Laboratory Procedures

Andrology Laboratory

Homapage / Andrology Laboratory

Infertility affects 15% of couples worldwide and for approximately half of them, infertility has a male origin. The andrology laboratory, with the use of special sperm analysis, evaluation and preparation techniques, helps the vast majority of male infertility cases...

Infertility affects 15% of couples worldwide and for approximately half of them, infertility has a male origin. The andrology laboratory, with the use of special sperm analysis, evaluation and preparation techniques, helps the vast majority of male infertility cases.
There are two separate laboratories specializing in male infertility in Memorial Hospital ART and Reproductive Genetics Center: the semen analysis laboratory and the andrology laboratory. Our andrology laboratory has experienced staff and all technical facilities necessary for the detailed evaluation, analysis and preparation of sperm samples. We share with our patients the outcomes of this analysis and discuss the options for a future IVF procedure.

Merkezimizdeki Semen Verme Odası Merkezimizdeki Semen Verme Odası
Private room for semen collection

 

  

Evaluating the semen sample

Semen analysis is a basic test performed in the evaluation of infertile couples as it gives valuable information on the function of the male reproductive system and the quality of sperm produced. Although semen can be rapidly assessed, the full analysis is complex and is carried out by experienced personnel.

Basic semen analysis parameters are:

  • Sperm concentration
  • Motility
  • Morphology 

The best time for semen analysis

Semen analysis is performed after a 3-5 day sexual abstinence. This time interval is critical to obtain accurate information on semen. If the abstinence is longer than 5 days, the number of spermatozoa increases, but the motility and vitality decrease and the risk of DNA damage is increased. On the other hand, if sexual abstinence is shorter than 3 days, the number of spermatozoa decreases and the motility increases. In both cases, the analysis results would be misleading.

Sperm production is affected by smoking, alcohol consumption, heat, medications and infections. If an abnormality is detected in the sperm analysis, physical and hormonal examinations are carried out and further samples are taken 3-4 weeks later and evaluated. Spermatozoa production in male testes takes around 2-3 months; hence, the sperm produced will be secreted in the semen 2-3 months later. Because of this, damage, caused by long term exposure to toxic substances and/or abnormal conditions affecting sperm production and the effects of drugs taken during infertility treatment can only be observed 2-3 months later. This is taken into account when semen is being evaluated.

INSTRUCTIONS FOR SEMEN SAMPLE COLLECTION

Please follow the instructions below.

It is important to ensure that the specimen does not become contaminated, which could yield incorrect results.

  1. Refrain from any sexual activity, including masturbation, for 3-5 days prior to the appointment date. Longer or shorter periods are not recommended.
  2. The collection must be done exclusively by masturbation.
  3. It can be difficult for some men to give a semen sample. If you become tense when you know you have to give a semen sample, please inform the assistant and your doctor.
  4. Urinate before giving a semen sample.
  5. Please ensure good hygiene practice at all times- wash and rinse hands and genital area. Dry thoroughly with a clean towel. 
  6. Do not use lubricants of any type.
  7. Collect the semen specimen in the sterile collection cup. 
  8. If the container is cracked or broken, please inform the lab personnel. 
  9. If any semen is spilled, please inform the laboratory staff. DO NOT attempt to transfer it to the cup. 
  10. Give the specimen directly to the laboratory staff or the receptionist to ensure immediate attention.

It can be difficult for some men to give a semen sample via masturbation. If your wife will be assisting with the collection, please follow the instructions below:

  1. Please inform your doctor and the laboratory staff.
  2. Sexual intercourse is not appropriate for semen collection because the sample can become contaminated by bacteria found in the human body and on the skin. This contamination could result in the failure of IVF or ICSI. To prevent this, the laboratory will provide a special semen collection condom.
  3. Your wife may not be able to assist you for semen collection on the procedure day.

If you produce your semen sample at home or in a hotel, please follow the above instructions. In addition:

You must be within 15 minutes travel from the clinic, as the sample should not be left at room temperature for an extended period of time. The sample must not be heated or refrigerated, but should be kept as close to body temperature as possible. In winter, it can, for example, be kept under the arm. It should not be exposed to sunlight.

Interpreting the spermiogram report: the normal parameters

Parameters Reference Limits
  (According to WHO manual 5th edition, 2010)
Volume (ml) 1.5
pH ≥7.2
Total sperm number (106 /ml) 39
Sperm concentration (106 /ml) 15
Total motility (%) 40
Normal morphology (%) 4
Vitality 58
Leucocyte (106 /ml) <1
    

Diagnosis after sperm analysis: a glossary

Aspermia: no semen (retrograde or no ejaculation)

Asthenozoospermia: percentage of progressively motile spermatozoa below the lower reference limit

Asthenoteratozoospermia: percentages of both progressively motile and morphologically normal spermatozoa below the lower reference limits

Azoospermia: no spermatozoa found in the ejaculate

Cryptozoospermia: spermatozoa absent from fresh preparations but observed in a centrifuged pellet

Haemospermia (haematospermia): presence of erythrocytes (red blood cells) in the ejaculate

Leukospermia (leukocytospermia, pyospermia): presence of leukocytes (white blood cells) in the ejaculate above the threshold value

Necrozoospermia: low percentage of live and high percentage of immotile spermatozoa in the ejaculate

Normozoospermia: total number or concentration of spermatozoa, and percentages of progressively motile and morphologically normal spermatozoa, equal to or above the lower reference limits

Oligoasthenozoospermia: total number or concentration of spermatozoa, and percentage of progressively motile spermatozoa below the lower reference limits

Oligoasthenoteratozoospermia: total number or concentration of spermatozoa, and percentages of both progressively motile and morphologically normal spermatozoa below the lower reference limits

Oligoteratozoospermia: total number or concentration of spermatozoa, and percentage of morphologically normal spermatozoa below the lower reference limits

Oligozoospermia: total number or concentration of spermatozoa below the lower reference limit

Teratozoospermia: percentage of morphologically normal spermatozoa below the lower reference limit 

Normal spermatozoa

A sperm cell is composed of three regions having different functions: the head, the mid-piece and the tail. The head contains the genetic material (DNA), the mid-piece contains mitochondria, which provide the required energy for the motility of sperm, and the tail enables helical movements using the energy obtained. A normal spermatozoa’s head should be 4-5µm long and 2.5-3.5µm wide. The length to width ratio should be 1.50-1.75. The cylindrical mid-piece should be 0.5-1µm thick and 7-8µm long, it should also be axially attached to the head. The tail should be thinner than the mid-piece, have a regular shape with no twist, and be 40-50µm long.

Normal spermatozoa

Assessing sperm morphology

Spermatozoa morphology is a very good indicator of sperm quality. In our center, spermatozoa morphology assessment is done according to criteria in the “WHO Manual for Examination and Processing of Human Sperm, 5th edition” using “Spermac stain”, which stains the nucleus red and the acrosome, neck and tail green. After staining, spermatozoa morphology is analyzed under a microscope at 100X magnification. Both abnormal and normal forms are recorded and over 200 spermatozoa are evaluated. Thirty-eight distinct types of anomaly in the head, neck and tail of the sperm cell, can be detected. If the ratio of spermatozoa with a normal morphology in a sample is below 4%, this condition is diagnosed as teratozoospermia. Although rates below or above 4% are considered to have some significance with regard to fertilization and pregnancy, every laboratory has to determine its own thresholds. For example, according to Kruger strict criteria, if this value is below 4%, the sub-distribution of anomalies is scrutinized: head anomalies may be severe or mild, affecting fertilization differently. In our center, we consider that severe head defects are significant for the ART outcome. This issue is clarified during counseling. 

Morphological defects in sperm

Studies have established a relationship between sperm morphology, fertility, embryo development and pregnancy outcomes. Sperm morphology analysis is not only important for the evaluation of male fertility but also for the decision regarding which ART strategy should be adopted.

Severe morphological anomalies affect the fertilization capacity of the sperm cell. The most important anomalies are: macrocephaly (or megalohead), globozoospermia (or roundhead), and tail-stump. If the majority of spermatozoa are morphologically abnormal, the best-morphology spermatozoa are selected from among the whole sample. However, the treatment may nevertheless result in a low fertilization rate, slow-growing embryos and poor quality day5 embryos


Round head

 


Ridged


Free head

 


Triple, double heads


Bent neck

 


Coiled tail


Dag defect

 


Double tail


Pinhead

 


Tail-Stump

Identifying abnormalities in the semen sample: Motile Sperm Organelle Morphology Examination (MSOME)

In our laboratory, if the normal sperm rate is below the expected value or if severe male infertility is suspected, as well as the routine semen analysis and morphology evaluation, semen is also examined by Motile Sperm Organelle Morphology Examination (MSOME), a technique using a high magnification microscope. MSOME is also used for couples with repeated ART failures and/ or with unexplained infertility.

The advantage of the MSOME technique is its magnification power (8050X). This enables the observation of major sperm anomalies and also defects in the head of the sperm (e.g. vacuoles -small sacs filled with liquids-). The presence of vacuoles provides a clue to the structure of the DNA in the nucleus. The major difference between the initial semen analysis and MSOME is the sperm preparation technique. For standard sperm morphology evaluation, sperm is fixed onto the microscope slide and stained with special dyes; therefore motile and immotile spermatozoa are all assessed together. However, for MSOME, only motile spermatozoa are analyzed, giving a better indication of the quality of the sperm that will be selected for microinjection on oocyte pick-up (OPU) day.

When sperm is subjected to MSOME, various parameters are investigated: spermatozoa with a normal mid-piece and tail are preselected, and the presence of vacuoles in the head is scrutinized. Accordingly, the sperm is graded from 1 to 4, with 1 being spermatozoa with normal morphology and no vacuoles and 4, spermatozoa with an abnormal head and/ or numerous vacuoles.

 
 
Grading of sperm cells under high magnification (8050x)

Performing immunological tests on the semen sample

Agglutination, in which sperm cells are attached to each other at the head/ midpiece/ tail, is related, in the majority of cases, to the presence of anti-sperm antibodies. These may attach to the surface of the cell, preventing sperm motility in the uterus. Agglutinated samples have been associated with unexplained infertility.

Immunologic factors are present in 8% of infertile couples. If infertility is thought to be associated with immunological problems, as in case of agglutination or decreased motility of the sperm, immunological tests are performed. Immunological tests performed at our clinic are the Direct Immunobead Test and the Sperm-Mar Test. 

Direct Immunobead Test

The Direct Immunobead Test detects immunoglobulins (Ig) attached to the spermatozoa’s surface. Motile sperm are mixed with antigen-coated latex beads and if antibodies are present on the cell surface, the beads bind to the antibody, forming visually distinct structures.

Sperm-Mar Test

The Sperm-Mar Test (mixed agglutination reaction test) is used to identify anti-sperm antibodies and to discriminate their specific subclass. Using this test, IgG and IgA antibodies can be detected in fresh semen. Antigen-coated beads that can attach to different regions of the motile spermatozoa (head, mid-piece and tail) are used in the test. The sample is observed using a light microscope. Sperm cells tagged with beads are counted, noting the region of the attachment. Binding between sperm cells and latex beads demonstrates the presence of anti-sperm antibodies. A rate of 15-39% indicates a suspected infertility. A rate above 40%, indicates a severe immunological factor.

Assessing sperm vitality for immotile sperm

To assess for sperm vitality, two tests are carried out in Memorial Hospital Andrology Laboratory: 

Eosin-Y Test

When motility is reduced or completely absent, and thus cannot be of any help in assessing vitality, eosin-Y allows the detection of living sperm cells. This procedure is used for diagnostic purposes only, as dyed vital spermatozoa cannot afterwards be used for microinjection.

Sperm cells after eosin-Y staining

Hypoosmotic Swelling Test (HOST)

The HOS test is used only when no motile spermatozoa are found. The test can be used for diagnostic purposes but has a major advantage over the eosin-Y test: the analyzed sperm cells can be used for microinjection. This means that vital spermatozoa for microinjection can be selected from an immotile sample.

Cells that swell under hypoosmotic conditions (those with a coiled tail) are alive whereas those with straight tails are dead

Identifying other cells 

Leucocytes

A normal semen sample should not contain more than 106 leucocytes/ ml. An increased number of leucocytes in the ejaculate (leucospermia) indicates an infection in the genital tract. As the presence of leucocytes affects sperm values, in our center, microscopic leucocyte identification is always confirmed with a detection kit.

 Leucocyte in the ejeculate

Immature germ cells

The number of immature germ cells should be ≤106/ ml. It is extremely important to identify immature germ cells in patients with a low number of sperms or with azoospermia to determine precisely the extent of the sperm production defect. In our center, we use Bryan-Leishman staining and Test-simplet kit containing pre-dyed slides to detect immature germ cells.

  Inmature germ cells in the ejeculete

Preparing the ejaculated sperm for insemination and ART

The sperm preparation method plays an important role in the outcomes of IUI (intrauterine insemination), IVF (in vitro fertilization) and ICSI (intracytoplasmic sperm injection). Studies have shown that a long waiting period in seminal plasma affects the fertilization capacity of spermatozoa. Therefore, preparation techniques need to be fast, efficient and thorough:

  • Microorganisms are discarded by washing the semen sample with antibiotic-containing solutions.
  • Seminal plasma, debris and contaminants are also removed.
  • Sperm with the best morphology and good motility are selected. Sperm are graded and those with abnormal morphology are excluded.
  • After this preparation, sperm are ready to fertilize an oocyte (egg).
Androloji laboratuarı görüntüsü
The andrology laboratory

Sperm preparation techniques

In Memorial Hospital ART Center Andrology Laboratory, the following techniques are used, alone or combined:

  • Sperm washing
  • Swim-up
  • Density gradient
  • Mini-gradient 

Sperm washing

Sperm washing is the first technique applied. The principle of this method is to mix the semen with culture media in defined amounts and to centrifuge it. Although large concentrations of sperm are retrieved, debris and leucocytes cannot be removed by this technique alone. We therefore use this procedure in combination with those described below.

Swim-up

Swim-up is applicable when enough forward moving (progressive) sperm cells are present in the sample. Sperm can be prepared this way for insemination, IVF and microinjection. Semen samples with normal viscosity and containing high motility sperm are divided into several centrifuge tubes and washed by two centrifugations. Sperm washing medium is added onto the pellet and the tubes left at a 45° angle, allowing sperm cells to “swim” to the upper phase.

Combining sperm washing and swim-up

Liquefied semen samples are mixed with sperm washing media in defined ratios, divided between 2 or 4 tubes, and centrifuged. The supernatant lying in the upper part of the tubes is discarded and sperm wash medium is added, yielding a ready-to-use sperm sample. Depending on the properties of the semen sample, this step may be repeated many times. After the washing step, sperm wash media is added to the pellet and the tubes are inclined at a 45° angle and incubated at 37°C with 5% CO2 for 45-60 minutes, allowing sperm cells to swim to the upper layer. At the end of this period, the supernatant, containing viable motile sperm, is collected and directly used for IUI, IVF or ICSI.

 

Density gradient

Density gradient is used to prepare sperm for microinjection. We use solutions that separate sperm from the rest of the semen sample. Semen is dropped on colloidal silica suspension and centrifuged, forcing cells to fall to the bottom of the tube. Sperm with normal morphology and motility move towards the bottom, and are then collected. With this method, Immotile or abnormal sperm cells, leucocytes and cell debris are thoroughly cleared from the final solution, which contains only sperm with normal morphology. This method, by the elimination in particular of dead cells and leucocytes, also leads to a decreased reactive oxygen species (ROS) leak that can cause DNA damage. Furthermore, since the obtained solution is non-toxic, it will not damage the sperm itself, the endometrium or the oocyte (egg). Viral contamination (Hepatitis B and C, HIV) can be reduced and even eliminated by this procedure.

 

Mini-gradient

The mini-gradient procedure allows the collection of good quality sperm from a semen sample with a low number of sperm and a high rate of abnormality. Mini-gradient yields a sample that can be used for microinjection directly. This method is applied to testicular sperm rather than ejaculated sperm because only a very low amount of sperm is usually obtained through testes surgery.

 

Determining sperm DNA damage

Studies have shown a close connection between sperm DNA damage and male infertility. It is therefore important to determine the extent of sperm DNA damage, first to predict the fertilization capacity of the sperm, and second to predict the risks that the future embryo will be exposed to.
Recent studies have proposed a correlation between the presence of vacuoles in the head of the sperm and sperm DNA damage. In the classical microinjection used for infertility treatment, sperm cells are evaluated according to their structural appearance. However, the presence of vacuoles cannot be observed using this technique. Instead, observation under higher magnification is required. We use Intracytoplasmic Morphologically selected Sperm Injection (IMSI) (please click for further details) in our center to overcome this problem by pre-selecting and microinjecting sperm with little or no DNA damage.

The causes of sperm DNA damage are not well understood. However, many studies have demonstrated that various factors may affect sperm DNA integrity by causing damage to DNA strands: abnormal cell metabolism, oxidative stress, genetic and environmental factors, long sexual abstinence, advanced age, smoking, elevated scrotal temperatures, varicocele and air pollution.
Damage to the sperm DNA structure may trigger an arrest in embryonic development or a slow/ poor embryonic development, thus affecting pregnancy outcomes. Testes sperm has been shown to include less DNA damage than ejaculated sperm, therefore testes sperm may be used instead of ejaculate in some patients.
The main tests used for the detection of sperm DNA integrity are the acridine orange test and TUNEL assay (Terminal deoxynucleotidyl transferase dUTP nick end labeling).

Acridine Orange Test

The Acridine Orange Test is a diagnostic detection method for sperm with DNA damage.

Acridine orange dyes native DNA containing sperm cells in green (left panel) and those containing DNA damage, because of acid-induced denaturation, in orange-red (right panel)
   

TUNEL Test

The TUNEL test (Terminal deoxynucleotidyl transferase dUTP nick end labeling) detects DNA fragmentation arising from apoptosis (or programmed cell death).

Sperm cells labeled with TUNEL: sperm with DNA damage are in green and those free of DNA damage are in blue

Testicular sperm retrieval methods

When no spermatozoa are found in the ejaculate, sperm may be retrieved from the testes. Many methods exist for sperm extraction but the two most widely used and preferred methods in our center are TESA and microTESE. While TESA (testicular sperm aspiration) is performed in obstructive azoospermia, microTESE (microdissection testicular sperm extraction) is done in non-obstructive azoospermia. 

TESA (Testicular Sperm Aspiration)

TESA is performed under local anesthesia. A needle is inserted in the testes and fluid and tissue are aspirated. This procedure is performed when spermioducts are obstructed or to increase pregnancy chances when ejaculate has been used in unsuccessful previous attempts. As testes are the organ in which the sperm are produced, the chance of finding sperm cells is nearly 100%.

MicroTESE ile alınan parçadaki sperm görüntüsü
 

Microdissection Testicular Sperm Extraction (MicroTESE)

MicroTESE is a microsurgical intervention that should be performed by an experienced surgeon to maximize the chances of obtaining spermatozoa. The operation is performed under general anesthesia.
The tubules in which sperm production takes place are magnified 30 times under a microscope. Whitish, opaque tubules are retrieved by the urologist as there is a greater probability of their containing spermatogenically active germ cells. The retrieved tubule is disintegrated by the biologist to release the sperm cells. The presence of sperm cells is investigated under a higher magnification microscope (200-400X). When present, spermatozoa are used for microinjection (ICSI).

MicroTESE procedure

Advantages of MicroTESE over TESE

MicroTESE and TESE are basically the same surgical procedures: However, in TESE, a microscope is not used and the surgeon collects the samples randomly. Yhe advantages of MicroTESE are:

  • Observing the sample under the microscope increases the chances of finding sperm from 30% to 52%.
  • Testes are responsible for testosterone (male hormone) production. The microTESE procedure reduces tissue loss by up to 70% compared to TESE, thereby minimizing interference with testosterone production.
  • As the surgery is done with the help of a microscope, incisions can be made while avoiding capillaries, thus reducing bleeding.
  • For pure Klinefelter patients (47XXY), who do not have spermatozoa in the ejaculate because of their genetic background, and for patients for whom spermatozoa cannot be found on a first attempt with TESE, the chances of finding sperm cells are increased by up to 50%.
  • Because of all these advantages, we perform microTESE instead of TESE in our center for non-obstructive azoospermia cases. 

Important Points before MicroTESE

  • Micro-TESE should be performed before the oocyte pick up procedure. 
  • Please be at the hospital on the morning of the operation at the appointed time.
  • The operation is performed under general anesthesia, it is important that you do not eat and drink anything after 12.00 pm the previous night.
  • The operation can take almost 2 hours, depending on how long it takes to find sperm. The chance of finding sperms is related to the testicle’s capacity to produce sperms. If the testicle is not producing sperms, then it will be impossible to find sperms.
  • After the operation you will stay in hospital for 3-4 hours and will be served a meal there. After you have been examined by your doctor, your medication will be prescribed and you will be discharged.  
  • As Micro-TESE is an open surgery, some complications such as hematoma (subcutaneous blood accumulation), infection, or opening of the scar may develop. However, all of these complications are very rare (Less than 1%)
  • You should avoid strenuous movement, walking for a long time or carrying heavy things in the post-operative period during your home rest. You will be called to the hospital for dressing your wound two days later. Until that day, do not take a shower.
  • You should take the medications prescribed by your doctor (antibiotics and analgesic) for 5 days after the operation. There is no diet limitation. 
  • After the operation, if you have swollen testicles, strong pain or fever, you should visit your doctor.

Sperm cryopreservation

Spermatozoa may be cryopreserved to maintain their viability and thawed for use in a future ART attempt. The first successful birth from a cryopreserved sperm sample occurred in 1973.

For the freezing process, solutions known as cryoprotectants are used:  to avoid the formation of water crystals inside the cell, which would inevitably damage the cellular content, the intracellular water is replaced by cryoprotectant.

Frozen sperm samples are stored in liquid nitrogen tanks at -196°C. During the thawing process, the cryoprotectant is removed and cells are refilled with water. Some vitality and motility loss is expected because of the different capacity of each sperm cell to tolerate the freezing/ thawing stress, and the mechanical stress engendered by the procedure itself. Nevertheless, studies have shown that when motile frozen/ thawed sperm cells are used for microinjection, neither the fertilization capacity nor the pregnancy outcome is negatively affected.

For azoospermic males for whom microTESE is mandatory, it is planned for the OPU (oocyte pick-up) day. As methods used for sperm retrieval have physiological, psychological and financial implications for the patient, if there are surplus vital/ motile sperm cells after microinjection, sperm freezing is recommended. However, if a very small number of sperm cells is retrieved from the testes, the probability of finding vital sperm after the freezing/ thawing process will be reduced. In this case, testicular sperm should again be obtained from the patient by microsurgery.

Sperm freezing

Circumstances under which sperm-freezing is advised

  • In azoospermic males, sperm retrieved from the testes or the epididyme and remaining surplus after the microinjection procedure, and the raw sample obtained from testicular biopsies may be cryopreserved.
  • In testes cancer, leukemia, or other type of cancers, sperm is frozen for fertility preservation, as chemotherapy or radiotherapy will have a negative effect on sperm production.
  • In cases with testes diseases that require surgery, or before vasectomy, sperm freezing is advised.
  • Patients may have difficulty in giving a semen sample on OPU day, for psychological, socio-cultural or religious reasons. Freezing sperm samples may thus be an option.
  • For genetic analysis, pooling cryopreserved sperm samples is important to increase sperm concentration and obtain reliable results. 

Important information for sperm and testicular tissue freezing

Sperm freezing is performed for men with a very limited number of sperm, or in some samples no sperm, in their semen and that this procedure will prevent unnecessary surgery to the testis. Chemotherapy and/or radiotherapy for the treatment of cancer may destroy sperm cells, and that sperm should be frozen and stored before these therapies. 

The sperm obtained from the testis by surgery are frozen and stored.

There is no guarantee of pregnancy when embryos obtained by using frozen-thawed sperm samples are transferred. Sperm and testis tissue samples might not be suitable for freezing-thawing in all cases and that, in some cases, most or all of the sperm and tissue samples may lose viability. 

The couples are required to call the center annually to confirm whether you wish to have the frozen sperm samples stored or destroyed. If you never call the center, your sperm samples will be destroyed at the end of the third year. 

The couples are required to inform the center if your addresses or phone numbers change and accept responsibility if you are not contactable.

 
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