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Azoospermia is defined as an absence of spermatozoa in the ejaculate (WHO, 2010), and it is classified as either obstructive or non- obstructive. Obstructive azoospermia is the result of a blockage in the male reproductive tract; therefore, sperm production in the testicle is normal, but the sperm are trapped inside the epididymis.

The absence of sperm in the ejaculate can be due to an abnormality in the epididymis, vas deferens, or ejaculatory ducts. The obstruction can be caused by many factors, such as infection, surgery (vasectomies), or an absent vas deferens. Non- obstructive azoospermia is the result of severely impaired or non-existent sperm production. Approximately 10% of male factor infertility has been attributed to complete azoospermia, which presents either in an obstructive or a non-obstructive form (Gardner et.al, 2001).

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There are four different harvesting techniques that can be used to obtain sperm for in vitro fertilization (IVF). Each procedure has advantages and disadvantages and is described below. Sperm-harvesting techniques used to obtain sperm from men with obstructive azoospermia are described in the following paragraphs.

MESA (microsurgical epididymal sperm aspiration): Using an intraoperative stereomicroscope, a cut is made in a single tubule of the epididymis. The contents therein, which should contain sperm, are then aspirated with a fine-gauge needle.

An embryologist examines the sample for the presence of motile sperm. If no motile spermatozoa are found at the first site, the maneuver is repeated. Typically, only a few microliters of epididymal fluid are retrieved because sperm are highly concentrated in the epididymal fluid (approximately 1×10 sperm/μl).

A MESA approach should provide more than adequate numbers of sperm for immediate use, as well as for cryopreservation. As reported by Dr. Shlegel and colleagues, who used MESA and ICSI in a group of men with obstructive azoospermia, clinical pregnancies were detected by a fetal heartbeat in 75% (57/76) of attempts, and healthy deliveries occurred in 64% (49/76) of attempts (Schlegel, et.al, 1994.)

PESA (percutaneous epididymal sperm aspiration): This simple technique can be performed under local anesthesia or mild sedation. Unlike MESA, where the surgeon is able to visualize the exposed epididymal tubules, PESA is a blind procedure. A fine needle is used to puncture the epididymis, and sperm are aspirated and given to an embryologist to examine. Again, if no motile spermatozoa are found at the first site, the surgeon will repeatedly draw samples for an embryologist to examine. TESA (testicular sperm aspiration): For obstructive azoospermia patients in whom sperm cannot be found in the epididymis, it is always possible to find sperm in the testis. TESA may be performed as a primary harvesting technique if there is an absent.

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epididymis or severe epididymal fibrosis. This blind procedure is usually performed under local anesthesia or mild sedation. A wide-bore needle is pushed into the testis through the skin, suction is applied, and the contents of the needle are flushed into a petri dish containing culture media. Then, an embryologist examines the aspirate for motile or immotile spermatozoa with a stereomicroscope. Using fine needles, sperm are released from within the seminiferous tubules, where sperm are produced, and they are then dissected from the surrounding tissue. Next, the embryologist determines whether there is a sufficient number of sperm for treatment or cryopreservation for future use. If not, several more needle biopsies will be attempted by the surgeon in each testis to obtain a sufficient amount. TESE (testicular sperm extraction): This technique is reserved for men with non- obstructive azoospermia. This procedure is also routinely carried out under mild sedation or local anesthesia. The surgeon exposes a small area of the testis by making an incision in the scrotum. The seminiferous tubules are forced out through the incision by gently squeezing the testis, the tubules are excised and the biopsy sample is placed in a petri dish and given to an embryologist to examine.

An approximately 500-g biopsy sample is rinsed in culture media to remove red blood cells and divided into small pieces, using fine needles, under a stereomicroscope to check for sperm. If no sperm are found, the surgeon will continue taking biopsies from different areas of the testis, and the embryologist will continue to examine the samples.

Sperm can usually be easily obtained from infertile men with obstructive azoospermia for intracytoplasmic injection (ICSI); however, individuals who exhibit non-obstructive azoospermia have historically been difficult to treat. Examining biopsies for sperm under a microscope can be very time-consuming and difficult. Finding sperm in the testicular tissue can be a laborious 2- to 3-hour process depending on the degree of sperm production and the etiology of testicular failure, especially in men with partial testicular failure.

For men with non-obstructive azoospermia, some IVF clinics ask that a TESE be performed a day before the egg retrieval process because they believe culturing the testicular tissue in an incubator overnight will help sperm to acquire motility. When sperm are not found after surgery, the couple must decide whether they want to cancel the egg retrieval, cancel the fertility cycle, or proceed with donor sperm. Alternatively, oocyte collection can be performed even if no sperm are found in the TESE process and there is no donor sperm back-up.

In this case, eggs can be cryopreserved to enable the couple to make future fertility decisions. Importantly, this new option is available because vitrification has significantly improved oocyte survival rates. For men who must undergo sperm- harvesting procedures to recover testicular or epididymal sperm, it is now possible to cryopreserve the excess sperm for future cycles if needed, thus eliminating the need for repeat surgery. However, some clinics still prefer to use freshly retrieved epididymal or testicular sperm instead of frozen-thawed samples.

INTRACYTOPLASMIC SPERM INJECTION (ICSI)

Two major breakthroughs have recently occurred in the area of male infertility.

The first is the development of the intracytoplasmic sperm injection (ICSI) technique for the treatment of male factor infertility due to severely abnormal semen quality. The second is the extension of ICSI to azoospermic males and the demonstration that spermatozoa derived from either the epididymis or the testis are capable of producing normal fertilization and pregnancy. ICSI is now the primary technique used to treat male-factor infertility. ICSI has achieved consistent fertilization and high pregnancy rates in couples with suboptimal spermatozoa (David, et.al, 2011).

A concern associated with the use of suboptimal spermatozoa for ICSI is the potential for transmitting the genetic abnormalities responsible for male infertility to the offspring (Gardner et.al, 2004). Despite this concern, ICSI is the customary therapeutic approach, although genetic screening and patient counseling are still imperative for these patients. In IVF clinics, surgically retrieved sperm samples are most often prepared for use before a patient progresses through an ICSI cycle. This is a safe course of action in cases of obstructive azoospermia, but in cases of non-obstructive azoospermia, it is beneficial to undergo the surgery before the cycle because the female has already been stimulated with medication. At that time, a couple would have to decide whether to proceed with donor sperm or cryopreserve the oocytes for use at a later time. Epididymal aspirates are relatively easy to prepare in the lab, either for ICSI or for cryopreservation.

Typically, an embryologist washes the sample with culture media to dilute any epididymal fluid or blood contamination. The most common issues are a high level of red blood cells, an absence of observed sperm, poor sperm motility, and slow progression (David et.al, 2011).

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REFERENCES
  1. WHO. WHO Laboratory Manual for the Examination and processing of human semen: 2010 World Health Organization.
  2. Gardner DK, Weissman A, Howles CM, Shoham Z. Textbook of Assisted Reproductive Techniques: Laboratory and Clinical Perspectives, First Edition: Martin Dunitz. 2001
  3. Schlegel PN, Berkeley AS, Goldstein M, Cohen J, Alikani M, Adler A, et al. Epididymal micropuncture with in vitro fertilization and oocyte micromanipulation for the treatment of unreconstructable obstructive azoospermia. Fertil Steril. 1994;61(5):895–901. [PubMed ]
  4. Gardner DK, Lane M, Watson AJ. New York: Oxford University Press; 2004. A laboratory guide to the mammalian embryo; pp. p.76–85.
  5. David K. Gardner B R M B R, Tommaso Falcone, editors. , Human Assisted Reproductive Technology: Future Trends in Laboratory and Clinical Practice 2011Cambridge: Cambridge University Press


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