The hemizona assay for assessment of sperm function.

The hemizona assay (HZA) has been developed as a diagnostic test for the tight binding of human spermatozoa to the human zona pellucida to predict fertilization potential. In this homologous bioassay, the two matching hemizona halves are functionally equal surfaces allowing controlled comparison of binding from a fertile control versus a test sample, with reproducible measurements of sperm binding obtained from a single oocyte. Oocytes from different sources (surplus from IVF treatment or recovered from ovarian tissue) are salt-stored and used after microbisection. Extensive clinical data have demonstrated excellent predictive power of the HZA for the outcomes of intrauterine insemination and IVF, and therefore the assay has relevance in the clinical diagnostic setting in infertility.

Fragmentation of DNA in morphologically normal human spermatozoa.

OBJECTIVE: To evaluate DNA fragmentation in spermatozoa with normal morphological appearance. DESIGN: Prospective study. SETTING: Academic tertiary center. PATIENT(S): Fertile, subfertile, and infertile men were studied. INTERVENTION(S): Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-fluorescein nick-end labeling assay and morphology assessment by phase contrast in the swim-up fractions. MAIN OUTCOME MEASURE(S): Simultaneous assessment of the percentage of normally shaped sperm and DNA fragmentation. RESULT(S): No DNA fragmentation was found in spermatozoa with normal morphology in any of the samples from the fertile group. In only one sample from the subfertile group did we observed normally shaped sperm cells exhibiting DNA fragmentation. However, in all the samples from the infertile group, we observed normal spermatozoa with DNA fragmentation. Spermatozoa from this late group exhibited a high proportion of DNA damage. CONCLUSION(S): In infertile men with moderate and severe teratozoospermia, the spermatozoa with apparently normal morphology present in the motile fractions after swim-up may have DNA fragmentation.

Apoptose e peroxidação lipídica antes e após criopreservação / Apoptosis and lipid peroxidation before and after cryopreservation

Objetivo: availar a existência da associação entre os parâmetros da análise seminal básica e a porcentagem de apoptose e peroxidação lipídica antes e após criopreservação. Material e médotos: foram avaliados ejaculados de 16 homens portadores de infertilidade primária atendidos no Laboratório de Andrologia do Jones Institute for Reproductive Medicine nos Estados Unidos. A porcentagem de apoptose foi avaliada pela aderência à anexina, a peroxidação lipídica espontânea por meio de análise espectofotométrica e os parâmetros da motilidade por meio da análise computadorizada. Resultados: antes do congelamento, houve maior porcentagem de espermatozóides apoptóticos (41,4%) e menor porcentagem de espermatozoides vivos (49,7%) em homens com menor concentração espermática (p menor que 0,05). Após criopreservação, a porcentagem de espermatozoides vivos foi menor nos sujeitos com menor concentração espermática (42,6%; p igual 0,06). Não houve diferenças significativas da morfologia e motilidade antes e após criopreservação. Antes do congelamento, houve correlação entre espermatozoides vivos e sua concentração (p menor que 0,05); uma correlação negativa entre a concentração de espermatozoides e apoptose (p menor que 0,05). Conclusões: antes e após criopreservação, houve maior proporção de espermatozoides apópticos em homens com menor concentração espermática; não foi encontrada associação entre a motilidade e morfologia com a apoptose peroxidação lipídica não apresentou associação com qualquer parâmetro da análise seminal básica

Characterisation of a human sperm cell subpopulation marked by the presence of the TSH2B histone.

During the process of mammalian spermiogenesis, a significant reorganisation of the chromatin structure occurs involving the sequential substitution of somatic histones with protamines. In the human sperm nucleus, approximately 15% of the basic nuclear protein complement is maintained as histones. Human testis/sperm-specific histone H2B (hTSH2B) is a variant of the histone H2B expressed exclusively in spermatogenic germline cells and present in some mature sperm cells. Thus, this protein marks a subpopulation of sperm cells in the ejaculate. Using indirect immunofluorescence, we examined the influence of hTSH2B on zona pellucida binding and sperm head decondensation in amphibian egg cell-free extract. As suggested by previous studies, we found that hTSH2B can be localised in only approximately 30% of sperm cells within a given ejaculate. We established that the presence of hTSH2B does not influence sperm zona pellucida binding capacity. Finally, we found that decondensation occurred more rapidly and to a greater extent in those cells containing hTSH2B. We propose that the presence or absence of hTSH2B within spermatozoa influences pronuclei formation and the activation of paternal genes following fertilisation and during early embryonic development.

Predictive value of the hemizona assay for pregnancy outcome in patients undergoing controlled ovarian hyperstimulation with intrauterine insemination.

OBJECTIVE: The hemizona assay (HZA) is an established functional test that examines in vitro sperm-zona pellucida binding capacity with high predictive power for fertilization outcome in IVF. The objective of this study was to evaluate the value of the HZA as a predictor of pregnancy in patients undergoing controlled ovarian hyperstimulation (COH) and intrauterine insemination (IUI). DESIGN: Prospective clinical study. SETTING: Academic center. PATIENT(S): Eighty-two couples with unexplained or male factor infertility that underwent 313 IUI cycles. INTERVENTION(S): Basic semen analysis and HZA were performed within three months of starting COH/IUI therapy. MAIN OUTCOME MEASURE(S): Hemizona index (HZI) and clinical pregnancy. RESULT(S): Overall, patients with an HZI of <30 had a significantly lower pregnancy rate compared to patients with an HZI of > or =30 (11.1% vs. 40.6%, respectively; P<.05; relative risk for failure to conceive: 1.5 (confidence interval 1.2-1.9)). In all patients combined, and in the range of HZI 0-60, the duration of infertility (P=.000) and the HZI (P=.004) were significant determinants of conception (receiver operating characteristics (ROC) analysis). In couples with male infertility, the average path velocity and HZI were significant predictors of conception (P=.001 and P=.005, respectively, ROC analysis). The negative and positive predictive values of the HZA for pregnancy were 93% and 69%, respectively. Logistic regression analysis provided models of HZI (P=.021) and duration of infertility (P=.037) with highest predictability of conception in male factor and unexplained infertility groups, respectively. CONCLUSION(S): The HZA predicted pregnancy in the IUI setting with high sensitivity and negative predictive value in couples with male infertility. Results of this sperm function test are useful in counseling couples before allocating them into COH/IUI therapy.

Mitochondrial membrane potential integrity and plasma membrane translocation of phosphatidylserine as early apoptotic markers: a comparison of two different sperm subpopulations.

OBJECTIVE: To examine whether a relationship exists between loss of mitochondrial transmembrane potential and plasma membrane translocation of phosphatidylserine (PS) in subpopulations of human spermatozoa of men consulting for infertility. SETTING: A tertiary institutional research center. DESIGN: Prospective observational study. PATIENT(S): Twelve infertile men and five fertile controls were compared. INTERVENTION(S): Sperm subpopulations were compared after density gradient separation. MAIN OUTCOME MEASURE(S): Mitochondrial membrane potential was measured with a cationic dye, translocation of PS was evaluated with Annexin-V binding, and motion parameters were assessed manually. RESULT(S): In both the study and control groups and compared with the high-motility fraction, the low-motility fraction had significantly lower sperm motility and normal morphology, and significantly higher percentage of cells with disrupted mitochondrial membrane potential and plasma membrane PS translocation. There was a positive and significant correlation in both subfertile and control groups between the percentages of Annexin-V+ live cells and cells with mitochondrial membrane potential disruption (r = 0.82 and r = 0.85, respectively). CONCLUSION(S): The correlation of plasma membrane PS translocation and loss of mitochondrial membrane potential is suggestive of an early apoptosis phenotype, as is typically observed in somatic cells identified in sperm subpopulations with percentage of low-motile cells. We speculate that such changes might be used as diagnostic markers of sperm dysfunction(s) and that increased levels found in subfertile men might be indicators of reduced fertility potential.

Presence and significance of somatic cell apoptosis markers in human ejaculated spermatozoa.

Ejaculated spermatozoa, particularly in infertile men, have been shown to display morphological and biochemical features that are typical of an apoptotic phenotype in somatic cells. Deregulation of apoptosis is known to play roles in a number of disease processes, but roles for apoptosis in ejaculated spermatozoa and male infertility are poorly defined or have not been studied. Preliminary data demonstrated that populations of ejaculated spermatozoa express: (i) various degrees of plasma membrane translocation of phosphatidylserine and DNA fragmentation; and (ii) active caspase-3, the main executor of apoptosis in somatic cells, with an apparent exclusive cellular location to the mid-piece. Tests are currently being carried out on the effects of well-known apoptosis agonists and caspase inhibitors on such markers using purified populations of leukocyte-free ejaculated human spermatozoa. The main objective is to determine if somatic cell apoptosis markers are relevant indicators and/or causative factors of male infertility.

Optimizing use of assisted reproduction.

Infertile couples who have failed to achieve pregnancy now have new options that have become available in the last two decades. The costs are generally large and frequently not covered by insurance. Risks include multiple pregnancy and hyperstimulation syndrome. Evidence concerning results of several treatment options is presented. The large difference in success between age groups is displayed. Results to be expected from donor eggs to overcome the low success in older age groups (>38 years) are presented.

Efficacy and pregnancy outcome of two methods of semen preparation for intrauterine insemination: a prospective randomized study.

OBJECTIVE: To examine pregnancy outcome with two methods of semen preparation for intrauterine insemination (IUI). DESIGN: Prospective and randomized study. SETTINGS: Academic tertiary center. PATIENT(S): Three hundred eleven couples undergoing 676 consecutive cycles of assisted conception using IUI. INTERVENTION(S): Semen samples collected for IUI were randomized to wash only or density gradient centrifugation (DGC) processing. MAIN OUTCOME MEASURE(S): Various prepreparation and postpreparation semen parameters were used for IUI. The influence of the method of semen processing, and impact of various semen parameters and female factors on pregnancy were examined by receiver operating characteristics (ROC) curves, logistic regression, and life table analysis. RESULT(S): Of the 676 cycles, 88 resulted in conception leading to an overall clinical pregnancy rate of 13.0% per cycle and 28.3% per patient with a miscarriage rate of 34.0%. Eighty-eight percent of pregnancies occurred in the first three cycles of IUI and 95.5% within the first four cycles. The pregnancy rate for wash only was 11.6% (37 of 319) and the rate for DGC was 14.3% (51 of 356). However, in samples with <22 million motile sperm in the inseminate, pregnancy rates were 4% for wash and 18% for DGC. The woman's age for both methods and the percentage of sperm in the original semen with a velocity of > or =80 micro m/s for the wash method influenced pregnancy outcome. CONCLUSION(S): Although samples with an acceptable number of motile sperm can be processed efficiently by wash only, poor quality semen samples should be processed using DGC.

Caspase activity and apoptotic markers in ejaculated human sperm.

The objectives of this study were to determine if human ejaculated sperm exhibit active caspases and if caspase-dependent apoptosis markers are identifiable. Sperm from fertile donors and infertile patients were examined after gradient separation into leukocyte-free fractions of high and low motility. Sperm were evaluated for motion parameters, morphology, caspase activation, and apoptosis markers including phosphatidylserine (PS) translocation (annexin V binding) and DNA fragmentation (TUNEL). Active caspase-3 was detected by immunofluorescent microscopy in a small proportion of sperm in situ, in fractions of high and low motility sperm of patients and donors, but low motility fractions had significantly higher numbers of positive sperm. Immunoblot analysis detected inactive procaspase-3 (32 kDa) in all fractions of low sperm motility from patients and donors, while active caspase-3 (17 kDa) was only detected by immunoblotting in a limited number of low motility fractions from patients and in even fewer fractions from donors. Caspase enzymatic activity, as measured using the fluorogenic substrate DEVD-afc, was higher in patients than in donors in both low and high motility fractions. Annexin V staining and DNA fragmentation were detected in a proportion of sperm, with a higher frequency in the low motility fractions. A significant positive correlation between in-situ active caspase-3 in the sperm midpiece and DNA fragmentation was observed in the low motility fractions of patients, suggesting that caspase-dependent apoptotic mechanisms could originate in the cytoplasmic droplet or within mitochondria and function in the nucleus. These data suggest that in some ejaculated sperm populations, caspases are present and may function to increase PS translocation and DNA fragmentation.

Intrauterine insemination: a systematic review on determinants of success.

Intrauterine insemination (IUI) is a frequently indicated therapeutic modality in infertility. Here, a systematic review of the literature was performed to examine the current status of clinical and laboratory methodologies used in IUI and the impact of female and male factors on pregnancy success. Emphasis was centred in questioning the following: (i) the value of IUI against timed intercourse; (ii) IUI application with or without controlled ovarian hyperstimulation; (iii) timing and frequency of IUI; and (iv) impact of various parameters (male/female) on the prediction of pregnancy outcome. The odds of multiple pregnancy occurrence and its risk factors, as well as the cost-effectiveness of IUI treatment compared with more complex assisted reproductive technologies are discussed. A computerized literature search was performed including Medline and the Cochrane library, as well as a crossover search from retrieved papers. It is concluded that although IUI is a successful contemporary treatment for appropriately selected cases of female and/or male infertility, further research is needed through well-designed studies to improve the methodologies currently utilized. Importantly, the clinical management of the infertile couple should be performed in an expedited manner taking into consideration the age of the woman, the presence of multifactorial infertility and cost-effectiveness of the available treatment alternatives.

Zona pellucida-induced acrosome reaction in human sperm: dependency on activation of pertussis toxin-sensitive G(i) protein and extracellular calcium, and priming effect of progesterone and follicular fluid.

In these studies, we aimed to characterize the effects of the physiological, homologous agonists of the acrosome reaction, i.e. the zona pellucida (ZP) and progesterone/follicular fluid, on human sperm. The specific aims of our studies were: (i) to examine the dependency of the solubilized ZP-induced acrosome reaction on G(i) protein activation and presence of extracellular calcium; and (ii) to determine whether progesterone/follicular fluid exert a priming or synergist effect on the solubilized ZP-induced acrosome reaction. Highly motile sperm from fertile donors were exposed to the agonists in a microassay and the acrosomal status of live sperm was determined by indirect immunofluorescence using PSA-FITC/Hoechst double-staining. Pretreatment with pertussis-toxin (100 ng/ml) and EGTA (2.5 mmol/l) significantly inhibited the ZP-induced acrosome reaction without affecting the spontaneous rate of exocytosis. Progesterone (1.25 microg/ml) and human follicular fluid (10%) exerted a priming, time-dependent effect on the ZP-induced acrosome reaction. These studies demonstrated that: (i) acrosomal exocytosis of capacitated human sperm triggered by the homologous ZP is dependent on the activation of G(i) proteins (pertussis toxin-sensitive) and the presence of extracellular calcium; and (ii) progesterone and follicular fluid exert a priming effect on the ZP-induced acrosome reaction.

Effect of different incubation conditions on phosphatidylserine externalization and motion parameters of purified fractions of highly motile human spermatozoa.

The objective of this study was to assess the temporal effects of sperm incubation at body temperature with various amounts of human serum albumin (HSA) on motion parameters and phosphatidylserine externalization, an expression of membrane integrity. Purified sperm populations were prepared by discontinuous gradient separation, incubated at 37 degrees C in 3 different culture conditions (human tubal fluid [HTF] alone, HTF plus 0.3% HSA, and HTF plus 3% HSA) and evaluated at 0, 1, 3, 6, and 24 hours. Annexin V binding was used to monitor membrane translocation of phosphatidylserine and a computer-assisted semen analyzer was used to evaluate motion parameters. All incubation conditions led to a time-dependent, significant decline in sperm motion parameters and an increase in exposure of phosphatidylserine (annexin V+, live cells) to the outer leaflet of the plasma membrane in both patients and donors. Patients had a higher degree of motility loss and externalization of phosphatidylserine than donors. The decline in the percentage of normal cells (annexin V-, live) was greater in HTF alone up to 6 hours, and the decline in the percentages of motile and rapid sperm were greater in HTF alone throughout 24 hours when compared with HSA supplementation. We conclude that prolonged incubation of purified populations of highly motile human spermatozoa at body temperature was associated with significant motility loss and membrane changes as revealed by phosphatidylserine translocation. A higher concentration of HSA resulted in a relative protective effect against such impairments, particularly during the first 6 hours of incubation. Under the experimental conditions tested, significant differences were observed between infertile men and fertile controls.