SEMEN ANALYSIS

The most common reasons for laboratory semen analysis in humans are as part of a couple's infertility investigation. It is also commonly used for testing human donors for sperm donation.

Semen Analysis is the single most important test of male fertility, what we looking for?

  • Sperm count
  • Ability of sperm to swim (motility)
  • Velocity or forward progression of the sperm
  • Size and shape of the sperm (morphology)
  • Total semen volume
  • The liquefaction of the semen (the ability to go from normal gel-like state at ejaculation to a liquid state)

TRAIL WASH

Trail wash is necessary to assess the basic sperm parameters (i.e. count; motility & morphology) after removing the seminal fluid and dead sperms.
Sometimes the raw sperm’s parameters and post wash sperm’s parameters differ and it help us to categorise that sample for future treatment (IUI/IVF/ICSI).
It can be done with the help of Density Gradient Technique or Sperm Swim up Technique.

SPERM SLOW

Sperm Slow is a viscous medium containing hyaluronic acid, which is naturally occurring and biodegradable. Hyaluronic Acid (HA) acts as a "physiological selector" for spermatozoa prior to intracytoplasmic sperm injection (ICSI). As the sperm suspension mixes with Sperm Slow, the more mature and viable sperm slow down and the immature sperm keep moving.
It has been suggested that mature sperm possess less fragmented DNA than immature forms, and as well as helping to identify these Sperm Slow also makes the actual sperm injection procedure smoother.
That Injection of HA-bound spermatozoa (HA-ICSI) significantly improves embryo quality and implantation. The use of Sperm Slow does appear to lower the early pregnancy loss. This may be because there was a reduction in the DNA fragmentation.
In nature, human oocytes are surrounded by Hyaluronic Acid (HA), which is then involved in the mechanism of sperm selection. In fact, only mature spermatozoa which have extruded their specific receptors to bind to and digest HA can reach the oocyte and fertilize it. HA’s role as “physiologic selector” is now well recognized also in vitro: it has been demonstrated that spermatozoa able to bind HA in vitro are those that have completed plasma membrane remodelling, cytoplasmic extrusion and nuclear maturation. Furthermore, these HA-bound spermatozoa show low chromosomal aneuploidies and DNA fragmentation, and good nuclear morphology.

MICROFLUIDICS SPERM SORTING

Sorting of Sperms based on DNA damage made possible by Microfluidics Sperm Sorting Technology:
Microfluidics Sperm Sorting Technology can help filter sperms that are more likely to carry DNA damage from normal sperms. This technique works on the principle that Sperms with lower motility have higher DNA damage and motile sperms are less like to carry DNA damage.
Microfluidic sperm sorter separates morphologically normal sperms with high motility. Sperm selection in this method is done by two gravity driven laminar flows within a central microfluidic channels.
The Sperm Sorting Principle:
  • The Microfluidic Sperm Sorter Qualis (MSSQ) contains 4 chambers A, B, C and D
  • These four chambers are connected by two parallel channels A – D and B - C.
  • Media is added in chambers B, C, D and Laminar flow channels are allowed to form between the chambers.
  • Sperm sample is added to Chamber A.
  • The Laminar flow will help separate motile sperms form immotile sperms. The immotile sperms would passively flow in the direction of laminar flow A to D and thus collect in Chamber D. While the motile sperms that can swim will swim up into the chamber C.
  • After some time, only motile sperms would collect in chamber C while immotile sperms would collect in chamber D.

Why is Microfluidic sperm sorting Test recommended for IVF?

While the natural method of conception has an evolutionary advantage of screening damaged Sperms from normal sperms, IVF doesn’t have that advantage.
Human body has a way of screening out sperms with high DNA damage and preventing them from reaching the egg and passing on the damaged DNA to the embryo. This mechanism allows only the best sperm to reach the egg. A test tube baby however is created in a petri- dish, where the eggs and sperms are collected from the couple and just mixed up in a Petri-plate allowing fertilization to take place. This technique by passes the natural selection mechanism of human body making it equally likely for DNA damaged sperms to fertilize the egg. In such a case the embryos would be of poor quality.
Although ICSI, a widely used advanced IVF tool can help select morphologically normal embryos from abnormal sperms, it doesn’t screen for DNA damage. MSSQ however is rapidly gaining favour with IVF laboratories because it can sort immotile sperms from motile sperms there by giving them a way of screening DNA damage in sperms. As it has been established1 that sperms with higher DNA damage index are poor swimmers.

DNA Fragmentation Test - Flowcrtometry

The genetic integrity of the spermatozoan is essential for normal embryo development. A high level of DNA fragmentation in sperm cells may represent a cause of male infertility that conventional examinations - sperm concentration, motility analysis, morphology assessment - cannot detect.
  • High sperm DNA fragmentation does not appear to affect fertilisation or the first or second embryo cleavage stages
  • High sperm DNA fragmentation can affect embryo cleavage once the paternal genome is switched on, and subsequent blastocyst development
  • Men with poor semen parameters are more likely to have high DNA fragmentation
  • High sperm DNA fragmentation is also found in men with normal semen parameters

High Rates of Sperm DNA Fragmentation and Pregnancy

Embryos derived from sperm whose DNA is highly fragmented have a poor prognosis. Evidence suggests that this could result in initiation of apoptosis and mutations resulting in blastocyst arrest, miscarriage, abnormalities in the offspring and an increased susceptibility to childhood cancer. Protection against high DNA fragmentation may be afforded by younger oocytes which are much more efficient at DNA repair of defective sperm than older oocytes.

Causes of Sperm DNA Fragmentation

  • Oxidative Stress
  • Abnormalities in the regulation apoptosis
  • Defective topoisomerase
  • Infection
  • Leucocytospermia
  • Sperm cytoplasmic droplets
  • Febrile illness
  • Elevated testicular temperature
  • Diet
  • Drug use
  • Cigarette smoking
  • Exposure to environmental and occupational pollutants
  • Advanced age
  • Varicocoele

Indications for male patients who may benefit from the Test

  • Unexplained infertility
  • Arrested embryo development
  • Poor blastocyst development
  • Multiple failed IVF/ICSI treatment
  • Recurrent miscarriage in partner
  • Advanced age
  • Varicocoele
  • Poor semen parameters
  • Exposure to harmful substances
Initial reports suggest that DNA damage occurs at the post-testicular level, so that testicular sperm may have a healthier DNA integrity than ejaculated sperm. Furthermore, studies show that ICSI may be a more effective treatment than IVF for sperm with a high DNA fragmentation.

Surgical Sperm Retrieval (TESA / PESA)

In vitro fertilization can be performed either in situations where surgery is unlikely to succeed, or as a first option. Males, who have had a vasectomy, undergo sperm collection under local or general anesthesia from either the epididymis (adjacent to the testicle) or the testicle itself.
Microinjection of a single sperm into oocytes (ICSI) achieves a high fertilization rate (60-70%) and implantation rate of up to 30% on transfer of embryos to the uterus. Sperm collection can be achieved in virtually all men who had been fertile before vasectomy no matter how long after vasectomy.

What is TESA / PESA?

Testicular Sperm Aspiration (TESA) and Percutaneous Epididymal Sperm Aspiration (PESA)) are procedures used in the management of obstructive and no obstructive azoospermia (no sperm in the ejaculate). A fine needle is passed into the tubes leading out of the testicles, or the testicles themselves, to retrieve sperm. The retrieved sperm are injected directly into an egg (ICSI) in a similar fashion to sperm retrieved from the ejaculate. This micromanipulation of a single sperm into an egg is the treatment of choice for couples with severe male factor infertility. However sperm return following reconstructive surgery occurs in approximately 50% of cases, and in these, pregnancy may not result because of the presence of sperm antibodies. The testes do not usually stop producing sperm, which become “pent-up” in the epididymis, located on the side of the testis, leading to the vas deferens. There may also be a relationship between the interval of time since the vasectomy and pregnancy prospects, with the possibility of lower success rates if the vasectomy was performed eight or more years previously. PESA and TESA offer a simple, quick, cheap and comfortable way of accessing sperm for use in assisted reproductive procedures post vasectomy, without resorting to vasectomy reversal. Until recently there were only a few ways for men with irreversible obstructions to become biological fathers. One way was for the man to undergo a complex open surgical operation on the scrotum using a magnifying microscope to allow for retrieval of sperm from the epididymis adjacent to the testis. The other was for the couple to use donor sperm.

What is the success rate of these procedures?

Treatment with IVF/ICSI/PESA carries a success rate similar to routine IVF per treatment cycle. This has been a tremendous advance for men with both obstructive and non-obstructive oligospermia. Prior to the development of these techniques very little help was available for men with these problems.

Who are these procedures most suited to?

To summarize, IVF and ICSI after PESA is of value for:
  • Men that have had a vasectomy or failed vasectomy reversal.
  • Those born with an absent vas deferens (often carriers of a genetic condition called cystic fibrosis).
  • Those with other irreversible obstructions of the genital tract.
  • Men with primary testicular problems, with deficient spermatogenesis.

Post Vasectomy Check

Post-vasectomy testing is carried out to check that a vasectomy has been successful. A vasectomy is a surgical procedure undertaken to render a man sterile (unable to father children). The procedure involves cutting the tubes (vas deferens) that carry sperm from the testicles, preventing it from mixing with semen, the fluid produced in the seminal vesicles and then ejaculated.
The success or failure of a vasectomy can be determined after the operation by testing the semen ejaculated by the patient to see whether it contains sperm. If the vasectomy has been successful, there will be no sperm in the semen. Testing for sperm after a vasectomy is called post-vasectomy semen analysis (PVSA).