PGD ( Pre- Genetic Implantation Diagnosis )

Procedure

First requirement for establishing PGD procedure would obviously be optimization of Day 5 (blastocyst) cultures in the IVF lab. Once the lab is producing decent blastocyst formation rates on day 5 embryo cultures, setting up PGD will not be difficult. Next most important step would be to identify and select an outside Genetic Testing Laboratory that will perform FISH (for aneuploidy screening) or PCR (for single gene mutations analysis). You would need to choose a genetic laboratory in your geographical area that can accept your day-3 embryo biopsy slides via overnight courier service and send back the results to your lab on early day 5, so you can select the desired healthy embryos to put back into the patient coming for day 5 transfer. There are different sets of probes for chromosomal abnormalities that can be used (anywhere from 3 or 5 or 8 chromosomes can be tested, including X and Y). I am sure you have some local labs that perform PGD analysis on routine basis in Bombay that you have connections set up. Once this can be arranged at your end, rest will be easy to set up. Of course you need a reliable courier service that can pick up and deliver slides to the genetic lab even on weekends.  

As far as embryo biopsy is concerned, Zona hydrolysis using Acid Tryodes, partial zona dissection (PZD) or laser-assisted zona breaching can all be used easily for performing day 3 embryos biopsies. I prefer PZD. All embryos for PGD are produced by ICSI fertilization and should be at least 6-8 cell on Day 3 in vitro development. Standard micromanipulation used for ICSI can be adapted for PGD by changing the single tool holder to double tool holder

Equipments

• Inverted Microscope with Micromanipulation for ICSI and PGD
• All Standard Functional Embryology Lab equipment for Day 5 embryo Culture Facilities
• Micro tools for ICSI and PGD (Humagen or Cook)
• Timers
• Electric pipettors (Fisher or Eppendorf)
• P-20, P-200 and P-1000 micropipets (Ranin or Eppendorf)
• Slide Warmer
• Dissection Microscope with zoom facility (for fixation of blastomeres) like Nikon SMZ 1500
• Light Microscope with 10 X phase objective, slide holder clamp and coordinate locator (i.e., Olympus #CH2);
• 37 Degree Centigrade water bath.
• Micro centrifuge (Bench top)
• Refrigerators
• Tri gas incubator preferred but 6% CO 2 will work (a bench incubator will be ideal in addition to IVF lab incubators)
• Stripper pipettes (Mid Atlantic or similar)
• All standard culture dishes and plastic ware standard for IVF cultures

Consumables:

• Silanated Slides (CEL Associates)
• 1.5 ml Eppendorf tubes
• Organ Culture dishes
• 0.2 µl Syringe Filters
• 1 ml Syringes
• 3 ml Syringes
• 50-60 ml Syringes
• 15 ml Conical Tubes
• 50 ml Conical Tubes
• 1 ml, 5 ml and 10 ml Pipettes
• Transfer Pipettes
• Sterile Glass Pasteur Pipettes
• Pipette Tips for P-20 up to P-1000 (VWR or Ranin)
• Sharps Biohazard Container
• Paper towels
• Kimwipes or KayDry XL
• Disposable gloves
• Biopsy micro tools (holding, PZD and biopsy needles)

Reagents

• SigmaCote (Sigma, #SL-2) 
• Fetal Bovine Serum or Human Serum Albumin (Gibco)
• HCl (0.1 N stock) (Sigma, #2104)
• HPLC grade water (VWR, #MK679510)
• Tween-20 (Sigma, #P-9416)