H.W. Gordon Baker

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.

A randomized, double-blind, placebo-controlled trial of heparin and aspirin for women with in vitro fertilization implantation failure and antiphospholipid or antinuclear antibodies.

OBJECTIVE To investigate whether heparin and low-dose aspirin increase the pregnancy rate in antiphospholipid antibody or antinuclear antibody-seropositive women with IVF implantation failure. DESIGN A double-blind, randomized, transfer-by-transfer of fresh or cryopreserved embryos, crossover trial.A hospital infertility clinic and associated IVF service. PATIENT(S) Women seropositive for at least one antiphospholipid (APA), antinuclear (ANA), or beta(2) glycoprotein I autoantibody and >or=10 embryos transferred without achieving pregnancy (n = 143). INTERVENTION(S) Subcutaneous unfractionated heparin (5000 IU b.i.d.) and aspirin (100 mg daily) (158 transfers of 296 embryos) or placebo (142 transfers of 259 embryos) from the day of embryo transfer. MAIN OUTCOME MEASURE(S) Fetal heart per embryo transferred (implantation rate). RESULT(S) There was no significant difference in pregnancy rates or implantation rates between treated and placebo cycles; for example, fetal hearts per embryo transferred implantation rates were 6.8% (20/296) and 8.5% (22/259), respectively, and the generalized estimating equation covariate adjusted relative pregnancy rate was 0.65 (95% confidence interval, 0.33-1.28). The implantation rate for seropositive trial participants (42/555, 7.6%) compared favorably with that for IVF implantation-failure patients continuing treatment outside the trial (147/3237, 4.5%). CONCLUSION(S) Heparin and aspirin did not improve pregnancy or implantation rates for APA-positive or ANA-positive patients with IVF implantation failure.

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

Adverse obstetric and perinatal outcomes in subfertile women conceiving without assisted reproductive technologies.

OBJECTIVE To determine whether adverse perinatal outcomes are increased in subfertile women. DESIGN Cohort study. SETTING Two tertiary assisted reproductive technologies (ART) centers; Victorian births register. PATIENT(S) Records of women who registered with the clinics (1991-2000), but did not have an infant using ART, were linked to the birth register (1991-2004) to identify singleton non-ART births within 5 years of registration (N = 2171). Controls, matched by maternal age and year of infants birth, were selected randomly from birth records (N = 4363). INTERVENTIONS None. MAIN OUTCOME MEASURE(S) Adverse obstetric and perinatal outcomes. RESULT(S) After adjusting for confounders, compared with controls, subfertile women had increased odds of hypertension or preeclampsia (adjusted odds ratio [OR] 1.29, 1.02-1.61), antepartum hemorrhage (adjusted OR 1.41, 1.05-1.89), perinatal death (adjusted OR 2.19, 1.10-4.36), low birth weight (adjusted OR 1.44, 1.11-1.85), preterm birth <37 weeks (adjusted OR 1.32, 1.05-1.67) or <31 weeks (adjusted OR 2.37, 1.35-4.13), and cesarean delivery (adjusted OR 1.56, 1.37-1.77). There was weak evidence for increased birth defects (adjusted OR 1.30, 0.98-1.72) and gestational diabetes (adjusted OR 1.25, 0.96-1.63). No increased risk was found for prelabor rupture of membranes, small for gestational age, or postpartum hemorrhage. CONCLUSION(S) Subfertile women with singleton births are at increased risk of several adverse outcomes. These risks should be considered during their antenatal care and when analyzing adverse effects of ART.

Increased risk of blastogenesis birth defects, arising in the first 4 weeks of pregnancy, after assisted reproductive technologies

BACKGROUND The reasons for increased birth defect prevalence following in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are largely unknown. Classification of birth defects by pathology rather than organ system, and examination of the role of embryo freezing and thawing may provide clues to the mechanisms involved. This study aimed to investigate these two factors. METHOD Data on 6946 IVF or ICSI singleton pregnancies were linked to perinatal outcomes obtained from population-based data sets on births and birth defects occurring between 1991 and 2004 in Victoria, Australia. These were compared with 20,838 outcomes for singleton births in the same population, conceived without IVF or ICSI. Birth defects were classified according to pathogenesis. RESULTS Overall, birth defects were increased after IVF or ICSI [adjusted odds ratio (OR) 1.36; 95% CI: 1.19-1.55] relative to controls. There was no strong evidence of risk differences between IVF and ICSI or between fresh and thawed embryo transfer. However, a specific group, blastogenesis birth defects, were markedly increased [adjusted OR 2.80, 95% CI: 1.63-4.81], with the increase relative to the controls being significant for fresh embryo transfer (adjusted OR 3.65; 95% CI: 2.02-6.59) but not for thawed embryo transfer (adjusted OR 1.60; 95% CI: 0.69-3.69). CONCLUSION Our findings suggest that there is a specific risk of blastogenesis birth defects arising very early in pregnancy after IVF or ICSI and that this risk may be lower with use of frozen-thawed embryo transfer.

Pregnancies after intracytoplasmic injection of sperm collected by fine needle biopsy of the testis

OBJECTIVE To investigate the use of intracytoplasmic sperm injection with sperm collected by fine needle biopsy of the testis as a treatment for male genital tract obstruction. DESIGN Sperm isolated from a fine needle biopsy of the testis were used to inseminate oocytes by intracytoplasmic sperm injection. SETTING A hospital-based tertiary referral infertility service. PATIENTS Case studies of two couples in whom the male partner had a genital tract obstruction. MAIN OUTCOME MEASURES Fertilization and pregnancy. RESULTS In the first case, 9 oocytes fertilized normally out of 13 injected. After the transfer of fresh (one cycle) and frozen (four cycles) embryos, a single intrauterine fetal heart pregnancy was achieved. In the second case, five oocytes fertilized normally from nine oocytes injected; two embryos were transferred fresh and three were frozen. A single fetal heart intrauterine gestation was obtained after the initial transfer of two fresh embryos. CONCLUSIONS A high normal fertilization rate and pregnancies are possible with intracytoplasmic sperm injection using sperm collected directly from the testis. Sperm retrieval by fine needle biopsy offers a viable alternative to microsurgical aspiration and is also suitable for treating patients with intratesticular blockage.

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.

Intracytoplasmic sperm injection for treating infertility associated with sperm autoimmunity

Objective: To determine whether intracytoplasmic sperm injection (ICSI) can be used to achieve normal fertilization, embryo cleavage, and pregnancies in cases of sperm autoimmunity. Design: A retrospective analysis of ICSI results in sperm antibody-positive and randomly selected antibody-negative groups. Setting: University- and hospital-based reproductive research laboratory and tertiary referral IVF program. Patient(s): Thirty-nine couples selected on the basis of a strongly positive result for sperm antibodies of immunoglobulin (Ig) G and/or IgA immunoglobulin class in the male partner and a control group of 140 antibody-negative couples. Intervention(s): Human menopausal gonadotropin, hCG and Lucrin (Abbott Australasia, Kurnell, NSW, Australia) were given by injection. Oocyte collection was by transvaginal ovarian puncture. Blood was collected for β-hCG measurement. Main Outcome Measure(s): Normal fertilization, embryo cleavage, establishment of clinical pregnancy, and delivery. Result(s): There were no significant differences in fertilization rates (62% versus 58%) or clinical pregnancy rates (19% versus 12%) between sperm antibody-positive and sperm antibody-negative patient groups. Conclusion: Intracytoplasmic sperm injection is an effective treatment for patients with severe sperm autoimmunity.