Practical Management of Male Infertility Manish Banker, Juan A García-Velasco, Rupin Shah
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Understanding the New WHO Semen ParametersCHAPTER 1

Cristina González Ravina,
Alberto Pacheco Castro
 
INTRODUCTION
As it is well known, the first Laboratory Manual for the Analysis of Human Semen and Its Interaction with the Cervical Mucus was published in 1980. This was as a response to the high necessity of standardizing the protocols to carry out this analysis.1 This manual has subsequently been reviewed three times. In 2010, the 5th edition of the Laboratory Manual for the Examination and Processing of Human Semen was published. In this edition, the latest so far, the reference values, the algorithms for diagnosis, the clinical interpretation of diagnosis, and other relevant aspects have been reviewed. Until last year, every andrology or assisted reproduction laboratory worked according to the guidelines recommended in the latest manuals published by the WHO.13
After making a revision of these manuals and the reference values that existed up to the year 2010, it can be observed that they were useful when established in 1999, but nowadays an update of these was necessary. It should be remembered that the studies performed to establish the reference values in 1999 were based in the analysis of two semen samples from a not-very-high number of males in different laboratories where the work method had not yet been standardized nor the pertinent quality systems had yet been introduced.
 
THE PREVIOUS WHO MANUAL
In the manual published in 1999, spermatozoa were classified in four categories regarding their motility: type a [rapid progressive motility (PM)], type b (slow PM), type c [nonprogressive motility (NP)], and type d [immotility (IM)]. One of the main restrictions from this classification is the acknowledgement that sperm PM (types a + b) studies and morphology studies were not only subjective but also they did not follow any standard protocol in the laboratories.462
Table 1.1   The reference values established in the WHO Laboratory Manual of 1999.
Seminal parameter
Lower reference value
• Volume
2.0 mL
• pH
7.2
• Concentration
20 million spermatozoa per milliliter
• Total number of spermatozoa
40 million spermatozoa per ejaculation
• Motility
50% type a + b spermatozoa per ejaculation
25% type a spermatozoa per ejaculation
• Vitality
75% spermatozoa alive per ejaculation
• Morphology
15% normal shape
However, the values obtained regarding sperm concen-tration and total motility (a + b + c) were considered acceptable. This is because they could be measured in a reproducible way in most centers. For this reason, it was recommended that each laboratory established their own limit values of normality for these variables, and they were also advised to include the appropriate internal and external quality assurances. These quality assurances are fully developed in the new manual of 2010.
In spite of this, reference ranges for the main seminal parameters were defined through a statistics analysis by comparing the sperm characteristics of a group of males. These reference ranges distinguished two different populations studied (fertile vs subfertile or subfertile vs infertile).7,8
The reference values established in 1999 are shown in Table 1.1.
Regarding morphology, the WHO Manual discusses the start of a multicenter study for the analysis of seminal parameters. The edition of 1999 specifies that the data obtained in centers with assisted reproduction programs suggest a connection between morphology values < 15% and a decrease in IVF (in vitro fertilization) rate.9
Regarding the nomenclature, these are the terms used to express seminogram values lower than the reference values:
  • Asthenozoospermia: PM < 50% types a + b or < 25% type a
  • Oligozoospermia: Concentration < 20 million/mL
  • Teratozoospermia: Morphology < 15% normal shapes
  • Necrozoospermia: Vitality < 75%
  • Cryptozoospermia: Absence of spermatozoa in the first analysis of the ejaculation sample and presence of spermatozoa after centrifugation
  • Azoospermia: Absence of spermatozoa in the first analysis of the ejaculation sample and absence of spermatozoa after centrifugation; concentration = 0
  • Aspermia: Absence of ejaculation; volume = 03
Based on the male's seminal classification, his medical history and his physical exploration, as well as any other additional information, a diagnosis of the male could be given through a diagram based on different sperm concentration ranging from azoospermia, cryptozoospermia, and oligozoospermia samples.13 It should be emphasized that in the manual of 1999 a subclassification for the different seminal alterations (oligo-, asteno-, terato-, necrozoospermia) is used, with a range from mild to moderate to severe.
 
CHANGES IN THE NEW 2010 WHO MANUAL
Eleven years after the edition of 1999, a review and update of the reference values and the established work protocols became more and more necessary. Therefore, the new online edition of the WHO manual of 2010 presents much more detail regarding seminal analysis: work solutions, procedures, calculations, and results interpretation. Furthermore, it recommends using a specific method in those cases in which there is more than one possible analysis technique.
Considering this, the main changes in the fifth edition include the following:
  • A new work protocol to determine sperm concentration. The recommended semen dilutions and the counting chamber areas necessary to establish the number of spermatozoa (concentration) in order to count 200 spermatozoa by replicate have been modified. The goal of reducing sampling errors has been intensified, giving more importance to the sample volume, which was not so relevant before. This sample volume must be measured with precision and using the method recommended by the WHO manual.
  • Regarding azoospermia, there is a change in the work protocols to be followed on this type of samples, in which there are no spermatozoa in the ejaculate. It differentiates the necessity to obtain a concentration value and/or an accurate motility value from centrifuging the sample to increase the chances of finding spermatozoa after washing.
  • There are significant changes in the sperm motility classification. From now on, spermatozoa should be only differentiated according to three categories: PM—combining former types a + b, NP—former type c, and IM—former type d.
  • The morphology analysis is dealt with in more detail in this edition. It includes pictures of spermatozoa considered normal and spermatozoa with one or several morphological anomalies, as well as accurate explanations to improve the technician's training regarding the study of this seminal parameter.
  • On the other hand, the manual includes an improved chapter on semen cryopreservation, with a well-described section on sperm preparation. 4It also dedicates a full chapter to quality assurance that, apart from being the novelty, involves a recommendation from the WHO to have quality assurance in our laboratories. This includes both the external and the internal quality assurance.
Therefore, the fifth edition of the Manual is divided into three parts:
 
METHODOLOGY AND INCLUSION CRITERIA
For this study, semen samples from 4,500 males from 14 countries and four continents were analyzed. They were divided into three wide groups: fertile males, males with unknown fertility, and normozoospermic males according to the manual of 1999. The group of males whose partner had become pregnant in a time period ≤12 months was selected as the control group to establish the reference values of seminal parameters (1,953 males, five studies in eight countries from three continents).
All the laboratories participating in this study obtained their data by using the standard methods of semen analysis.12 Each laboratory was taken into account at the moment of assessing the results because this manual offers different methods to determine volume, sperm concentration, and morphological stain. Moreover, many laboratories implemented with delay the internal and external quality assurances. For this reason, the data were reviewed to calculate the reference distribution considering that fact.10
For this study, only one ejaculated semen sample per male, which was gathered with a sexual abstinence period of 2–7 days, was used. Concentration was measured in most cases with the improved Neubauer hemocytometer. Motility data were gathered on total motility (WHO 1999: a + b + c) and PM (WHO 1999: a + b).11 Morphology data were taken into account only in those centers using Tygerberg's strict criteria.13Finally, vitality, analyzed by eosin-nigrosin staining, got a significantly lower number of samples studied than the other seminal parameters, only two centers provided data. The advantage of having standard work protocols is that the analytic error has been minimized and the values presented are considered representative of the seminal characteristics of a fertile male.
 
NEW REFERENCE VALUES AND DIAGNOSIS
This edition reviews old reference values and establishes new ones with which we have to work nowadays (Table 1.2).5
Table 1.2   Distribution of values, lower reference limits, and their 95% Cl for semen parameters from fertile men those partners had a time-to-time pregnancy of 12 months or less.
Seminal parameter
95% Cl
• Volume
1.5 milliliters
• pH
7.2
• Concentration
15 million spermatozoa per milliliter
• Total number of spermatozoa
39 million spermatozoa per ejaculation
• Motility
40% total motility (progressive + nonprogressive)
32% progressive motility
• Vitality
58% spermatozoa alive per ejaculation
• Morphology
4% normal shape
These are available in a free access article published in May 2010 in Human Reproduction Update,12 offering, with more clarity, lower reference limits of the fifth percentile of the values for a fertile male reference population.
This is accompanied by some changes in the samples diagnosis. Therefore, despite maintaining the nomenclature, there are some changes:
  • Teratozoospermia: Normal shape by strict criteria (Tygerberg) <4%
  • Asthenozoospermia: PM <32% does not take into account total motility (% PR + NP) even if under the reference value.
  • Oligozoospermia: Total number of spermatozoa <39 million. The WHO prefers this parameter to sample concentration, which was the selectable parameter in the Manual of 1999.
  • Necrozoospermia: Vitality <58% and very high IM percentage. The second criterion is arbitrary since the WHO does not specify a number, but it does talk about much IM. This way, fake necrozoospermia, which can alarm unjustifiably, is avoided.
  • Cryptozoospermia/azoospermia: There are no changes from 1999.
 
PROBLEMS AND IMPROVEMENTS OF THE NEW WHO CLASSIFICATION
As it occurs in every change or review, we have found not only significant improvements but also some issues that we will need to solve as we get familiarized with the modifications. Some of these issues were as follows:
  • New concept of oligozoospermia. In 1999, the WHO assigned this diagnosis to those samples with concentration under the reference value of this parameter (20 million/mL). However, it has become clear that this concentration depends on the final volume of the sample. That is the reason why the manual of 2010 links preferably the oligozoospermia 6diagnosis to the total number of spermatozoa, provided that it is under 39 million. This is an improvement in the assessment of this parameter.
  • New categories of sperm motility. The old category “a” from the manual of 1999 was difficult to justify without a computerized system to count motility in the sample. It was actually observed that the relevant information from this parameter was in the total number of PM (a + b). For these two reasons, sperm motility categories were modified. Now, it is recommended to differentiate spermatozoa in three categories or types: PM, NP, and IM. Some centers consider the new classification as an advantage.
  • Decrease of the percentage of normal shapes in morphology analysis. It is well known that this is not only the most subjective parameter of the semen analysis but also the one which creates the most controversy when choosing one treatment type or another in assisted reproduction clinics. Numerous articles have been published for and against the importance of morphology in many key aspects of these treatments: pregnancy rate, implantation rate, fertilization success, etc.1317 Anyway, it has always been debated not only in andrology and IVF laboratories but also between these departments and the clinical departments. This is due to the fact that, apart from extreme morphology cases with many anomalies, it is difficult to unify morphological assessment criteria to evaluate the samples, even when performing the appropriate internal and external quality assurances.
  • Sperm vitality and necrozoospermia. It looks like this parameter, not included in the WHO as a “compulsory study” in the seminogram but as something very advisable, is gaining importance in this new edition. Because this parameter is linked to the percentage of sperm IM but there is not an exact fixed value from which necrozoospermia can be diagnosed, each group can decide that value individually depending on the methodology employed in their center and the information expected from the diagnosis. It is well known that the vitality study is essential in those cases with a percentage of sperm IM over 50%. It is not the same to have a high percentage of immotile spermatozoa that are alive after the stain test, which would guide toward possible structural flaws in the tail,18 than a high percentage of immotile spermatozoa which are also dead, more related to an epididymis pathology.19,20
  • The disappearance of the “adjectives” mild, moderate, and severe results in diagnosis of the samples being incomplete and obliges to take into account numerical values of volume, concentration, motility, and morphology to get a real idea of the state of the sample. Depending on the number of millions of progressive motile spermatozoa, each center or laboratory, can decide the type of treatment for a couple.7
    Table 1.3   Different male seminogram reference values and parameters in 1999 and 2010.
    Seminal parameter
    Semen 1999
    Semen 2010
    • Volume
    2.0 milliliters
    1.5 milliliters
    • Concentration
    20 × 106 spermatozoa per milliliter
    15 × 106 spermatozoa per milliliter
    39 × 106 total spermatozoa
    • Progressive motility
    50%
    32%
    • Morphology
    15% normal shape
    4% normal shape
    • Millions of progressive motile
    20
    7.2
    12
  • Finally, the most remarkable aspect is probably the different male seminogram reference values and parameters in 1999 and 2010. In Table 1.3, a comparison of the two possible situations is described.
As it can be observed, the number of millions of progressive motile spermatozoa is reduced by 64%—or 40% if we use the total spermatozoa criterion that is now advised—to the number obtained for a semen sample of a male according to the reference values of 1999. The change of this aspect could cause more problems at clinical consultation level, since males now diagnosed as normal may still receive the recommendation to be submitted to an intrauterine insemination (IUI) or even an IVF cycle. Obviously, each center should decide whether they change their criteria or they explain to every patient that with the current reference values there is the possibility that, even being “normal”, they could have subfertility that would need a specific-assisted reproduction treatment.
 
DEBATE
First of all, it is important to remember that the seminogram does not have diagnosis capacity per se—sensitivity and specificity—to indicate male infertility. Seminogram diagnosis are better understood merely as a sample description to check that they “are similar” to the values of a fertile population. Nonetheless, they could be useful in the clinical diagnosis to detect cryptozoospermia or azoospermia.
On the other hand, for people who work in assisted reproduction it is important to differentiate between the male seminal diagnosis and the “real” use of the sample in an infertility treatment. We know that WHO establishes clearly that being under the reference values or having a sample not “normozoospermic” does not necessarily mean male infertility but it shows that seminal parameter is under the fifth percentile in a reference population of fertile males, i.e. it means that this value is under 95% of 8the values presented by these fertile males. Theoretically, it is likely that the reference values keep on updating as population studies get widened, especially due to the way those values were obtained—statistics of parameters in 2,000 fertile male. Apart from its utility for the diagnosis of an infertile couple, the seminogram is, together with the other tests and examinations, a useful tool to make therapeutic decisions. Its main goal is to evaluate if the sample, once prepared, allows obtaining the minimum number of millions of progressive motile spermatozoa to follow one reproduction treatment or another: artificial insemination or IUI, in vitro fertilization or IVF, or intracytoplasmic sperm injection (ICSI). We could think that for standard IVF the new reference values should not modify the internal values established by every laboratory according to their insemination protocols. However, we should be careful with normozoospermic samples containing values under the lower limits of the different parameters, as aforementioned. Finally, morphology determination is still a parameter with doubtful clinical utility to recommend a patient directly to IVF or ICSI treatment, and therefore should be considered with caution.
REFERENCES
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