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Andrology Lab

Andrology Lab

At ManipalAnkur, we are the pioneers in the field of Andrology. Andrology is the medical specialty that deals with male health, particularly relating to the problems of the male reproductive system and urological problems that are unique to men. At ManipalAnkur we offer several diagnostic tests that can aid this treatment process, including semen analysis, sperm penetration assay, and an anti-sperm antibody test.

  • ASA
  • DNA Fragmentation Index
  • ROS
  • Annexin V Staining
  • Sperm Processing
  • Sperm Cryobanking

Advanced Semen Analysis - ASA

    Manipal Ankur'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 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.

DNA Fragmentation Index - Science and Physiology

  • DNA Fragmentation Index (DFI)

    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. 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.

    Advantages of the Sperm DNA Fragmentation test

    This test provides a reliable analysis of sperm DNA integrity that may help to identify men who are at risk of failing to initiate a healthy ongoing pregnancy. Information about sperm DNA integrity may help in the clinical diagnosis, management and treatment of male infertility and may be of prognostic value in assessing outcome of assisted conception treatment.

    High Rates of Sperm DNA Fragmentation and Pregnancy

    Normal, healthy pregnancies do occur in couples where the male partner has a high percentage of sperm with fragmented DNA, although the chances are significantly reduced, as the percentage of sperm bearing low levels of DNA fragmentation is much lower. 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. Couples undergoing fertility treatment where sperm DNA fragmentation is high have a better prognosis if the female partner is young.

Reactive oxygen species assay

  • 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.

    Accurate assessment of ROS levels and, subsequently, OS is vital, as this will help clinicians both elucidate the fertility status and identify the subgroups of patients that respond or do not respond to these therapeutic strategies.

Annexin V staining

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.

Sperm Processing for Intrauterine Insemination (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.

Sperm Cryofreezing - Semen Banking

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

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),
is not able to 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.

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