De Yi Liu

Tests of human sperm function and fertilization in vitro

Objective To review recent studies on the development of new tests of human sperm function and evaluation of which sperm characteristics are most important for fertilization in vitro by logistic regression analysis. Study Selection Recent studies on the relationship between putative and new tests of human sperm function and fertility in vitro or in vivo are discussed in this review. Some physiological and technical aspects are included. Main Outcome Measures Fertilization rates in vitro and sperm tests including standard semen analysis, improved morphology assessment, objective assessment of sperm motility and movement characteristics, nuclear maturity, hypo-osmotic swelling, the acrosome and the acrosome reaction, acrosin activity, human sperm-hamster oocyte penetration assay, and sperm-zona pellucida (ZP) and sperm-oolemma binding. Results The percentages of sperm with normal morphology and a normal intact acrosome, mean linearity, and the number of sperm binding to the ZP were highly significantly related to fertilization rates in vitro. Other sperm tests evaluated usually provided no additional information about fertilization rates. The human ZP is highly selective for binding of morphologically normal sperm. Acrosome-reacted human sperm have little or no ability to bind to the ZP. Conclusion Results of in vitro fertilization can be used to evaluate tests of human sperm function. Logistic regression analysis is a powerful method for determining which groups of sperm characteristics are independently related to fertilization rates. Normal morphology, linearity, acrosome status, and sperm-ZP binding are the most important characteristics for fertilization in vitro.

A human sperm-zona pellucida binding test using oocytes that failed to fertilize in vitro

A test for human sperm binding to the zona pellucida (ZP) was developed using oocytes which failed to fertilize in vitro. Heterospermic insemination with equal numbers of test and fertile donor sperm differentially labeled with fluorescein isothiocyanate or tetra-methylrhodamine B isothiocyanate controlled for variability in ZP-sperm binding capacity. The number of sperm bound to the ZP was independent of previous sperm binding in in vitro fertilization (IVF), preservation of the ZP in salt solution, and fluorochrome labeling but increased linearly with time and sperm concentration. Sperm from men who had one or more failed attempts at IVF with no or few oocytes fertilized usually displayed very low ZP binding ratios of test to normal sperm. This test may predict the ability of sperm to fertilize human oocytes in vitro and should be useful in studies of human gamete interaction.

Human seminal clusterin (SP-40,40). Isolation and characterization.

Molecular cloning of the human complement inhibitor SP-40,40, has revealed strong homology to a major rat and ram Sertoli cell product, sulfated glycoprotein-2, known also as clusterin. This study reports the purification and characterization of human seminal clusterin. Two-dimensional gel electrophoresis revealed charge differences between clusterin purified from semen and the serum-derived material. Both preparations demonstrate comparable hemagglutination (clustering) activity and inhibition of C5b-6 initiated hemolysis. The average clusterin concentration in normal seminal plasma is considerably higher than that found in serum. Mean seminal plasma clusterin concentrations were significantly lower in azoospermia caused by obstruction or seminiferous tubule failure than with oligospermia or normospermia. Only men with vasal agenesis had undetectable seminal clusterin, suggesting that some of the seminal clusterin is produced by the seminal vesicles. Immunofluorescence of human spermatozoa revealed that clusterin was detected on 10% of spermatozoa, predominantly those that were immature or had abnormal morphology. A pilot study of 25 patients suggests that seminal clusterin concentration, together with sperm motility and morphology, is correlated with the fertilization rate in vitro. The function of seminal clusterin is unknown. Its extensive distribution in the male genital tract and its high concentration in seminal plasma suggests an important role in male fertility.

The use of in vitro fertilization to evaluate putative tests of human sperm function

Results of 106 in vitro fertilization procedures were used to evaluate the usefulness of tests of human sperm function for predicting fertilization rates. Sperm tests included concentration, motility, morphology, vitality (eosin Y exclusion), nuclear immaturity (aniline blue stain), and hypo-osmotic swelling. Only the number of sperm in the insemination medium, percentage normal morphology, and vitality were statistically significant in logistic regression models of fertilization rates. The other tests, such as the hypo-osmotic swelling test, did not give additional information about fertilization rates in this study. It is concluded that logistic regression analysis of factors affecting results of fertilization in vitro provides a powerful tool for evaluating some clinical tests of sperm function.

A sperm-zona pellucida binding test and in vitro fertilization.

Sperm binding to the zona pellucida was studied in 106 in vitro fertilization (IVF) patients. Oocytes that failed to fertilize in vitro were inseminated with a mixture of equal numbers of test and fertile donor sperm differentially labeled with fluorescein or rhodamine to control for variability in the sperm-zona pellucida binding capacity of oocytes. The ratio of the number of test and control sperm bound to four to six zonae pellucidae was significantly correlated with sperm morphology, viability, motility, motility index, and normal intact acrosomes in semen. The sperm-zona pellucida binding ratio was the most significant factor related to IVF rates by logistic regression analysis. But the proportions of sperm with normal morphology and intact acrosomes in semen also were significant. In patients with

Associations between andrological measures, hormones and semen quality in fertile Australian men: inverse relationship between obesity and sperm output

BACKGROUND The World Health Organization developed a time to pregnancy (TTP) study (number of menstrual cycles taken to conceive) to determine whether the average TTP is increasing and semen quality decreasing with time. The present study describes clinical, semen and hormone characteristics obtained from male partners of pregnant women in Melbourne, Australia, and examines the associations between these characteristics. METHODS Male partners (n = 225) of pregnant women (16-32 weeks) who conceived naturally had physical examination, health and lifestyle questionnaires, semen and hormone (FSH, LH, sex hormone-binding globulin, testosterone and Inhibin B) analyses. RESULTS Previously known associations between semen, hormone and clinical variables were confirmed as significant: sperm numbers (concentration and total sperm count) correlated positively with Inhibin B and inversely with FSH and left varicocele, while total testicular volume correlated positively with sperm numbers and Inhibin B and inversely with FSH. However, only abstinence, total testicular volume, varicocele grade and obesity (BMI > 30 kg/m2) were independently significantly related to total sperm count. Compared with those with BMI < 30 (n = 188), obese subjects (n = 35) had significantly lower total sperm count (mean 324 versus 231 million, P = 0.013) and Inhibin B (187 versus 140 pg/ml, P < 0.001) but not FSH (3.4 versus 4.0 IU/l, P = 0.6). CONCLUSIONS Obese fertile men appear to have reduced testicular function. Whether this is cause or effect, i.e. adiposity impairing spermatogenesis or reduced testicular function promoting fat deposition, remains to be determined.

A simple method for assessment of the human acrosome reaction of spermatozoa bound to the zona spellucida: lack of relationship with ionophore A23187-induced acrosome reaction

Acrosome reactions induced by the calcium ionophore A23187 and zona pellucida (ZP) were studied. Sperm samples were obtained from fertile men or men with normal semen analysis and normal sperm-ZP binding. Oocytes were obtained, with the consent of the patients, after the failure of fertilization in vitro. Motile spermatozoa selected by a swim-up technique were incubated with 10 microM A23187 for 1 h, four oocytes for 2 h or solubilized ZP (4 ZP/microliters) for 2 h. Spermatozoa bound to the ZP were dislodged and collected in a small volume of phosphate-buffered saline by aspirating the oocytes with a glass pipette with an inner diameter (120 microns) slightly smaller than the diameter of the oocyte. The acrosome status of the spermatozoa was determined using fluorescein-labelled Pisum sativum agglutinin. The proportion of spermatozoa undergoing the acrosome reaction on the ZP at 2 h varied over a wide range (5-99%), but the agreement between results for the same semen sample exposed to different groups of oocytes was good: the standard deviations of the differences being 9%. Pre-incubation of spermatozoa for 2 h did not increase the ZP-induced acrosome reaction. Re-incubation of ZP with the same sperm suspension for 2 h after removing ZP-bound spermatozoa from the first 2 h incubation produced a significantly lower ZP-induced acrosome reaction in the second incubation (22 +/- 16%) than in the first incubation (30 +/- 14%; P < 0.001, n = 20). There was no significant difference in the ZP-induced acrosome reaction with oocytes with ZP which had or had not been penetrated by spermatozoa during the in-vitro fertilization insemination. Pre-incubation of spermatozoa with solubilized ZP blocked sperm-ZP binding. However, the acrosome reaction induced by solubilized ZP (4 ZP/microliters) was significantly lower than the acrosome reaction induced by intact ZP (10 +/- 5 and 30 +/- 13% respectively, n = 11, P < 0.001), but there was a high correlation (Spearman r = 0.822, P < 0.01) between the results. On the other hand, although the average of the acrosome reaction was similar for A23187 (42%) and for ZP (43%), there was no significant correlation between the results for the two stimuli (n = 60). In conclusion, a useful method for assessing the ZP-induced acrosome reaction has been developed using oocytes which failed to fertilize in vitro. The lack of a relationship between the result of the chemical (A23187) and physiological (ZP) stimuli for the acrosome reaction in the same subjects questions the biological basis of using A23187 for tests of sperm function. Solubilized human ZP in a concentration that blocks sperm-ZP binding is a less efficient inducer of the acrosome reaction than is intact ZP. It is possible that the three-dimensional structure of the ZP is important for induction of the acrosome reaction or that spermatozoa which bind to the ZP are more likely to acrosome react. Assessment of the physiological acrosome reaction for diagnosis of sperm defects which interfere with the fertilization process should be concentrated on the spermatozoa which are capable of binding to the ZP.

The Molecular Chaperone HSPA2 Plays a Key Role in Regulating the Expression of Sperm Surface Receptors That Mediate Sperm-Egg Recognition

A common defect encountered in the spermatozoa of male infertility patients is an idiopathic failure of sperm–egg recognition. In order to resolve the molecular basis of this condition we have compared the proteomic profiles of spermatozoa exhibiting an impaired capacity for sperm-egg recognition with normal cells using label free mass spectrometry (MS)-based quantification. This analysis indicated that impaired sperm–zona binding was associated with reduced expression of the molecular chaperone, heat shock 70 kDa protein 2 (HSPA2), from the sperm proteome. Western blot analysis confirmed this observation in independent patients and demonstrated that the defect did not extend to other members of the HSP70 family. HSPA2 was present in the acrosomal domain of human spermatozoa as a major component of 5 large molecular mass complexes, the most dominant of which was found to contain HSPA2 in close association with just two other proteins, sperm adhesion molecule 1 (SPAM1) and arylsulfatase A (ARSA), both of which that have previously been implicated in sperm-egg interaction. The interaction between SPAM1, ARSA and HSPA2 in a multimeric complex mediating sperm-egg interaction, coupled with the complete failure of this process when HSPA2 is depleted in infertile patients, provides new insights into the mechanisms by which sperm function is impaired in cases of male infertility.

Disordered zona pellucida–induced acrosome reaction and failure of in vitro fertilization in patients with unexplained infertility

OBJECTIVE To determine the relationship between the zona pellucida (ZP)-induced acrosome reaction (AR) and fertilization rate and pregnancy rate in standard IVF and the frequency of disordered ZP-induced AR (DZPIAR) in patients with unexplained infertility. DESIGN Prospective study. SETTING Academic research and teaching tertiary hospital. PATIENTS Patients with unexplained infertility with normal semen analysis. INTERVENTION None. MAIN OUTCOME MEASURE Semen analysis, the ZP-induced AR, and measurements of fertilization rate and pregnancy rate with in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). RESULT(S) A statistically significant correlation was found for the ZP-induced AR and fertilization rate with standard IVF (n = 65). Patients with DZPIAR (ZP-induced AR of <or=16%) had an average fertilization rate of 23%, in contrast to patients with ZP-induced AR of >16%, who had an average fertilization rate of 61%. The sensitivity and specificity of DZPIAR for prediction of IVF rates <30% and >or=30% were 80% and 86%, respectively. Of 260 patients screened, the frequency of DZPIAR was 29%. Ten patients with DZPIAR had an average fertilization rate of 15% and no pregnancy with initial IVF cycles, and a fertilization rate of 61% (with three live-birth pregnancies) with subsequent ICSI cycles. Another 33 patients with DZPIAR were treated with ICSI alone, with an average fertilization rate of 71% and a live-birth pregnancy rate of 17% per embryo transfer. Sixteen of the patients had live-birth pregnancies (including one set of twins) after undergoing an average of 3.2 embryo transfers. CONCLUSION(S) Patients with DZPIAR have a low or zero fertilization rate with standard IVF but high fertilization and pregnancy rates with ICSI. Up to 29% of patients with unexplained infertility with normal semen analysis may have this condition, which should be diagnosed and treated with ICSI rather then standard IVF.

Normal fertilization and embryo development by intracytoplasmic sperm injection of round-headed acrosomeless sperm

OBJECTIVE To investigate the ability of round-headed acrosomeless sperm to bind to the human zona pellucida (ZP) and oolemma and to fertilize human oocytes by intracytoplasmic sperm injection. DESIGN Oocytes that had failed to fertilize in IVF were used for sperm-ZP and spermoolemma binding tests. Sperm from a fertile donor was used as a control for oocyte variability. Intracytoplasmic sperm injection was used for assisted fertilization. SETTING University- and hospital-based reproductive research laboratory and tertiary referral IVF program. PATIENTS Case study of a couple in which the man has 100% round-headed acrosomeless sperm in the ejaculate. MAIN OUTCOME MEASURE Fertilization and embryo development and the ability of sperm to bind to the ZP and oolemma. RESULTS No ZP or oolemma binding was achieved, but normal fertilization and embryo development was obtained after intracytoplasmic injection of round-headed acrosomeless sperm. However, no pregnancy was achieved after the transfer of two cleaving embryos. CONCLUSIONS Normal fertilization and embryo development from round-headed acrosomeless sperm is possible with intracytoplasmic sperm injection. However, it remains to be reported whether pregnancy can result from fertilization with this type of sperm defect.