Technology

Andrology Labs -

Andrology Test

Manipal Fertility Andrology laboratory uses the Advanced Semen Analysis (ASA) system. Ours is the first system in the country. This automated system provides a detailed analysis of density, percent motility, curvilinear and straight-line velocities beat cross frequency and amplitude of lateral head displacement of spermatozoa. Besides the computerized report, information on morphology, number of immature cells and white blood cells and other routine assessment is provided. This powerful tool helps us in counseling the couple as to which method of treatment is best suited for them.

Couples undergoing preliminary work up for infertility, patients with male factor infertility, unexplained infertility and repeated failed attempts at AdvancedReproduction benefit from ASA.
ASA system is a necessary instrument to standardize all the parameters of sperm analysis, and also to approve the internal and external quality controls.

Computer-assisted techniques are most often used for the assessment of sperm concentration and motility following the W.H.O criteria. To assess motility, different types of spermatozoa have to be analysed: “a” (fast progressive motility), “b” (low progressive motility), “c” (non progressive motility) and “d” (immotile).

For the morphology analysis there are different options of classification criteria such as W.H.O, Kruger or user defined and also different staining solutions can be used for a better visualization and capture of images in different species.
Nowadays ASA systems are not only considered as an eventuality but as a reality.


computer_assisted_semen_analysis2

 

  • Sperm concentration in mill/ml
  • Concentration of progressively motile sperm in mill/ml
  • WHO motility grades a, b, c, d in percent (old classification)
  • WHO motility progressive / local / immotile in percent (new classification)
  • VCL (curvilinear velocity) in μm/sec
  • VSL (straight-line velocity) in μm/sec
  • VAP (average path velocity) in μm/sec
  • WOB (wobble = VAP/VCL)
  • ALH (amplitude of lateral head displacement) in μm


Andrology
Display / calculation of:

  • Exact morphometry data for each individual sperm
  • Total number of analyzed sperm cells
  •  normal forms
  •  head abnormalities
  • midpiece abnormalities
  •  tail abnormalities
  •  abnormalities of head and midpiece
  •  abnormalities of head and tail
  •  abnormalities of midpiece and tail
  •  abnormalities of head, midpiece and tail


Advanced Semen Analysis System

Manipal Fertility’s andrology laboratory uses the Advanced Semen Analysis (ASA) system. Ours is the first system in the country. This automated system provides a detailed analysis of density, percent motility, curvilinear and straight-line velocities beat cross frequency and amplitude of lateral head displacement of spermatozoa. Besides the computerized report, information on morphology, number of immature cells and white blood cells and other routine assessment is provided. This powerful tool helps us in counseling the couple as to which method of treatment is best suited for them. Couples undergoing preliminary work up for infertility, patients with male factor infertility, unexplained infertility and repeated failed attempts at Advanced Reproduction benefit from ASA. ASA system is a necessary instrument to standardize all the parameters of sperm analysis, and also to approve the internal and external quality controls. Computer-assisted techniques are most often used for the assessment of sperm concentration and motility following the W.H.O criteria. To assess motility, different types of spermatozoa have to be analysed: “a” (fast progressive motility), “b” (low progressive motility), “c” (non progressive motility) and “d” (immotile). For the morphology analysis, there are different options of classification criteria such as W.H.O, Kruger or user defined. Also, different staining solutions can be used for a better visualization and capture of images in different species. Nowadays, ASA systems are not only considered as an eventuality but as a reality.


Sperm DNA Fragmentation Test and Treatment

The primary function of a sperm is to deliver male DNA to the egg, and therefore the quality of the DNA delivered is of clear importance to the developing embryo and pregnancy success. Standard semen analysis generally provides information about the quality of sperm as a delivery vessel (i.e., motility, concentration, morphology) but does not directly assess what is being delivered, namely the DNA. One way of assessing the quality or “integrity” of sperm DNA is by measuring the fragmented sperm DNA (DNA breaks). The quantity of breaks is reflected by the amount of DNA that exists as a single strand (ssDNA) rather than in the intact double strand form (dsDNA).


Sperm quality is dependent on the amount of damage to the sperm DNA or DNA fragmentation. Sperm DNA fragmentation has little or nothing to do with the parameters that we measure on the routine semen analysis. However, the degree of DNA fragmentation correlates very highly with the inability of the sperm to initiate a birth regardless of the technology used to fertilize the egg such as insemination, IVF or ICSI.
By testing for sperm DNA fragmentation, many cases of formally “unexplained” infertility can now be explained. To test for sperm DNA fragmentation; we use the Sperm Chromatin Structure Assay (SCSA). A stress is applied (low pH). The sperm are then labelled with a special orange coloured dye that only attaches to the ends of broken DNA within the sperm cell. If the DNA is intact then no dye will attach to the sperm. The sperm are passed single file by a beam of light that hits the dye inside the sperm cell and reflects light at a specific wavelength causing the sperm to appear either orange (damaged) or green (normal). A computer counts the percentage of green versus orange-labelled sperm and software allows for creation of a graphic plot of the percent of damaged sperm giving an index known as the DNA fragmentation Index (DFI).
A normal sample has less then 15% of the sperm with DNA damage. Men with poor fertility potential have greater then 30% of their sperm damaged. The causes of high DNA fragmentation are those same causes of male factor infertility that we have known about for years such as chemical/toxin exposure, heat exposure, varicocele, infection, age, smoking, testicular cancer, radiation, and anything that increases the free radical levels in the semen among a list of many other things. It is very important to understand that sperm DNA fragmentation can change with time and it can be improved in many cases. The goal of a male factor evaluation is to seek out the causes of poor sperm quality and try to correct them so conception can occur naturally or to improve the sperm quality for IVF and maximize the chances of success. In situations where DFI can’t be improved there is evidence to suggest that removing the sperm directly from the testicle via biopsy and using it with ICSI may lead to better outcomes then using poor quality ejaculated sperm.
All men with an abnormal semen analysis are candidates for this test as well as men with normal semen analyses who have failed IVF for unexplained reasons. Those couples using egg donors or surrogates may also benefit from screening prior to going thru the procedures because the effort and costs are so great. Men with poor DFI should have a male factor evaluation including a physical examination by a male reproductive specialist.


Cryo Freezing Sperm

We routinely have a back-up semen sample frozen for couples who are undergoing treatment in case of inability to give a sample on the day of the IUI, IVF or ICSI. Men undergoing orchidectomy /cancer treatment prior to radiation and chemotherapy can also avail of our cryopreservation facilities. Those who are undergoing ICSI are encouraged to freeze their sperm so that we have adequate number of sperm at the time of ICSI. We routinely cryopreserve testicular and epididymal sperm, so that repeated attempts at obtaining fresh epididymal and testicular sperm may be avoided.
Donor Insemination

Sperm Freezing

In this case the sperm is contributed by a donor male whose identity is withheld. The procedure for placing of sperms into the female uterus is same as for intra uterine insemination. The following interesting points may be noted:

  • The female is the biological mother and genetic contributor, but the husband is not the biological father, but for all other concerns (psychological and legal), he is the accepted father. He is involved in the pregnancy preparation right from the pre pregnancy counselling to insemination to the pregnancy, delivery and rearing of the child thereafter.
  • The procedure is simple and no surgery is involved in insemination.
  • The procedure may be treated as adoption right from conception with one partner being sharing the genes with the full support and agreement of the husband. Both the husband and wife must be attitudinally ready to accept an anonymous sperm. Presently there is a debate whether the child must be informed of his biological father after he / she reaches adulthood.
  • With the increasing spread of HIV and other sexually transmitted diseases (STD, the importance of using properly tested and quarantined semen, for the treatment of infertility, has gained great importance. In most developed countries, the use of fresh untested semen for donor insemination has been discontinued to prevent the spread of these diseases through insemination procedures. These samples are screened for HIV, hepatitis B and C and then quarantined in deep freeze (liquid nitrogen) to ensure safe donor insemination. Donor semen is used only after re-screening after 6 months for HIV. Stringent criteria are used to select donors- for e.g. blood cross match is one such to prevent Rh incompatibility. All the records of semen samples and donors are meticulously maintained under strict confidentiality.

Therapeutic donor insemination is indicated in couples where the male partner:

  • Is not able to produce sperms at all ( irreversible azoospermia, severe oligospermia),
  • Does not have the right quality of sperms (poor motility and abnormal morphology), or
  • Has known genetic disorders (Huntington’s disease), haemophilia, non correctable ejaculatory dysfunctions, as a reproductive option after radio or chemotherapy for carcinoma testis, and in severely Rh sensitized Rh negative female partner with a Rh positive male partner.


The plasma membrane is one of the key structures in spermatozoa of infertile men displaying apoptotic feature. When the cell membrane is disturbed, phospholipid phosphatidylserine (PS) is translocated from the inner to the outer leaflet of the plasma membrane. This is one of the earliest signs of apoptosis and can be monitored by the calcium-dependent binding of annexin V. Annexin V is characterized by high affinity for PS and does not have the ability to pass the intact sperm membrane. Therefore, annexin V binding to spermatozoa characterizes disturbed integrity of the sperm membrane.
Colloidal super-paramagnetic microbeads (~50 nm in diameter) conjugated with annexin V separate the dead and apoptotic spermatozoa by magnetic-activated cell sorting (MACS). Cells exposing PS bound to these microbeads (annexin positive) are enriched to high extent within a column containing iron balls when placed in a very strong magnetic field. Cells with intact membranes remain unlabelled (annexin negative), and pass freely through the column. The binding of paramagnetic annexin V microbeads (ANMB) during MACS is an effective method to eliminate spermatozoa at early apoptotic stages from fresh and cryopreserved samples.
Magnetic-activated cell sorting (MACS) is used to provide a high-quality sperm fraction and as sperm preparation technique prior to assisted reproduction. Separating a distinctive population of non-apoptotic spermatozoa with intact membranes and subjecting it to IVF or ICSI is a step further in optimizing the outcome of assisted reproduction.


There is a dynamic interplay between pro- and anti-oxidant substances in human ejaculate. Reactive Oxygen Species (ROS)in low, controlled levels in the extracellular space play an important physiological role, modulating gene and protein activities vital for sperm proliferation, differentiation, and function. Excessive Reactive Oxygen Species (ROS) generation can overwhelm protective mechanism and initiate changes in lipid and/or protein layers of sperm plasma membranes. Additionally, changes in DNA can be induced.

Oxidative Stress (OS) is defined as a cellular condition associated with an imbalance between the production of free radicals, mainly ROS, and their scavenging capacity by antioxidants. When the production of ROS exceeds the available antioxidant defence, significant oxidative damage occurs to many cellular organelles by damaging lipids, proteins, DNA, and carbohydrates, thus ultimately leading to cell death. The OS-induced sperm damage has been suggested to be a significant contributing factor in 30–80% of all cases of male infertility. The generation of ROS can be exacerbated by environmental, infectious, and lifestyle aetiologies.
Luminol is used for quantification of redox activities of spermatozoa in the laboratory.

Reactive Oxygen Species (ROS)


Sperm Processing For IUI

Sperm washing is the process which prepares a semen sample for an intrauterine insemination (IUI). For an IUI to be performed, the semen sample must be washed free of debris, white blood cells, and prostaglandins, which can cause the uterus to contract. The processing also removes dead sperm and concentrates the sperm into a small volume which can easily be handled by the uterus.
The semen sample is processed by washing, centrifugation and migration. A suitable culture medium is used. The final migration is done under stringent culture conditions using CO2 incubation. The spermatozoa thus obtained are free of debris and bacteria, are energetic and improve the success rates of intrauterine insemination. We have a special room with privacy for semen collection. We also prepare the enriched sample for other clinics so that they can perform the insemination in their clinic. Women with ovulatory dysfunction, treated endometriosis with patent tubes, luteal phase defects, cervical factor infertility and polycystic ovarian disease can try IUI first before trying the more advanced techniques of assisted reproduction. Men with slightly compromised semen analysis parameters can benefit from IUI.
Magnetic-activated cell sorting (MACS) is used to provide a high-quality sperm fraction and as sperm preparation technique prior to assisted reproduction. Separating a distinctive population of non-apoptotic spermatozoa with intact membranes and subjecting it to IVF or ICSI is a step further in optimizing the outcome of assisted reproduction.