IVF IVEP

In-Vitro Fertilization or In Vitro Embryo Production (IVF/IVEP) Amit Sharma (Associate Professor) Veterinary Gynaecology & Obstetrics College of Veterinary & Animal Sciences, CSK HPKV, Palampur Himachal Pradesh India 176 062 E-mail [email protected]; [email protected] Introduction Ovum pick up (OPU) In Vitro Maturation (IVM) In Vitro Fertilization (IVF) In Vitro Culture (IVC) Embryo Transfer (ET) In Vitro Fertilization became entirely In Vitro systems, called “In Vitro Embryo Production”(IVEP). In-Vitro Fertilization In Vitro Fertilization (IVF) is the process of fertilization of oocyte by a sperm in a test tube, outside the animal body is known as in vitro fertilization (IVF). For effective or successful IVF, it is imperative to prepare or capacitate sperms, so that they can gain fertilization potential, besides in vitro maturation of oocytes. Embryos can be derived by IVEP from;  Cow that do-not respond properly to superovulatory treatments.  Cows have abnormalities in their reproductive tracts. Significance  Conservation of germplasm of genetically superior individuals and endangered species  To unreveal different physiological, biochemical, immunological, developmental and other changes occurring during maturation of oocytes  To determine and standardize essential requirements for IVF  To study oocyte as well as embryo metabolism  To obtain large no. of oocytes for scientific investigation or for subsequent transfer to recipients  Can fertilize multiple donors with a single straw of semen  There is less cost for boarding per procedure for IVF (because of short interval time period)  Reduces the duration of progeny testing programme  Most cost effective on donors that either failed to produce or produce low number of embryos per collection in ET  Can be used on healthy or clinically infertile cows that are not responding to conventional ET IVF Steps Involved in IVF  In vitro maturation of oocytes and its evaluation  In vitro sperm capacitation  Sperm oocyte co-culture and its evaluation Ovum pick up (OPU) /Abattoir ovaries In Vitro Maturation (IVM) In Vitro Sperm Capacitation In Vitro Fertilization (IVF) In-Vitro Embryo Production IVEP includes three main procedures :  In Vitro Maturation (IVM)  In Vitro Fertilization (IVF)  In Vitro Embryo Culture (IVC) IVEP In-Vitro Embryo Production IVEP Application of IVEP Provide an excellent source of low cost embryos for basic research on developmental biology and physiology  Commercial application of emerging biotechnologies like SCNT & Transgenics  Proposed as a strategy for rescue of some endangered species To facilitate safe worldwide movement of germplasm Cryopreservation of embryos could avoid germplasm losses in the event of infectious disease, thus helps in conservation of germplasm 8 IVEP Oocyte Collection Techniques for Oocytes Collection from Abattoir Ovaries  Aspiration (Most efficient tech. in term of time to obtain oocytes)  Puncturing (Best method in term of quality of oocytes and ease of exam. in medium)  Slicing (Highest no. of oocytes)  Mincing Techniques for Oocytes Collection from Live Donors  Surgically by laparotomy and aspiration using syringe and needle  Transvaginal ovum pick-up technique Oocyte Collection Oocyte Collection from Abattoir Collection of Ovaries : 1. 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 2. Then, transfer ovaries to the second container and place the containers in the thermos 3. Transport the ovaries to the lab immediately 4. Ovaries are generally transported to the laboratory in physiological saline that contains some form of antibiotic to help prevent bacterial contamination at approximately 22-24ºC 5. The best results are obtained when oocytes are collected within 4-6 hrs after slaughter 10 Oocyte Collection Aspiration Using Syringe and Needle  Aspirate all follicles (upto 3-7 mm), using an 18 gauge needle in a 5-10 cc syringe  Expel the fluid collected into a 15 cc centrifuge tube & centrifuge tube at 500 rpm for 3 min  Remove the debris from the bottom of the tube with a pasteur pipette  Place it in a petri dish along with some wash media for oocyte scanning (zoom stereomicroscope) Puncturing  Place ovaries in petridish containing 5ml of TL-HEPES medium or NSS at 370C  Visible follicles punctured with 18 gauge needle and oocyte released freely in medium  Collect the media into 50 cc centrifuge tube & centrifuge tube at 500 rpm for 3 min  Repeat the procedure of scanning debris kept in media 11 Oocyte Collection Slicing  Ovaries are placed in petridish containing 5ml of TL-HEPES medium or NSS at 370C, held with artery forcep  All visible follicles are dissected and finally whole ovary is sliced with scalpel blade into thin pieces.  Large ovarian tissues removed by thorough washing .  Repeat the procedure of centrifugation followed by scanning Mincing 12 Oocyte Collection Oocyte Collection by Laparoscopy  LOPU must be conducted under general anesthesia. Different anesthesia protocols can be used.  Induction to allow intubation was done with a mixture composed of 0.05 mg/kg Xylazine, 2 mg/kg Ketamine and 0.1mg/kg Diazepam, administered intravenously, and maintained under anesthesia with 2% isoflurane.  Under laparoscopic observation, all follicles of ≥2mm diameter are aspirated using a 20G needle mounted on an acrylic pipette connected to a collection tube and a vacuum pump.  The laparoscopic equipment consists of a 5mm/0° laparoscope, 3 trocar/cannula ports, an atraumatic grasping forceps, and a cabled light source. 13 Oocyte Collection Transvaginal USG Guided Ovum-Pickup Techniques  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.  An 18-gauge, 550 or 600-mm long needle is inserted through the needle guide in the plastic handle. OPU  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 phosphatebuffered saline (PBS) with 10% Bovine serum albumin, 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. ADD A FOOTER 15 OPU 16 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 Oocyte Grading Categorization of Oocytes Based on Cumulus Layers CATEGORY I CATEGORY II CATEGORY III CATEGORY IV  Category I : Oocyte with ≥ 4 layers of cumulus oophorus and uniform cytoplasm  Category II: Oocyte with less than 4 cumulus layers or with partially cumulus mass but with uniform cytoplasm  Category III: Partially naked oocytes with thin cumulus or ramnants of cumulus cells or only corona radiate and with uniform cytoplasm  Category IV: Includes oocytes without cumulus cells i.e Zona pellucida but with uniformly distributed cytoplasm Oocyte Grading Culture Media & Supplements Simple media: – They are usually bicarbonate-buffered systems containing physiological saline with pyruvate, lactate and glucose, and they differ in their ion concentration and conc. of energy sources. • eg. Kreb’s–Ringers bicarbonate medium, Modified Dulbecco’s medium (DPBS), Whitten medium and Synthetic oviduct fluid (SOF) Complex Media: – Contains Amino acids, Vitamins & Purines in addition to basic components. • eg. TCM-199, Modified Ham’s F-10/F-12, TALP, Modified minimum essential media with Earl’s salts (MEM) Media & Supplements Culture Media & Supplements  Protein supplements like BSA decrease surface tension, thereby reducing embryo tendency to float or adhere to plastic or glass surface. – BSA or FCS- inactivated by being held at 560C/30 Min – BSA added @ 0.3-1%, but conc. (0.1-50%) – FCS added @ 10% (5-20%)  Antibiotics: – – – – Streptomycin sulphate @ 50mg/lt or 100 µg/ml Potassium Pencilium G @1lac IU/lt or 100 IU/ml Gentamicin @ 50µg/ml Kanamicin @ 50µg/ml  All media except modified PBS are bicarbonate buffered so require an atmosphere of 5% CO2 to maintain pH  pH of media range from 7-8 (best results 7.2-7.6)  Osmolality: 270-300 mOsm/kg mostly used for embryos Media & Supplements Culture Media Transportation and Washing medium Dulbecco’s Phosphate buffered saline (DPBS) Holding medium Tissue culture medium (TCM 199)+ 1% Fetal calf serum (FCS) + 1% Bovine serum albumin (BSA) Maturation and Fertilization medium TCM alone or supplemented with either 10% FCS or 10% Estrus cow serum (ECS) Capacitation medium Brackett and Oliphant (BO) Media & Supplements Basics of Oocyte Maturation  In vivo meiosis begins during early fetal life, which is then arrested at diplotene stage at birth and again resumed by preovulatory surge of gonadotropins  While meiosis is arrested, oocytes are continuously selected to grow from the pool of primordial oocytes  After preovulatory surge of gonadotropins, full grown oocytes in the follicle resume meiosis, completes the first meiotic division and then enter a second period of arrest at Metaphase II  Sequence of events in the oocytes between the two periods of meiotic arrest is called maturation.  It consists of nuclear, cytoplasmic and membrane changes. The completion of these maturational changes leads to normal fertilization and embryonic development IVM Culture for In Vitro Maturation  Bovine oocytes are matured in TCM-199 with 10% FCS and Gonadotropins (FSH, LH)  4-5 oocytes or group of COC (Cumulus oophorus cells) are placed in microdroplet (60 – 80 µl) of maturation medium in petridish  Cover with warm (37oC) light liquid paraffin oil  Placed in CO2 incubator kept at 39oC, 5% CO2, 95% air, with high relative humidity for 24 hrs IVM In Vitro Maturation  In vitro maturation is a key step to provide good quality oocytes for in vitro fertilization and determines the potential developmental competence of the oocytes.  Oocyte are transferred into a dish containing maturation medium.  Cumulus-oocyte complexes (COCs) are washed several times and placed in microdroplets of the same medium under paraffin oil in petri dishes. Species Duration of oocyte maturation Cow 21-24 hrs Pig 40-44 hrs Horse 24-48 hrs Human 28-36 hrs Mouse 28-36 hrs Incubated at 39°C in 5% CO2 and 95% air with high humidity for 24 h. Most widely used media used in IVM  Tissue culture medium (TCM) 199 with or without serum and Synthetic oviductal fluid (SOF). Maturation medium will include several components including ; Nutrients (Pyruvate, Glucose, Glutamine, Serum), Hormones (Estrogen, LH, FSH) and Antibiotics (Penicillin/Streptomycin or Gentamicin). 23 IVM Assessment of Maturation  Cumulus cell expansion Metaphase -II and / or extrusion of first polar body (1% Acetorcein stain) Extrusion of 1st polar body  Flouroscent dye: Hoechest 33342 (Flouroscent microscope)   IVM Sperm Capacitation  Spermatozoa acquire maturity during epididymal transit, but maturation changes do not render sperm completely fertile.  It refers to modification of the ejaculated sperms in the female reproductive tract making them capable of fertilizing eggs. It allows sperm to undergo a normal acrosome reaction before fertilization.  Capacitation starts in uterus, however major site is oviduct, specifically isthmus  Capacitation changes includes;  Removal of surface components by genital tract secretions causing phospholipid bilayer to destabilized  Removal of sperm cholesterol at sperm surface  Alteration in glycosaminoglycan and changes in ion (Ca) Sperm Capacitation Sperm Capacitation  Sperm capacitated is caused by high ionic strength (HIS) media like Brackett and Oliphant (BO) medium (osmolarity 360-390 mOsm) containing heparin and its combinations in in vitro such as; Hypotaurine, Ca++ ionophore, Caffeine, long incubation (18-24h), high pH and washing with percoll gradient.  To capacitate bovine sperm, the required number of sperm is diluted in bicarbonate buffered medium containing 10µg/ml of heparin.  Medium containing sperm is dispensed in microdroplets under paraffin oil, or in wells without oil, and incubated for 4h Sperm Capacitation Sperm Preparation & Capacitation  Sperm purification is necessary so that sperm cells can be washed from the extender + cryopreserves (if frozen is used) /seminal plasma (if fresh semen is used) If frozen semen is used  Different physical methods for separation of good motile sperm:  Swim-up Technique  Percoll based separation system  Glass-wool Filtration  With unfrozen semen, most sperm are motile/viable so simply determining the concentration of sperm and adding a set amount is sufficient. 27 Sperm Capacitation Sperm Preparation Percoll Purification Method A-The straw are expelled onto the top of the sperm purification gradient. B-The layer of semen on top of the gradient before centrifugation. C-Live sperm at the bottom of the gradient after centrifugation.  Place the conical tube containing the semen and sperm purification gradient into a centrifuge carrier that has been pre-warmed to 38.5°C, and centrifuge at 1000 x g for 10 min.  After centrifugation, aspirate sperm pellet from the bottom of the conical tube with a plastic Pasteur pipette .  Place the sperm pellet into a 15 ml conical tube containing 10 ml HEPES-TALP and place in a warm centrifuge carrier before centrifuging for 5 min at 200 x g.  Remove the supernatant with a Pasteur pipette.  Determine dilution required to bring sperm to a concentration of 17 x 106/ml. This will produce a final concentration of sperm in the fertilization plate of approximately 1 x 106/ml.  Bring the semen pellet to a volume of 600 µl using pre equilibrated IVF-TALP medium. Swim-Up technique for production of hypermotile sperms for IVF In-Vitro Fertilization DAY –2 Preparation of Media DAY -1 Collection of ovaries from slaughter house DAY 0 Preparation of media for fertilization (2h prior to fertilization) The in vitro fertilization process can be divided in three main steps:  COC washing  Sperm purification  Fertilization 30 IVF Cumulus-Oocyte-Complexes (COCs) washing Necessary so that hormones, nutrients and metabolites present in the maturation micro-drop are not carried over to the fertilization drop. Procedure  Transfer COCs from each maturation micro drop to the X-plate containing the buffer HEPESTALP (hydroxyethyl piperazineethanesulfonic acid )-(Tyrode's albumin lactate pyruvate).  Transfer 10 COCs from the X-plate to each well of the 4-well fertilization plate. Oocyte maturation medium (OMM) Microdispensor pipette Washing of COCs with buffer Fertilization plate IVF Fertilization In Drops 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 known to induce sperm hyper activation).  Place the 4-well fertilization plates in a incubator (5% CO2 in air). Make sure that all media used for sperm are warmed to 38.5°C before use. Media necessary for fertilization should be prepared at least 2 h prior to IVF (HEPES-TALP, IVF-TALP, Pure Sperm) Incubation in 5% CO2 at 38.5°C for 8-20 h IVF Assessment of Fertilization      Presence of spermatozoa within vitellus Presence of sperm tail in the vitellus Presence of male & female pronuclei in the egg Presence of two polar bodies in the perivitalline space Clevage and formation of two blastomeres with equal size, shape and no fragmentation IVF In-Vitro Culture (IVC)  Following IVF the zygotes must be cultured for further development before they are transferred into uterus or cryopreserved.  Three systems of IVC of embryos:  Transferring to the ligated oviducts of a temporary recipient. eg. sheep, or rabbit. After 4 5 days later embryos are recovered , graded and frozen or transferred.  Zygotes co-cultured in-vitro with somatic cells like Oviductal epithelial cells, granulosa cells, in medium (TCM-199)  Cultured in simple medium such as Synthetic Oviduct Fluid (SOF) in microdrops under paraffin oil under 5% CO2 at 390C in CO2 incubator for 6-8 days (Most common method) Mammalian oviduct has ability to support development of embryos across many species 34 (eg. Bovine embryos can develop vivo Sheep & rabbit oviduct co-culture) IVC In-Vitro Culture (IVC)  Presently the commonly used system for culturing of embryos is SOF with Amino acid and BSA in the absence of serum at are embryo at 38.50C in 5% CO2, 5% O2, 90 % N2 under humidified atmosphere.  However, 5-10% FCS at 2-3 day post insemination is added to promote higher viability after transfer of such IVP embryos  Prepare embryo culture medium (SOF-BE 2 or other culture medium) at least 2 h before removing zygotes from the fertilization plate.  Make 50 ul micro drops of culture medium (upto 30 zygotes per drop) in petri dishes and cover with mineral oil.  For 60 X 15 mm dishes cover drops with 9 mL mineral oil.  For 35 x 10 mm dishes use 4 mL mineral oil to cover drops  To culture small groups of embryos (<15 per drop) we typically prepare 25 ul microdrops of culture medium Cultured for 9 days in IVC35 medium in a CO2 incubator (5% CO2 in air, 90-95% humidity) at 38.5°C IVC 36