that these two phenomena correspond with each other and, thus, is responsible for defining sperm DNA integrity, fertility, and sterility.

With the technological advancements in assisted reproduction treatment, it is now feasible to cause fertilization and pregnancy by injection of a single spermatozoon into the oocyte via the ICSI method. The ultimate goal in this respect is the selection of a spermatozoon that has genetic and cellular attributes comparable to those sperm that interact with the zona pellucida under physiological or conventional IVF fertilization conditions. In light of this aim, this review focuses largely on objective biochemical markers within the nuclear and cytoplasmic compartment of spermatozoa, which define and reflect sperm developmental and genetic characteristics that are optimal for fertilization as well as for paternal contribution to the developing embryo. The ICSI sperm selection methods, including those based on hyaluronic acid binding and high-magnification visualization, are discussed along with the research evidence that supports the interrelated developmental and genetic integrity of the selected sperm. Furthermore, the review examines the use of other sperm diagnostics which may guide us in the treatment of male factor infertility patients. The diagnostic techniques examined include those focusing on the sperm nuclear integrity, in particular the Sperm Chromatin Structure Assay (SCSA). The fundamental information being generated from more molecular and biochemical-based analysis of sperm will create a basis for identifying key deficiencies in spermatozoa that might be implicated in the defective sperm function observed in a significant proportion of infertile males and may aid in both diagnosis and treatment.

Our previous creatine phosphokinase (CK) activity studies in human sperm revealed differences among men and among sperm populations within the same specimen. Samples with low sperm concentrations, high incidence of abnormal sperm morphology, and diminished fertility had higher per sperm CK activity. In the present work, we demonstrated, with 14C-FDNB covalent CK active site modification and with direct CK immunocytochemistry, that the higher CK activity is related to an increased content of CK and of other proteins in sperm. Also, sperm heads with higher CK content were significantly larger and rounder and showed a higher incidence of amorph configuration. We suggest that these biochemical and morphological irregularities are related and are due to a failure of spermatogenesis, more specifically, to a higher retention of cytoplasm, which in normal sperm development is lost to the Sertoli cells as residual bodies. Thus higher CK activity and larger or irregular head size in human sperm signify cellular immaturity and a failure to complete spermatogenesis.

Objective To examine the value of sperm creatine phosphokinase M-isoform (CK-MM) measurements toward predicting fertilizing potential of men. Design In 84 in vitro fertilization (IVF) couples without knowing the semen parameters, reproductive history or the outcome of the IVF cycles, we determined the sperm CK-MM ratios (the proportion of sperm CK-MM versus CK-MM + CK-BB). Husbands with Results Both the CKMM-Infertile and CKMM-Fertile groups (CK-MM ratios: 4.9% ± 0.6% versus 31.1% ± 1.8%) were in the normospermic range (31.5 ± 6.9 versus 78.4 ± 5.9 × 106 sperm/mL and 45.6% ± 5.0% versus 54.0% ± 2.0% motility). The fertilization rates (6.2 versus 4.9 oocytes inseminated) were 14.2% versus 53.4%, and 72.7% versus 25.8% of the couples failed to achieve any oocyte fertilization. All 14 pregnancies (16.7% rate) occurred in the CKMM-Fertile group. The pregnancy rate in the 62 CKMM-Fertile couples was 22.6%, and considering only the 46 CKMM-Fertile women in whom oocyte fertilization occurred, it was 30.4%. Among the 22 CKMM-Infertile men, 9 were normospermic and 9 of the 62 CKMM-Fertile men were Oligospermic. Within the CKMM-Fertile group, 12 and 2 of the 14 pregnancies occurred by the 53 normospermic and 9 oligospermic men (22.6% versus 22.2% rate). Conclusions Sperm CK-MM ratios, a measure of normal sperm development, predict fertilizing potential independently from sperm concentrations. Sperm CK-MM ratios also detect unexplained male infertility (infertile men with normospermic semen), a diagnosis that until now could not be substantiated.

Objective To study the potential relationship between two sperm nuclear attributes: persistence of histones and occurrence of chromosomal aneuploidies. Design The two variables were examined by double probing of the same spermatozoa. Setting Academic Andrology Laboratory. Patient(s) Semen samples subjected for analyses were examined. Intervention(s) We studied >58,000 spermatozoa, in seven men, first with aniline blue histone staining, graded as light (mature sperm), intermediate (moderately immature), and dark (severely arrested maturation). After recording the staining patterns and destaining, the same spermatozoa were subjected to fluorescence in situ hybridization (FISH), using centrometric X, Y, and 17 chromosome probes. Main Outcome Measure(s) Proportions of sperm with light, intermediate, and dark staining were assessed, and ploidy of these sperm was evaluated. Result(s) The aneuploidy frequencies in intermediate versus light (mature) spermatozoa were increased four- to sixfold. In addition, aneuploidy frequencies and proportions of intermediate sperm were related. There was no FISH signal detectable in the darkly stained, severely arrested mature sperm. Conclusion(s) The data suggest that in sperm with arrested maturity and DNA fragmentation, the binding of FISH probes is diminished. DNA damage is further aggravated by the decondensation and denaturation steps of FISH. Thus, there is a strong likelihood that in oligozoospermic men, with a higher proportion of sperm with arrested maturation, the sperm disomy frequencies are historically underestimated.

Individual spermatozoa were assessed with pairs of probes for persistent histones and cytoplasmic retention, persistent histones and DNA fragmentation, and persistent histones and apoptotic markers. The individual spermatozoa were treated sequentially with combinations of probes for these cytoplasmic and nuclear biochemical markers. Sperm fields were recorded with computer-assisted imaging, and staining patterns with the two probes in the same spermatozoa were examined and scored as light, intermediate or dark (mature to arrested-maturity spermatozoa). The effects of arrested sperm maturation were similar with respect to the cytoplasmic and nuclear characteristics of spermatozoa in 84% of cells, indicating that cytoplasmic and nuclear attributes of arrested sperm maturation are related. However, there were moderate (intermediate-dark or intermediate-light patterns, 14.5% of cells) or major (light-dark patterns, 1.6% of cells) discrepancies in the intensity of the double staining patterns. Thus, testing with single maturity markers may not be fully reliable. These findings are important with respect to: (i) arrested sperm maturation; (ii) potential efficacy of antioxidant and similar therapeutic strategies in subfertile men, as spermatozoa with infrastructure defects due to mismaturation or maturation arrest are unlikely to respond to interventions; and (iii) detection of adverse male environmental exposures.

The effects of Sperm Select (Pharmacia AB, Uppsala, Sweden), a hyaluronic acid medium, on the motility and membrane integrity properties of sperm were studied. In 15 normospermic specimens after overnight incubation, the motility parameters in the control versus the Sperm Select group were as follows (mean +/- SEM): motility, 18.8% +/- 2.8% versus 27.4% +/- 2.9%; velocity, 21.5 +/- 2.4 versus 27.2 +/- 2.2 microns/s; linearity, 3.8 +/- 0.3 versus 4.4 +/- 0.2; lateral head displacement, 1.5 +/- 0.2 versus 1.9 +/- 0.1 microns; and tail beat/cross frequency, 8.8 +/- 1.3 versus 10.8 +/- 1.4 Hz. The density of motile sperm was 10.8 +/- 2.3 versus 18.5 +/- 2.5 X 10(6) sperm/mL. Finally, the velocity coefficient, the multiple of the sperm motility and linear velocity, was 4.6 +/- 1.1 versus 8.1 +/- 1.4. However, we found no Sperm Select related differences when testing sperm membrane integrity with hypoosmotic swelling and supravital staining. Thus, Sperm Select improves the retention of sperm motility (most prominently velocity) apparently due to a direct action of hyaluronic acid on sperm metabolism or contractility rather than to preservation of sperm membrane integrity. In 20 oligospermic specimens, Sperm Select caused similar improvements in sperm motility, and the duration of motility could be predicted from the degree of enhancement in sperm velocity after short-term Sperm Select exposure. A modified Sperm Select protocol is described that further increases motile sperm yield without a centrifugation step.

The testis-expressed chaperone protein, HspA2 (previously creatine kinase M isoform) was established as a measure of human sperm cellular maturity, function and fertility. The presence of HspA2 in the synaptonemal complex is likely to link low HspA2 expression and increased frequency of chromosomal aneuploidies in arrested-maturity spermatozoa. A relationship also exists between HspA2 expression in elongating spermatids and the associated spermatogenetic events, including plasma membrane remodelling and the formation of zona pellucida and hyaluronic acid (HA) binding sites. The HA receptor of mature spermatozoa, when coupled with HA-coated slides and/or Petri dishes, allows visual observation of sperm–HA binding, providing a basis for sperm maturity testing, a major improvement in semen evaluation, and selection of mature spermatozoa for intracytoplasmic sperm injection (ICSI). Thus, in HA-selected spermatozoa the frequency of chromosomal disomy and diploidy is reduced 4- to 6-fold compared with semen sperm fractions. This reduction is similar to the increase in numerical chromosomal aberrations in ICSI children. Combined studies of sperm shape and chromosome probes demonstrated that sperm morphology does not aid selection of haploid spermatozoa. The HA-mediated sperm selection is a novel and efficient technique that may alleviate potential problems related to ICSI fertilization with visually selected spermatozoa.

The current concepts of sperm biochemical markers and the central role of the HspA2 chaperone protein, a measure of sperm cellular maturity and fertilizing potential, are reviewed.Because HspA2 is a component of the synaptonemal complex, low HspA2 levels and increased frequency of chromosomal aneuploidies are related in diminished maturity sperm. We also suggest a relationship between HspA2 expression in elongating spermatids and events of late spermiogenesis, such as cytoplasmic extrusion and plasma membrane remodeling that aid the formation of the zona pellucida binding and hyaluronic acid binding sites. The presence of hyaluronic acid receptor on the plasma membrane of mature sperm, coupled with hyaluronic acid coated glass or plastic surfaces, facilitates testing of sperm function and selection of single mature sperm for intracytoplasmic sperm injection. The frequencies of sperm with chromosomal disomy are reduced approximately fourfold to fivefold in hyaluronic acid selected sperm compared with semen sperm, comparable to the increase in such abnormalities in intracytoplasmic sperm injection offspring. Hyaluronic acid binding also excludes immature sperm with cytoplasmic extrusion, persistent histones, and DNA chain breaks.Hyaluronic acid mediated sperm selection is a novel technique that is comparable to sperm zona pellucida binding. Hyaluronic acid selected sperm will also alleviate the risks related to intracytoplasmic sperm injection fertilization with sperm of diminished maturity that currently cause worldwide concern.

Objective: To test a newly invented intracytoplasmic sperm injection (ICSI) sperm selection method based on sperm hyaluronic acid (HA) binding. Design: Comparison of chromosomal disomy and diploidy frequencies in sperm arising from semen and in HA-bound sperm. Setting: Academic andrology laboratory. Patient(s): Men presenting for semen analysis. Intervention(s): Washed sperm fractions of 32 semen samples were applied to Petri dishes or glass slides coated with immobilized HA. The unbound sperm were rinsed gently, and the HA-bound sperm were removed with an ICSI pipette. The control sperm population was the unselected sperm. Both HA-selected and unselected sperm were treated with fluorescence in situ hybridization with centromeric probes for the X, Y, and 17 chromosomes. Main Outcome Measure(s): Chromosomal disomy and diploidy frequencies. Result(s): In the HA-bound sperm (495‐2,079 per man, 41,670 in all) compared with unselected sperm (4,770 per man, 162,210 in all), the chromosomal disomy frequencies were reduced to 0.16% from 0.52%, diploidy to 0.09% from 0.51%, and sex chromosome disomy to 0.05% from 0.27% (a 5.4-fold reduction vs. 4-fold respective increase in ICSI offspring). Conclusion(s): The HA sperm selection method for ICSI, which is based on a relationship between sperm receptors for zona pellucida and HA, will likely reduce the potential genetic complications and adverse public health effects of ICSI. (Fertil Steril 2005;84:1665‐73. ©2005 by American Society for Reproductive Medicine.)

We tested several approaches that can be used topreserve sperm attributes and the objective biochemical markers ofsperm maturity and function for assessment in a remote centralizedlaboratory after overnight shipping of semen samples. Addition ofphenyl-methyl-sulfonyl-fluoride (PMSF) to a final concentration of 20mg/mL semen at 48C has preserved sperm concentrations andHspA2 isoform ratios, even at room temperature, simulating a ship-ping delay in moderate ambient temperatures. Regarding the attri-butes of individual spermatozoa, the patterns of CK-immunocyto-chemistry (demonstrates cytoplasmic retention in diminished-matu-rity spermatozoa); aniline blue staining pattern (tests chromatin ma-turity); sperm shape assessed by both Kruger strict morphology andcomputer assisted morphometry; and sperm DNA integrity, as testedby DNA nick translation, all remained unchanged. Thus, the PMSF-48C conditions preserved sperm concentrations and the cytoplasmicand nuclear biomarkers of sperm cellular maturity and function fornext-day analysis. This shipping method will facilitate the early de-tection of subtle changes in semen quality that can affect spermfunction, even when there has been no decline in sperm concentra-tions to signal possible toxic effects. Furthermore, sample preser-vation will enable investigators to evaluate semen for toxicologystudies and for diagnosis of male infertility from remote locations.Home collection of semen should enhance study participation, andsemen assessment in centralized laboratories will address concernsregarding interlaboratory variations and quality control.Key words: Reproductive toxicity, male infertility, cytoplasmic andnuclear biomarkers.J Androl 2004;25:593–604

This chapter presents an overview of our ongoing work focusing upon the relationship between diminished sperm maturity and the frequencies of chromosomal aneuploidies, and on the efficiency of the various sperm preparation methods utilized in assisted reproduction for elimination of immature sperm. This chapter will also extend to the major new breakthrough of hyaluronic acid (HA) binding by mature sperm for ICSI sperm selection. First we will discuss research carried out by the Huszar laboratory in the past 15 years relating to objective biochemical markers of sperm function and fertility. Subsequently, we will review the current data and will evaluate the relative utilities of swim-up, gradient centrifugation and HA binding for selection of sperm with low levels of chromosomal aneuploidies and high levels of DNA integrity for IVF and ICSI. The key points to be emphasized are the following: (1) Cytoplasmic retention as a marker of sperm immaturity, and the two-wave expression pattern of the testis specific HspA2 chaperone protein in developing sperm during meiosis and late spermiogenesis. (2) Cellular maturation, plasma membrane remodeling and their contributions to the fertilization function of human sperm. (3) The relationship between sperm immaturity and increased frequencies of chromosomal aneuploidies. (4) The relative efficiencies of density gradient and swim up methodologies in eliminating aneuploid and diploid sperm. (5) The use of HA binding for selection of mature individual sperm with low levels of both chromosomal aneuploidies and DNA degradation.

The expression of a 70 kDa chaperone protein, HspA2 (formerly called CK-M), has been identified in mature human spermatozoa. The central role of HspA2 has been established, as the expression level of this protein is related to sperm cellular maturity, DNA integrity, chromatin maturity, chromosomal aneuploidy frequency and sperm function, including fertilizing potential. The spermiogenetic events of cytoplasmic extrusion and remodelling of the plasma membrane, which facilitate the formation of zona pellucida binding site(s) in human spermatozoa, are related. Finally, the presence of the hyaluronic acid (HA) receptor on the plasma membrane of mature sperm coupled with the HA-coated slide sperm-binding assay, facilitates the testing of infertile men and the selection of single mature spermatozoa for ICSI. Because mature spermatozoa have no residual cytoplasm, the HA-bound sperm fraction is also enriched in spermatozoa that are normal by the Kruger strict morphology method.