Invitro-fertilization (IVF)

Invitro-fertilization (IVF)
Oocyte Collection,Maturation & Fertilization
Reproductive biotechnology

1. Invitro-fertilization (IVF) O ocyte Collection, Maturation & Fertilization Dr Muhammad Awais Tatari

2. IVF In Vitro Fertilization (IVF) is the process of creating embryos from oocytes (unfertilized egg cells) by fertilizing them with semen in a Petri dish.

3. Oocytes are first collected from the ovaries of donors by ultrasound-guided follicular aspiration. They are then matured in a Petri dish and fertilized 20-24 hours later. Conventional or sexed frozen semen may be used for fertilization.

4. Oocyte Collection Immature oocytes in the form of cumulus-oocyte complexes (COC; figure) can be collected from live donor animals or directly from ovaries obtained from a abattoir.

5. There are 2 main ways in which oocytes can be collected from live donors, S urgically by laparotomy and aspiration using a syringe and needle. T ransvaginal ovum-pick-up techniques as shown.

6. Transvaginal ovum-pick-up technique In cattle, the donor female is restrained in a suitable holding chute and administered an epidural block A convex ultrasound 5-MHz sector transducer is fitted onto the distal end of a specially fitted 500-mm plastic handle to visualize the ovaries on the ultrasound monitor. The plastic handle is inserted into the vaginal canal, and then the ovary is grasped per rectum and placed against the transducer. Follicles are identified as black (hypo echoic) circular shapes on the monitor screen.

7. An 18-gauge, 550 or 600-mm long needle is inserted through the needle guide in the plastic handle. This needle is connected to a suction pump by means of polyethylene tubing, passing into a 50-ml conical-shaped test tube for collection of the follicular fluid containing the oocytes. The flushing medium used for this procedure is phosphate-buffered saline (PBS) with 10 % bovine serum, antibiotics and heparin. Using this aspiration method, 60 to 70% of the medium to large-size follicles punctured result in oocytes recovered, with an average of 3 to10 oocytes per non stimulated donor female.

8. Ovaries from abattoir Remove ovaries from the reproductive tract of cows immediately after internal organs are extracted from the carcass and place the ovaries into one of the saline containers.   Then , transfer ovaries to the second container and place the containers in the thermos. Transport the ovaries to the lab immediately. Ovaries are generally transported to the laboratory in physiological saline that contains some form of antibiotic to help prevent bacterial contamination at approximately 22-24 ºC. The best results are obtained when oocytes are collected within 4-6 hrs after slaughter.

9. Oocytes can be collected from abattoir derived ovaries by following methods. A spiration using a syringe and needle By “slashing” the surface of the ovary with a scalpel blade and collection of oocytes into a beaker by cell strainer method.

10. Oocyte recovery by slashing Slashing of ovaries to recover oocytes ovary to hold the ovary firmly in place. The excess tissue from the ovarian stalk is removed. O vary is held above the beaker and 2-3 mm incisions made in a downward direction with a rapid but firm movement across follicles.

11. Cell Strainer Method Add ~75 ml oocyte collection medium (OCM) to a sterile 400 ml beaker . OCM contain Hank’s salts, L-glutamine and HEPES ( hydroxyethyl piperazineethanesulfonic acid ) . Add NaHCO3 to maintain media ph. Submerge ovary into OCM and swirl vigorously. One can anticipate a yield of about 10 usable oocytes/ovary. Sometimes as many as 20-30 oocyte can be obtained.

12. After slashing ovaries, the medium containing the oocytes is poured into sterile 50-ml centrifuge tubes .   Place the tubes containing the oocytes and media into a water bath and allow oocytes to settle to the bottom of the centrifuge tubes for about 5 minutes. While oocytes are settling, fill a 10 ml syringe with warmed OCM and pour ~ 2 ml OCM into an integrid petri-dish to prevent oocytes from sticking to the bottom of the plate. Also , pour OCM into 3 wells of an X-plate. 

13. Use a forceps to hold a 100 μm cell strainer in position over a sterile 100 ml beaker. Using a plastic pasteur pipet and sterile technique, aspirate the pellet of oocytes at the bottom of each tube. Slowly pour the aspirated pellet into the cell strainer . Up to 3 tubes of oocytes can be processed through a single filter. Immediately with a 10 ml syringe fitted with an 18 g needle filled with OCM, rinse the oocytes into an integrid petri-dish. Place the integrid dish on a plate warmer until ready for searching

14. Collect cumulus oocyte complexes (COCs) as fast as possible to prevent adverse effects of cold shock. Only COCs which have at least a couple of layers of compact cumulus cells and an evenly granulated cytoplasm with no clear spaces should be used for subsequent steps. After completing COC search, transfer oocytes from one well to the next leaving all debris behind This can be completed using a microdispensor pipet for handling oocytes. After oocytes have been cleaned of debris, transfer groups of 10 to a 50 ml microdrop of OMM

15. Grading of oocytes Grade A: Compact cumulus‑oocyte‑complexes (COCs ) with an unexpanded cumulus mass having ≥ 4 layers of cumulus cells, and with homogenous evenly granular ooplasm Grade B: COCs with 2-3 layers of cumulus cells and a homogenous evenly granular ooplasm Grade C: Oocytes partially or wholly denuded or with expanded or scattered cumulus cells or with an irregular and dark ooplasm

16. Oocyte Maturation Following collection, cumulus-oocyte complexes are washed several times P laced into maturation medium for a specified amount of time depending on the species .

17. During this culture period, the oocyte will resume meiosis and arrest at metaphase II so that it is ready for fertilization. The COC also undergoes other morphological changes during maturation, including the expansion of the cumulus cells .

18. M aturation medium TCM-199 Maturation medium will include several components including N utrients (pyruvate, glucose, glutamine, serum) H ormones (estrogen, LH, FSH) A ntibiotics (penicillin/streptomycin or gentamicin)

19. Collected COC are generally matured in 50 μL microdrops (10 COC/drop) or in wells of a 4-well plate (40-50 COC/well) overlaid with mineral oil to help prevent evaporation of the maturation medium. Once placed into maturation drops, COC are placed in an incubator set at 38.5-39 ºC for the desired amount of time depending on the species .

20. Fertilization The in vitro fertilization process can be divided in three main steps COC washing Sperm purification Fertilization

21. a. COC washing Necessary so that hormones, nutrients and metabolites present in the maturation microdrop are not carried over to the fertilization drop. Procedure 1. Transfer COCs from each maturation microdrop to the X-plate containing the buffer HEPES-TALP ( hydroxyethyl piperazineethanesulfonic acid   ) ( Tyrode's albumin lactate pyruvate) 2. Transfer 10 COCs from the X-plate to each well of the 4-well fertilization plate .

22. b. Sperm purification Necessary so that sperm cells can be washed from the extender + cryopreserves ( if frozen is used ) / seminal plasma (if fresh semen is used )

23. Methods Three methods of sperm purification Percoll Purification Sperm Swim-up Glass- wool Filtration

24. Percoll Purification Method Place 1.5 ml of 90% Percoll and 1.5 ml of HEPES-TL to one 15 ml conical tube. Mix to make a solution of 45% Percoll . In another 15 ml conical tube, add 3 ml of 90% Percoll . Make a Percoll gradient (45% over 90%) by slowly layering the 90 % Percoll on the bottom of the tube containing the 45% Percoll using a plastic Pasteur pipet If using frozen semen thaw enough straws of semen in a citothaw for 45-60 seconds or in another thermo with water pre-warmed to 37 C. C ut the tip of the straw with a scissors and expel contents of the straw onto the top of the Percoll gradient. Care must be taken so that the gradient is not disturbed and the semen lie on top of the 45% layer.

25. Place the conical tube containing the semen and Percoll gradient into a centrifuge carrier that has been pre-warmed to 38.5°C, and centrifuge at 1000 rpm for 10 min. After centrifugation, collect sperm pellet from the bottom of the conical tube.

26. Place the sperm pellet into a 15 ml conical tube containing 10 ml Sp-TALP(sodium pyruvate) and place in a warm centrifuge carrier before centrifuging for 5 min at 200rpm. Remove the supernatant with a Pasteur pipet while being careful not to disturb the pellet. Use IVF-TALP media to dilute the sperm solution (Usually 2 ml of IVF-TALP( Tyrode's albumin lactate pyruvate) is enough to bring the content of 3 semen straws to desired 26 x sperm cells/ml).  

27. Sperm Swim-up Procedure Thaw 6 to 8 straws of frozen semen in the cito -thaw for 60 seconds. Combine contents of straws in 5 ml SP-TALP. Place sample into the incubator (38.5°C) for 5 minutes . Centrifuge semen ( 200 rpm; 5 min) and discard all but the bottom 1 ml of supernatant. Prepare 4 to 5 test tubes containing 1 ml SP-TALP. Add approximately 250 μ l of sperm suspension very slowly to the bottom of each tube using a 20 gauge needle and 1 ml syringe. Place tubes in incubator (38.5°C) for 1 h. At the end of sperm swim-up, aspirate the top 800 μ l from each tube and combine samples. Centrifuge (1000 rpm) the combined sample for 5 minutes. Discard all but the bottom 500 μ l of supernate .

28. c. Fertilization At this point, sperm cells can be added to the wells containing the COCs so that fertilization can take place. Procedure Add 25 µl sperm preparation (the IVF-TALP contains heparin which will help in capacitation of the sperm cells); 25 µl PHE mix into each well (PHE is the acronym for penicillamine, hypotaurine and epinephrine which are molecules know to induce sperm hyper activation ); Place the 4-well fertilization plates in a incubator (5% CO2 in air) at 38.5C for 8-20 h.

29. Intracytoplasmatic sperm injection (ICSI) ICSI is used when the male has poor sperm quality (low concentration, motility etc .) In this technique, fertilization is assisted by injecting one selected sperm cell into one mature oocyte.

30. Procedure ICSI is generally performed following a transvaginal oocyte retrieval procedure to extract one to several oocytes. Sperm from epididymis or testicle. The procedure is done under a  microscope  using multiple micromanipulation devices ( micromanipulator ,  microinjectors  and  micropipettes ).

31. A holding pipette stabilizes the mature  oocyte  with gentle suction applied by a micro injector. From the opposite side a thin, hollow glass micropipette is used to collect a single sperm, having immobilized it by cutting its tail with the point of the micropipette . The oocyte is pierced through the  oolemma  and directed to the inner part of the oocyte (cytoplasm). The sperm is then released into the oocyte. After the procedure, the oocyte will be placed into cell culture and checked on the following day for signs of  fertilization .

32. Thanks