Better sperm selection for intracytoplasmic sperm injection with the side migration technique.

The side migration technique (SMT) is a recent method for preparing very poor-quality semen samples to be used in intracytoplasmic sperm injection (ICSI). In most centres, the washing swim-up and Percoll gradient columns techniques have been routinely used. The present study is aimed at comparing the quality of oligozoospermic semen samples selected after these three methods. All three methods were found to select better percentage motility, normal morphology, viability, functional integrity of plasma membrane and nuclear chromatin integrity compared with the original semen samples. Among the three methods, however, SMT yielded better sperm quality, including morphology, viability, membrane integrity and nuclear chromatin integrity. The results of this study and our experience have confirmed that SMT is an effective and physiological method to prepare sperm for ICSI.

Evaluation of the immunological cause of male infertility.

The presence of antisperm antibodies was considered as one of the main causes of male infertility. This study was conducted on 776 semen samples of the male partners of infertile couples in order to evaluate the immunological factor. The detection of antisperm antibodies was performed by means of the direct Mixed Antiglobulin Reaction (MAR) test. A positive test was found in 35 samples. Hence, the prevalence of immunological infertility in this study is 4.5% among the male partners of infertile couples. Further explorations were performed on men with positive results, in order to detect the presence of antibodies in the sera by means of the indirect MAR tests, and sperm immobilization tests for quantitating the cytotoxic activity of the antibodies. The indirect MAR tests showed that 97% of the antibodies were present in sera, whereas 51% of the samples were cytotoxic. Routine semen analysis indicated that around 60% of the semen samples with antisperm antibodies were good. Semen culture showed that positive results were detected in 40% of samples. Clinical evaluations revealed that the presence of antisperm antibodies may have been the cause of long-standing infertility, on average 5 years, and were not all primary. Evaluations of all clinical and laboratory findings indicated that unilateral testis obstruction and male accessory gland infection were the main pathologies found.

Objective semen analysis: has the target been reached?

Objectivity in assessing critical sperm characteristics such as concentration, motility and morphology, has been the target of many systems and devices. Methods based on physical principles such as turbidimetry, spectrophotometry, laser Doppler technology, are too imprecise or technically too complicated to be applied routinely in the laboratory. Microscopic images providing photographical, cinematographical or video data acquisition followed by manual or computer-assisted image analysis (CASA) suffer from practical and/or device deficiencies, making them tedious and time-consuming, or unreliable because of confounding factors and uncontrolled artefacts. A single-step computer system using visual evaluation of the microscopic field and manual tracking of sperm movement yields more reliable results, but technician expertise remains essential for correct performance. The scientific basis of multiple errors in semen aspiration, chamber quality, data acquisition and analysis is given. Biased interpretations of results or incorrect handling of statistical data are criticised.

Follicular fluid steroid levels in relation to oocyte maturity and in vitro fertilization.

Steroid levels in follicular fluid (FF) obtained from stimulated ovaries in patients undergoing in vitro fertilization (IVF) were measured by capillary gas chromatography. The correlation between these levels and the maturity of the oocyte, judged from the morphology of the oocyte corona cumulus complex (OCCC) and the fertilizability of the oocytes was analysed. Oocyte maturity was associated with higher FF levels of progesterone, 17-hydroxyprogesterone, 16 alpha-hydroxyprogesterone and 20 alpha-dihydroprogesterone. Follicular fluids containing oocytes that became fertilized had significantly higher levels of 20 alpha-dihydroprogesterone and progesterone and lower levels of androstenedione. Of all the steroids determined, 20 alpha-dihydroprogesterone provides the most significant group differences. Enhanced 20 alpha-dihydrogenation in the presence of decreased 16 alpha- and 17-hydroxylation appears to be an important characteristic of the ultimate ripening stages and early luteinization, at least in stimulated cycles.

Possibilities and limitations of techniques of assisted reproduction for the treatment of male infertility.

Since relatively few spermatozoa are needed for oocyte fertilization during gamete intra-Fallopian transfer (GIFT) or in-vitro fertilization (IVF), these methods have been applied in couples with infertility due to male causes. Forty-six couples with male factor infertility were enrolled in this study and results were compared with those attained in 48 couples treated with the same techniques for other than male causes. Overall, GIFT resulted in 26% ongoing pregnancies. GIFT seems to be particularly successful when the sperm concentration is 20 x 10(6)/ml or more, but sperm motility and/or morphology are poor. Nine pregnancies occurred out of 26 GIFT cycles in 18 cases selected on this basis. The ongoing pregnancy rate after IVF was 16% per patient. The latter treatment should be attempted in male immune infertility and in cases with a low sperm concentration, with or without abnormal sperm motility and/or morphology. In these circumstances, five pregnancies were attained out of 28 cycles in 14 cases. For similar sperm concentrations, the conception rate per cycle attained with techniques of assisted reproduction was more than twice that attained with conventional treatment of male infertility.

Value of sperm characteristics and the result of in-vitro fertilization for predicting the outcome of assisted reproduction.

The outcome of procedures for assisted reproduction, namely in-vitro fertilization (IVF) and gamete intrafallopian transfer (GIFT) were used as end-points to assess the capacity of particular sperm characteristics to predict the fertilizing potential of semen. In addition, the result of IVF performed with spare oocytes from the GIFT procedure, was used to predict the pregnancy outcome of GIFT. The power of sperm characteristics to predict the outcome of assisted reproduction procedures was poor. With regard to the outcome of IVF, sperm morphology was the best discriminant. The threshold value of 16% normal sperm morphology could be used as a good predictor of successful IVF, but could not be applied as a criterion to reject an IVF trial. For the latter purpose, the limit of 5% sperm with normal morphology should be used. The proportion of sperm with grade A motility in native semen provided the best discrimination between GIFT-fertile and GIFT-infertile cases. A negative result from IVF performed with spare oocytes was 93% accurate in predicting unsuccessful outcome of GIFT. Hence, diagnostic IVF is the most sensitive assay for male fertility. The occurrence of oocyte fertilization during IVF predicts a successful outcome of GIFT in only 50% of cases. Hence, a negative result in IVF indicates that semen is almost certainly infertile, but a positive result does not guarantee the successful outcome of GIFT.

Steroid determinations in human ovarian follicular fluid using capillary gas chromatography.

A method is presented based on capillary GLC using both a thermionic and a flame ionization detector to simultaneously analyse all major unconjugated steroids in ovarian follicular fluids (FF). Although specificity can not always be guaranteed for the smaller concentrations of androstenedione and cortisol, accuracy and reproducibility are excellent for the major progestagens and estrogens (progesterone, 17- and 16 alpha-hydroxyprogesterone, pregnenolone, 20 alpha-dihydroprogesterone, estradiol and estrone). Above all the analysis is performed with relatively cheap instrumentation and products. Apart from the "profiles" of unconjugated steroids, a semi-quantitative analysis of steroid conjugates is possible if a preliminary group separation with disposable anion exchanger columns is included.

Effect of different procedures of semen preparation on antibody-coated spermatozoa and immunological infertility.

To assess whether procedures of semen preparation can reduce the proportion of antibody-coated spermatozoa, semen samples with positive direct mixed antiglobulin reaction (MAR) were washed in media supplemented with 10% or 50% fetal cord serum (FCS). Washing reduced the MAR to a negative level, but the MAR was identical to that in the native semen when spermatozoa were resuspended in serum-free medium. Donor spermatozoa, recovered after swim-up in media supplemented with 10% or 50% FCS or after passage through a column with 7.5% human serum albumin (HSA), were incubated in serum samples with both agglutinating and cytotoxic antisperm antibodies. Cytotoxic activity was significantly reduced against sperm filtered over the albumin column.

Pregnancy after in vitro fertilization and embryo transfer in severe immune male infertility.

The male partner of an infertile couple was found to have anti-spermatozoal antibodies (ASA) of the IgG and IgA class in his semen as well as agglutinating and cytotoxic antibodies in serum. His wife conceived twice after IVF-ET with ongoing pregnancies.

Determination of adenosine triphosphate in human semen to estimate the fertilizing potential and to quantify sperm antibodies.

The measurement of the ATP content of fresh semen is as accurate as the estimation of sperm motility by conventional methods in discriminating between semen of fertile versus subfertile men. The ATP content of frozen thawed donor semen is correlated with the probability of conception per cycle of insemination. Exact quantification of cytotoxic sperm antibodies in serum is possible with the adenosine-triphosphate-release-cytotoxicity test, since measurement is free of the bias of microscopic examination. The procedure has been simplified by testing only one serum dilution and calculating the 'sperm toxicity index'.

Accuracy of sperm characteristics in predicting the in vitro fertilizing capacity of semen.

Based on the results of in vitro fertilization (IVF) in 56 couples, the power was assessed of traditional sperm characteristics of native semen to discriminate between in vitro fertile and in vitro infertile semen. The number per ejaculate of spermatozoa with regular oval heads was the best discriminant, followed by the concentration of progressively motile spermatozoa. This contrasts with the in vivo fertilizing capacity, which depends mostly on the proportion and concentration of spermatozoa with rapid linear progression. The lower limit of sperm characteristics was assessed as the fifth percentile of in vitro fertile semen and was compared with the lower limit of semen of fertile men and of subfertile men who achieved spontaneous or treatment-related conception in vivo. It appeared that the semen quality needed for in vitro fertilization is inferior to that of fertile men but not remarkably different from that of subfertile men who achieved spontaneous conception during 1-year follow-up after consultation. If conventional methods for semen preparation are used, there seems to be no major advantage in favor of IVF for the treatment of male infertility due to sperm deficiency. An increased success rate may, however, be attained, thanks to improved techniques of semen collection, semen preparation, and oocyte insemination.

Capacity of objectively assessed sperm motility characteristics in differentiating between semen of fertile and subfertile men.

A simple and inexpensive computer-assisted method for the objective assessment of sperm motility characteristics was employed to evaluate semen samples of 42 fertile men and 70 subfertile patients. The capacity of each motility parameter to discriminate between semen of the two groups was evaluated by receiver operating characteristic curve analysis. Velocity, linear velocity, and "angular" velocity are reasonably accurate, whereas linearity and angularity index had a very poor discriminating power. The best discrimination between the two groups was the proportion and concentration of sperm with rapid linear progressive motility, based on the cut-off value of linear velocity greater than or equal to 22 microns/sec. This parameter was 90% accurate in discriminating semen of infertile men from that of subfertile patients.

Validation of a single-step procedure for the objective assessment of sperm motility characteristics.

A relatively cheap method is described for the objective assessment of sperm concentration and motility characteristics. The method uses a digitizing tablet with cursor, a micro-computer and a phase-contrast or dark-field microscope equipped with a drawing tube. With this technique the following are accurately assessed: sperm concentration, percentage motility, motility grading, concentration of grade a motile spermatozoa, sperm velocity, linear velocity, linearity and angularity. The data are acquired in less than 5 min. Validation studies reveal this method to be accurate, reproducible (coefficient of variation of motility characteristics = less than 7%) and clinically useful.

Evaluation of a simplified adenosine triphosphate release cytotoxicity test for the detection of sperm antibodies in human serum.

The adenosine triphosphate release cytotoxicity test (ARCT) is an objective method for the detection of cytotoxic sperm antibodies in serum providing measurements without the bias of microscopic estimation. The procedure has been simplified by testing only one serum dilution and calculating the "sperm toxicity index". The latter is closely correlated with the sperm cytotoxic titer. Immunoglobulin class determination of 32 sera with sperm cytotoxic activity was performed by means of the indirect mixed antiglobulin reaction (MAR test) and the indirect immunobead (IB) test, and revealed that all sera were positive for IgG, 17% for IgM and 20% for IgA. Of 32 sera without sperm cytotoxic activity, 19% contained sperm specific IgG, 6% had IgM class antibodies belong mainly to the IgG class since the results of the ARCT and the indirect MAR test for IgG are closely correlated. The ARCT appears to be useful as a simple and objective method for the detection and quantitation of cytotoxic antisperm antibodies.

The indirect mixed antiglobulin reaction test using a commercially available kit for the detection of antisperm antibodies in serum.

A simple procedure is described for the detection of antisperm antibodies of the IgG class in human serum using the indirect mixed antiglobulin reaction (MAR) test. The test uses only one dilution of serum (1/16) and no washing procedures. The test was performed in 361 sera. Comparative studies were conducted with the tray agglutination test (TAT) in 164 sera and with the direct MAR test on semen in 110 cases. In 24 sera with borderline test results, and in 13 sera with discordant findings, evidence of antisperm antibody activity was investigated by means of additional testing, namely, the adenosine triphosphate release cytotoxicity test (ARCT) and the indirect immunobead tests (IB) for IgA and IgM. Using the value of 40% reaction between motile spermatozoa and coated latex particles as the lower limit of significant activity, the indirect SpermMAR test has a sensitivity of 96% and specificity of 87%. The two false-negative indirect MAR results occurred in cases with IgM, the clinical significance of which is doubtful. Hence, the indirect MAR test appears to be an ideal screening procedure for sperm antibodies in serum since it is easy to perform and sensitive. Considering the false-positive rate of 13%, additional tests should, however, be performed on sera with a reaction of 40% or more in order to assess the relevance of the antibodies as a cause of infertility.

Evaluation of the direct and indirect mixed antiglobulin reaction with latex particles for the diagnosis of immunological infertility.

The direct Mixed Antiglobulin Reaction (MAR test) is a simple method for the detection of auto-antibodies attached to spermatozoa in fresh semen. The test was improved by using IgG-coated latex particles (SpermMAR) instead of coated red blood cells. A positive direct MAR test with adherence of latex particles to 40% or more of the motile spermatozoa was found in 16 out of 312 men (5%) consulting for infertility, but never in fertile controls. The percentage of motile spermatozoa reacting with the coated latex particles was correlated significantly with the serum titre of sperm agglutinins assessed by the tray agglutination test. The direct MAR test on semen was highly specific but rather insensitive, particularly if the serum titre of agglutinins was low. The same kit (SpermMAR) can be used to detect circulating sperm antibodies in serum of male or female patients with the indirect mixed antiglobulin reaction. The indirect MAR test discriminated clearly between serum with low (less than 1/32) or high (greater than 1/32) titres of circulating agglutinins assessed by the tray agglutination test. Considering their simplicity and accuracy, both the direct test on semen and the indirect spermMAR test on serum should be included in the routine evaluation of the infertile couple.