When couples apply to our center with the desire to have children, the situation of both spouses is discussed together. While the woman is being examined and examined, the sperm status of the man is examined. Previous semen analysis values are checked.
In addition, before starting the application, semen analysis may be requested to be repeated 1-2 times in our center. It is true that differences can be observed in semen analyzes performed at different times. This will give us an idea about the method of preparing sperm during in vitro fertilization. In fact, it will help in determining the possibility of no sperm being seen on the day of egg collection and in choosing the necessary treatment and sperm retrieval methods.
On the day of egg collection, sperm is also collected from the man. The duration of sexual abstinence should be at least 3 and at most 5 days. Special containers that are sterile and non-toxic to sperm are used for sperm collection. The patient is given the necessary information to obtain sperm under sterile conditions. Soap or similar lubricants should not be used during the semen sample collection process. The penis and fingers should not touch the inside of the lid of the supplied container. It is necessary to put all the incoming semen into the container. In case the inside of the container is accidentally touched or some of the semen spills out, laboratory staff must be informed of the situation. The patient is taken to a special room prepared in our center. Men who cannot produce sperm under laboratory conditions should inform their doctors, laboratory staff or IVF department patient consultants about their situation. Men who will donate sperm outside the laboratory must deliver the container containing the semen to the laboratory within 20 minutes at most, protected at body temperature and protected from sunlight. The semen sample is received from the patient with a name written on the container.
Human sperm coagulates immediately after ejaculation and dissolves and liquefies again within approximately 20 minutes. It is examined after 20-30 minutes. Volume, liquefaction time, viscosity, color, odor and pH values are noted. Ejaculate volume is considered normal between 2-6 ml. Indicator paper is used for pH measurement. The ejaculate is measured within an hour after collection and should be between 7.2-8.0.
Sperm concentration, motility, agglutination and the presence of non-sperm cells are evaluated under a phase contrast microscope with a Makler camera.
Normal Sperm Parameters:
1. Quantity (volume) 2.0 ml and above
2. pH 7.2 and above
3. Sperm count (concentration) in ml is 20 million/ml and above
4. Total sperm count is 40 million/ml and above
5. Mobility: 50% and above at grade A+B or 25% and above at grade A
6. Shape (morphology) 4% and above
7. Viability (viability 75% and above)
8. Leukocyte (inflammatory cell) below 1 million/ml
In cases where sperm motility is low, Eosin Y vitality test and HOS test can be performed to determine the number of live sperm.
The basis of the Eosin Y vitality test is that dead sperm absorb dye because their membrane structure is disrupted and appear stained.
The HOS test is based on the principle that the sperm absorbs water and swells when placed in a hypoosmolar environment due to the semi-permeability of intact cell membranes.
One of the most important parameters of semen analysis is the morphological evaluation of sperm. The effect of sperm morphology on the fertilization rate is large and direct. Sperms are motile cells consisting of head, neck and tail. The characteristics that these different anatomical regions should have should be determined and the morphology evaluation should be made according to these criteria. In our center, morphological evaluation is made according to the criteria defined by Kruger.
Morphology assessment can be roughly done during direct examination with a phase-contrast microscope. However, the distinction between the nucleus and acrosome in the head, the characteristics of the acrosomal cap, the localization of the middle part and the differences in the wall of the sperm head cannot be adequately examined. For this reason, after the semen is liquefied, it must be spread in a very thin layer on a clean slide, detected and stained after it dries, and the dyed preparations must be evaluated. Diff-Quik or Spermac dyes are used for sperm morphology. According to Kruger's strict criteria, the normal sperm rate is expected to be over 14%. However, it is observed that the success rate in IVF applications with normal sperm findings above 4% is high.
Sperms are extremely sensitive to oxidative stress. Because the spermatozoa membrane is very rich in unsaturated fatty acids. Unsaturated fatty acids are very sensitive to free oxygen radicals. When they are in excess, they cause oxidative damage to cell membrane fats, proteins and DNA.
There are two sources of oxidative stress in the ejaculate. Seminal leukocytes and abnormal spermatozoa. Therefore, leukocyte concentration should be taken into consideration when examining a sperm sample. High leukocyte concentration has a significant relationship with fertilization. DNA breaks, parallel to the increase in oxidative stress level, cause deterioration in sperm functions and reduce fertilization potential. Single cell electrophoresis (COMET) and Tunnel Tests can be applied to demonstrate DNA damage.
The process that the sperm goes through after entering the female genital system in order to gain the ability to fertilize the oocyte is called capacitation. Although sperm entering the female genital tract following ejaculation is motile, it does not have fertilization capacity. Here, it gains fertilization potential under the influence of some factors arising from its environment. In this process, known as encapsidation, some changes occur in the sperm membrane and inside the cell, and the sperm begins to receive signals from the environment for the acrosome reaction, hyperactivation and fusion with the oocyte membrane.
The sperm sample is washed using balanced culture media that will trigger capacitation and the beginning of the chain of events associated with it in in vitro conditions and ensure their continuity. Sperm washing is done with culture media containing antibiotics, and sperms that are morphologically good and have proper movement are selected for processing. It is decided which method to use by considering all the characteristics of the semen sample. In the swim-up method, the sperm sample is subjected to a double-stage centrifugation and washing process and incubated for a certain period of time to ensure that the highest quality sperm with proper morphology and motility swim up. Gradient method is based on concentration difference. It consists of collecting the sperm at the bottom in a chromatographic manner by creating layers with media of different concentrations.
In cases where sperm motility is low, pentoxifylline is used to increase motility. However, this procedure must be performed carefully due to the possibility of pentosyphylline damaging sperm.
TESTIS BIOPSY: If no sperm is found in the semen analysis performed at the patient's first admission, the reasons for the absence of sperm are first investigated. There may be two situations. The first is the situation where the patient produces sperm but there is no sperm in the ejaculate due to a blockage in the ducts or any other reason, which is called obstructive azoospermia. In obstructive azoospermia, it is used in the microinjection process by obtaining sperm by aspiration (TESA) on the day of application. In the second case, there is azoospermia caused by the lack of sperm production even though there is no blockage in the ducts, and this is called non-obstructive azoospermia. In cases of non-obstructive azoospermia, sperm is obtained by taking a biopsy from the testicle (TESE). Biopsy can be performed by viewing the testicular structure in more detail under a microscope at x20 magnification. This process is also called microtease. In this way, there is no need to take a piece from each area of the testicle. If there is enough sperm left after the microinjection process, it can be frozen and stored for use in subsequent attempts when necessary.
Regardless of the sperm collection and preparation method, the aim is to obtain good quality embryos.
Ultrasound and vasography The testicles and the blood flow therein can be examined with scrotal ultrasound and Doppler ultrasound technique. During vasography, a radioactive dye is injected into the vas deferens (the connection between the sperm storage area and the excretory ducts) and an X-Ray (film) is taken to detect any blockage in these pathways.
Hormonal Tests Hormonal tests in male infertility are performed only if a disorder is detected in semen analysis or physical examination. The results of these tests, especially in men whose semen analysis does not detect sperm, can determine the levels of reproductive hormones with a simple blood test to investigate whether the cause is testicular. The most important hormone tests are Testosterone, FSH and LH.
Genetic investigation of sperm in severe male infertility
(sperm FISH) In cases where rare and serious deformities occur in sperm or when the man is a carrier of a genetic disease called translocation; Chromosomal errors in sperm are examined with the FISH technique. It provides the opportunity to identify possible genetic disorders in the sperm to be used during microinjection, and healthy embryos can be selected thanks to Preimplantation genetic diagnosis.
ICSI: "Intracytoplasmic Sperm Injection" is the abbreviated name of the process of introducing sperm into the cytoplasm of the female egg cell 'oocyte'. The term microinjection, which is common in our country, is used.
TESE: "Testicular Sperm Extraction" is the process of obtaining sperm from testicular tissue taken through open surgery.
TESA: Obtaining sperm from testicular tissue by inserting a "Testicular Sperm Aspiration" needle.
MESA: "Micro-Epididymal Sperm Aspiration" Obtaining sperm from the epididymal organ with the help of an operating microscope.
PESA: "Percutaneous Epididymal Sperm Aspiration" Obtaining sperm by entering the epididymal organ with a needle. The organs of the male reproductive system are named as shown in the figure.
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