HA-bound Sperm Research Articles

IZUMO1 Receptor Localization during Hyaluronic Acid Selection in Human Spermatozoa.

IZUMO1 is an acrosome transmembrane protein implicated in the adhesion and fusion of gametes. This study aims to describe the distribution of IZUMO1 in human sperm under different physiological conditions: before capacitation (NCS), at one-hour capacitation (CS1), after a hyaluronic acid (HA) selection test (mature, MS1 and immature, IS1), and induced acrosome reaction from one-hour-capacitated sperm (ARS1). The data obtained in NCS, CS1, and MS1 significantly highlight dotted fluorescence in the acrosomal region (P1) as the major staining pattern (~70%). Moreover, we describe a new distribution pattern (P2) with a dotted acrosomal region and a labelled equatorial region that significantly increases in HA-bound spermatozoa, suggesting the onset of the migration of IZUMO1. In contrast, unbound spermatozoa presented an increase in P3 (equatorial region labelled) and P4 (not labelled). Finally, costaining to observe IZUMO1 distribution and acrosome status was performed in ARS1. Interestingly, we reported a variety of combinations between the IZUMO1 staining patterns and the acrosomal stages. In conclusion, these data show as a novelty the diffusion of the IZUMO1 protein during different physiological conditions that could contribute to the improvement in sperm selection techniques.

The Efficacy of Hyaluronic Acid Binding (HAB) in the Treatment of Male Infertility: A Systematic Review of the Literature

Hyaluronic acid (HA)-binding is reported to predict the fertilising capacity of spermatozoa, while HA-bound sperm selection is reported to reduce the incidence of miscarriage. However, the clinical effectiveness of these techniques remains uncertain. This work investigated the prognostic value of sperm-HA binding (HAB) as a predictor of treatment outcomes, and whether HAB-sperm selection for Invitro fertilisation (IVF)/intracytoplasmic sperm injection (ICSI) improves clinical outcomes or reduces miscarriage rates. A systematic review of the literature was carried out. A modified version of the Downs and Black Checklist was used to assess bias and study quality on eleven selected studies. No significant correlations were found between HAB score and fertilisation, clinical pregnancy, or live birth rates (low-quality evidence). Three studies reported a significant reduction in the incidence of miscarriage, including a Cochrane review (low-quality evidence). While the prognostic value of HAB scores is currently undetermined, there is evidence that HAB-sperm selection prior to insemination reduces the incidence of miscarriage following ART. Moreover, there are no reports of detrimental effects of HAB-sperm selection on treatment outcomes when compared with conventional IVF or ICSI. Therefore, it is unclear why it is assigned as a treatment “add-on” with a red light by the HFEA, and why its routine use is not recommended.

Hyaluronic acid-sperm selection significantly improves the clinical outcome of couples with previous ICSI cycles failure.

Beneficial effects of hyaluronic acid (HA)-based selection of spermatozoa for intracytoplasmic sperm injection (ICSI) are still controversial, and further studies are needed to categorize patients that might benefit from such a method. We investigated whether HA sperm selection improved ICSI outcome of couples with previous ICSI cycle failure. In this retrospective study, we prospectively collected data of (i) Group 1: 96 couples who performed one failed ICSI cycle ("1st procedure," n=96) followed by another ICSI cycle ("2nd procedure," n=101); ii) Group 2: 87 couples who performed one failed ICSI cycle (n=87) followed by an HA-ICSI cycle (n=104). Differences between procedures and groups were measured by paired and independent statistical tests, respectively. A generalized linear mixed model analyzed the effect of procedure on the outcomes and the interaction between procedures and groups. Injection of HA-bound sperm significantly improved cleavage rate with respect to standard ICSI (p=0.026). No evolutive pregnancies were obtained in the 1st ICSI attempts. The 2nd ICSI cycles resulted in successfully seven pregnancies. In HA-ICSI cycles, the better quality of embryos with respect to ICSI (p=0.034) increased the choice of day 5 embryo transfer (p=0.030), which resulted in successfully 28 pregnancies. No differences were observed in clinical outcomes of the two ICSI procedures in Group 1, while pregnancy and implantation rates were significantly higher in HA-ICSI with respect to ICSI cycles (p=0.001, p<0.0001, respectively). No negative perinatal outcomes were recorded. In couples where previous 1st ICSI failed, selection of HA-bound spermatozoa significantly improved clinical outcomes with respect to further standard ICSI. This study identified couples with previous ICSI cycles failure as a category of infertile patients that really may benefit from HA sperm selection before ICSI.

P–255 Hyaluronic acid-sperm selection significantly improves the clinical outcome of couples with previous ICSI cycles failure

Abstract Study question Does hyaluronic acid (HA) sperm selection improve the intracytoplasmic sperm injection outcome of couples with previous ICSI cycles failure? Summary answer In couples where previous first ICSI failed, selection of HA-bound spermatozoa significantly improved clinical outcomes respect to further standard ICSI. What is known already HA is the major component of the matrix surrounding the human oocyte and in physiological fertilization it plays an important role in sperm selection since only mature spermatozoa express specific binding protein and are able to bind to HA. Although several studies demonstrated better outcomes of ICSI with selection of mature HA-bound spermatozoa, such a beneficial effect of HA-ICSI is still controversial and to date no firm clinical guidance for the routine use of HA can be drawn. Further studies are needed to categorize patients that really might benefit from HA sperm selection before ICSI. Study design, size, duration A retrospective, longitudinal cohort study performed at a tertiary level public infertility center. We selected 164 couples who performed one or more failed ICSI cycles with low fertilization rate and poor embryo quality in the period 2010–2020 (n = 164 cycles, group A), followed by other standard ICSI (n = 99, group B) and/or HA-ICSI (n = 96 group C) cycles. We included only fresh ejaculated sperm and fresh oocytes. Participants/materials, setting, methods Endpoints were fertilization, cleavage, top quality embryo, implantation (IR), clinical pregnancy (CPR), pregnancy loss (PLR), and live birth (LBR) rates. Comparisons among groups were performed using a generalized estimating equation model performed at patient level, to take into account the correlation between observations originating from the same woman. A p-value &amp;lt;0.05, after correction by female age at oocyte retrieval, was considered statistically significant. Main results and the role of chance The three groups were similar for number of retrieved, MII and injected oocytes. As regarding embryological outcomes, there was no difference in fertilization and cleavage rates between group A and C (fertilization: 47.55 + 29.88% versus 54.10 + 28.51%, p = 0.096; cleavage: 96.19 + 12.70% versus 97.52 + 10.50%, p = 0.519), nor between group B and C (fertilization: 60.30 + 30.73% versus 53.71 + 28.61%, p = 0.112; cleavage: 92.26 + 20.540% versus 97.55 + 10.44%, p = 0.106). Selection of HA-bound spermatozoa in ICSI significantly improved the embryo quality rate (63.78 + 35.55% versus 51.42 + 34.31% p = 0.024) and the blastulation rate (43.44 + 25.55% versus 17.93 + 25.52%, p = 0.001) respect to standard ICSI. Comparisons of clinical outcomes between group B and group C highlighted significant higher IR (26.16 + 40.47% versus 7.34 + 22.16%, p = 0.0001), CPR/cycle (32.29% (31/96) versus 12.12% (12/99), p = 0.0007, chi-square test), and lower PLR (12.90% (4/31) versus 41.67% (5/12), p = 0.0398, chi-square test) in HA-ICSI respect to standard ICSI cycles. The LBR/cycle in group B was 10.10% (10/99) and in group C was 32.29% (31/96) (p = 0.0029, chi-square test). No stillbirths as well as no malformations in newborns were recorded. Limitations, reasons for caution We are aware of the retrospective nature of the study performed in a single ART center. Wider implications of the findings: This study identified couples with previous ICSI cycles failure as a category of infertile patients that really may benefit from HA sperm selection before ICSI. Trial registration number Not applicable

Effect of erythrocyte-sperm separation medium on nuclear, acrosomal, and membrane maturity parameters in human sperm.

The purpose of this study is to investigatewhether erythrocyte-sperm separation medium (ESSM) has effects on human sperm motility, morphology, viability, membrane maturity, acrosome integrity, and nuclear attributes before and after cryopreservation. Semen samples from normozoospermic (n = 36) and oligozoospermic (n = 9) patients were analyzed. Samples from the same patient were divided into three aliquots: group 1 and group 2 were resuspended in sperm washing media and ESSM, respectively. Group 3 was resuspended in ESSM with blood sample to mimic the extensive number of erythrocytes in the testicular sperm extraction (TESE) material. All groups were evaluated for sperm concentration, motility, Kruger/Tygerberg strict morphology, viability by eosin-nigrosin staining, membrane maturity by hyaluronic acid-binding assay (HBA), acrosomal integrity by Pisum sativum lectin staining, chromatin maturity by aniline blue staining, and DNA integrity by TUNEL assay before and after cryopreservation. No significant difference was determined between ESSM-treated and ESSM-untreated sperm samples for the sperm parameters tested (p > 0.05). After cryopreservation, total sperm motility and viability decreased regardless of ESSM used. The percentages of sperm with Tygerberg normal morphology, intact acrosome, and HA-bound sperm were found to be lower in oligozoospermic samples before cryopreservation in each group. However, no statistically significant differences were found between oligozoospermic and normozoospermic samples when all groups were compared. Thus, ESSM treatment did not cause a significant change on sperm motility, normal morphology, viability, HA-binding capacity, chromatin maturity, and DNA fragmentation. ESSM can enhance the efficiency of sperm retrieval protocol and can also decrease the time required to collect spermatozoa while not affecting sperm morphogenetic properties.

The efficiency of conventional microscopic selection is comparable to the hyaluronic acid binding method in selecting spermatozoa for male infertility patients

ObjectiveTo evaluate if hyaluronic acid (HA)-bound spermatozoa surpassed conventional microscopy-selected spermatozoa in the status of sperm DNA integrity by acridine orange (AO) fluorescence staining. Materials and methodsSpermatozoa obtained from couples with indication for the intracytoplasmic sperm injection (ICSI) procedure due to male infertility (n = 34) and control males with normal sperm parameters (n = 12) were analyzed using AO fluorescence staining after density-gradient centrifugation (DGC), polyvinylpyrrolidone (PVP)-microscopic selection, and HA-binding selection to determine sperm DNA integrity. ResultsPercentages of DNA intact spermatozoa with green fluorescence were significantly higher in both PVP-microscopic selected spermatozoa (82.1 ± 24.0%) and HA-bound spermatozoa (83.9 ± 21.1%) than in spermatozoa prepared by DGC (66.8 ± 24.0%). However, there was no significant difference between the PVP-sperm and HA-sperm groups. When the percentage of green fluorescent spermatozoa prepared by DGC fell initially below 68%, both PVP-microscopic and HA-binding selection failed to select over 90% spermatozoa with intact DNA for ICSI in the male infertility group. Compared to control males with normal sperm parameters (99.3 ± 1.8%), the proportion of green fluorescence sperm after HA-binding selection from couples with male infertility (83.9 ± 21.1%) did not reach the range of > 99% reported by Yagci et al. ConclusionThe percentages of DNA intact spermatozoa between the PVP-sperm and HA-sperm groups were not significantly different. In an ICSI procedure, a well-trained embryologist will have the same ability to choose sperm with intact DNA by conventional microscopic selection as with HA-bound spermatozoa selection.

Correlation study between sperm concentration, hyaluronic acid-binding capacity and sperm aneuploidy in Hungarian patients

Infertile men with low sperm concentration and/or less motile spermatozoa have an increased risk of producing aneuploid spermatozoa. Selecting spermatozoa by hyaluronic acid (HA) binding may reduce genetic risks such as chromosomal rearrangements and numerical aberrations. Fluorescence in-situ hybridization (FISH) has been used to evaluate the presence of aneuploidies. This study examined spermatozoa of 10 oligozoospermic, 9 asthenozoospermic, 9 oligoasthenozoospermic and 17 normozoospermic men by HA binding and FISH. Mean percentage of HA-bound spermatozoa in the normozoospermic group was 81%, which was significantly higher than in the oligozoospermic (P<0.001), asthenozoospermic (P<0.001) and oligoasthenozoospermic (P<0.001) groups. Disomy of sex chromosomes (P=0.014) and chromosome 17 (P=0.0019), diploidy (P=0.03) and estimated numerical chromosome aberrations (P=0.004) were significantly higher in the oligoasthenozoospermic group compared with the other groups. There were statistically significant relationships (P<0.001) between sperm concentration and HA binding (r=0.658), between sperm concentration and estimated numerical chromosome aberrations (r=–0.668) and between HA binding and estimated numerical chromosome aberrations (r=–0.682). HA binding and aneuploidy studies of spermatozoa in individual cases allow prediction of reproductive prognosis and provision of appropriate genetic counselling.Infertile men with normal karyotypes and low sperm concentrations and/or less motile spermatozoa have significantly increased risks of producing aneuploid (diminished mature) spermatozoa. Selecting spermatozoa by hyaluronic acid (HA) binding, based on a binding between sperm receptors for zona pellucida and HA, may reduce the potential genetic risks such as chromosomal rearrangements and numerical aberrations. In the present study we examined sperm samples of 45 men with different sperm parameters by HA-binding assay and fluorescence in-situ hybridization (FISH). Mean percentage of HA-bound spermatozoa in the normozoospermic group was significantly higher than the oligozoospermic, the asthenozoospermic and the oligoasthenozoospermic groups. Using FISH, disomy of sex chromosomes and chromosome 17, diploidy and estimated numerical chromosome aberration frequencies were significantly higher in the oligoasthenozoospermic group compared with the three other groups. A significant positive correlation was found between the sperm concentration and the HA-binding capacity, and significant negative correlations between the sperm concentration and the estimated numerical chromosomes aberrations as well as between the HA-binding ability and the estimated numerical chromosome aberrations were identified. We conclude that HA-binding assay and sperm aneuploidy study using FISH may help to predict the reproductive ability of selected infertile male patients and to provide appropriate genetic counselling.

The effect of the swim-up and hyaluronan-binding methods on the frequency of abnormal spermatozoa detected by FISH and SCSA in carriers of balanced chromosomal translocations

The swim-up and hyaluronan (HA)-binding methods are used for the selection of good quality spermatozoa to improve pregnancy rates and embryo quality and to reduce the number of miscarriages after IVF. We evaluated whether the processing of sperm by these methods reduces the frequency of spermatozoa with abnormal karyotypes and altered chromatin quality in balanced translocation carriers. Semen samples of 12 carriers of balanced chromosomal translocations were analysed for the frequency of spermatozoa, which are chromosomally unbalanced due to the segregation of balanced translocations, aneuploidies for chromosomes 7, 8, 13, 18, 21, X or Y, diploid sperm or sperm with fragmented DNA and poorly condensed chromatin. Results obtained by fluorescence in situ hybridization (FISH) and sperm chromatin structure assay were compared between ejaculated (n = 12), swim-up (n = 12) and HA-binding processed (n = 6) semen samples of the translocation carriers and with the control group (n = 10). The mean frequencies of unbalanced segregation products were 17.5 and 16.5% in neat and swim-up processed samples from Robertsonian translocation carriers, and 55.4, 54.5 and 50.9% in neat, swim-up and HA-bound sperm samples from reciprocal translocation carriers. Significant decreases in the frequency of sperm showing chromosome 18 and XY disomy and of diploidy, and in the rates of high-density staining sperm were observed in the motile swim-up fractions. There were significantly more sperm showing fragmented chromatin in the group of translocation carriers than in the control group, but no differences in the aneuploidy and diploidy rates were observed. The swim-up method is suitable for selection of sperm with condensed chromatin and a lower frequency of some aneuploidies and of diploidy. The frequency of spermatozoa chromosomally unbalanced due to the segregation of reciprocal (but not Robertsonian) translocations is significantly lower in HA-bound sperm. However, the advantages of either method for selecting normal sperm are limited.

Application of Sperm Selection Using Hyaluronic Acid Binding in Intracytoplasmic Sperm Injection Cycles: A Sibling Oocyte Study

The purpose of this study was to investigate whether sperm selection by hyaluronic acid (HA) binding could improve fertilization rate and embryo quality in intracytoplasmic sperm injection (ICSI) cycles. Two hundred nineteen oocytes obtained from eighteen women were injected with either HA-bound (n = 107) or conventionally selected spermatozoa (n = 112) in a randomized way. All of the participants were infertile couples who had normal sperm parameters but low fertilization rate in previous in vitro fertilization (IVF) cycle (n = 5) or experienced multiple IVF failures (n = 13). Lower fertilization (75.7% vs 83.0%) and cleavage rate on day 2 (72.9% vs 83.0%) was observed in oocytes injected with HA-bound spermatozoa than the conventional group, but the difference was not significant. Significantly lower cleavage rate was observed on day 3 in HA group (56.0% vs 69.6%, P = 0.038). Blastocyst formation rate and the number of transferred embryos were similar in both groups. In multiple IVF failure patients, significantly reduced fertilization rate (71.8% vs 85.3%, P = 0.046) and cleavage rate on day 2 (70.4% vs 85.3%, P = 0.029) and day 3 (53.5% vs 77.3%, P = 0.002) were noticed in HA group. Five women achieved pregnancy continuing more than 12 weeks after transfer (27.8%). Success of ICSI was not related with the number of embryos fertilized by HA-bound spermatozoa. Application of ICSI by sperm selection using HA binding is not helpful in couples with repeated poor fertilization or implantation despite normal sperm parameters.

Efficacy of hyaluronic acid binding assay in selecting motile spermatozoa with normal morphology at high magnification

BackgroundThe present study aimed to evaluate the efficacy of the hyaluronic acid (HA) binding assay in the selection of motile spermatozoa with normal morphology at high magnification (8400x).MethodsA total of 16592 prepared spermatozoa were selected and classified into two groups: Group I, spermatozoa which presented their head attached to an HA substance (HA-bound sperm), and Group II, those spermatozoa that did not attach to the HA substance (HA-unbound sperm). HA-bound and HA-unbound spermatozoa were evaluated according to the following sperm forms: 1-Normal morphology: normal nucleus (smooth, symmetric and oval configuration, length: 4.75+/-2.8 μm and width: 3.28+/-0.20 μm, no extrusion or invagination and no vacuoles occupied more than 4% of the nuclear area) as well as acrosome, post-acrosomal lamina, neck, tail, besides not presenting a cytoplasmic droplet or cytoplasm around the head; 2-Abnormalities of nuclear form (a-Large/small; b-Wide/narrow; c-Regional disorder); 3-Abnormalities of nuclear chromatin content (a-Vacuoles: occupy >4% to 50% of the nuclear area and b-Large vacuoles: occupy >50% of the nuclear area) using a high magnification (8400x) microscopy system.ResultsNo significant differences were obtained with respect to sperm morphological forms and the groups HA-bound and HA-unbound. 1-Normal morphology: HA-bound 2.7% and HA-unbound 2.5% (P = 0.56). 2-Abnormalities of nuclear form: a-Large/small: HA-bound 1.6% vs. HA-unbound 1.6% (P = 0.63); b-Wide/narrow: HA-bound 3.1% vs. HA-unbound 2.7% (P = 0.13); c-Regional disorders: HA-bound 4.7% vs. HA-unbound 4.4% (P = 0.34). 3. Abnormalities of nuclear chromatin content: a-Vacuoles >4% to 50%: HA-bound 72.2% vs. HA-unbound 72.5% (P = 0.74); b-Large vacuoles: HA-bound 15.7% vs. HA-unbound 16.3% (P = 0.36).ConclusionsThe findings suggest that HA binding assay has limited efficacy in selecting motile spermatozoa with normal morphology at high magnification.

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.

Efficiency of hyaluronic acid (HA) sperm selection

Hyaluronic Acid (HA) has a role as "physiologic selector" for spermatozoa prior to intracytoplasmic sperm injection (ICSI). The objective of this study is to analyze the results achievable by the introduction of a routine HA-ICSI programme. We retrospectively observed 293 couples treated with HA-ICSI versus 86 couples treated with conventional PVP-ICSI (historical control group). ICSI was performed on a limited number of oocytes per patient (1-3) according to Italian IVF law at the time of the study. Main outcome measures observed were: fertilization, embryo quality, implantation and pregnancy. This study showed that Injection of HA-bound spermatozoa (HA-ICSI) significantly improves embryo quality and implantation. If wider multi-center randomized studies will confirm these beneficial effects on ICSI outcome, HA could be considered as a routine choice for "physiologic" sperm selection prior to ICSI.

“Physiologic ICSI”: Hyaluronic acid (HA) favors selection of spermatozoa without DNA fragmentation and with normal nucleus, resulting in improvement of embryo quality

To evaluate the role of hyaluronic acid (HA) for sperm selection before intracytoplasmic sperm injection (ICSI). Three prospective studies. Private assisted reproduction center in Italy. Study 1: 20 men. Study 2: 15 men. Study 3: 206 couples treated with ICSI on a limited number of oocytes per patient (1-3) in accordance with Italian IVF law. Study 1: determination of sperm DNA fragmentation of HA-bound spermatozoa versus spermatozoa in polyvinylpyrrolidone (PVP). Study 2: assessment of nuclear morphology of HA-bound spermatozoa versus spermatozoa in PVP. Study 3: randomized study comparing conventional PVP-ICSI to ICSI in which the spermatozoa are selected for their capacity to bind to HA (HA-ICSI). Study 1: sperm DNA fragmentation rate. Study 2: sperm nucleus normalcy rate according to motile sperm organellar morphology examination criteria. Study 3: fertilization, embryo quality and development, and implantation and pregnancy. Spematozoa bound to HA show a significant reduction in DNA fragmentation (study 1) and a significant improvement in nucleus normalcy (study 2) compared with spermatozoa immersed in PVP. Furthermore, injection of HA-bound spermatozoa (HA-ICSI) significantly improves embryo quality and development (study 3). Hyaluronic acid may optimize ICSI outcome by favoring selection of spermatozoa without DNA fragmentation and with normal nucleus. Furthermore, HA may also be used to speed up the selection of spermatozoa with normal nucleus during intracytoplasmic morphologically selected sperm injection (IMSI).

P-630: Similarity in tyrosine phosphorylation patterns in human sperm bound to hyaluronic acid and to zona pellucida of oocytes

During spermiogenesis there is a sperm plasma membrane remodeling that facilitates the formation of zona pellucida and hyaluronic acid (HA) binding sites. Sperm with arrested maturation can fail to bind to the zona pellucida and HA. Also, the sperm selection attributes of HA and zona pellucida are similar: HA-bound sperm show no cytoplasmic retention, persistent histones or DNA fragmentation. Further, in HA-selected sperm the frequencies of chromosomal aneuploidies are normal, regardless of their frequencies in semen (Fert.Ster., 2005). A number of previous studies have shown specific protein tyrosine phosphorylation (TP) changes in spermatozoa related to events associated with capacitation and zona-binding. In the present study, we have studied TP patterns in spermatozoa isolated from media or bound to HA. We have also explored whether sperm with arrested maturation and surplus cytoplasm would undergo TP changes. Time related study of TP patterns in sperm suspended in media or bound to HA. Immunofluorescence was performed with antisera for TP and CK. We prepared sperm smears and HA-bound mature sperm from each semen sample suspended in HTF medium (Irvine Scientific) and using HA-coated slides (MidAtlantic Diagnostics). The sperm were fixed with 3.7% formaldehyde after 0 time and 4hr incubations. The slides were treated with either a monoclonal anti-phosphotyrosine antibody (Sigma) or with polyclonal anti-creatine kinase(CK) antiserum (Chemicon Co., USA), or with both. These steps were followed with FITC or Rhodamine labeling for sperm assessment with fluorescence microscopy. Statistical evaluation by paired T-test and Wilcoxon ranked sum test were carried out with SigmaStat (Jandel, CA). All values are mean±SEM. TP occurred in the equatorial segment, acrosomal area, neck, and principal piece of sperm. To investigate whether TP may occur in sperm of diminished maturity we explored the potential relationship between sperm maturity and TP by double-labeling sperm for TP and CK (a marker of surplus cytoplasm). Double staining revealed that the TP changes occur exclusively in mature spermatozoa, and there were no TP changes in sperm with cytoplasmic retention. When we assessed TP in sperm at 0 time and 4h in HTF medium, and after HA-binding, the proportion of sperm with TP at 0 time and 4h in HTF were 41.7±2.6% and 54.7±4.2% (NS, N=7873 sperm). The respective TP values in the HA-bound sperm fractions were 54.7±4.2% and 67.0±3.8% (NS, N=7508 sperm). We observed major TP changes, with time and HA-binding, only in the neck region of mature sperm. The % sperm with neck phosphorylation in HTF at 0 time and 4 hours were: 14.4±5.3 and 41.5±8.8 (p=0.02, N=3777 sperm) compared to 21.9%±4.9% and 51.5±5.5% (p=0,01, N=3555 sperm) in the HA-bound sperm fraction, respectively. The extent of TP in the sperm neck, a marker of sperm activation, increases with time and contact with HA. We have demonstrated for the first time that TP does not occur in diminished maturity sperm with cytoplasmic retention. Moreover, in line with other sperm attributes, the sperm TP pattern changes are also similar in sperm that are bound to HA or to zona pellucida of oocytes.

P-628: Sperm bound to hyaluronic acid or to human zona pellucida exhibit similar levels of improvement in tygerberg strict morphology scores

During spermiogenesis, a plasma membrane remodeling step facilitates formation of zona pellucida and hyaluronic acid(HA) binding sites in sperm. Thus, diminished maturity sperm, which contain surplus cytoplasm and show consequential abnormal morphology, are unable to bind to HA. We have tested the hypothesis that there will be an improvement in the proportion of sperm with normal Kruger strict morphology in HA-bound sperm fractions of semen samples, similar to the 4.2x increase reported by the Tygerberg, South Africa (TSA) group for zona pellucida bound sperm fractions of teratozoospermic specimens (Mol. Repr. Dev., 1991). Strict sperm morphology was determined in semen and in their respective HA-bound fractions in 30 men (Sperm conc. range: 7.4-115x106/mL, motility: 45.6±3.1%) by three independent investigators. Two readers were from the USA (US1 and US2) and the third, TSA, who was blinded to the A and B surface of slides, was from South Africa. METHODS: For sperm evaluation, a two-chamber version of the HBA slide (MidAtlantic Diagnostics) was used. An aliquot of semen was smeared on the uncoated A side. In another aliquot, the sperm were allowed to bind to the HA-coated surface of B side, to determine HA-binding score. After Diff-Quik staining, 200 sperm were evaluated for strict morphology by each investigator on the A and B sides. The sperm maturity markers, CK-activity (cytoplasmic retention) and HspA2 chaperone were assessed, as published previously. Statistical analysis by paired T-test, Wilcoxon ranked sum test, and Spearman correlations were by Sigma Stat (Jandel). All values are mean±SEM. Comparing the three readers, US1 and US2 used stricter criteria than TSA. The % normal scores by the three investigators for A semen and the respective B HA-bound sides were: US1: 6.6±0.7% (range:14.7) and 13.5±1.4% (range: 33.2): US2: 8.9±0.9 (range:20.7) and 14.5±1.4 (range:29.8); TSA: 11.0±1.1 (range: 23) and 26.5±2.1 (range: 50), A vs. B p<0.003 in all readers. There were similar variations in Kruger strict morphology score improvements from the A to B sides: US1: 2.2x, US2:1.8x and TSA: 3.1x. However, despite the differences in “strictness,” the evaluations were internally consistent among USA1, USA2, and TSA between r=0.38 and 0.66 (p=0.039 - <0.001). The rationale for the morphology improvements is supported by the relationships between HA-binding scores and sperm maturity markers: HA-binding vs. CK activity: r= -0.56, p=0.004; HA-binding vs. HspA2 levels: r=0.55, p=0.005. Most revealing are the % increase in sperm with normal morphology bound to HA from the 19 teratozoospermic (<14% normal) samples: 226% and 194% for the stricter US1 and US2. However, TSA found a 376% improvement, a value nearly identical to the 420% improvement found in zona pellucida-bound sperm by the TSA group in 1991. CONCLUSIONS: Consistent with our hypothesis, there was a significant improvement in normal strict morphology scores in the HA-bound vs. semen sperm fractions. The improvement was comparable to those reported earlier for zona pellucida-bound sperm. The relationships between sperm maturity, HA-binding and normal morphology are further supported by the HA-binding scores and sperm maturity markers. This selection/separation method holds promise for semen evaluation and ICSI programs, as mature normal forms are highly selected.

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

To test a newly invented intracytoplasmic sperm injection (ICSI) sperm selection method based on sperm hyaluronic acid (HA) binding. Comparison of chromosomal disomy and diploidy frequencies in sperm arising from semen and in HA-bound sperm. Academic andrology laboratory. Men presenting for semen analysis. 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. Chromosomal disomy and diploidy frequencies. 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). 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.

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.

Hyaluronic acid binding by human sperm indicates cellular maturity, viability, and unreacted acrosomal status

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.

Biochemical markers of sperm function: male fertility and sperm selection for ICSI

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.