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Evolution of the World Health Organization semen analysis manual: where are we?

Abstract

The WHO (World Health Organization) manuals provide state-of-the-art guidance on how a semen analysis should be carried out. The much anticipated sixth edition of the WHO semen analysis manual has been released 10 years after its previous version and includes essential updates, such as new reference standards for semen volume and microscopic sperm characteristics of recent fathers. A well-conducted semen analysis remains an essential foundation of the infertility evaluation process and affects patient referral, diagnosis and treatment. However, a male infertility work-up primarily based on routine semen analysis does not provide men with an optimal fertility pathway; the primary reasons for routine semen analysis inadequacy in this context include its low predictive value for natural and assisted conception success, its inability to detect sperm DNA and epigenetic deficiencies that might negatively affect embryo development, implantation and offspring well-being, and the substantial overlap between semen parameters of fertile and subfertile individuals. Ideally, a full andrological assessment should be carried out by reproductive urologists in all men dealing with couple infertility and should include a detailed history analysis, physical examination, semen analysis, endocrine assessment and other tests as needed. Only through a complete male infertility work-up will relevant underlying medical and infertility conditions be revealed and potentially treated or alleviated. The ultimate goals of a comprehensive andrological assessment are to positively influence overall male health, pregnancy prospects and offspring well-being.

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Fig. 1: Central components and main goals of male infertility evaluation.

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Correspondence to Sandro C. Esteves.

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Glossary

AZFa microdeletion

A structural chromosomal aberration affecting the AZFa subregion of the long arm of the Y chromosome. AZFa deletion typically includes the loss of DEAD-box protein 3, Y-chromosomal (DBY) and ubiquitin specific peptidase 9 Y-linked (USP9Y) genes and results in a total loss of germ cells (Sertoli cells only). The AZFa microdeletion is the least frequent microdeletion (0.5–4%) among men affected by AZF microdeletions.

AZFb microdeletion

A structural chromosomal aberration affecting the AZFb subregion of the long arm of the Y chromosome. AZFb microdeletion is found in ~1–5% of men affected by AZF microdeletions. The complete deletion of the AZFb region affects the Y-chromosome RNA recognition motif 1 (RBMY1) and PTPN13-like Y-linked (PRY) clusters, leading primarily to meiotic arrest at the primary spermatocyte stage, although other phenotypes can be observed.

AZFc microdeletion

A structural chromosomal aberration affecting the AZFc subregion of the long arm of the Y chromosome. AZFc is the most frequent microdeletion (~80%) among men affected by AZF microdeletions. Deletions within this region result in severe oligozoospermia or azoospermia. Patients with azoospermia harbouring AZFc deletions can have residual spermatogenesis. The retrieved sperm might be used for intracytoplasmic sperm injection (ICSI) and generate a healthy child. However, the male offspring of fathers with AZFc microdeletions will inherit the Yq microdeletion.

Azoospermia factor

(AZF). A region of the long arm of the Y chromosome (Yq11) including 26 genes involved in spermatogenesis regulation. The AZF interval has three subregions — AZFa, AZFb and AZFc — each enclosing crucial genes for the control of spermatogenesis. Microdeletions of this region are usually associated with azoospermia or severe oligozoospermia.

Comet assay

A sperm DNA fragmentation (SDF) test that relies on DNA decompaction and protein depletion coupled with single-cell electrophoresis in agarose microgel. Removal of protamines and histones creates a nucleoid-like structure containing supercoiled loops of DNA. Alkaline or neutral pH conditions allow the uncoil of double-stranded DNA, which under electrophoresis results in migration of fragments of single-stranded and double-stranded DNA towards the anode, forming a comet tail that can be observed under fluorescence microscopy. The relative fluorescence in the tail of the comet compared with the head reflects the level of SDF; spermatozoa with increased fluorescence intensity in the comet tails have high levels of chromatin damage.

Klinefelter syndrome

A chromosomal aberration (47,XXY or 46,XY/47,XXY) resulting in hypogonadism and severe oligozoospermia or azoospermia. This alteration induces impaired testicular growth, premature degeneration of the primordial germ cells before puberty, and early or late maturation arrest of spermatogenesis at the primary spermatocyte stage. This condition is the most common genetic cause of infertility and hypogonadism in men, affecting ~1 in 500 individuals.

Magnetic-activating cell sorting

A method of sperm processing that relies on specific antibodies and super-magnetic microspheres and magnets. Under an activated magnetic field, the non-apoptotic, healthy sperm cells flow through a selection column, whereas apoptotic sperm cells are retained in the column through the binding to micromagnetic beads coated with annexin V. This technique enables the removal of apoptotic sperm, retaining live undamaged sperm, which is then used for assisted reproductive technology.

Sperm aneuploidy test

A test to assess the presence of an abnormal number of chromosomes (aneuploidy and diploidy) in sperm using fluorescence in situ hybridization. The test scores the percentage of spermatozoa with chromosomal abnormalities in a sperm sample. Typically, the analysis is performed on the chromosomes most frequently associated with spontaneous miscarriages and birth of children with chromosomal abnormalities (chromosomes 13, 18, 21, X and Y).

Sperm chromatin dispersion test

A sperm DNA fragmentation test that relies on the principle that spermatozoa with DNA fragmentation fail to produce a characteristic halo of dispersed DNA loops (observed in spermatozoa with non-fragmented DNA) following acid denaturation and removal of nuclear proteins. Sperm suspensions are prepared and stained with 4′,6-diamidino-2-phenylindole (DAPI) or Diff-Quik, and spermatozoa with non-dispersed and dispersed chromatin loops are identified by fluorescence or bright-field microscopy examination to compute the percentage of sperm with DNA fragmentation. The halos corresponding to relaxed DNA loops attached to the residual nuclear structure are seen in spermatozoa with low or no sperm DNA fragmentation (SDF). Conversely, spermatozoa with very small or no halos correspond to those exhibiting SDF.

Sperm chromatin structure assay

A sperm DNA fragmentation test that relies on acid denaturation of DNA at the sites of existing single-strand or double-strand breaks. Acridine orange is used for staining; the dye penetrates the sperm chromatin and intercalates into double-stranded DNA (intact DNA), which emits green fluorescence when exposed to a blue laser light. Conversely, acridine orange attachment to sites of single-strand or double-strand breaks creates a complex that produces a metachromatic shift to red fluorescence. The fluorescence patterns emitted by spermatozoa are captured using a flow cytometer. The ratio of red:total (green + red) fluorescence intensity is used to calculate the percentage of spermatozoa with DNA fragmentation (DNA fragmentation index).

Sperm penetration assay

A sperm function test to measure the ability of human sperm to fertilize a hamster oocyte. Hamster oocytes from which the zona pellucida was removed are incubated with sperm specimens. The zona-free eggs are examined using phase-contrast microscopy. The test is scored by calculating the percentage of penetrated ova or the average number of sperm penetrations per ovum.

Sperm vitrification

A technique of ice-free and cryoprotectant-free cryopreservation by direct plunging sperm suspensions into liquid nitrogen. Sperm vitrification relies on ultra-fast freezing of a small sample volume with direct contact with liquid nitrogen, which should prevent the formation of ice and reduce osmotic damage. The technique is typically used to cryopreserve a single or a low number of spermatozoa.

TUNEL assay

A sperm DNA fragmentation test that relies on a terminal deoxynucleotidyl transferase (TdTA) enzyme for the direct labelling of 3′ free ends of DNA. The sites of DNA breaks are identified with optical fluorescence microscopy or flow cytometry.

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Esteves, S.C. Evolution of the World Health Organization semen analysis manual: where are we?. Nat Rev Urol 19, 439–446 (2022). https://doi.org/10.1038/s41585-022-00593-2

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