In vitro Fertilization for Undergraduate Medical Students

This is a slide on in vitro fertilization and everything you need to know about it in your medical school. All data and information are validated and extracted from authentic resources.

1. In-Vitro Fertilization (IVF) Presented by: Anish Dhakal (Aryan)

2. Patient presents with infertility Establish by relevant history (unable to conceive in 1 year despite adequate frequency of unprotected coitus (4 to 5 times/week) Menstrual history, Obstetric history, Past Medical History, Occupation, Coital act in fertile period or not (Safe: 7 days before and after menstruation) Detect male infertility factor by semen analysis (volume, count, motility) Approach to Infertility

3. Detect female infertility factors USG. If USG shows PCOS, start treatment including Clomiphene Citrate Tubal, uterine, Cervical causes Check tubal patency by HSG by urografin dye Intrauterine insemination IVF-D (Donor) IVF-H (Husband)

4. Assisted Reproductive Technology (ART) ART encompasses all the procedures that involve manipulation of gametes and embryos outside the body for the treatment of infertility

5. Assisted Reproductive Technology (ART) IVF-ET : In vitro fertilization and embryo transfer GIFT : Gamete intra-fallopian transfer ZIFT : Zygote intra-fallopian transfer POST : Peritoneal oocyte and sperm transfer TET : Tubal embryo transfer zone SUZI : Subzonal insemination ICSI : Intracytoplasmic sperm injection AH : Assisted hatching IVM : In vitro maturation of oocyte

6. Introduction to IVF IVF is an advanced process by which egg is fertilized by sperm outside the body Once an embryo or embryos form, they are then placed in the uterus

7. History World first: Louise Brown was the first test tube baby born in 25 July, 1978 in London Nepal first: Om Mani Tamang ; March 3, 2005 Robert G. Edwards, the physiologist, was awarded the Nobel Prize in Physiology or Medicine in 2010

8. Indications Tubal factor (IVF is primary therapy if tubes are completely blocked) Severe male factor infertility (mild male factor may be treated with inseminations) Diminished ovarian reserve (time to conception is critical and success with other therapies is low) Ovarian failure (although donor eggs must be used in this case) Uterine factor ( Asherman syndrome or irreparable distortion of the uterine cavity, gestational surrogacy may be needed) All other causes of infertility, after failing treatment with less invasive therapies ( eg : ovulatory dysfunction, endometriosis, unexplained infertility)

9. Patient Selection (Ideal) Age < 35 years Presence of ovarian reserve (serum FSH <10 IU/L) Husband: normal seminogram Couple must be screened negative for HIV and hepatitis Normal uterine cavity as evaluated by hysteroscopy/ sonohysterography

10. Down Regulation of Pituitary function Use of GnRH analogues To prevent premature LH surge, premature ovulation, higher number of oocyte retrieval, gives higher pregnancy rates Therapy is continued either subcutaneously or intranasally Examples: Cetrorelix , L euprolide

11. Down Regulation of Pituitary function Long protocols: I nvolve starting medications in the menstrual cycle (21 days of previous cycle) before the IVF cycle This can be done with either a GnRH agonist or antagonist or oral contraceptive pills Because of cost, GnRH agonists are preferred over GnRH antagonists for the long protocol

12. Down Regulation of Pituitary function Short protocols: M edications are started at the time of the natural menstrual cycle Stimulation is achieved with hMG or FSH and spontaneous ovulation is blocked with either a GnRH agonist or with a GnRH antagonist GnRH antagonists are preferred over GnRH agonists for the short protocol

13. Steps in IVF: Ovarian stimulation Monitoring of follicular growth Egg retrieval Fertilization Embryo culture Embryo transfer

14. 1. Ovarian Stimulation Also known as ovulation induction Medications including fertility drugs are used Stimulate multiple eggs to grow in the ovaries rather than the single egg Some eggs will not fertilize or develop normally after fertilization

15. Ovarian Stimulation Regimen Clomiphene Citrate (CC) CC + human menopausal gonadotropin ( hMG ) CC + pure FSH CC + Recombinant FSH hMG FSH GnRH analogues + hMG or pure FSH

16. Ovarian Stimulation Regimen Clomiphene citrate (CC): anti-estrogenic as well as weakly estrogenic It blocks the estrogen receptors in the hypothalamus. This results in increased GnRH pulse amplitude causing increased gonadotropin secretion from the pituitary Starting dosage is 50 mg/day, begin within the first 5 days after the onset of a spontaneous or progesterone induced menses and is continued for 5 days Ovulation is expected to occur 5 to 10 days after the last day of therapy

17. Ovarian Stimulation Regimen Combination of CC 1 hMG : CC 50 to 100 mg/day from day 2-6 of the cycle for 5 days. Injecting hMG 75 units IM is added on day 3, 5 and 7, and more if so required

18. 2. Monitoring of Follicular Growth Ovum development is monitored by using Sonographic measurement Cervical mucus study Serum estradiol estimation Commencing on the 8th day of treatment cycle

19. 2. Monitoring of Follicular Growth When two or more follicles are 17–18 mm in diameter and serum E2 levels > 250 pg/ml/per follicle: 5,000–10,000 IU of hCG or 250 mg of recombinant hCG is given IM hCG replaces the natural LH surge and causes the final stage of egg maturation

20. 3. Egg Retrieval Oocyte retrieved before ovulation occurs, usually 34 to 36 hours after the hCG injection is given Done by transvaginal ultrasound aspiration An ultrasound probe is inserted into the vagina to identify the follicles, and a needle is guided through the vagina and into the follicles Eggs are aspirated from the follicles through the needle connected to a suction device

21. 4. Fertilization and Embryo Culture After the eggs are retrieved, they are examined in the laboratory for maturity and quality Mature eggs are placed in an IVF culture medium and transferred to an incubator to await fertilization by the sperm

22. Sperm Collection Semen is collected just prior to ovum retrieval Best specimen is one obtained by masturbation in the vicinity of the laboratory Sperm used for insemination in vitro is prepared by the wash and swim-up or density gradient centrifugation technique

23. 4. Fertilization and Embryo Culture Fertilization may be accomplished by insemination or by intracytoplasmic sperm injection (ICSI) Insemination: motile sperm are placed together with the oocytes and incubated overnight Approximately 50,000 to 100,000 capacitated sperm are placed into the culture media containing the oocyte within 4–6 hours of retrieval IVF-Husband or IVF-Donor

24. Intracytoplasmic Sperm Injection (ICSI) One single spermatozoon or even a spermatid is injected directly into the cytoplasm of an oocyte by micropuncture of the zona pellucida Oocyte is stabilized at 6 or 12 O’clock position and entered at the 3 O’clock position Injecting pipette pierces the zona and the sperm is injected directly into the ooplasm

25. 4. Fertilization and Embryo Culture Visualization of two pronuclei the following day confirms fertilization One pronucleus is derived from the egg and one from the sperm Two days after the egg retrieval, the fertilized egg has divided to become a 2-4 cell embryo

26. 4. Fertilization and Embryo Culture By the third day, a normally developing embryo will contain approximately 6-10 cells By the fifth day, a fluid cavity forms in the embryo, and the placenta and fetal tissues begin to separate, called blastocyst

27. 5. Embryo Transfer Transferred to the uterus at any time between one and six days after the egg retrieval Technique: One or more embryos suspended in a drop of culture medium are drawn into a transfer catheter with a syringe on one end Gently guides the tip of the transfer catheter through the cervix and places the embryos into the uterine cavity 1 cm below the fundus is performed

28. 5. Embryo Transfer

29. Embryo Transfer

30. 5. Embryo Transfer The maximum number of embryos transferred is based on the patient’s age embryo characteristics High-order multiple pregnancy (three or more implanted embryos) is an undesirable consequences of ART Multiple gestation lead to increase risk to complications in both mother and fetuses Justification for transferring additional embryos beyond recommended limits should be clearly documented in the patient's medical record

31. Other favorable include any ONE of these criteria: For fresh cycle: (1) expectation of one or more high-quality embryos available for cryopreservation, or (2) previous live birth after an IVF cycle. For frozen embryo transfer cycle: (1) availability of vitrified day 5 or day 6 blastocysts, (2) euploid embryos, (3) first frozen embryo transfer cycle, or (4 ) previous live birth after an in vitro fertilization cycle .

32. Cryopreservation Extra embryos remaining after the embryo transfer may be cryopreserved (frozen) for future transfer Makes future ART cycles simpler, less expensive, and less invasive than the initial IVF cycle Once frozen, embryos may be stored for prolonged periods, and live births have been reported using embryos that have been frozen for almost 20 years

33. Luteal phase support Progesterone supplement Started on the day after oocyte retrieval or embryo transplant up to the pregnancy test is positive or 1 st trimester hCG can be used with progesterone Micronized progesterone 200mg thrice a day oral or vaginal suppository Progesterone in oil injection 50mg IM daily for 14 days

34. Follow up Pregnancy is diagnosed by identification of rising serum hCG levels after transfer Implantation occurs no less than 7 days , late implantation can also occur so first blood test is done no earlier than 12 days. A negative hCG level 14 days after egg retrieval is a strong indication of a failed IVF cycle If hCG is positive , ultrasound evaluation is done at 6 th week.

35. SURROGACY/GESTATIONAL CARRIER Pregnancy may be carried by the egg donor ( traditional surrogate) or by another woman who has no genetic relationship to the baby (gestational carrier) The surrogate will be biologically related to the child Gestational carrier will not be genetically related to the child

36. Gamete intrafallopian transfer (GIFT): Zygote intrafallopian transfer (ZIFT) or tubal embryo transfer (TET): A laparoscope is used to aspirate one or more mature oocytes from ovarian follicles and transfer the oocytes and sperm to the fallopian tube This procedure involves placement of fertilized eggs (zygotes) or embryos into the fallopian tube More invasive than IVF usually for patients with religious or personal reasons, do not wish to have embryos in the laboratory ZIFT is analogous to GIFT in that laparoscopy is needed to place the zygotes in the fallopian tubes It is also appropriate for those who have failed donor insemination or require laparoscopy for other reasons Patients with difficult trans-cervical embryo transfer, uterine abnormalities (such as those caused by DES exposure), or recurrent failure with standard IVF

37. RISKS OF IVF Ovarian stimulation: risk of hyperstimulation , ovaries become swollen and painful Accumulate of free fluid in abdominal cavity and chest, and woman may feel bloated, nauseated, and experience vomiting or lack of appetite Removing eggs: risk of bleeding, infection, and damage to the bowel, bladder, or a blood vessel

38. RISKS OF IVF The chance of multiple pregnancy : when more than one embryo is transferred The risk of preterm delivery in multiple pregnancies is high Miscarriage Increased chance of ectopic pregnancy with ART Premature ovarian failure Psychological stress is common : high expectations despite high failure rate Couples are encouraged to consider psychological counseling as an additional means of support and stress management

39. References: Shaw’s Textbook of Gyanecology , 16 th edition P. Richard, In vitro fertilization, https :// www.uptodate.com/contents/in-vitro-fertilization L. Charles et al., Pregnancy outcomes after assisted reproductive technology, https ://www.uptodate.com/contents/pregnancy-outcome-after-assisted-reproductive-technology DC Dutta’s Textbook of Gynaecology , 7 th edition Assisted Reproductive Technology, A Guide for Patients, American Society for Reproductive Medicine