Peak retrograde flow a potential objective management tool to identify young adults with varicocele 'at risk' for a high sperm DNA fragmentation.

INTRODUCTION: Determining which patients are negatively affected by varicocele would enable clinicians to better select those men who would benefit most from surgery. Since conventional semen parameters, have been limited in their ability to evaluate the negative effects of varicocele on fertility, specialized laboratory tests have emerged. OBJECTIVE: To identify clinical and ultrasound parameters (including PRF) which would negatively influence standard and functional semen variables in young adults with a varicocele. DESIGN: Prospective, cross-sectional observational study. SETTING: Antwerp University Hospital, Belgium. PATIENT(S): Young volunteers between 16 and 26 years, Tanner 5, were recruited. INTERVENTION(S): Every participant had a scrotal ultrasound to calculate testicular volumes. If a varicocele was present, the grade, vein diameter, peak retrograde flow (PRF) in supine position and spontaneous reflux in standing position were measured. All participants provided a semen sample. Standard semen parameters were analyzed and sperm DNA fragmentation. MAIN OUTCOME MEASURE(S): Of all clinical and ultrasound parameters tested, PRF was an objective tool identifying young adults with a varicocele. PRF was highlighted by the prevalence of SDF, both in the total and vital fractions of the spermatozoa, providing opportunities to manage such 'at-risk' adolescents/young adults. RESULT(S): Total SDF was significantly increased in grade 3 varicocele compared to grade 1 and 2 but no significant difference with vital SDF or standard descriptive semen parameters was seen. Total and vital SDF on the other hand were significantly increased when PRF was above 38.4 cm/s. Standard semen analysis showed no difference with PRF as an independent predictor. Testicular atrophy index, varicocele vein diameter and spontaneous reflux revealed no significant differences in both the descriptive and functional semen variables. DISCUSSION: Descriptive semen parameters showed no significant difference between the non-varicocele controls and the varicocele group with low and high PRF. Increased PRF negatively influenced sperm quality via increased DNA fragmentation both in the total as in the vital fractions of the semen. CONCLUSION(S): Of all clinical and ultrasound parameters tested, PRF was an objective non-invasive tool to identify varicocele patients at risk for a high SDF.

Time intervals between semen production, initiation of analysis, and IUI significantly influence clinical pregnancies and live births.

PURPOSE: Does IDEF mapping help monitor the technical process of IUI and explore the potential improvements which might contribute to increased pregnancy and live birth rates? METHOD: Retrospective analysis of 1729 homologous IUI cycles of couples attending a fertility clinic in a university hospital setting. Standardized conventional semen parameters were analyzed and the semen samples prepared via discontinuous density gradient centrifugation. RESULTS: There was no significant association between sperm concentration, motility and morphology (analysis phase), and pregnancy outcome. Only female and male ages were significantly associated with the pregnancy outcome. There was a significant difference in the odds on clinical pregnancies and live births when analysis was ≤ 21 min initiated, and < 107 min between sample production and IUI, adjusted for male and female age. CONCLUSIONS: Adjusting for the couple's age, we could show that time intervals between semen production and analysis and IUI when kept low significantly influenced clinical pregnancies and live births.

DNA fragmentation in concert with the simultaneous assessment of cell viability in a subfertile population: establishing thresholds of normality both before and after density gradient centrifugation.

PURPOSE: TUNEL assay is the most common, direct test for sperm chromatin integrity assessment. But, lack of standardized protocols makes interlaboratory comparisons impossible. Consequently, clinical thresholds to predict the chance of a clinical pregnancy also vary with the technique adopted. This prospective study was undertaken to assess the incidence of sperm DNA fragmentation in a subfertile population and to establish threshold values of normality as compared to a fertile cohort, both before and after density gradient centrifugation in the total and vital fractions. METHOD: Men presenting at a university hospital setup for infertility treatment. DNA damage via TUNEL assay was validated on fresh semen samples, as conventional semen parameters, to reduce variability of results. RESULTS: Total DNA fragmentation in the neat semen was significantly higher in the subfertile group, but the vital fraction was not significantly different between the two cohorts. After gradient centrifugation, DNA fragmentation increased significantly in the total fraction of the subfertile group but decreased significantly in the vital fraction. In the fertile cohort, there was a non-significant increase in total fragmentation and in the vital fraction the trend was unclear. CONCLUSIONS: Estimating total and vital sperm DNA fragmentation, after density gradient centrifugation, increased both the sensitivity and the specificity, thereby lowering the number of false negatives and false positives encountered. These findings provide opportunities to investigate the significance of the total and the vital fractions after different assisted reproductive technologies.

Sperm DNA fragmentation in the total and vital fractions before and after density gradient centrifugation: Significance in male fertility diagnosis.

BACKGROUND: Sperm DNA fragmentation measured by different techniques make comparisons impossible due to lack of standardization. Induction of DNA damage after sperm preparation in the entire fraction has been observed on independent occasions but findings are not consistent. METHODS: Men presenting at a University hospital setup for infertility treatment. DNA damage via TUNEL assay was validated on fresh semen samples, as conventional semen parameters, to reduce variability of results. RESULTS: Sperm motility in neat semen inversely correlated with sperm DNA fragmentation in the total fraction, but, total count, leukocytes and immature germ cells significantly affected the vital fraction. Sperm DNA fragmentation was observed both in normal and subnormal semen samples, but was significantly different in the total fraction of astheno-, asthenoterato- and oligoteratozoospermic men. After density gradient centrifugation, sperm DNA fragmentation increased significantly in the total but decreased in the vital fraction. Advancing male age significantly influenced damage in the total but not in the vital population. CONCLUSIONS: These findings provide opportunities to investigate the significance of the total and the vital fractions both in natural conception and after different assisted reproductive technologies.

Validating semen processing for an intrauterine program should take into consideration the inputs, actions and the outputs of the process.

To validate semen preparation via density gradient centrifugation, we took into account the input via the semen sample, the action generated by technical and equipment characteristics and the output measured by the level of performance. Longer periods of abstinence reduced % yield, but increased viscosity and incomplete samples collected had no effect. Under controlled technical and equipment characteristics, precision and reproducibility were validated for density gradient. Additionally, as a good laboratory practice, internal and external quality control measures were implemented to guarantee the level of performance. Inseminating motile sperm count is an important predictive parameter for IUI success. In our group of patients, a yield of an absolute lower limit of 2 million motile spermatozoa was sufficient to contemplate IUI. Pregnancy rate of 13.8% where >2 million rapid progressive spermatozoa were inseminated was significantly higher than the pregnancies (4.4%) obtained with <2 million rapid progressive spermatozoa. This percentage was even higher than the national data registered for IUI (12.2%). To make IUI an attractive first-line treatment, standardization and proper validation of semen preparation procedure are mandatory.

Fifteen years of Belgian experience with external quality assessment of semen analysis.

Semen analysis is difficult to standardize, quality control and quality assurance are necessary to ensure that results are accurate and precise. This Belgian EQA survey over a 15-year period, involving 121 laboratories, attempted to reduce interlaboratory variability and at the same time, encouraged participating laboratories to implement correct techniques as advised by the WHO. Over the total period, the median coefficient of variation (CV) for sperm count, irrespective of the method used was 19.2%, while using improved Neubauer chamber resulted in a significantly (p < 0.001) lower median CV (14.4%). The overall median CV for rapid progressive motility was high (37.1%), but progressive motility (15.1%) and total motility (13.8%) were acceptable. Sperm morphology revealed a large variability in 79.4% irrespective of the staining procedures or evaluation criteria used. Participation in the Belgian EQA is on voluntary basis. Both, participation and implementation of the correct techniques should be made mandatory for accreditation and benefit of patient treatment. The existing Belgian EQA program should now be harmonized with other existing EQA schemes in Europe.

The relation between levels of selected PCB congeners in human serum and follicular fluid.

Individual congener and total PCB concentrations were determined in serum and follicular fluid obtained from women undergoing assisted reproductive technologies (in-vitro fertilization and embryo replacement). Although the mean individual PCB levels revealed varying degrees of contamination, the results fall in the same range as that observed by other investigators. Except for PCB 118, correlations between levels in serum and follicular fluid were strong, and statistically significant at p < 0.05. Moreover PCB 153, a major and very stable PCB congener has been shown to correlate to the total amount of PCBs (r = 0.994, and r = 0.987, for serum and follicular fluid, respectively). The same accumulation patterns of PCBs for serum and follicular fluid have been observed.

Multicenter study on reproducibility of sperm morphology assessments.

Sperm morphology has always been considered an important tool in evaluating a man's fertilizing potential. The objective of this multicentric study was to evaluate intra- and interindividual variability and between-laboratory variation using the same or different criteria of sperm morphology assessment. Semen samples were obtained from 20 males and 32 smears were made of all samples. Eighty coded smears (4 per patient) were sent to 8 laboratories for morphology assessment. The centers applied different classification systems (strict criteria, WHO 1987, Düsseldorf criteria) and participants were asked to analyze the 80 smears twice, with an interval of 1 week between each participant's two analyses. Intraclass correlations between repeats showed that sperm morphology can be assessed with acceptable within observer reproducibility. Expected increases in imprecision were observed up to coefficients of variation of >30% with decreasing morphology scores, regardless of the classification system used. Agreement in correct classification of samples as normal/abnormal was obtained in 80% of cases. Differences in reproducibility between slides may reflect an important source of heterogeneity due to smear preparation. These results emphasize the importance of external quality control systems to improve the value of sperm morphology assessments in the investigation of the male partner in a subfertile couple.

Cytokines in semen of normal men and of patients with andrological diseases.

PROBLEM: The potential value of assessment of cytokine concentrations for the diagnosis of certain pathological conditions of male reproduction has not been fully evaluated. METHOD: The concentrations of interleukin 6 (IL-6, pg/mL), its soluble receptor (IL-6 sR, ng/mL), and of interleukin 1 beta (IL-1 beta, pg/mL) have been measured in semen samples of 114 men and 12 corresponding blood sera. RESULTS: The concentration of IL-6 was unrelated to that of its receptor. Both IL-6 and IL-6 sR were higher in the first (mean: 69 and 31 resp.) than in the second fraction (39 and 13) of split ejaculates, and were within normal limits in vasectomised men. The Interleukin 1 beta concentration presented a strong positive correlated with that of IL-6 (r = 0.74, P < 0.001). The concentrations of IL-6 and IL-1 beta were unrelated to sperm concentration, motility and morphology, and they were within normal limits in immunological cases. Both IL-6 and IL-1 beta were higher (P < 0.01) in cases with accessory gland inflammation. CONCLUSIONS: Measurement of in particular IL-6 in semen may contribute to the diagnosis of inflammatory disease of the accessory sex glands (positive predictive value = 98%, sensitivity = 72%, specificity = 94%), but it is not relevant for the diagnosis of immunological disease.

Embryo-derived platelet activating factor, a marker of embryo quality and viability following ovarian stimulation for in vitro fertilization.

Embryo-derived platelet-activating factor (PAF) may be an important mediator of early maternal recognition of pregnancy. PAF was higher in media associated with clinical pregnancies when compared to preclinical pregnancies but not higher in pregnant vs nonpregnant groups. The production of PAF by the preimplantation embryo was not related to follicle size or embryo morphology. However, differences in PAF concentrations in the culture media were related to the age of the embryo culture medium and the developmental stage of the embryo.

Comparison between different pre-treatment techniques for sperm recovery prior to intrauterine insemination, GIFT or IVF.

An attempt was made to compare different pre-treatment techniques in 16 normal and proven fertile and 30 subnormal and hitherto infertile semen samples. The techniques used were (i) standard, (ii) layering, (iii) discontinuous Percoll density gradient and (iv) albumin columns. Percoll gradient was most effective in separating a high fraction of progressive motile spermatozoa (75 and 57% in normal and subnormal semen samples, respectively). The albumin columns, as well as the standard techniques, were equally effective in recovering 45 and 24% respectively of progressive motile spermatozoa in normal and subnormal semen samples. The layering method was the least effective of the four techniques (4% recovery in normal and subnormal semen samples). In cases of contamination with inflammatory cells, the standard and layering methods were significantly (P less than 0.001) more advantageous than isolations with Percoll gradient and albumin columns. The percentage of ideal forms of spermatozoa recovered from a normal semen sample was significantly higher with the standard (P less than 0.01), layering (P less than 0.05) and Percoll gradient (P less than 0.05) techniques. In subnormal samples, only the Percoll gradient gave a significantly (P less than 0.02) higher percentage of ideal forms, whereas the other techniques were less effective. The significance and practical use of the various pre-treatment techniques are discussed in relation to the characteristics of the pre-treatment semen sample.

The value of intrauterine insemination with washed husband's sperm in the treatment of infertility.

Twenty-nine infertile couples were treated by intrauterine insemination (IUI) of washed sperm from a sub-fertile husband (n = 16), in cases of gynaecological (n = 3), combined (n = 4) or idiopathic (n = 6) infertility; 116 treatment cycles resulted in 11 ongoing pregnancies. Between 0.25 and 0.45 ml of capacitation medium, containing at least 500,000 pretreated spermatozoa, were inseminated. Pre-treatment of first split fractions was performed by centrifugation and swimming up of motile spermatozoa. The pregnancy per cycle index (P/C) for IUI was 9.5% for a total of 37.9% of all couples treated achieving pregnancy. These results suggest a substantial benefit compared with a calculated six months' cumulative pregnancy rate of 4.2% independent of treatment, for this infertile population. The value of IUI in selected cases of infertility seems obvious but needs further investigation.

Luminometric measurement of ATP concentrations in human semen.

A positive correlation is found between the ATP concentration and the sperm motility characteristics. So ATP measurement may contribute to a better selection of patients; in vases of asthenospermia, a differential diagnosis between ultrastructural abnormalities and problems related to energy supply is possible without the expensive electron microscopy.

Testicular blood flow in young and old rats and influence of hCG.

To evaluate the influence of age, testicular capillary blood flow (TCBF) was measured, using the microsphere technique, in rats 3 to 24 months old, under basal conditions and after hCG stimulation (10 IU/d for two days). Despite a decline in plasma T levels, testicular capillary blood flow did not decrease with age, and hCG stimulation resulted in similar increases of approximately 50% (P less than 0.01) in testicular capillary blood flow in all age groups. We concluded that the age-associated decrease in testosterone secretion in rats is not the consequence of a decreased testicular blood flow.

Androgen and precursor levels in serum and testes of adult rats under basal conditions and after hCG stimulation.

In adult male Wistar rats, serum and testicular concentration of testosterone (T), estradiol (E2), dihydrotestosterone (DHT), 5 alpha-androstane 3 alpha, 17 beta-diol (Adiol), and their precursors were measured under basal conditions as well as 4 and 8 h respectively after i.m. injection of 100 I.U. of hCG. Under basal conditions T (203 +/- 30 (SE) ng/dl) was quantitatively by far the most important serum steroid, followed by progesterone (P) (76.5 +/- 12 ng/dl), 17-hydroxyprogesterone (170HP) (37.3 +/- 4.1 ng/dl), androstenedione (A) (24.6 +/- 2.5 ng/dl) and pregnenolone (P5) (22.9 +/- 4 ng/dl). Estradiol (E2) was present in a low concentration (1.06 +/- 0.26 ng/dl). In the testes, T was quantitatively the most important steroid (89 +/- 7.2 ng/g), followed by 5 alpha-androstane-3 alpha-17 beta-diol (Adiol) (26.5 +/- 2.8 ng/g) whereas 170HP (11.8 +/- 1.0 ng/g), P (11.5 +/- 1.0 ng/g) and P5 (16.6 +/- 1.8 ng/g) were present in roughly the same concentration, concentrations of A, DHT, dehydroepiandrosterone (DHEA), 17 hydroxy-pregnenolone (170HP5) and androst-5-ene-3 beta-17 beta-diol (D5diol) being much lower; E2 (0.06 +/- 0.01 ng/g) was hardly detectable. Within 4-8 h after hCG stimulation, serum androgen concentrations increased by a factor of 4-12, except for DHEA and D5diol (X2), and E2 (X 1.5). Intratesticular androgens and delta 4 steroid precursors increased by a factor of 5-10, delta 5 precursors by a factor of 2-4 and E2 by a factor of 2, the data tending to confirm that the delta 4 pathway is preferred over the delta 5 pathway. After hCG the relative increase of T precursors was the most important for P, suggesting that 17 hydroxylation might be the rate limiting step in T biosynthesis.