Chapter 1 – Standard Semen Examination: Manual Semen Analysis




Abstract




The aim of this chapter is not to provide all practical details necessary for proper semen examination. There already exist other sources for that type of information [1, 2]. The purpose of this chapter is therefore to focus on biological and physiological aspects that are relevant to the examination of human ejaculates. It is aimed to give the clinician a proper background to set requirements for a qualitative laboratory service needed to diagnose and treat men with disorders in the male reproductive organs contributing to couple infertility.





Chapter 1 Standard Semen Examination: Manual Semen Analysis



Lars Björndahl



1.1 Introduction


The aim of this chapter is not to provide all practical details necessary for proper semen examination. There already exist other sources for that type of information [1, 2]. The purpose of this chapter is therefore to focus on biological and physiological aspects that are relevant to the examination of human ejaculates. It is aimed to give the clinician a proper background to set requirements for a qualitative laboratory service needed to diagnose and treat men with disorders in the male reproductive organs contributing to couple infertility.


The investigation of the human ejaculate includes observations (e.g. color, odor, viscosity and liquefaction) as well as measurements and assessments (volume, concentration, motility, vitality and morphology). It is therefore adequate to refer to semen examination that is considered to be a concept that is wider than analysis, also including observations and not only measurements.


The examination of the ejaculate is an essential cornerstone in the evaluation of the reproductive functions of the human male. The ejaculate is different from all other body fluids possible to analyze in a modern medical laboratory, and many delusions still exist concerning what the ejaculate is and what it can tell about the man and his fertility. Since the rise of Assisted Reproductive Technologies (ART) with the introduction and the development of In Vitro Fertilization (IVF) and Intra Cytoplasmic Sperm Injection (ICSI), the main focus in studies of ejaculate examination has been on its usefulness to predict the probability for spontaneous and assisted conceptions, pregnancies and live births of children. However, since many factors affecting a couple’s fertility are independent from semen examination results, most isolated male or female factors cannot be expected to solely be a major determinant of a couple’s probability for a successful pregnancy. Still, if simplistic testing of isolated ejaculate parameters is abandoned in favor of multifactorial ejaculate evaluations, useful predictive information regarding spontaneous and assisted pregnancies can be achieved [3, 4]. Thus, although not completely without interest, this predominant focus on ejaculate examination as a pregnancy predictor has drawn the attention from the information that can be gained about the functions of the male reproductive organs. In the light of a possible decrease in general human male reproductive function [5] it is essential that ejaculate examination for the evaluation of the function of the male reproductive organs is in focus.


Another aspect that is essential to the understanding of semen examination is that the “semen sample” only exists in the laboratory. Semen does not even exist in the body: the ejaculate is formed instantly during the process of ejaculation: spermatozoa are transported from the cauda epididymides to the urethra where they are suspended in prostatic fluid concomitantly emptied from the small prostatic gland acini and expelled in the first ejaculate fractions. The seminal vesicles empty into the urethra after the bulk of spermatozoa has been ejaculated [6]. In general, the seminal vesicular fluid appears to be hostile to sperm function (motility), survival and DNA protection [7]. There is no evidence that the seminal vesicular fluid is in any way beneficial for sperm function in vivo and there is no evidence that exposure of spermatozoa to seminal vesicular fluid ever occurs in vivo [6, 8]. In contrast, examination of ejaculates in vitro is based on collection of all parts of the ejaculate in one device, where the entire ejaculate is included in the gel-like substance originating from the seminal vesicles. This structure is then subsequently decomposed by enzymes of prostatic origin. The effect of this process – called liquefaction – is not only that the ejaculate becomes more watery in appearance, but the process also means that the osmolality of ejaculates increase in vitro [9, 10].



1.2 Pre-Examination Aspects


The composition and quantity of each ejaculate depends on several factors, among them the rate of sperm production but not limited to that. The procurement of an ejaculate for examination is highly dependent on the man himself. Frequency of ejaculation – and not only time of sexual abstinence before collection of the examination sample – will have influence on characteristics like sperm number, motility and morphology. Also the duration and quality of sexual arousal during sample collection is important [11]. In addition, truthful information on the completeness of sample collection is required for correct interpretation of ejaculate examination results.


Due to the continuous changes (osmolality, pH) occurring in the ejaculate in vitro leading to deterioration of sperm motility and changes in morphological appearance, standardized temperature and time to initiation of assessments is required.



1.3 Examination Aspects



1.3.1 Ejaculate Volume


The ejaculate volume is important to achieve reliable data on total sperm number as well as measures of secretory contributions from the epididymides, prostate and seminal vesicles.


There is a highly variable and significant volume loss when using pipettes for measuring ejaculate volume or transferring to reliable measuring devices [12]. Practically, a reliable and more correct volume is best obtained by weighing the collection device before and after sample collection. The specific weight (density) of human semen has been assessed to be 1.03–1.04 g/mL [13], indicating that the error in volume, based on ejaculate weight, would be less than 4 percent even when assuming a specific weight of 1.0 g/mL. A 4 percent error is much less than can be expected from measurements where the ejaculate must be removed from the collection device and an unknown volume is left in the collection device.



1.3.2 Sperm Concentration


The accuracy of sperm concentration assessment depends on several factors. The most basic is that the examined aliquots are representative of the entire ejaculate. Macroscopically well mixed ejaculates quite often still show considerable variation between different fields of vision in the microscope (10 µL aliquot). Based on this typical finding the recommendation is that aliquots of at least 50 µL should be used to reduce the risk for poor representation of the entire ejaculate. Furthermore, comparison of two separate aliquots should be examined and compared, to further reduce the risk for poor representation and other errors that may occur in the process of establishing the correct number of spermatozoa.


Another essential aspect is the use of dilutions. One important reason is that it is much easier to assess non-moving objects than live motile spermatozoa. Another equally important reason is that with adequate dilutions counting is easier when spermatozoa are evenly spread within and between the microscopic fields; densely packed spermatozoa or too diluted makes counting more difficult, time consuming, exhausting and less reliable.


A third important aspect is to reduce the influence of random factors that cause a spermatozoon to occur or not occur in the area of observation. If only a few spermatozoa are observed, the influence of random factors can influence the final result considerably. Therefore, the number of observations is crucial. As can be seen in Figure 1.1, the uncertainty of a cell counting result varies with the number of observations. The recommendation of assessing at least 400 spermatozoa is based on the fact that statistically a total of 400 observations reduces the random variability to ±10 percent of the observed value [2].





Figure 1.1 Range of uncertainty of sperm concentration results based on different numbers of observations (100 represents calculated value; lines show range of 95 percent Confidence Interval for the different numbers of observations).


Further sources of possible causes of significant errors and variability lie in the accuracy of the volume assessed in the counting chamber. While the area examined can be very precisely determined, the depth can vary. A shallow chamber (10 µm) would cause a 10 percent volume error if the cover is only 1 µm wrong. However, in a 100-µm deep hemocytometer, the same absolute error would only cause a 1 percent error in the volume.



1.3.3 Sperm Motility


The only practical way of assessing sperm motility “manually” in the microscope means using wet preparations with a depth of approximately 10–20 µm, meaning that a 10 µL aliquot under a 22 mm × 22 mm cover slip is appropriate. It is necessary also for the representativity of the aliquots to examine at least two aliquots and compare results to minimize random error.


Sperm velocity is very dependent on the temperature. While “room temperature” is not well defined and can vary significantly in most laboratories, the recommendation is to standardize microscope stage temperature to 37°C and preferably to use pre-warmed slides.


The same statistical consideration as for sperm concentration assessment is relevant for the motility assessment. Thus, at least 400 spermatozoa should be included in the assessment of each sample to reduce the random error to ±10 percent of the observed value.



1.3.4 Sperm Vitality


Vitality assessment is only important when many spermatozoa are immotile. The investigation is essential to identify samples with many live but immotile spermatozoa to distinguish from disorders where reduced sperm motility is due to poor sperm survival.


Since the assessment of sperm vitality by eosin-nigrosine staining [14, 15] does not appear to be sensitive to aliquot representativity (compared to sperm concentration and sperm motility assessment), replicate assessment and comparison is not considered necessary for the purpose of distinguishing between samples with immotile dead spermatozoa and samples with immotile live spermatozoa [16]. It is more important to use adequate equipment (bright field, high magnification (x1000), high resolution microscopy) and count at least 200 spermatozoa in each ejaculate.

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Apr 6, 2021 | Posted by in GENERAL & FAMILY MEDICINE | Comments Off on Chapter 1 – Standard Semen Examination: Manual Semen Analysis
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