Sperm Preparation For IVF / ICSI.

Emobileclinic Trending Topic

Emobileclinic Specialist

Intra Uterine Insemination (IUI) offers a simple and cost effective method of isolating the most functional spermatozoa from the ejaculate. It helps obtain an enriched fraction of motile spermatozoa and also removes of seminal plasma and other cellular components from ejaculate. This is important because the cellular debris, prostaglandins and any micro organisms if present must be removed so that the sample can be used for IUI procedure.

semen quality pic

However, there is no technique, which is superior to all other techniques. Ideally, at the end of a sperm wash, we should aim to be able to attain minimum criteria of sperm quality in order to enhance the chances of pregnancy.

SPERM PREPARATION TECHNIQUE
Method: these are various methods for preparation of semen for IUI/IVF/ICSI:
-Simple washing
-Swim up from pellet
-Swim up from ejaculate
-Swim up from ejaculate with hyaluronic acid (sperm –select)
-Discontinuous gradient centrifugation
-Sedimentation method/Layering under paraffin
-Sperm incubation with pentoxifylline
Glass wool filtration

Sample washing:
Washing is oldest method described. The aim is to remove only the seminal plasma, and not the cellular debris, bacteria or non-motile spermatozoa. After liquefaction, the semen is mixed with culture medium (1:1) and centrifuged at 300-400 g for 10 minutes. The supernatant is discarded and pellet is suspended in 2 ml of culture medium. This is centrifuged again at 300-400 g for 5-10 minutes and supernatant against discarded. The final pellet is suspended in 0.4-0.5 ml of medium and immediately used for insemination. This is especially useful when sperm density is very low and you are doing an IUI.

Swim –up form Pellet
This is the most widely used technique for separation of most sperms from non-motile sperms and cellular debris. It is used with normal semen samples and is based on principle active self-migration. After liquefaction, the ejaculate is mixed with culture medium and then centrifuged at 300-400 g for 10 minutes. The supernatant is discarded and the sperm pellet is gently over lay with 1 ml of culture medium and stored inside incubator at 37°C for 30-45 minutes. The 0.4-0.5 ml of the supernatant is then gently aspirated and used for insemination.

Swim-up from Ejaculate:
This is the ideal method because it obviates the needs for centrifugation. There are some workers who still prefer to centrifuge the recovered sperms.

Discontinuous Gradient Centrifugation:
The layers are made with 1-2 ml of gradient solution (80% lower and 40% upper layer) 2 ml of semen is then carefully layered on top of 40% and centrifuged at 400 600 g for 15-20 min. cell debris, immobile an abnormal sperm all accumulate at interfaces and a soft pellet is formed at the bottom of the tube. This pellet is aspirated and suspended in 2 ml of culture medium (Depending on sperm density either simple washing or wash with swim-up technique is used). This is centrifuged at 300 g for 5-10 min. the pellet is now suspended in 0.3-0.5 ml of culture medium and used for insemination. The final wash with culture medium to remove the gradient is drawback of this method.

Sedimentation Method / Layering under paraffin.
Only method to succeed if the number and motility of sperm is very low and is now usually used only in patients undergoing IVF with ICSI. It is very effective in removing cells and debris. The disadvantage is that the preparation takes a long time.
The initial steps are similar to the simple washing procedure. Suspend final pellet in the form of a single droplet and put it under paraffin oil in a petridish at 37°C in 5 percent Co2 incubator. Leave it for 1-24 hours depending on progression of sperms. All the cellular debris will settle at the bottom of droplet and using a fine drawn pipette under dissecting microscope one can ensure that only the upper part of droplet is picked up. Especially useful for very low counts during ICSI.

Sperm Incubation with Pentoxifylline:
It is well known that adding pentoxyfylline to the semen will enhance the motility of the spermatozoa and improved pregnancy rates. Equal volumes of pentoxifylline solution is added to semen and centrifuged at 200 g for 5 minutes. The pellet is suspended in culture medium and centrifuge is repeated. The final pellet is suspended in 0.3-0.5 ml of culture medium and used for insemination immediately.

semen quality pic

Treatments
Intracytoplasmic Sperm Injection (ICSI) is a specialized form of in vitro fertilization (IVF) most often used for men who have poor sperm quality or low sperm counts. During an ICSI procedure, a single sperm is injected directly into each mature egg, instead of adding sperm to the dish containing the egg.

Who qualifies for ICSI?
ICSI is typically reserved for couples with severe male factor infertility. Previously, therapeutic donor insemination (TDI) or adoption was the only option for these couples. However, ICSI now provides couples with severe male factor infertility with the opportunity to conceive their own biological offspring.

The ICSI Procedure:
Female patients undergo the same ovarian stimulation and egg retrieval as those patients undergoing a routine IVF treatment cycle. The best quality eggs are selected
after careful analysis under a microscope. At around the same time as the egg retrieval, semen specimens are collected. A conventional semen analysis is performed to identify the healthiest sperm, which is the carefully prepared for injection directly into the egg. The sperm suspension is kept at 37°C in an incubator until the moment of injection. Approximately 16 to 18 hours after injection, the eggs are assessed for fertilization. If successful fertilization has occurred, the newly formed embryos are then transferred approximately 72-120 hours later.

Failed fertilization with ICSI:
This is an uncommon scenario. However, for cases with low or failed fertilization with ICSI, special techniques like oocyte activation with calcium ionophore or puromycin are implemented at our center to remedy this challenging problem.

Embryo Transfer procedure:
After fertilization, the embryos are cultured for a number of days in order to select the best ones. The number of embryos transferred depends on several factors including: patient age, cause of infertility, previous treatment outcomes, embryo quality, and the stage of development. A pregnancy test is usually done 10-12 days following the embryo transfer.



Leave a Reply