Thinus F. Kruger

Predictive value of abnormal sperm morphology in in vitro fertilization.

In patients with acceptable sperm count and motility, two patterns of abnormal morphology, judged with strict criteria, were identified and described. Patients with less than 4% normal forms and less than 30% morphology index (summation of normal and slightly amorphous forms) had a fertilization rate of 7.6% of the oocytes (P pattern, poor prognosis). Patients with normal morphology between 4 and 14% had a significantly better fertilization rate of 63.9% of the oocytes (P less than 0.0001). Cases with greater than 14% normal forms fertilized within the normal range for the laboratory. By evaluating sperm morphology with the proposed strict criteria, its predictive value in in vitro fertilization is enhanced.

World Health Organization reference values for human semen characteristics

BACKGROUND Semen quality is taken as a surrogate measure of male fecundity in clinical andrology, male fertility, reproductive toxicology, epidemiology and pregnancy risk assessments. Reference intervals for values of semen parameters from a fertile population could provide data from which prognosis of fertility or diagnosis of infertility can be extrapolated. METHODS Semen samples from over 4500 men in 14 countries on four continents were obtained from retrospective and prospective analyses on fertile men, men of unknown fertility status and men selected as normozoospermic. Men whose partners had a time-to-pregnancy (TTP) of < or =12 months were chosen as individuals to provide reference distributions for semen parameters. Distributions were also generated for a population assumed to represent the general population. RESULTS The following one-sided lower reference limits, the fifth centiles (with 95th percent confidence intervals), were generated from men whose partners had TTP < or = 12 months: semen volume, 1.5 ml (1.4-1.7); total sperm number, 39 million per ejaculate (33-46); sperm concentration, 15 million per ml (12-16); vitality, 58% live (55-63); progressive motility, 32% (31-34); total (progressive + non-progressive) motility, 40% (38-42); morphologically normal forms, 4.0% (3.0-4.0). Semen quality of the reference population was superior to that of the men from the general population and normozoospermic men. CONCLUSIONS The data represent sound reference distributions of semen characteristics of fertile men in a number of countries. They provide an appropriate tool in conjunction with clinical data to evaluate a patients semen quality and prospects for fertility.

DNA fragmentation of spermatozoa and assisted reproduction technology

Despite the ever-increasing knowledge of the fertilization process, there is still a need for better understanding of the causes of sperm DNA fragmentation and its impact on fertilization and pregnancy. For this reason, human sperm DNA fragmentation was investigated by means of the terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) assay and the production of reactive oxygen species (ROS) in the ejaculate and in the spermatozoa themselves. These data were correlated with fertilization and pregnancy data from IVF and intracytoplasmic sperm injection (ICSI) patients. Sperm DNA fragmentation did not correlate with fertilization rate, but there was a significantly reduced pregnancy rate in IVF patients inseminated with TUNEL-positive spermatozoa. ICSI patients exhibited the same tendency. This implies that spermatozoa with damaged DNA are able to fertilize an oocyte, but at the time the paternal genome is switched on, further development stops. The determination of ROS in the ejaculate and the percentage of ROS-producing spermatozoa revealed markedly stronger correlations between sperm functions (i.e. motility) and the percentage of ROS-producing spermatozoa. The influence of seminal leukocytes, known to produce large amounts of oxidants, on sperm DNA fragmentation should not be neglected.

The hemizona assay (HZA): A predictor of human sperm fertilizing potential in in vitro fertilization (IVF) treatment

The hemizona assay (HZA) was developed to assess human sperm fertilizing potential. This blinded study investigated the relationship between sperm binding to the hemizona and in vitro fertilization (IVF) success (36 patients). Nonliving human oocytes were recovered from excised ovaries and stored. Each zona pellucida was cut into equal hemispheres by micromanipulation. For the HZA, one droplet exposed a hemizona to abnormal spermatozoa, while the control droplet contained the matching hemizona and spermatozoa from normal semen. After 4 hr, the number of tightly bound spermatozoa was counted. Binding to the hemizona was significantly higher for those having IVF success (mean of 36.1±7, versus 10.4±4 from the failure group;P<0.05). Fewer sperm from the failure group had a strictly normal morphology (3,2 versus 12.7%;P<0.05, Kruger method). Tight zona binding was significantly correlated with the percentage motile sperm, percentage normal morphology, and seminal sperm concentration. These results enhanced our confidence that the HZA is diagnostic for identification of patients at high risk of failing to achieve fertilization in vitro.

A quick, reliable staining technique for human sperm morphology.

The evaluation of sperm morphology is still an important parameter in the diagnosis of the infertile male. Most techniques used for staining human sperm are very time-consuming. A routine stain used for determining differential count of leucocytes (Diff-Quik stain) was evaluated against the standard Papanicolaou stain. Morphology results from 20 duplicate semen smears using both staining methods were determined separately by 2 technicians using a blind protocol. No significant differences were observed when comparing the two staining methods (paired Students t-test). The advantages of the Diff-Quik stain are: a) complete staining-to-reading time under 7 min, b) commercially prepared reagents, and c) case of staining procedure.

Sperm morphology: assessing the agreement between the manual method (strict criteria) and the sperm morphology analyzer IVOS * †

OBJECTIVES To correlate the percentage normal morphology reported by different observers and a computerized method (IVOS; Hamilton-Thorne Research, Beverly, MA) on a slide-by-slide basis using strict criteria: [1] Experienced observer (T.F.K.) versus experienced observer (R.M.), [2] experienced observer (T.F.K.) versus sperm morphology analyzer (IVOS), and [3] repeatability of normal and abnormal cells (IVOS). DESIGN SETTING, PATIENTS: Slides from 30 different patients from the Tygerberg IVF program were selected randomly. Microscopic fields and sperm cells were chosen randomly and percent normal morphology was recorded (objectives 1 and 2). The same slides were used and a cell-by-cell repeatability was done as outlined (objective 3). RESULTS Experiment 1 (objective 1): there was no significant bias between T.F.K. and R.M. The limits of agreement were 8.6% and -7.3%. The SDs were not significantly different (P = 0.1283). The Spearman correlation coefficient between readers was 0.83. Experiment 2 (objective 2): the same findings were reported but the limits of agreement were 12.1% and -15.5%. The Spearman correlation coefficient was 0.85. The limits of agreement was tighter below 20% normal forms (+8.4 and -6.6). Experiment 3 (objective 3) (repeatability): 255 cells were analyzed three times in succession. Estimating pairwise agreement, the kappa statistic for the pairs are 0.85, 0.80, and 0.85, respectively, which compares favorably with the second canonical moment of 0.8329 (kappa = 0.83). DISCUSSION The computers ability to classify normal morphology per slide is promising. Below 20% normal forms, the limit of agreement is tight. Because of the 6% higher reading compared with the manual method, different thresholds possibly will be developed to identify subfertile from fertile patients. The computer gives excellent repeatability of normal and abnormal cells. Based on results obtained, this system can be of clinical value both in IVF units and andrology laboratories but more clinical data is required in this field.

Failure of fertilization in in vitro fertilization: The “Occult” male factor

Failure of fertilization in patients undergoing in vitro fertilization (IVF) deserves extensive analysis for better prediction of the success or failure of this therapeutic modality. Consequently, we retrospectively studied the 52 couples in whom fertilization failed during Norfolk series 18 to 25, in an effort to establish the precise causes of failure. In the initial evaluation, pure oocyte abnormalities were identified in 19.2% of the cases; 32.6% showed sperm abnormalities, and a combination of oocyte and sperm anomalies was found in 7.7%. In 40.4% of the cases, failure of fertilization could not be explained. Re-assessment of sperm morphology by new, strict criteria increased the identification of sperm abnormalities to 61.5% and of combined sperm and oocyte anomalies to 13.4%, for a total of 74.9% of sperm factors involved, as opposed to 40.3% in the original evaluation. The incidence of unexplained failed fertilization was substantially reduced, to 11.5%. In a control group (tubal infertility) matched by age, date, and stimulation, in whom fertilization occurred, 83.3% had normal sperm parameters as judged by the new criteria for morphology evaluation. This paper emphasizes the need for a more accurate diagnosis of sperm abnormalities to establish the true incidence of this factor in failed fertilization and to obtain information of prognostic value to patients and clinicians.

Recurrent failure of in vitro fertilization: role of the hemizona assay in the sequential diagnosis of specific sperm-oocyte defects.

The results of predictive fertilization bioassays (hemizona assay, hamster ova-human sperm penetration assay), in vitro fertilization treatment, fertile donor cross-match tests with either sperm or oocytes, and oocyte micromanipulation for assisted fertilization were used to establish a pathophysiologic diagnosis in cases of recurrent failed fertilization in vitro. Disorders of sperm function manifested at the level of zona binding, zona penetration, oolemma fusion, and pronuclear decondensation as well as oocyte anomalies were considered to represent the specific gamete defects that led to abnormal sperm-oocyte interactions (i.e., failed fertilization). Our findings show that the sequential application of bioassays can elucidate specific sperm or oocyte defects that characterize functional abnormalities among sperm populations.

Luteal phase support in in vitro fertilization : Meta-analysis of randomized trials

Objective: To determine if luteal phase support improves the pregnancy rate in in vitro fertilization (IVF) cycles. Design: A meta-analysis of randomized trials of luteal phase support was carried out with the main outcome measure being the pregnancy rate per cycle. Results: Fifty-nine trials were evaluated. Eighteen trials met the inclusion criteria. Five main themes were identified: human chorionic gonadotropin (hCG) versus progesterone; progesterone versus progesterone and hCG; progesterone versus placebo; hCG versus placebo, and hCG versus progesterone versus no support. Conclusion: Luteal phase support is definitely indicated in IVF treatment cycles. This meta-analysis favored hCG above progesterone as luteal phase support with respect to pregnancy rates. Further prospective randomized trials are needed to determine a definite consensus with respect to the duration of luteal phase support in IVF cycles.

The use of semen parameters to identify the subfertile male in the general population

Aims: To present a structured review of the literature published on semen parameters and in vivo fertility potential and to establish fertility/subfertility thresholds for sperm morphology using Tygerberg strict criteria, sperm concentration, and sperm motility. Method: The published literature comparing fertile and subfertile populations between 1983 and 2002 was reviewed. Results: A total of 265 articles were identified by the sourcing methodology, but only four articles provided data that could be tabulated and analyzed. Using receiver-operating characteristics curves, morphology proved to be the best predictor of subfertility in 2 of the 4 articles, with concentration and motility also showing good predictive power. The thresholds calculated ranged between 4 and 10% for morphology, between 13.5 × 106/ml and 34 × 106/ml for concentration, and between 32 and 52% for motility. A second set of much lower thresholds was calculated in three of the articles using either a 15 or 50% prevalence of subfertility in the population or the tenth percentile of the fertile population. The adjusted thresholds were between 3 and 5% for morphology, between 9 × 106/ml and 20 × 106/ml for concentration, and between 20 and 30% for motility. Conclusions: Because these lower thresholds have a much higher positive predictive value, we suggest that thresholds of <5% normal sperm morphology, a concentration <15 × 106/ml, and a motility <30% should be used to identify the subfertile male. The lower threshold for morphology also fits in vitro fertilization and intrauterine insemination data calculated previously. Using the parameters in combination increases the clinical value of semen analysis.