​Sperm Analysis 100: UNDERSTANDING AZOOSPERMIA AND CRYPTOZOOSPERMIA DIAGNOSIS.

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CRYPTOZOOSPERMIA OR CRYPTOSPERMIA

 Is a severe condition affecting sperm concentration. The World Health Organization (WHO) considers that the sperm concentration should reach at least the amount of 15million spermatozoa/ml to achieve a natural pregnancy. When sperm concentration is lower than 15 million spermatozoa/ml, the condition is called oligospermia, also known as oligozoospermia. But if the quantity is even lower than that established as a reference value, the condition is known as cryptozoospermia. When there is cryptozoospermia, the ejaculated semen contains less than 100,000 spermatozoa per ml. In order to achieve a natural pregnancy, the presence of sperm in the ejaculate is necessary. Thanks to assisted reproductive technologies (ART) it is possible to get pregnant having solely a viable spermatozoid per extracted egg.

Thanks to the innovative technique called ICSI, achieving pregnancy now is possible even if we only have a single, viable spermatozoon per retrieved egg. Thus, we could almost say that ICSI is the assisted reproductive technology related per se to cryptozoospermia.

CRYPTOZOOSPERMIA- HIDDEN SPERMS

Male factor is responsible for about 30-40% of couples having infertility treatment. The issue varies from having a low sperm count, motility, morphological abnormality of sperm, and no sperm at all, or sperms incapable of fertilizing eggs. The majority of these sperm problems can be solved by ICSI (INTRACYTOPLASMIC SPERM INJECTION) where a single sperm is injected into each oocyte with help of micromanipulator. 

CRYPTOZOOSPERMIA, the word CRYPTO MEANS HIDDEN and as word suggests, patients with cryptozoospermia have occasional or a few sperms in their semen that is 4 – 40 sperms in an entire ejaculate while a normal male semen sample has around 15 million sperms in it. In many cases , these men come to us having been told in the past that they have no sperm,( azoospermia) as sperm number is very less, they are very difficult to find and easy to miss. This is quite a common problem when semen analysis is done in a general medical laboratory, where the laboratory scientists are not experienced enough and do not know how to do accurate semen analysis in this type of critical conditions. A lot of scientists do microscopic analysis of the semen sample quite casually and if they cannot see sperm in the first glance they report it as having a zero sperm count or AZOOSPERMIA.

This is why it is important that before labelling a semen sample as being azoospermia, the laboratory scientists should observe sample very carefully for the presence of even a single sperm. At our laboratory in these conditions semen sample is collected and allowed to liquefy naturally for about 30 minutes before we begin to work on it. Next this sample is centrifuged to concentrate any sperm in the sample into a smaller volume. For example, if a sample has a volume of 3ml, we concentrate it down to 0.5 ml or less to make our search easier. This concentrated sample is distributed into tiny drops in a petri dish and an embryologist scan through each drop in an INVERTED MICROSCOPE WITH MAGNIFICATION OF 200X TO 400X. This search usually lasts for a minimum of 2 hours, but time may increase depending on the difficulty of finding sperm.

CRYPTOZOOSPERMIA OR AZOOSPERMIA?

It is not unusual that, when seeing a zero sperm count in the ejaculate, semen samples are classified as azoospermic instead of cryptozoospermic. The difference between azoospermia and cryptozoospermia is decisive, since a wrong diagnosis may lead to the performance of a testicular biopsy or a epididymal sperm aspiration to obtain sperm when actually there are sperm in the ejaculate, but only a few. The difficulty here is being able to find this quantity of sperm in order not to be mistaken. To avoid errors and provide the patient with the appropriate diagnosis of cryptozoospermia in cases where the first semen analysis shows a zero sperm count, usually a second seminogram is requested. This time, the semen sample will be examined after having undergone a centrifugation process in which the sperm are concentrated and separated from the seminal fluid so that identifying them becomes easier.

LABORATORY PROCEDURE OF CRYPTOZOOSPERMIA

If no spermatozoa are observed in the replicate wet preparations, azoospermia can be suspected. Although it has been suggested that the definition should be change, azoospermia remains a description of the ejaculate rather the statement of its origin or basis for the diagnosis and therapy. It is generally accepted that the term azoospermia can only be used if no spermatozoa are found in the sediment of a centrifuged sample.

NOTE THAT 

1. Whether or not spermatozoa are found in the pellet depends on the centrifugation time and speed and on how much of the pellet is examined.

2. Centrifugation at 3000g for 15minutes does not pellet all spermatozoa from a sample.

3. After centrifugation, motility can be lost and concentration will be underestimated.

WHEN AN ACCURATE ASSESSMENT OF LOW SPERM NUMBER IS NOT REQUIRED.  

If the number of spermatozoa per HPF in the initial wet preparation is low (0-4 per x400 HPF or 0-16 per x200HPF) several options are available.

1. Taking no further action: if the number of spermatozoa per x400 HPF is <4 (i.e approximately 1million per ml, it is sufficient for most clinical purposes to report the sperm concentration as < 2 million per ml.

2. Examination of the centrifuged samples to detect spermatozoa. : When no spermatozoa are observed in either wet preparation, the sample can be centrifuged to determine if any spermatozoa are present in a larger sample.

Mix the semen sample well. Remove a 1-ml aliquot of the semen and centrifuge at 3000g for 15 mins.

Decant most of the supernant and resuspend the sperm pellet in the remaining approximately 50ul of the seminal plasma.

Place one 10ul aliquot of the pellet on each of the 2 slides. Examine the slide with phase-contrast optics at x200 

Scan the entire cover slip systematically field by field.
REPORT

A. The presence of spermatozoa in either replica          

indicates cryptozoospermia,

B. The absence of spermatozoa from both replicates suggest azoospermia.
SEMEN ANALYSIS  REFERENCES
Semen analysis

Characteristics Normal findings

Volume 1.5 – 5 mL 

Color Whitish, opalescent

Liquefaction Complete within 30 minutes 

pH Basic 7.2–8.0 

Total number of sperm per ejaculate > 39 million per ejaculate

Sperm concentration per mL > 15 million per mL

Vitality (percentage of live sperm) > 58% live sperm

Morphology (percentage of normal forms) > 4% sperm are morphologically normal. 

Total motility (progressive and non-progressive sperm) > 40% 

Progressive motility > 32%

Fructose in seminal plasma > 13 μmol/L 

Leukocytes per mL of semen

< 1 million 

Pathological findings
Conditions Characteristics

Aspermia No ejaculate

Hypospermia Low ejaculate volume (< 1.5 mL)

Azoospermia No spermatozoa in the ejaculate

Cryptozoospermia < 1 million spermatozoa/mL of ejaculate

Oligospermia < 15 million spermatozoa/mL of ejaculate

Asthenozoospermia < 32% of spermatozoa show progressive motility (category PR)

Teratozoospermia Increased amorphous spermatozoa 

Oligoasthenoteratozoospermia (OAT syndrome) Low concentration, insufficient motility, and increased amorphous spermatozoa
REFERENCES

http://emedicine.medscape.com/article/436829-overview

https://dx.doi.org/10.1093/humupd/dmp048

https://www.uptodate.com/contents/approach-to-the-male-with-infertility

Cryptozoospermia

https://www.amboss.com/us/knowledge/Semen_analysis

https://web.facebook.com/IvfCentreIndia/posts/678006775597654?_rdc=1&_rdr

WHO lab. Manual for the examination and processing of human semen. 5TH edition 

Compiled by.
WOJUADE KEHINDE (ACC)

Chairman Andro scientific /Ikeja Chapter AMLSN Chairman

www.androscientific.com.ng

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