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Cited by 271Sperm and Testicular FunctionMale Reproductive Health StudiesReproductive Health and Technologies

The Sixth Edition of the WHO Manual for Human Semen Analysis: A Critical Review and SWOT Analysis

Open access
Boitrelle, Florence, Shah, Rupin, Saleh, Ramadan, Henkel, Ralf, Kandil, Hussein, Chung, Eric, Vogiatzi, Paraskevi, Zini, Armand +2 more·2021·Life
DOI 10.3390/life11121368PMC PMC8706130
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Abstract

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.

AI snapshot

Written from the paper’s full text.

TL;DR

This critical review summarizes key changes in the 6th Edition of the WHO Manual for Human Semen Analysis and evaluates its clinical utility using SWOT analysis. It finds the 6th Edition improves basic semen analysis and adds SDF/oxidative stress testing, but abandons reference thresholds without providing replacement decision limits.

Key findings

  • The 6th Edition, published July 2021, abandons reference thresholds and suggests replacing them with “decision limits,” yet no decision limits are proposed for basic, extended, or advanced semen parameters.
  • Its reference data combine the 5th Edition and new data from 2010–2020, totaling 3589 fertile men (1800 from the 5th Edition and 1789 new subjects); the 5th centiles are not significantly different from the 2010 WHO 5th Edition.
  • SDF and seminal oxidative stress tests are well-described, but SDF has no thresholds and each laboratory is recommended to develop its own reference range; clinical indications and utility are not discussed.
  • Basic semen analysis changes include re-adopting grade a/b progressive motility, recommending vitality assessment when total sperm motility is below 40%, requiring 200 spermatozoa per replicate, and more precise assessment of concentrations <2 × 10^6/mL.

Methods

  • Design: narrative critical review of the WHO 6th Edition combined with SWOT (strengths, weaknesses, opportunities, threats) analysis.
  • Data/sample: comparison of the 5th and 6th Editions, including the 6th Edition’s combined semen data from 3589 fertile men and discussion of extended/advanced tests, sperm preparation, and cryopreservation.
  • Main analysis: qualitative evaluation of methodological changes, clinical utility, and external/internal factors affecting global use.

Limitations

  • The paper states that some geographical areas, such as South America and Sub-Saharan Africa, remain under-represented in the 6th Edition.
  • The paper states that the 6th Edition lacks decision limits, SDF thresholds, clinical guidance for SDF/genetic tests, and accepted cut-off values for seminal oxidative stress.
  • Evident from the text: this is a narrative critical review and SWOT analysis without a stated systematic search strategy or primary data collection; the SWOT assessment is qualitative.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.3390/life11121368

Cites

63 of 63 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

21 of 45 cited sources checked · 10 supported · 0 flagged · accuracy not yet measured

  • Findingcitation

    The WHO manual cites older papers stating that rapid motility (grade a) has clinical value.

    “To justify this choice, the WHO manual cites older papers stating that rapid motility (grade a) has clinical value [21,22,23,24,25,26,27,28,29,30].”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites [21] Aitken, 1985, Relationship between the movement characteristics of human spermatozoa and their ability to penetrate cervical mucus and zona-free hamster oocytes, Reproduction source

      The Discussion (page unavailable) identifies linear velocity of progression as one factor associated with cervical-mucus penetration, giving some support to the clinical relevance of sperm movement. The paper does not identify rapid motility as WHO grade a or establish the clinical value of that grade; its Discussion also says sperm movement had no consistent, clinically useful relationship with fertilizing potential in normospermic males. The claim about what the WHO manual cites cannot be established from this paper. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [22] Mortimer, 1986, Relationship between human sperm motility characteristics and sperm penetration into human cervical mucus in vitro, Reproduction source

      The Discussion (page unavailable) says that detailed evaluation of sperm motility may contribute to infertility diagnosis, and the discriminant-analysis section links movement velocity and progressively motile sperm concentration to cervical-mucus penetration test results. The paper does not identify these measures as WHO grade-a rapid motility or establish that the WHO manual cites it. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
    • cites [23] Barratt, 1992, Prognostic value of two putative sperm function tests: Hypo-osmotic swelling and bovine sperm mucus penetration test (Penetrak), Hum. Reprod. source
  • Premisecitation

    Seminal OS develops due to production of excessive amounts of ROS in numerous medical conditions, including varicocele, leukocytospermia, diabetes mellitus, and obesity.

    “Due to the production of excessive amounts of ROS in numerous medical conditions, including varicocele [43], leukocytospermia [44], diabetes mellitus [45], and obesity [46], seminal OS develops.”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites [43] Agarwal, 2006, Relationship between oxidative stress, varicocele and infertility: A meta-analysis, Reprod. Biomed. Online source
    • cites [44] Saleh, 2002, Leukocytospermia is associated with increased reactive oxygen species production by human spermatozoa, Fertil. Steril. source
    • cites [45] Amaral, 2008, Diabetes and the Impairment of Reproductive Function: Possible Role of Mitochondria and Reactive Oxygen Species, Curr. Diabetes Rev. source
  • Premisecitation

    Sperm DNA integrity is a pre-requisite for proper embryo development, implantation, and pregnancy.

    “Sperm DNA integrity is a pre-requisite for proper embryo development, implantation, and pregnancy [31]”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [31] Ward, 2009, Function of sperm chromatin structural elements in fertilization and development, Mol. Hum. Reprod. source

      The Implications section (page unavailable) says sperm chromatin structures are probably required for proper embryonic development and recommends preserving paternal DNA integrity in assisted reproduction. The Introduction also says DNA integrity affects ART outcomes. The paper does not establish DNA integrity as a prerequisite for implantation or pregnancy, and its developmental conclusion is qualified rather than absolute. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
  • Premisecitation

    Recent progress has been made in terms of identification of relevant patients for improving diagnostic capabilities of antioxidant capacity or redox balance by means of ORP.

    “For the latter, recent progress has been made in terms of the identification of relevant patients [60,66,67].”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites [60] Nakamura, 2018, Prediction of pregnancy after frozen-thawed embryo transfer via in vivo intrauterine oxidation-reduction potential measurements: A pilot study, Reprod. Med. Biol. source
    • cites [66] Agarwal, 2017, Determination of seminal oxidation-reduction potential (ORP) as an easy and cost-effective clinical marker of male infertility, Andrology source
    • cites [67] Tanaka, 2020, Seminal oxidation–reduction potential and sperm DNA fragmentation index increase among infertile men with varicocele, Hum. Fertil. source
  • Premisecitation

    Luminometric determination of seminal OS resulted in a significant prediction of good embryo cleavage and blastocyst quality after ICSI in the low OS group.

    “Similarly, the luminometric determination of seminal OS resulted in a significant prediction of good embryo cleavage and blastocyst quality after intracytoplasmic sperm injection (ICSI) in the low OS group [61].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [61] Kuroda, 2020, Early and late paternal effects of reactive oxygen species in semen on embryo development after intracytoplasmic sperm injection, Syst. Biol. Reprod. Med. source
  • Classified as probably not a substantial claim. 59
  • May combine two claims. 28

  • Classified as probably not a substantial claim.

    Interpretationcitation

    The adoption of actual epigenetic assays by a large number of fertility laboratories will require robust research and clinical trials proving their value in diagnostic andrology.

    “These assays may have a use in the evaluation of male infertility, however, their adoption by a large number of fertility laboratories will require robust research and clinical trials proving their value in diagnostic andrology [11,38].”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [11] World Health Organization (2021). WHO Laboratory Manual for the Examination and Processing of Human Semen, WHO Press. [6th ed.]. Available online: https://www.who.int/publications/i/item/9789240030787.

      Not checked in the free run.

    • cites [38] Donkin, 2018, Sperm epigenetics and influence of environmental factors, Mol. Metab. source

      The Introduction discusses sperm epigenetics as a possible mechanism of inheritance, and the Concluding remarks discuss confounders in that research. Neither section addresses adoption of epigenetic assays by fertility laboratories or a need for clinical trials to establish their diagnostic value in andrology. Searches for clinical validation, infertility diagnostics, and laboratory adoption found no passage making that point. (LLM, conf=0.72)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Unselected infertile men, as well as men with SDF, Robertsonian and reciprocal translocation, and those with a history of recurrent pregnancy loss are at increased risk of producing aneuploid sperm.

    “Unselected infertile men, as well as men with SDF, Robertsonian and reciprocal translocation, and those with a history of recurrent pregnancy loss are at increased risk of producing aneuploid sperm [33,34].”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [33] Godo, 2015, Altered segregation pattern and numerical chromosome abnormalities interrelate in spermatozoa from Robertsonian translocation carriers, Reprod. Biomed. Online source

      The Introduction reports that more than half of Robertsonian translocation carriers produce significantly increased percentages of sperm with numerical chromosome abnormalities. The Biological samples section says this study included only carriers already known to have elevated aneuploid or diploid sperm rates, and the Conclusions limit its findings to that group. The paper does not establish the same increased risk for unselected infertile men, men with sperm DNA fragmentation, or men with recurrent pregnancy loss; its comparison with reciprocal translocation carriers does not establish that risk for them either. No page numbers were available in our copy. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [34] Wang, 2017, Analysis of meiotic segregation patterns and interchromosomal effects in sperm from 13 robertsonian translocations, Balk. J. Med. Genet. source

      The Results report increased aneuploidy in many Robertsonian translocation carriers and more unbalanced sperm in heterozygous carriers than in controls; the homozygous carrier did not show this increase. The Patients and Results sections do not establish increased risk for unselected infertile men, men with sperm DNA fragmentation or reciprocal translocations, or men with recurrent pregnancy loss as a separate group. Page numbers are unavailable in our copy. (LLM, conf=0.90)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    FISH is used in the assessment of the male partner's chromosomal abnormalities in cases of recurrent pregnancy loss or ART failure.

    “Additionally, FISH is used in the assessment of the male partner’s chromosomal abnormalities in cases of recurrent pregnancy loss or ART failure [37].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [37] Kohn, 2016, Genetic counseling for men with recurrent pregnancy loss or recurrent implantation failure due to abnormal sperm chromosomal aneuploidy, J. Assist. Reprod Genet. source

  • May combine two claims.

    Premisecitation

    OS negatively affects all sperm functions, including sperm DNA integrity, and thereby the fertilization process and reproductive outcomes.

    “Consequently, OS negatively affects all sperm functions, including sperm DNA integrity [47,48], and thereby the fertilization process and reproductive outcomes [49,50,51,52,53,54,55].”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites [47] Cicek, 2021, The association of seminal oxidation reduction potential with sperm parameters in patients with unexplained and male factor infertility, Int. Braz. J. Urol. source

      The Introduction says excessive ROS can cause sperm DNA damage and dysfunction, supporting the claim's general point. But the Results report no correlation between oxidative-stress measure sORP and total motility or immotility, so the source does not support the unqualified assertion that oxidative stress negatively affects *all* sperm functions. The later clause about fertilization and reproductive outcomes is attached to different citations in the citing sentence. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [48] Garcia-Segura, S., Ribas-Maynou, J., Lara-Cerrillo, S., Garcia-Peiró, A., Castel, A., Benet, J., and Oliver-Bonet, M. (2020). Relationship of Seminal Oxidation-Reduction Potential with Sperm DNA Integrity and pH in Idiopathic Infertile Patients. Biology, 9. source

      The Introduction reports that redox dysregulation alters sperm motility and can cause sperm DNA fragmentation. It does not establish that oxidative stress negatively affects *all* sperm functions. The fertilization and reproductive-outcome clause is attached to other citations, not this source. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [49] Aitken, 2010, On the possible origins of DNA damage in human spermatozoa, Mol. Hum. Reprod. source

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Semen analysis is the most basic evaluation of male infertility, and evaluation of semen parameters is currently based on the standards defined in the WHO laboratory manual for the examination and processing of human semen.

    “Semen analysis (SA) represents the most basic evaluation of male infertility. The evaluation of semen parameters is currently based on the standards defined in the laboratory manual for the examination and processing of human semen created by the World Health Organization (WHO) [8,9,10].”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites [8] Barratt, 2017, The diagnosis of male infertility: An analysis of the evidence to support the development of global WHO guidance—Challenges and future research opportunities, Hum. Reprod. Update source
    • cites [9] Guzick, 2001, Sperm Morphology, Motility, and Concentration in Fertile and Infertile Men, N. Engl. J. Med. source
    • cites [10] Schlegel, 2021, Diagnosis and treatment of infertility in men: AUA/ASRM guideline part I, Fertil. Steril. source

      The guideline’s section on initial infertility evaluation recommends one or more semen analyses and calls semen analysis an important component of the initial male evaluation. That supports its role as a basic evaluation, but the passages from our copy do not say that semen parameters are evaluated according to standards in the WHO laboratory manual. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    FISH is commonly used in the assessment of chromosomal aneuploidy involving chromosomes 13, 18, 21, X, and Y, which usually result in viable but defective births.

    “FISH is commonly used in the assessment of chromosomal aneuploidy involving chromosomes 13, 18, 21, X, and Y, which usually result in viable but defective births [36].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [36] Hwang, 2010, The use of fluorescent in situ hybridization in male infertility, Ther. Adv. Urol. source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingnone given

    Concerning epigenetic mechanisms of chromatin condensation, the aniline blue and chromomycin A3 assays have been described in the 6th Edition.

    “Concerning the epigenetic mechanisms of chromatin condensation, for example, the aniline blue and chromomycin A3 assays have been described.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Findingcitation

    The importance of SDF and genetic evaluation in the context of male infertility has been expanded in the 6th Edition, unlike the 5th Edition, which merely described sperm genetics and chromatin evaluation under the research procedures section.

    “The importance of SDF and genetic evaluation in the context of male infertility has been expanded in the 6th Edition, unlike its previous counterpart, the 5th Edition, which merely described sperm genetics and chromatin evaluation under the research procedures section [11,14].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [11] World Health Organization (2021). WHO Laboratory Manual for the Examination and Processing of Human Semen, WHO Press. [6th ed.]. Available online: https://www.who.int/publications/i/item/9789240030787.

      Not checked in the free run.

    • cites [14] World Health Organization (2010). WHO Laboratory Manual for the Examination and Processing of Human Semen, WHO Press. [5th ed.].

      Not checked in the free run.

  • Classified as probably not a substantial claim. May combine two claims.

    Findingreasoning

    Although the 6th Edition lists some correlates between abnormal genetic tests and clinical parameters, it does not adequately address the indications for sperm genetic testing.

    “Although the authors list some correlates between abnormal genetic tests and clinical parameters, they do not adequately address the indications for sperm genetic testing.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingreasoning

    The distinction of progressive motility in two categories was not evaluated like other parameters by comparing data from men with time to pregnancy ≤12 months with men that remain childless, since it was abandoned in the 2010 5th Edition.

    “Since this assessment was abandoned in the 2010 5th Edition, the distinction of progressive motility in two categories was not evaluated like the other parameters (by comparing the data obtained from men with time to pregnancy ≤12 months with men that remain childless).”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Findingcitation

    The 6th Edition re-adopted the distinction of progressive motility in two categories (grade a and b).

    “Concerning sperm motility, the 6th Edition re-adopted the distinction of progressive motility in two categories (grade a and b).”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Findingreasoning

    A discussion on the indications and how to apply the results of SDF and genetic tests in clinical practice is missing from the 6th Edition.

    “However, a discussion on the indications and how to apply the results of these tests in clinical practice is missing.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Findingnone given

    The 6th Edition acknowledges the scarcity of tools available in the general andrology laboratory and that most genetic tests require an advanced genetic testing facility.

    “Finally, the 6th Edition acknowledges the scarcity of tools available in the general andrology laboratory and that most tests require an advanced genetic testing facility.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Findingcitation

    The 6th Edition provides a detailed outline on the technical aspects of SDF and genetic tests and some guidance on interpretation of test results.

    “In addition, the 6th Edition provides a detailed outline on the technical aspects of these tests and some guidance on the interpretation of the test results.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Findingreasoning

    Throughout the 280 pages of the 6th Edition, the terms normozoospermia, asthenozoospermia, necrozoospermia, and teratozoospermia are not used at all.

    “Notably, throughout the 280 pages of the current Edition, the terms “normozoospermia” “asthenozoospermia”, “necrozoospermia”, “teratozoospermia” are not used at all.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Findingnone given

    The 6th Edition describes and elaborates on different methods of SDF testing, including the TUNEL assay, sperm chromatin dispersion assay, Comet assay and acridine orange flow cytometry assay.

    “The 6th Edition describes and elaborates on different methods of SDF testing, including the TUNEL assay, sperm chromatin dispersion assay, Comet assay and acridine orange flow cytometry assay.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Findingnone given

    Actual epigenetic assays, which assess post-translational histone modifications, the degree of sperm DNA methylation, or small RNA, are not described in the 6th Edition.

    “However, the actual epigenetic assays, which assess, for example, post-translational histone modifications, the degree of sperm DNA methylation, or small RNA are not described.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Findingnone given

    Except for the Comet assay, the SDF tests described in the 6th Edition are deemed useful for clinical testing.

    “Except for the Comet assay, these tests are deemed useful for clinical testing.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingcitation

    The 6th Edition has addressed an inconsistency surrounding the assessment of sperm vitality that exists in the previous 5th Edition.

    “Regarding the indications for sperm vitality assessment, the 6th Edition has addressed an inconsistency surrounding the assessment of sperm vitality that exists in the previous 5th Edition [11,14].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [11] World Health Organization (2021). WHO Laboratory Manual for the Examination and Processing of Human Semen, WHO Press. [6th ed.]. Available online: https://www.who.int/publications/i/item/9789240030787.

      Not checked in the free run.

    • cites [14] World Health Organization (2010). WHO Laboratory Manual for the Examination and Processing of Human Semen, WHO Press. [5th ed.].

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Findingreasoning

    In the 6th Edition, assessment of sperm vitality is recommended when total sperm motility is below 40%.

    “In the 6th Edition, the assessment of sperm vitality is recommended when total sperm motility is below 40%.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Findingnone given

    The 6th Edition provides no thresholds for SDF testing and recommends that each laboratory develop its own reference range based on appropriate controls.

    “However, the 6th Edition provides no thresholds and recommends that each laboratory develop its own reference range based on appropriate controls.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Findingnone given

    In the 6th Edition, it is specified that the 5th centile values are only one way to interpret SA results, and the use of the 5th centile alone is not sufficient to diagnose male infertility.

    “In this 6th Edition, it is clearly specified that the 5th centile values are only one way to interpret the results of SA, and the use of the 5th centile alone is not sufficient to diagnose male infertility.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Findingreasoning

    Mitochondrial membrane potential assessment tests are not described in the 6th Edition.

    “It is probably also for this reason that the mitochondrial membrane potential assessment tests are not described in this 6th Edition.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Findingnone given

    The tests assessing sperm DNA fragmentation and seminal oxidative stress are well-described in the 6th Edition.

    “The tests assessing sperm DNA fragmentation and seminal oxidative stress are well-described.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Findingnone given

    The evaluation of semen odor has been added in the 6th Edition.

    “The evaluation of semen odor has been added”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingnone given

    In the 6th Edition, seminal OS is described together with ROS in a separate subsection under Advanced examinations of semen.

    “In the new 6th Edition, seminal OS is described together with ROS in a separate subsection under “Advanced examinations of semen”.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Findingnone given

    The main novelty of the 6th Edition is that it abandons the notion of reference thresholds, suggesting instead to replace them with decision limits.

    “The main novelty is that this latest edition abandons the notion of reference thresholds, suggesting instead to replace them with “decision limits”.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingnone given

    No decision limits are proposed in the 6th Edition for either basic semen parameters, or for extended or advanced parameters.

    “While this seems attractive, no decision limits are proposed for either basic semen parameters, or for extended or advanced parameters.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Findingreasoning

    In the 6th Edition, evaluation of low sperm concentrations (<2 × 106/mL) must be assessed more precisely, noting that errors associated with counting a small number of spermatozoa may be very high.

    “Henceforth, the evaluation of low sperm concentrations (<2 × 106/mL) must be assessed more precisely, by noting that the errors associated with counting a small number of spermatozoa may be very high.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingnone given

    Sperm morphology assessment using a systematic approach is described in the 6th Edition with multiple and better-quality micrographs of spermatozoa from unprocessed semen samples considered normal, borderline, or abnormal.

    “Sperm morphology assessment using a systematic approach is described in the new 6th Edition with multiple and better-quality micrographs of spermatozoa from unprocessed semen samples that are considered normal, borderline, or abnormal.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingnone given

    Some tests concerning evaluation of seminal OS, sperm acrosome reaction, functional analysis of transmembrane ion flux, and transport in sperm or methods for evaluation of chromatin condensation have been described in the 6th Edition.

    “Some tests concerning the evaluation of seminal OS, sperm acrosome reaction, functional analysis of transmembrane ion flux, and transport in sperm or methods for the evaluation of chromatin condensation have been described in the 6th Edition.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingreasoning

    The newly added data in the 6th Edition originate from two countries in Southern Europe, two countries from Asia, and one country from Africa.

    “The newly added data originate from two countries in Southern Europe, which were under-represented in the previous 5th Edition, along with two countries from Asia and one country from Africa that lacked representation in the previous release.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingcitation

    In the 5th Edition, seminal OS has been mentioned under Research procedures.

    “In the 5th Edition of the WHO manual, seminal OS has been mentioned under “Research procedures” [14,20].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [14] World Health Organization (2010). WHO Laboratory Manual for the Examination and Processing of Human Semen, WHO Press. [5th ed.].

      Not checked in the free run.

    • cites [20] World Health Organization (1999). WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction, Cambridge University Press. [4th ed.].

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Findingreasoning

    For evaluation of sperm count, semen dilutions have been simplified, but 200 spermatozoa per replicate should be counted.

    “For the evaluation of sperm count, semen dilutions have been simplified, but 200 spermatozoa per replicate should be counted.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Findingreasoning

    The 6th Edition introduces the reader to sperm genetic tests and how they relate to male reproductive biology and associated fertility disorders, yet it fails to provide guidelines on the indications and utility of these tests.

    “The 6th Edition introduces the reader to sperm genetic tests and how they relate to male reproductive biology and associated fertility disorders, yet it fails to provide guidelines on the indications and utility of these tests.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingnone given

    The 6th Edition of the WHO manual was published in July 2021.

    “The 6th Edition was published in July 2021.”
    • Also stated in: § 2.1. Methodological Considerations in the New 6th Edition · plain
      “The new 6th Edition, released in July 2021, commended the importance of SA as a tool to:”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingnone given

    The 6th Edition mentions determination of ORP using MiOXSYS and total antioxidant capacity with a brief description of procedures and issues.

    “Additionally, the determination of oxidation–reduction potential (ORP) using the male infertility oxidative system (MiOXSYS), and total antioxidant capacity are mentioned with a brief description of procedures and issues.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S2 (checkable).

    Interpretationreasoning

    Without guidance as to the utility of sperm genetic assays, it is unclear how the tests will be used to guide the management of infertile couples.

    “Moreover, without some guidance as to the utility of these assays, it is unclear how the tests will be used to guide the management of infertile couples.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S2 (checkable), S4 (based). May combine two claims.

    Interpretationreasoning

    To rely on other reference values, clinicians will need to look for other sources in the literature, which can be time-consuming and difficult.

    “To rely on other reference values, clinicians will need to look for other sources in the literature. This can be time-consuming and difficult.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Clinicians and laboratories rely on WHO reference values in clinical practice.

  • Classified as probably not a substantial claim. May combine two claims.

    Interpretationreasoning

    Reference thresholds alone are meaningless and multiple criteria must be applied to establish a diagnosis of male infertility.

    “These terms have been voluntarily removed as the editors of the manual explain, quite rightly, that the reference thresholds alone are meaningless and that multiple criteria must be applied to establish a diagnosis of male infertility.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S4 (based).

    Interpretationreasoning

    The addition of semen odor evaluation is at odds with recommendations for the safety of laboratory personnel.

    “Furthermore, the addition of this parameter is at odds with recommendations for the safety of laboratory personnel.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Interpretationreasoning

    The omission of guidance on indications for sperm genetic testing can lead to inappropriate use of these tests in clinical practice.

    “This important omission can lead to inappropriate use of these tests in clinical practice.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Interpretationreasoning

    The 6th Edition's morphology micrographs are accompanied by explanations for classification, rendering this a helpful guide.

    “These are accompanied by explanations for the classification of each assessment, rendering this a helpful guide.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationreasoning

    It is unclear why the 6th Edition discusses sperm chromatin tests in the chapter Advanced examinations of semen when these tests are closely related to, and certainly no more advanced than, tests of SDF.

    “It is unclear why they have chosen to discuss and describe sperm chromatin tests in the chapter on “Advanced examinations of semen” when these tests are closely related to, and certainly no more advanced than tests of SDF.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationcitation

    The incorporation of additional areas and continents and addition of more participants and samples in the final analysis provide greater statistical power to the reference ranges reported, even though the 5th centiles are not significantly different from the 2010 WHO 5th Edition.

    “The incorporation of additional areas and continents and, importantly, the addition of more participants and samples in the final analysis provide greater statistical power to the reference ranges reported, even though the 5th centiles are not significantly different from the 2010 WHO 5th Edition [11,14] (Table 2).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [11] World Health Organization (2021). WHO Laboratory Manual for the Examination and Processing of Human Semen, WHO Press. [6th ed.]. Available online: https://www.who.int/publications/i/item/9789240030787.

      Not checked in the free run.

    • cites [14] World Health Organization (2010). WHO Laboratory Manual for the Examination and Processing of Human Semen, WHO Press. [5th ed.].

      Not checked in the free run.

  • Classified as probably not a substantial claim. May combine two claims.

    Interpretationreasoning

    The clinical utility of SDF testing is not discussed in the 6th Edition, so the reader is uncertain as to how to use the results in a clinically meaningful way.

    “Additionally, the clinical utility of SDF testing is not discussed, so the reader is uncertain as to how to use the results in a clinically meaningful way.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationcitation

    The evaluation of semen odor is subjective, which makes standardization of this parameter very complicated.

    “It should be noted that the evaluation of semen odor is subjective [11,19], which makes the standardization of this parameter very complicated.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [11] World Health Organization (2021). WHO Laboratory Manual for the Examination and Processing of Human Semen, WHO Press. [6th ed.]. Available online: https://www.who.int/publications/i/item/9789240030787.

      Not checked in the free run.

    • cites [19] Mortimer, D. (1994). Practical Laboratory Andrology, Oxford University Press. source

  • Probably not a substantial claim: may not pass S2 (checkable), S4 (based).

    Interpretationnone given

    The 6th Edition has made the analysis of basic semen parameters more robust, taking into account the criticisms and grey areas of the previous editions.

    “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.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S2 (checkable).

    Interpretationreasoning

    A possibility remains that clinicians will continue to use the 5th centile values, which were designed in the 5th Edition to compare fertile and infertile men with the criterion of time to pregnancy ≤12 months.

    “Therefore, a possibility remains that clinicians will continue to use the 5th centile values, which were designed in the 5th Edition to compare fertile and infertile men with the criterion of time to pregnancy ≤12 months.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Clinicians and laboratories rely on WHO reference values in clinical practice.

  • Probably not a substantial claim: may not pass S2 (checkable).

    Interpretationreasoning

    In actual practice, it is highly likely that clinicians may encounter some degree of confusion with the absence of reference values.

    “The latter statement is correct, but in actual practice, it is highly likely that clinicians may encounter some degree of confusion with the absence of reference values.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Clinicians and laboratories rely on WHO reference values in clinical practice.

  • Probably not a substantial claim: may not pass S2 (checkable).

    Interpretationreasoning

    Until individualized reference thresholds or decision limits are established, the current threshold issue will remain a problem for laboratories and clinicians.

    “Till then, the current threshold issue will remain a problem for laboratories and clinicians.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Clinicians and laboratories rely on WHO reference values in clinical practice.

  • Probably not a substantial claim: may not pass S4 (based).

    Premisenone given

    The new 6th Edition presents revised reference values of basic semen parameters based on the combined data of fertile men from the previous 5th Edition, released in 2010, and 5 additional studies published between 2010 and 2020.

    “The new 6th Edition presents revised reference values of basic semen parameters based on the combined data of fertile men from the previous 5th Edition, released in 2010, and 5 additional studies published between 2010 and 2020,”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisenone given

    Open and closed systems are detailed.

    “Open and closed systems are detailed.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    The 6th Edition does not describe MACS as clinically valuable.

    “this technique is not described as clinically valuable because the WHO states that a Cochrane review has not shown its effectiveness in increasing live birth rates [68].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [68] McDowell, 2014, Advanced sperm selection techniques for assisted reproduction, Cochrane Database Syst. Rev.

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Classified as probably not a substantial claim. May combine two claims.

    Interpretationnone given

    The manual neither provides guidance as to the indication for testing nor addresses the variability of test results with different currently available SDF assays.

    “although the manual neither provides guidance as to the indication for testing nor addresses the variability of test results with different currently available SDF assays.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The 6th Edition briefly describes the MACS technique as a technique for selecting sperm with potentially undamaged DNA.

    “In the chapter “Sperm preparation techniques”, this 6th Edition briefly describes the MACS technique as a technique for selecting sperm with potentially undamaged DNA (i.e., with intact DNA).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisenone given

    In the 5th Edition, sperm cryopreservation and sperm preparation techniques are described in a single chapter; in the 6th Edition, they are discussed in two separate chapters.

    “While sperm cryopreservation and sperm preparation techniques are described in a single chapter in the 5th Edition, they are discussed in two separate chapters in the 6th Edition.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisenone given

    In the chapter on "Cryopreservation of spermatozoa", the vitrification technique makes its appearance.

    “In the chapter on “Cryopreservation of spermatozoa”, the vitrification technique makes its appearance.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    The manual states that vitrification techniques have not been proven to be superior to conventional freezing techniques and should be considered experimental.

    “However, the manual states that these techniques have not been proven to be superior to conventional freezing techniques and should be considered experimental [11].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [11] World Health Organization (2021). WHO Laboratory Manual for the Examination and Processing of Human Semen, WHO Press. [6th ed.]. Available online: https://www.who.int/publications/i/item/9789240030787.

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisenone given

    The introduction of the SDF assay as an extended assessment of semen that can be requested in certain clinical scenarios.

    “An example is the introduction of the SDF assay as an extended assessment of semen that can be requested in certain clinical scenarios,”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Seminal radical buffering capacity is the best predictor of fertilization.

    “Earlier, it was shown that the seminal radical buffering capacity is the best predictor of fertilization [62].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [62] Rhemrev, 2001, The acrosome index, radical buffer capacity and number of isolated progressively motile spermatozoa predict IVF results, Hum. Reprod. source

  • Classified as probably not a substantial claim.

    Premisenone given

    The 6th Edition incorporates advances in laboratory techniques of semen examination.

    “In addition, the 6th Edition incorporates advances in laboratory techniques of semen examination.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    Currently there are insufficient well-designed studies evaluating the impact of seminal OS on reproductive outcome and providing normal values in order to identify men with abnormal seminal OS levels.

    “Most importantly, however, currently there are insufficient well-designed studies evaluating the impact of seminal OS on the reproductive outcome and providing normal values in order to identify men with abnormal seminal OS levels.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisenone given

    The 6th Edition highlights the recent developments of the techniques of sperm preparation and cryopreservation and recommends that the laboratories adhere to optimum quality control and quality assurance measures.

    “Additionally, the 6th Edition highlights the recent developments of the techniques of sperm preparation and cryopreservation and recommends that the laboratories adhere to optimum quality control and quality assurance measures.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisereasoning

    The addition of the MACS test represents a novelty compared to the 5th Edition.

    “But while the addition of this test represents a novelty, compared to the 5th Edition,”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    The two main novelties of the 6th Edition are the description of the MACS technique and the sperm vitrification technique.

    “Concerning sperm preparation techniques, the two main novelties of this 6th Edition are the description of the magnetic-Activated cell sorting (MACS) technique and the sperm vitrification technique [11].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [11] World Health Organization (2021). WHO Laboratory Manual for the Examination and Processing of Human Semen, WHO Press. [6th ed.]. Available online: https://www.who.int/publications/i/item/9789240030787.

      Not checked in the free run.

66 of 66

  • The paper relies on this without support.

    Clinicians and laboratories rely on WHO reference values in clinical practice.

    “To rely on other reference values, clinicians will need to look for other sources in the literature.”
    “Therefore, a possibility remains that clinicians will continue to use the 5th centile values, which were designed in the 5th Edition to compare fertile and infertile men with the criterion of time to pregnancy ≤12 months.”

  • The paper relies on this without support.

    Professional societies have not yet fully endorsed oxidative stress (OS) as a diagnostic tool.

    “Despite more evidence being published recently [55], professional societies have not yet fully endorsed OS as a diagnostic tool.”

  • The paper relies on this without support.

    There are a number of different methodologies for the determination of seminal OS with different results described.

    “However, as there are a number of different methodologies for the determination of seminal OS with different results described,”

  • The paper relies on this without support.

    Seminal plasma is regarded as the human body fluid with the highest amount of antioxidants.

    “which is regarded as the human body fluid with the highest amount of antioxidants.”

  • The paper relies on this without support.

    The MACS technique is not approved for human in vivo use in the United States due to the lack of safety studies on paramagnetic beads in the animal model.

    “Furthermore, the MACS technique is not approved for human in vivo use in the United States (and maybe in some other advanced countries as well) due to the lack of safety studies on paramagnetic beads (used in MACS columns to trap sperm with high SDF) in the animal model.”
5 of 5

Cites

Flagged first, then by citations

Research works printed but not linked: 3

  1. World Health Organization (2010). WHO Laboratory Manual for the Examination and Processing of Human Semen, WHO Press. [5th ed.].
  2. World Health Organization (1999). WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction, Cambridge University Press. [4th ed.].
  3. Morris, 2019, Prediction of successful ICSI cycles by Oxidation-reduction potential (ORP) and sperm DNA fragmentation (SDF) analysis. A Prospective study, Hum. Reprod., 34, i8

Cited by

Sorted by citations

Related

Authors
Boitrelle, F., Shah, R., Saleh, R., Henkel, R., Kandil, H., Chung, E., Vogiatzi, P., Zini, A., Arafa, M., & Agarwal, A.
Affiliations
  • Université de Versailles Saint-Quentin-en-Yvelines (FR)
  • Lilavati Hospital & Research Centre (IN)
  • Sohag University (EG)
  • Cleveland Clinic (US)
  • Fakih IVF Fertility Center, Abu Dhabi 31452, United Arab Emirates
  • The University of Queensland (AU)
  • Andromed Health & Reproduction, Fertility Diagnostics Laboratory, Maroussi, 15126 Athens, Greece
  • McGill University (CA)
Corresponding author
Agarwal, Ashok
ORCID
Venue
Life
Publisher
MDPI AG
Published
2021-12-09
Volume
11
Issue
12
Pages
1368
ISSN
2075-1729 (ISSN-L)
DOI
10.3390/life11121368
PMC
PMC8706130
OpenAlex
W4200457040
Semantic Scholar
e4691510fb9cccf4c0d68533be7fa8d52c795fe8
Cited by
271
References
63
Citations by year
2026: 40 · 2025: 53 · 2024: 96 · 2023: 59 · 2022: 23
FWCI
31.25
Type
journal article
Language
en
Open access
Licence
Locations
  • Life (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion, cc-by)
  • HAL (submittedVersion, other-oa)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • UWC Research Repository (University of the Western Cape) (submittedVersion)
  • Spiral (Imperial College London) (submittedVersion, cc-by)
Topics
  • Health Sciences › Medicine › Reproductive Medicine › Sperm and Testicular Function
  • Health Sciences › Health Professions › General Health Professions › Male Reproductive Health Studies
  • Health Sciences › Medicine › Reproductive Medicine › Reproductive Health and Technologies
Keywords
semen analysis, male fertility, semen parameters, reference thresholds, decision limits, sperm DNA fragmentation, seminal oxidative stress, semen quality assessment, clinical practice

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-26.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-26.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

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