In-Vitro Fertilization (IVF Treatment) & ICSI Treatment

What is IVF?

It is micromanipulation technique used in the process of IVF which involves injection a single sperm into the centre of mature oocyte under the microscope.

Indications of IVF

  • Tubal disease:- Tubal blocks, Rigid, Oedematous, Convoluted Tubes, Hydrosulpine,
  • Oligo-astheno-zoospermic
  • Diminished ovarian reserve
  • Stage III & IV Endometriosis
  • Multiple IUI
  • Oocyte donates/ Embryo donation
  • Obstructive/Non-obstructive azoospermia
  • Pre-implantation genetic diagnosis

Indications of ICSI

Indications of ICSI have now widely been expanded. In some indications, the sperms are collected from testes, while in others the sperm collection is done from ejaculated semen. Accordingly indications may be classified depending on the method of collection of spermatozoa.

Typical IVF cycle

  1. Stimulation for multiple follicular development
  2. Monitoring follicular growth and development
  3. Trigger of follicular maturation
  4. Oocyte recovery and identification
  5. Insemination
  6. Embryo culture
  7. Embryo replacement
  8. Luteal phase support
  9. Confirmation of pregnancy

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Luteal gonadotropin-releasing hormone agonist stimulation

  1. StartGnRH-a on day 21 of a regular menstrual cycle.The luteal start is confirmed by a serum progesterone level >4 ng/ml or by a
    transvaginal scan showing the presence of a corpus luteum. By starting GnRH-a in the luteal phase the risk of ovarian cyst formation is decreased.
  2. Spontaneous menses expected 10-12 days after the administration of GnRH-a.
  3. Transvaginal scan to evaluate the lining of the endometrium after menses started and serum estradiol to confirmed down-regulation before starting stimulation using gonadotropins(rec-FSH,or urinary FSh or combination)
  4. Serial ultrasound and E2 levels monitor follicular development;measuring the number and size of follicles and also the
    development of the endometrium and its morphology.
  5. Once follicular size reaches 18-22 mm,administer rec-hCG,typically 5000-10 000 IU SC or IM.
  6. Oocyte retrieval 34-36 hours after hCG.

Fixed,multi-dose GnRh antagonist protocol

  1. Transvaginal ultrasound and serum E2 are arranged on day 2/3 of the period after oral contraceptive pretreatment
  2. After confirmation of quiescent ovaries and low E2 level,SCrecFSH is commenced daily for ovarian stimulation.GnRH
    antagonist 0.25 mg daily is then given as a fixed protocol strting on day 6 of the stimulation until the dayofhCG administration.
  3. Ovulation triggering is achieved by subcutaneous injection of 10 000 IU of hCG when leading follicles reach 18-20 mm together with at least three mature follicles 16 mm detected on ultrasound scan. This is followed by transvaginal
    ultrasound-guided oocyte retrieval 34-36 h later.

Flare-up or BOOST GnRH agonist protocol

  1. Daily 0.05 mg/day GnRH-a from day 2 of the cycle.
  2. recFSH started from day 3.
  3. GnRh agonist is administered subcutaneously and continued daily until and including the day of human chorionic
    gonadotropin(hCG)administration.
  4. A total of 10 000 units of rec-hCG 250 mcg is administered SC when the leading follicle reaches 17-18 mm In diameter followed 34-36h later by an ultrasound-guided transvaginal oocyte retrieval.

Oocyte recovery in day Surgery Theater is now usually performed under general anesthesia or intravenous sedation an oocyte is carried out ultrasound guidance.
The patient is first placed in lithotomic positions, cleaned and draped. The Trans virginal probe with a needle guide attached is then inserted into the vagina to visualize both ovaries and their follicles meanwhile the aspiration system is readies and visualize pressures are checked (100 mm Hg) by aspiring culture media with the specially designed disposable needles.

The lower more accessible ovary is targeted first and the needle is inserted under ultrasound guidance into the first follicle. The follicular fluid is then aspirated into a test tube placed in a warmer block and test tube is handed over to the wafting embryologist located in the adjoining IVF laboratory. All the follicles on one side are systematically aspirated without flushing, with care at all times to avoid the nearby pelvic side –wall vessel. Once all the follicles on one side have been aspirated, the needle is withdrawn and used to aspirate warm culture media in a test tube to flush the system through.

The Surgeon then moves to the opposite ovary and needle is again inserted under ultrasound guidance to target the remaining follicles. Once again, the follicles are system apically punctured and aspirated (without flushing) until all the follicles are tapped. Once completed, the needle is withdrawn and the system flushed again with warm cultured media to ensure no egg is left behind in the needle system.
The uterus is scanned and the endometrial thickness measured before the ultrasound probe is removed.

The vagina is cleaned with sterile guaze swabs and homeostasis is confirmed before the procedure is deemed completed. Postoperatively, the patient is monitored in the recovery ward and reviewed 2-6 hours later before discharge. When the embryologist receives the test tube containing the follicular fluid, she/he pours the contents into a Petri dish and examines the contents under a binocular dissecting microscope for the oocyte cumulus complex (OCC).

Once the OCC is identified, it is quickly transferred into a buffered medium. it is washed thrice in the medium to remove red blood cells before it is transferred into a culture dish containing culture medium. The dish containing all the OCCs is placed in the incubator until the time of insemination, which is about 4 hours post oocyte retrieval.

The Husband semen sample is processed in the morning of the oocyte retrieval. The density gradient technique followed by washing in culture medium is the most common method used for recovery in the incubator until the time of insemination or intracytoplasmic spermatozoa injection (ICSI). Fertilization is achieved either insemination or ICSI. This is preformed 4 hours post oocyte retrieval. Insemination is done for patient with high sperm count and normal morphology sperm. Each OCC is placed in a droplet containing 150 000 mottle sperm/ml. ICSI is done for patient who have poor sperm quality or anti sperm antibodies and also for patients who had fertilization failure in a prior cycle .Each mature oocyte is injected with one sperm each.

After insemination or ICSI the oocytes are placed in the incubator until the next day.A fertilization check is done 18 hours post insemination or ICSI. The oocytes are checked for the presence of two pronuclei. The fertilized ooctyes are placed in the incubator until the next day, which is referred to as day 2. Embryos are scored daily from day 2 until day 6. On day 2 the embryos should be at the four-cell stage. Day 3 embryos should be at the eight cell stage, day 4 embryos should be at the compacted stage and day 5 and 6 embryos should be at the blastocyst stage. Embryos are graded based on the regularity of the blastomeres and presence of fragments. Good quality embryos have regular blastomeres with no fragmentation. Average quality embryos have regular blastomeres with moderate fragments. Poor quality embryos have irregular blastomeres with a lot of fragments.

After insemination or ICSI the oocytes are placed in the incubator until the next day. A fertilization check is done 18 hours post insemination or ICSI.

The oocytes are checked for the presence of two pronuclei. The fertilized ooctyes are placed in the incubator until the next day, which is referred to as day 2. Embryos are scored daily from day 2 until day 6. On day 2 the embryos should be at the four-cell stage. Day 3 embryos should be at the eight-cell stage, day 4 embryos should be at the compacted stage and day 5 and 6 embryos should be at the blastocyst stage.

Embryos are graded based on the regularity of the blastomeres and presence of fragments. Good-quality embryos have regular blastomeres with no fragmentation. Average-quality embryos have regular blastomeres with moderate fragments. Poor-quality embryos have irregular blastomeres with a lot of fragments.

Success rate of IVF treatment mainly depends on embryo quality & on endometrial receptivity. Embryo quality can get affected due to either available oocyte or sperm quality in that month. Endometrial receptivity is determined by endometrial thickness measurement & presence of adequate subendonetrial blood flow. Success rate of IVF increase by either minimal stimulation during IVF or routine ovarian stimulation followed by embryo vitrification & transfer of thawed embryo in frozen thaw embryo transfer cycle.

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