Semen analysis is the cornerstone of male fertility evaluation with WHO guidelines providing the basis for procedural standardization and reference values worldwide. The first WHO manual was published in 1980, and five editions have been subsequently released over the last four decades. The 6th Edition was published in July 2021. In this review, we identify the key changes of this 6th Edition. Additionally, we evaluate the utility of this 6th Edition in clinical practice using SWOT (strengths, weaknesses, opportunities, and threats) analysis. This new Edition has made the analysis of basic semen parameters more robust, taking into account the criticisms and grey areas of the previous editions. The tests assessing sperm DNA fragmentation and seminal oxidative stress are well-described. The main novelty is that this latest edition abandons the notion of reference thresholds, suggesting instead to replace them with “decision limits”. While this seems attractive, no decision limits are proposed for either basic semen parameters, or for extended or advanced parameters. This critical review of the 6th Edition of the WHO laboratory manual combined with a SWOT analysis summarizes the changes and novelties present in this new Edition and provides an in-depth analysis that could help its global use in the coming years.

Oxidative stress (OS) due to an imbalance between reactive oxygen species (ROS) and antioxidants has been established as an important factor that can negatively affect the outcomes of assisted reproductive techniques (ARTs). Excess ROS exert their pathological effects through damage to cellular lipids, organelles, and DNA, alteration of enzymatic function, and apoptosis. ROS can be produced intracellularly, from immature sperm, oocytes, and embryos. Additionally, several external factors may induce high ROS production in the ART setup, including atmospheric oxygen, CO₂ incubators, consumables, visible light, temperature, humidity, volatile organic compounds, and culture media additives. Pathological amounts of ROS can also be generated during the cryopreservation-thawing process of gametes or embryos. Generally, these factors can act at any stage during ART, from gamete preparation to embryo development, till the blastocyst stage. In this review, we discuss the in vitro conditions and environmental factors responsible for the induction of OS in an ART setting. In addition, we describe the effects of OS on gametes and embryos. Furthermore, we highlight strategies to ameliorate the impact of OS during the whole human embryo culture period, from gametes to blastocyst stage.

BACKGROUND An embryo's ability to grow and implant can be improved by selection of a normal spermatozoon with a vacuole-free head. However, large vacuoles in spermatozoa have yet to be fully characterized. The present study aimed to determine whether these vacuoles are of nuclear, membrane and/or acrosomal origin. METHODS We studied 15 infertile patients with differing sperm profiles. For each sperm sample, we used high-magnification (×10 000) contrast microscopy to select and assess 30 normal ‘top’ spermatozoa and 30 spermatozoa with a large sperm-head vacuole (≥ 25% of the head's cross-sectional area). We subsequently analysed the spermatozoa's degree of chromatin condensation (aniline blue staining), DNA fragmentation (terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling assay) and chromosome content (fluorescence in situ hybridization X,Y,18). Atomic force microscopy enabled us to map the plasma sperm membrane in detail. Three-dimensional deconvolution microscopy enabled us to reconstruct images of the nucleus and acrosome in ‘top’ and ‘vacuolated’ spermatozoa. RESULTS We studied a total of 450 ‘top’ spermatozoa and 450 vacuolated spermatozoa. The rate of non-condensed chromatin was higher for ‘vacuolated’ spermatozoa than for ‘top’ spermatozoa (36.2 ± 1.9 versus 7.6 ± 1.3%, respectively; P < 0.0001). ‘Top’ and ‘vacuolated’ spermatozoa did not differ significantly in terms of DNA fragmentation (0.7 ± 0.4 versus 1.3 ± 0.4% respectively; P = 0.25) or aneuploidy (1.1 ± 0.5 versus 2.2 ± 0.7% respectively; P = 0.21). The majority of aneuploid spermatozoa (9 out of 15) lacked chromatin condensation. In all vacuolated spermatozoa, the acrosome was intact, the plasma membrane was sunken but intact and the large vacuole was identified as an abnormal, ‘thumbprint’-like nuclear concavity covered by acrosomal and plasmic membranes. CONCLUSIONS The large vacuole appears to be a nuclear ‘thumbprint’ linked to failure of chromatin condensation.

Ghayda RA, et al. World J Mens Health. 2024 Jan;42(1):39-61. https://doi.org/10.5534/wjmh.230050

Kandil H, et al. World J Mens Health. 2021 Jun;39:e33. https://doi.org/10.5534/wjmh.210074

BACKGROUND In non-obstructive azoospermia (NOA), testicular sperm extraction (TESE) is successful in ∼50% of cases. A parameter for predicting TESE quality and pregnancy rates after ICSI of testicular spermatozoa is still lacking. METHODS We retrospectively evaluated the total testicular volume (TTV), hormone levels and TESE quality in 280 patients with NOA. After successful TESE, the characteristics of the ICSI cycles and the take-home baby rates were evaluated. RESULTS TESE was successful in 149 patients (53.2%). In a multivariate logistic regression analysis, only TTV, FSH and inhibin B were correlated with the TESE outcome. A score including these three parameters was the best predictor of successful TESE (positive likelihood ratio: +3.01). When the score was 100 spermatozoa) in 91.1% of cases; 42.8% of couples took a baby home. The take-home baby rate did not depend on whether the score was 3700, TESE was successful in 37.8% of cases and ‘sperm rich’ in 14.3% of cases; only one couple took a baby home (a rate of 7.7 versus 42.8% when the score was <18.5; P< 0.001). Owing to low sperm retrieval on the day of oocyte retrieval, fewer oocytes were injected when the score was >3700 than when <3700. CONCLUSIONS Thanks to better knowledge of TESE quality, the present score could help to improve care and pre-ICSI counseling for patients with NOA.