Who performs in vitro fertilization & How is it performed? Fertilization (IVF/ICSI) Conventional In Vitro Feritilization, Embryo Culture
Author Name: Dhiraj Singh Ranawat || Mentor Name: Dr. Naval Shah on April 21, 2020
According to ICMR Guidelines
Embryology: A science of creating embryos from gametes outside the human body.
Embryologist: The scientist who combines the sperm and egg and helps the resulting embryos to grow in a controlled environment is called an Embryologist.
Embryos: The origin of life which becomes the future baby.
They are highly trained professionals usually holding BSc or MSc or even PhD degree due to the specialized nature of their work. They are responsible for maintenance, handling and culturing of Gametes (sperm and ovum) and Embryo’s and the processes responsible for Gamete fertilization. Also responsible for assessing the genetic material present in the Gamete and Embryos and their Testing for any Genetic abnormality.
Qualification: A medical graduate or have a post graduate degree or doctorate in principle of Life Sciences, or a B.Sc. graduate with at least 5 years of Hands on Experience in ART.
• Andrology Training (Semen analysis and Sperm preparation techniques).
• Oocyte cytology and various maturation stages of the same.
• Developmental biology.
• Cell biology with Cell and Tissue culture.
• Molecular biology and Human genetics.
• Micromanipulation techniques of manipulating sperm and oocytes for performing ICSI and biopsies of embryos for preimplantation genetic diagnosis (PGD).
• Equipment and Instrument working.
• In vitro fertilization of oocytes.
• Embryo and Oocyte cryopreservation.
• Ensuring that all the equipments essentials are present, working and calibrated in the laboratory
• Performing Quality Assurance & Quality check of all the instruments, equipments, Culture media required for ART procedure.
• Should have knowledge about Microscopes, incubators, Laminar air flow, Micromanipulators, Media’s used in laboratory.
• Should be also able to take work of Andrologist in his absence i.e. well versed with Andrology Principles ie. Semen Analysis, DNA fragmentation Index, Semen Preparation techniques, Semen Cryopreservation and Thawing.
• Follicular fluid Scanning i.e. scanning and identifying Cumulus Oocyte Complex among the other follicular tissues present in the fluid.
• Denudation of these Cumulus oocyte complex with help of enzymes (Hylase) and mechanical removal of Cumulus cells.
• Capable of performing Fertilization procedures such as Conventional In Vitro fertilization or Intracytoplasmic Sperm Injection or even Intrauterine Insemination.
• Culturing of Gametes after fertilization, having knowledge about Culture media (One step, Sequential media),
• Assessing Embryo Grading through morphological assessment or Time lapse technology.
• Assisted Hatching technology using Laser assisted method.
• Embryo transfer techniques involving loading of Embryo in soft catheter.
• Embryo and Oocyte cryopreservation using Vitrification technology to Preserve the Gametes and Embryos for future use.
• Maintenance and documentation of records needed for ART procedure.
• Held responsible for fertilization failure and should be answerable and Troubleshoot any error occurred during the ART procedure.
• Should be able to counsel patients related to Embryology perspective.
• Should be able to perform Embryo biopsy procedures related to PGD cases.
• Knowledge about ethics and principles related to Human subject for proper counselling and working.
• B.Sc. or M.Sc. degree holders are considered over any other graduates because they have the knowledge about Reproductive biology, basic artificial reproductive technologies and Practical knowledge about Microbiology i.e. Handling of micron sized cells i.e. Gametes and Embryos related with embryology.
• These techniques require precise handling of Gametes and Embryos which can only be done by Embryologist and not a Laboratory level Technician.
• In vitro fertilization is a ART procedure in which a sperm and eggs are combined in vitro i.e. outside the body in a laboratory conditions.
• Embryos are transferred into the woman’s uterus, which are implanted in Endometrium for successful full term pregnancy.
• Embryos retained after transfer are cryopreserved (vitrified) for future use.
• Initially In vitro fertilization was only limited to Women with Tubal disorders such as blocked fallopian tubes.
• But Today, IVF deals uterine factors in female such as Endometriosis, male factors related to Semen parameters and Idiopathic (unknown) factors.
• Steps involved in In vitro fertilization are Ovarian Stimulation (Multi Follicular development), Oocyte pick up, Fertilization, Embryo culture, Embryo transfer.
Myths among Patients:
• Mixing of gametes among patients – There is no mixing of gametes as proper identification by Two embryologist is done or RFID technique is used in which label is attached to the dish to recognize the same given to patient.
• Test Tube baby – Common Laymen term used earlier, ART procedure has nothing to do with Test tube, all the procedures are carried in dish and Embryo is transferred into Uterus
• Damage to Gametes – Proper handling of Gametes by Skilled personnel is done ensuring viability of Gametes.
• Genetically malformed or structurally abnormal baby – ART does not have any Genetic malformation impact on Foetus. Patients with any previous Genetic disease history is advised to undergo Pre implantation genetic Diagnosis to prevent any Genetic Disease in New born.
(According to ICMR guidelines)
• Pathophysiology of Fallopian tube such as Blocked tube or any Trauma.
• Male factors.
• Idiopathic (unknown) infertility.
• Uterine factor such as Endometriosis.
• Immunologically related Infertility.
1. Ovarian Stimulation (Multifollicular growth)
2. Oocyte pick up
4. Embryo Culture
5. Embryo Transfer
Ovarian Stimulation (Multifollicular growth)
• In normal condition every menstrual cycle one dominant follicle develops which contains only one oocyte but
in stimulated cycle multifollicular growth is achieved.
• Need for stimulated cycle is to achieve multifollicular growth to increase the fertilization potential.
• Ovaries are evaluated with help of Ultrasound guidance to assess the Follicle development.
• Different protocols of stimulation are available which are given to patients consisting of primarily
Gonadotropins and HCG trigger to ovulate the egg.
• Medications for Ovarian Stimulation:
a. Human menopausal gonadotropin or Menotropin (hMG)
b. Recombinant Follicle-stimulating hormone (FSH)
c. Luteinizing hormone (LH)
d. Human chorionic gonadotropin (hCG)
e. Clomiphene citrate (CC)
f. Letrozole (Aromatase inhibitor)
• Earlier this procedure was performed Laproscopically but now performed by minimally invasive procedure using suction pump and tubes used to aspirate follicular fluid.
• A needle is inserted into the ovary through vagina to puncture the follicle and aspirate the follicular fluid guided by USG probe.
• Then the follicular fluid is scanned for the Oocyte Cumulus Complex.
• After oocyte pick up oocyte are scanned for maturity levels and kept in culture dish for fertilization procedure.
• Sperm is separated from Semen by Semen Preparation methods such as Density Gradient, Swim up or Microfluidics.
Conventional In Vitro Fertilization:
Fertilization procedure is performed by keeping the oocyte in drop of culture medium and motile sperm being inseminated around the oocyte and overlaid with culture oil.
In this Process a Single Spermatozoa is inserted with help of Micromanipulator device directly into the cytoplasm of Oocyte for fertilization.
* Fertilization rates are similar in Conventional IVF & ICSI.
• Confirmation of fertilization is done by checking pronuclei formation (one paternal & one maternal) or 2nd Polar body appearance.
• After successful fertilization procedure oocyte (now called embryo) are kept in culture dish for culturing i.e. Embryogenesis.
• By 2nd day 2-4 celled stage is achieved depending on the progression of Embryo.
• By 3rd day 8-16 celled stage is formed.
• By the fifth day, a fluid filled cavity is formed termed as Blastocyst containing Inner cell mass (Fetal cells) and Trophoblast (Placental tissue).
• Embryos are generally transferred in the uterus between 3rd to 5th day of culture.
• If successful development of Embryo continues then it gets hatched on 6th day to implant in the lining of Uterus.
• One or more Embryos are transferred to the Uterus depending on the morphological characteristics of Embryo with help of a Transfer catheter attached to Syringe picking desired Embryo’s from Culture dish.
• The clinician transfers the Embryo in the Transfer catheter to Uterus by Cervical passage guided by USG.
• Extra Embryos and Oocytes remaining after fertilization are Cryopreserved for future uses.
• Cryopreservation makes next cycle for the patient easier, quicker and convenient as Stimulation will not be require for transferring the Embryo.
• Once Cryopreserved Embryo’s and Oocyte’s can be preserved for indefinite time as long as Patient wants to without changing the Viability.
• Extra oocytes can also be cryopreserved for later use in case of failed IVF cycle or in the patients undergoing chemotherapy to preserve fertility.
• Most commonly used cryopreservation technique is Vitrification which is defined as solidification of a water based solution forming a glass like amorphous vitreous state without ice crystal formation.
• Temperature used for Cryopreservation is –196ºC.
• It should be noted that Theoretically there is no recorded risk of Cryopreserving the Gametes or Embryo’s provided Technical aspect is strong. The Implantation rate and Pregnancy rate ae similar with Cryopreserved Gametes and Embryo’s.
• Various cryoprotective media’s are used for protecting oocytes and embryos from cold shock of cryopreservation.
• Cryo-devices are used for loading oocytes or embryos for cryopreservation.
• These are specialized closed microscopic work stations which provide stable environment for handling of the Gametes and Embryo’s In vitro.
• This prevents Gametes and Embryo from shock to the Environmental conditions such as Temperature, Gas levels, Humidity which can hamper the Viability of these cells.
• Closed chambers provide optimum temperature of 37ºC and CO2 levels of 6% which is in synchrony with In vivo conditions.
• These conditions favour the Embryo development giving higher Implantation potential for the Embryo.
• It also consists of HEPA & CHARCOAL based Filters which maintain the sterility of the air inside the chamber and provides humidified environment to minimize evaporation thereby maintaining osmolarity and pH to the optimal conditions.
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