Evaluation of structural problems in the application of strict criteria for sperm morphology assessment.

BACKGROUND: The WHO manual for basic semen analysis and ISO 23162 describe sperm morphology assessment as a standard part of semen analysis. Older studies showed a correlation between morphology results and (artificial) conception. In more recent studies this relationship was less apparent and there is more emphasis on sperm morphology as a marker for healthy spermatogenesis (and general male health). Meantime, many laboratories ceased morphology assessment, probably due to unfamiliarity with this paradigmatic shift and to technical difficulties in the assessment, like the interpretation of morphological criteria. OBJECTIVES: The aim of this study was to identify morphological criteria with high variability in results in the Dutch External Quality Control (EQC) program. MATERIAL AND METHODS: Over the period 2015-2020, a total of 72 photos of sperm cells along with dichotomous propositions based on 14 criteria as defined in WHO5 (2010) were distributed in the Dutch EQC program for semen analysis. The EQC results were evaluated for variability per criterion and for trends in time. RESULTS: Between 2015 and 2020, 40 to 60 laboratories assessed the photos. Criteria with low variability between participants were related to acrosomal vacuoles, excessive residual cytoplasm, and tail metrics. In contrast, head ovality, regularity of head and midpiece contours, and alignment of the major axis of the midpiece and head led to the highest variability in outcomes. In general, there was a slightly positive trend (lower variability) in time, except for the criteria with the highest variability (stable or declining trend). DISCUSSION AND CONCLUSION: This study indicates that there are (high) variabilities in the interpretation of the morphological criteria, leading to inconsistent outcomes of morphology assessment. The results are discussed from the perspective of imperfections in definitions and examples of the criteria as given in the WHO manuals.

Hb Nouakchott [α114(GH2)Pro→Leu; HBA1: c.344C>T], A Second and Third Case Described in Two Unrelated Dutch Families.

We report two families, members of which are carriers of a hemoglobin (Hb) variant previously described as Hb Nouakchott [α114(GH2)Pro→Leu; HBA1: c.344C>T; p.Pro115Leu]. In the first family of Dutch origin, the proband, a 32-year-old male and his 65-year-old father, were both carriers of Hb Nouakchott. Of the second family we tested, only the proband, a 56-year-old Dutch female was a Hb Nouakchott carrier. Hematological analyses of these cases showed the anomaly behaves as a silent Hb variant without clinical consequences. The Hb variant remained unnoticed using high performance liquid chromatography (HPLC), while an additional peak was detected by capillary electrophoresis (CE). These independent findings of Hb Nouakchott indicate that this Hb variant might not be very rare, but simply remains under diagnosed depending on the Hb separation technique used.

Predictive value of sperm morphology and progressively motile sperm count for pregnancy outcomes in intrauterine insemination.

OBJECTIVE: To investigate the value of sperm parameters to predict an ongoing pregnancy outcome in couples treated with intrauterine insemination (IUI), during a methodologically stable period of time. DESIGN: Retrospective, observational study with logistic regression analyses. SETTING: University hospital. PATIENT(S): A total of 1,166 couples visiting the fertility laboratory for their first IUI episode, including 4,251 IUI cycles. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Sperm morphology, total progressively motile sperm count (TPMSC), and number of inseminated progressively motile spermatozoa (NIPMS); odds ratios (ORs) of the sperm parameters after the first IUI cycle and the first finished IUI episode; discriminatory accuracy of the multivariable model. RESULT(S): None of the sperm parameters was of predictive value for pregnancy after the first IUI cycle. In the first finished IUI episode, a positive relationship was found for ≤4% of morphologically normal spermatozoa (OR 1.39) and a moderate NIPMS (5-10 million; OR 1.73). Low NIPMS showed a negative relation (≤1 million; OR 0.42). The TPMSC had no predictive value. The multivariable model (i.e., sperm morphology, NIPMS, female age, male age, and the number of cycles in the episode) had a moderate discriminatory accuracy (area under the curve 0.73). CONCLUSION(S): Intrauterine insemination is especially relevant for couples with moderate male factor infertility (sperm morphology ≤4%, NIPMS 5-10 million). In the multivariable model, however, the predictive power of these sperm parameters is rather low.

Apparent loss of urinary albumin during long-term frozen storage: HPLC vs immunonephelometry.

BACKGROUND: Urinary albumin detection by immunonephelometry is decreased by approximately 30% in samples that have been frozen at -20 degrees C. An HPLC method for assessment of urinary albumin that detects immunoreactive and immunochemically nonreactive albumin has been introduced as an alternative to immunonephelometry. We investigated whether this technique is affected by sample temperature, particularly freezing. METHODS: Urine samples (n = 295) were collected from the general population (Prevention of Renal and Vascular End-Stage Disease Study). Samples were assessed by both immunonephelometry and HPLC when fresh and after storage at -20 degrees C for 4, 8, and 12 months and at -80 degrees C for 12 months. RESULTS: With immunonephelometry, storage for 4, 8, and 12 months at -20 degrees C resulted in mean (SD) urine albumin changes of -21% (29%), -28% (29%), and -34% (31) (P <0.001 for trend). Storage at -80 degrees C resulted in a 5% (19%) change after 12 months of storage (not significant). With HPLC, storage for 4, 8, and 12 months at -20 degrees C resulted in urine albumin changes of -33% (28%), -43% (24%), and -55% (21%; P <0.001 vs immunonephelometry). Storage at -80 degrees C resulted in a -29% (29%) change (P <0.001 vs immunonephelometry). CONCLUSION: Loss of albumin after freezing urine depends not only on freezing temperature but also on detection method. Detection of albumin by immunonephelometry appears to be significantly less influenced by freezing than detection by HPLC. Storage at -80 degrees C appears to prevent loss when using immunonephelometry, whereas HPLC still shows considerable loss even when urine is frozen at -80 degrees C. We propose that for reliable measurement of urine albumin, fresh samples should be used.

Update on microalbuminuria as a biomarker in renal and cardiovascular disease.

PURPOSE OF REVIEW: To discuss recently published papers on the potential use of albuminuria as a predictor of cardiovascular and renal disease. RECENT FINDINGS: Recent studies indicate that screening for microalbuminuria may not only be beneficial for detection and prevention of cardiovascular and renal disease in patients with diabetes, but also in the general population. The best method for this, however, is not yet clear. Findings indicate that it is preferable to assess albumin concentration in fresh urine samples rather than in samples that have been frozen. Furthermore, a new assay for albumin assessment has become available, which detects previously undetectable immuno-unreactive albumin above and beyond immunoreactive albumin detected by classic immunochemical assays. The pros and cons of this assay are considered. SUMMARY: Urinary albumin is a cheap, noninvasive, and easily assessable risk marker, that does not per se require a visit to a physician or health center. As such, it is a promising candidate for screening to identify subjects at high risk of cardiovascular and renal disease, even if albuminuria is not shown to be independent of other risk markers.

Which method for quantifying urinary albumin excretion gives what outcome? A comparison of immunonephelometry with HPLC.

BACKGROUND: Microalbuminuria has recently been identified as an independent risk factor for cardiovascular disease in the general population. Immunochemical urinary albumin assays only detect immunoreactive intact albumin. High performance liquid chromatography (HPLC) is able to detect both immunoreactive and immunounreactive intact albumin. We compared both measurement methods respectively in subjects with normo-, micro-, and macroalbuminuria in the general population. METHODS: We used 24-hour urine samples that were collected within the framework of the second screening for the PREVEND Study, a prospective cohort study on albuminuria in the city of Groningen, The Netherlands. RESULTS: With nephelometry as immunochemical reference method, we classified 986 subjects as normoalbuminuric, 283 as microalbuminuric, and 43 subjects as macroalbuminuric. The mean +/- SD albumin concentration was 6.8 +/- 4.3 mg/L for nephelometry in the urine samples of the 998 subjects with a concentration <20 mg/L according to nephelometry versus 17.6 +/- 10.3 mg/L for HPLC (P < 0.001, HPLC 159% higher). These values were 58.9 +/- 40.6 mg/L for nephelometry versus 74.0 +/- 51.8 mg/L for HPLC (P < 0.001, N = 280, HPLC 26% higher) in the concentration range between 20 to 200 mg/L, and 436.3 +/- 371.8 mg/L for nephelometry versus 399.1 +/- 329.2 mg/L for HPLC above 200 mg/L (P = 0.048, N = 34, HPLC 8.5% lower). Associations of 24-hour urinary albumin excretion with cardiovascular risk factors were generally somewhat stronger for nephelometry than for HPLC. Logistic regression analyses with an abnormal ankle-brachial index as outcome parameter revealed adjusted odds ratios of 1.78 (95%CI 1.01-3.12, P < 0.05) and 4.67 (95%CI 1.68-12.9, P < 0.05) respectively for micro- and macroalbuminuria as determined by HPLC, compared to 1.37 (95%CI 0.77-2.41, P = NS) and 3.85 (95%CI 1.53-9.67, P < 0.05) respectively for nephelometry. The ROC-curve showed similar sensitivity and specificity for both methods (P = 0.25). CONCLUSION: The use of HPLC for determination of urinary albumin concentrations reveals higher values compared to nephelometry, especially in the lower concentration range, resulting in a higher prevalence of microalbuminuria. With HPLC compared to nephelometry, we found a 21% higher independent odds ratio for microalbuminuria with the presence of peripheral vascular disease, and a 30% higher independent odds ratio for macroalbuminuria. This higher prevalence of microalbuminuria, accompanied with a similar absolute risk for peripheral vascular disease compared to patients with microalbuminuria detected by nephelometry, suggests HPLC to identify more people at risk, which is of great importance, especially when screening in large populations is concerned.