A new media without animal component for sperm cryopreservation: motility and various attributes affecting paternal contribution of sperm.

PURPOSE: 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Next day determination of ejaculatory sperm motility after overnight shipment of semen to remote locations.

PURPOSE: 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. METHODS: 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). RESULTS: 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. CONCLUSIONS: 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.

The pattern of tyrosine phosphorylation in human sperm in response to binding to zona pellucida or hyaluronic acid.

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.

Methodology of aniline blue staining of chromatin and the assessment of the associated nuclear and cytoplasmic attributes in human sperm.

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.

Spermatozoa bound to solid state hyaluronic acid show chromatin structure with high DNA chain integrity: an acridine orange fluorescence study.

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.

Double probing individual human spermatozoa: aniline blue staining for persistent histones and fluorescence in situ hybridization for aneuploidies.

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.

Selectivity of hyaluronic acid binding for spermatozoa with normal Tygerberg strict morphology.

During spermiogenesis, a plasma membrane remodelling step facilitates formation of sperm zona pellucida and hyaluronic acid (HA) binding sites. Enrichment of Tygerberg normal spermatozoa in HA-bound versus semen sperm fractions was postulated. Semen was placed on the uncoated A side and HA-coated B side of a semen chamber. After 15 min, the HA binding score (proportion of HA-bound motile spermatozoa) was assessed on the B side, and unbound spermatozoa were removed by gentle rinsing. Following Diff-Quick staining, sperm morphology of A and B sides was evaluated by three blinded investigators at Yale and Tygerberg. The proportion of Tygerberg normal spermatozoa was higher in HA-bound versus semen spermatozoa (n = 63 subjects) with a 3.04-fold improvement (95% confidence limits: 1.9-4.7) in 37 teratozoospermic men, comparable with a 4.2-fold enrichment in zona pellucida-bound spermatozoa reported earlier by the Tygerberg group. The morphology scores of three investigators were different but related, indicating that the variations reflect individual-to-individual differences in the perception of shape normality. The selection power of HA and zona pellucida for normal spermatozoa are similar. The sperm biomarkers of creatine phosphokinase (reflecting retained cytoplasm in arrested maturity spermatozoa) and chaperone protein HspA2 (heat shock protein) were proportional with sperm HA binding. As HA binding reflects sperm maturity and function, the combination of Tygerberg morphology and HA binding is likely to improve male infertility management.

Double probing of human spermatozoa for persistent histones, surplus cytoplasm, apoptosis and DNA fragmentation.

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.

Fertility testing and ICSI sperm selection by hyaluronic acid binding: clinical and genetic aspects.

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.

Hyaluronic acid binding ability of human sperm reflects cellular maturity and fertilizing potential: selection of sperm for intracytoplasmic sperm injection.

PURPOSE OF REVIEW: 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. RECENT FINDINGS: 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. SUMMARY: 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.

Increased IL-18 levels in seminal plasma of infertile men with genital tract infections.

PROBLEM: Interleukin (IL)-18 is a novel cytokine, previously known as interferon (IFN)-gamma inducing factor. We evaluated the levels of IL-18 and IFN-gamma 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-gamma 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 < 0.05). However, no significant differences could be detected for IFN-gamma. A significant positive correlations was found between IL-18 and IFN-gamma in total patient population (P < 0.001). Moreover, a negative correlation was observed between IL-18 and sperm concentrations, and motility (P < 0.01 and < 0.03, respectively). Furthermore, there was a positive and statistically significant association between IL-18 and IFN-gamma levels in SP of infertile men with genital tract infections (P < 0.0001). However, there was no relationship between IL-18 and IFN-gamma, and semen parameters in the same group. CONCLUSION: SP IL-18 levels were increased in men with urogenital infections. Thus, the elevated expression of IL-18 in SP may be used as a diagnostic marker in the male genital tract infections.

Dimensional assessment of X-bearing and Y-bearing haploid and disomic human sperm with the use of fluorescence in situ hybridization and objective morphometry.

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.

Intracytoplasmic sperm injection: a novel selection method for sperm with normal frequency of chromosomal aneuploidies.

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.

Sperm selection for ICSI: shape properties do not predict the absence or presence of numerical chromosomal aberrations.

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.

Semen characteristics after overnight shipping: preservation of sperm concentrations, HspA2 ratios, CK activity, cytoplasmic retention, chromatin maturity, DNA integrity, and sperm shape.

We tested several approaches that can be used to preserve sperm attributes and the objective biochemical markers of sperm maturity and function for assessment in a remote centralized laboratory after overnight shipping of semen samples. Addition of phenyl-methyl-sulfonyl-fluoride (PMSF) to a final concentration of 20 microg/mL semen at 4 degrees C has preserved sperm concentrations and HspA2 isoform ratios, even at room temperature, simulating a shipping delay in moderate ambient temperatures. Regarding the attributes of individual spermatozoa, the patterns of CK-immunocytochemistry (demonstrates cytoplasmic retention in diminished-maturity spermatozoa); aniline blue staining pattern (tests chromatin maturity); sperm shape assessed by both Kruger strict morphology and computer assisted morphometry; and sperm DNA integrity, as tested by DNA nick translation, all remained unchanged. Thus, the PMSF-4 degrees C conditions preserved sperm concentrations and the cytoplasmic and nuclear biomarkers of sperm cellular maturity and function for next-day analysis. This shipping method will facilitate the early detection of subtle changes in semen quality that can affect sperm function, even when there has been no decline in sperm concentrations to signal possible toxic effects. Furthermore, sample preservation will enable investigators to evaluate semen for toxicology studies and for diagnosis of male infertility from remote locations. Home collection of semen should enhance study participation, and semen assessment in centralized laboratories will address concerns regarding interlaboratory variations and quality control.

Cellular maturity and apoptosis in human sperm: creatine kinase, caspase-3 and Bcl-XL levels in mature and diminished maturity sperm.

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.

[Numerical chromosome anomalies in human sperm at low sperm concentration]. / Számbeli kromoszómaeltérések humán spermiumban alacsony spermiumkoncentráció esetén.

INTRODUCTION: With the assisted reproduction techniques the natural selection of sperm is bypassed on different levels. AIM: The aim of the present work is to determine the frequencies of sperm numerical chromosome aberrations in infertile men with low sperm count and to examine the relationship between the spermatogram parameters (sperm count and motility) and the aneuploidy and diploidy frequencies. METHODS: 32 men with low sperm count were investigated. Semen analysis was performed according to the WHO criteria. Disomy and diploidy frequencies were detected with fluorescence in situ hibridization using 17, X and Y centromeric probes. 200,969 sperm were scored, with the mean of 6272 cells in each subjects. The rate of numerical chromosome anomalies were also estimated using the detected disomy and the diploidy frequencies. RESULTS: Mean sperm concentration of the 32 men was 18.2 million/ml (SD: +/- 8.43, range: 8.0-45.5), mean motility 49.4% (+/- 9.32, 30-69.2). The X/Y ratio was 1.07. The mean frequencies of sex chromosome disomy, chromosome 17 disomy and diploidy were 0.36%, 0.16% and 0.56%, respectively. The most frequent disomy was XY disomy (0.14). Sex chromosome disomy frequencies were higher in oligospermic samples (0.37% vs. 0.32%, OR = 1.18, 95% CI = 1.01-1.39, p < 0.001). With special regard to XY disomy (0.08% vs. 0.17%, OR = 1.99, 95% CI = 1.48-2.67, p < 0.001). In subjects with oligoasthenozoospermia the diploidy frequency increased (0.96%, OR = 2.39, 95% CI = 2.13-2.69, p < 0.001), mostly due to the elevated rate of diploid sperm of meiosis II. origin (XX or YY diploids). Estimated frequency of the numerical chromosome anomalies was 8.3 +/- 5.3 in the study population. Neither the sperm count, nor the sperm motility showed correlation with the detected frequency of the chromosome aberrations. CONCLUSIONS: In oligospermic patients there is a risk for elevated frequency of sperm with sex chromosome aneuploidy, especially the XY disomy. Furthermore, the diploidy frequency is increased in oligozooastenospermic samples. Nevertheless, classical parameters of semen analysis (sperm count and motility) do not correlate with the frequency of numerical chromosomal anomalies. The risk can be determined using fluorescens in situ hybridization on sperm.

Efficacy of the swim-up method in eliminating sperm with diminished maturity and aneuploidy.

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.

Human sperm maintain their shape following decondensation and denaturation for fluorescent in situ hybridization: shape analysis and objective morphometry.

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 computer-assisted 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 = 115), asymmetrical (n = 115), irregular (n = 115), and amorphous (n = 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.