IVF is a method of assisted reproduction in which a man’s sperm and a woman’s eggs are combined outside of the body in a laboratory dish. One or more fertilized eggs called embryos are transferred into the woman’s uterus, where they may implant in the uterine lining and develop resulting in a pregnancy. The chance of becoming pregnant after IVF/ICSI is about 50- 60 % and the chance of having a live birth at term is about 40 – 50%.
Indications for IVF/ICSI
• Tubal occlusion or severe tubal disease
• Severe male factor infertility
• Grade III/IV endometriosis
• Unexplained infertility
• PCOS
• Poor responders
• Failed IUI cycles
• High DFI
Apart from the severe deficiency in sperm parameters the other factors which, influence the selection of treatment modality between IUI, IVF and ICSI include
Age of the women - above 40 years should receive GT for OI & should consider IVF early in the treatment protocol
Duration of sub fertility
Presence or absence of cervical factor
Presence of endometriosis or tubal pathology
Presence of anti-sperm antibody - male or female
History of previous pelvic or abdominal surgeries
Patients with advanced age, decreased ovarian reserve, severe endometriosis, tubal factor, unexplained infertility and severe male factor benefit with early IVF/ICSI and have a better chance of success. Other diagnoses may start with basic, ‘low-tech’ treatments like hormone therapies or IUI and then transition to IVF, if needed. However, with most of the more basic treatment options, data shows that after three to four treatment cycles, success rates begin to decline sharply. By moving to IVF treatment, a couple increases their chances of success dramatically.
IVF requires adequate number of selected functional sperms & normal gamete interaction. Gamete interaction may be deficient in patients with sperm disorder where ICSI will be more beneficial. ICSI is also treatment of choice for moderate to severe sperm dysfunction, failed fertilization at IVF, abnormal sperm function and absence of sperms in the ejaculate (Azoospermia). In azoospermia the sperms may be obtained from the testis or epididymis. If the testicular size is small and hormonal levels are elevated, the chance of obtaining sperms from the testis may be bleak.
IVF – Eggs and Sperms Mixed and natural fertilization allowed
ICSI – Single sperm injected into the Egg
Procedure of IVF
IVF involves ovarian stimulation with gonadotropins to get more oocytes. Either GnRH agonist or antagonist are used to prevent release of eggs before their collection 35 hours after administration of ovulation trigger either with hCG or GnRH agonist.
Oocyte retrieval is done by transvaginal aspiration of follicles under USG control , which is less invasive and associated with minimal complications
Selection of Sperm for IVF/ICSI
In IVF it is natural selection of Sperm after processing of the sample
In ICSI/ MESA/TESA, most morphologically normal looking sperms are selected, but these may or may not be normal.
To select the most normal sperm certain techniques have evolved which include IMSI - Real time fine sperm Morphology assessment , PICSI (Real time sperm binding ability assessment), Birefringence in sperm head, MACS etc. Though these methods help in selection of good quality sperms, there is no evidence to use it in all cases as no significant increase in pregnancy rates have been observed. It should be used only in indicated cases.
It is important that the semen sample be collected in the clinic itself. If the male partner is unable to collect the sample in the IVF clinic and gets the sample from home, it is mandatory that the couple signs a consent form for use of this sample collected outside the ART clinic premises for medico-legal purposes.
Embryogenesis
Expelled secondary oocyte is surrounded by the zona pellucida and several layers of the follicular cells -corrona radiate.
Development and quality of the embryo after fertilization will depend on Ovarian controlled stimulation, follicle size at trigger, follicular fluid volume and vascularization, abnormalities in the oocyte as well as the environment in the culture media where they are cultured.Fertilization, the process by which the male and female gametes fuse, marks the beginning of the pregnancy. With the creation of two daughter cells the fertilization is complete. 4 cell embryo is seen at 48 hours after fertilization and 8 cells after 72 hours
Different stages of embryo development
The cleavage embryos are graded by looking at the cell number and equality, rate of development and fragmentation. We can transfer the embryos on day 3 and freeze the remaining for further use. We can also culture these day 3 embryos to blastocyst . The blastocyst normally forms at 120 hours. The quality of blastocyst will depend on the expansion, quality and number of the inner cells mass which forms the baby and trophectoderm which forms the placenta.
Blastocyst graded depending on
degree of expansion and hatching status - numerically coded by an integer from 1 to 6
ICM and TE grades are represented by one letter each
with A representing the highest grade
Difference between cleavage stage embryo and blastocyst
Blastocyst transfer gives opportunity to select better embryos with temporal synchronization of embryo and endometrium at embryo transfer. Blastocyst culture allow genetically defective embryos to cease development and allow normal to survive which provides more selected population for replacement. Pregnancy & live birth rate per ovum pick up similar in 2 groups but higher per embryo transfer for blastocyst transfers. With blastocyst transfer the number of embryos available for cryopreservation are fewer compared to embryo transfer done on Day 3. Usually, a single embryo is transferred but at times one can transfer two especially if the age of the women in more than 35 years.
One must identify the best embryo for transfer
Today 95 % of the clinics use morphology to identify the best embryo, but morphology does not always reveal the truth. So, one could perform additional procedures, but this is going to add to the cost of IVF.
The additional procedures include culturing embryos in time lapse incubators or pre-implantation genetic screening of the embryos before transfer or assisted hatching. Time lapse is a method of embryo evaluation which, gives information about cleavage division timing and post cleavage stage dynamics of the embryo. Pre-implantation genetic screening (PGS), which assesses the chromosomes to determine if the embryo has normal number of chromosomes. One must also remember that both time lapse embryo imaging and PGS will increase the cost of IVF considerably and not be hundred percent accurate. Preimplantation Genetic testing can be done for Aneuploidy (PGT-A), Preimplantation Genetic testing for monogenic disorders (PGT-M), and PGT-SR (Preimplantation Genetic Testing - Structural Rearrangement). For pre-implantation testing the biopsy can be taken of the polar body, cell from 8 cell embryo or blastocyst as shown in below figure.
Assisted hatching where the outer cover of the embryo is cut using laser so that embryo comes out easily for implantation. This procedure may be helpful in a small group of women to improve achieve a pregnancy. It may be helpful in the women who are aged more than 38 years, have history of repeated IVF failures or thick zona and in frozen embryo transfer cycles.
Luteal phase support (LPS)
All IVF cycles require luteal phase support as the medicines used for producing more eggs and preventing their rupture result in inadequate functioning of corpus luteum, which may result in
· Decreased Progesterone levels
· Decreased duration of progesterone secretion
Moreover, removal of cells surrounding the oocyte/egg during IVF result in reduced functioning of the corpus luteum with decreased production of hormones required for establishment of pregnancy. Apart from that, high estrogen levels due to stimulation with gonadotropins for multiple folliculogenesis result in luteal phase insufficiency as well as interfere with implantation.
Luteal phase support may not be required in timed intercourse and IUI cycles if only oral drugs are used for ovulation required but is definitely required if ovulation is induced with gonadotropin injections. But we do administer progesterone for luteal phase support even in timed intercourse and IUI cycles empirically to increase the chance of pregnancy as if progesterone is deficient in the luteal phase the chance of conception decreases.
When to start LPS
In IUI cycles it is started 24 hours after the follicular rupture is documented, whereas in ART cycles it is started on the day of oocyte retrieval. The medicines used for luteal phase support include progesterone given either as intramuscular injection, vaginal progesterone or oral progesterone. Apart from that in some cases hCG or estrogen is also administered as luteal phase support. In patients with repeated failures with IVF addition of GnRH agonist to progesterone has shown benefit on the outcomes of live birth, clinical pregnancy and ongoing pregnancy
For how Long?
q ART cycles P4 supplementation (50 - 100 mg/day administered IM or 400–800 mg/day administered vaginally or Drydrogesterone 10 mg three times a day) yields significantly higher PRs, compared with treatment with placebo or no treatment
q The P4 supplementation is usually given till 10 – 12 weeks, when the placenta takes over the function of producing hormones
What are the risks of the egg retrieval?
During the egg retrieval, using vaginal ultrasound to guide the insertion of a long, thin needle through your vagina into the ovary and then into each follicle to retrieve eggs may have possible risks of
• Mild to moderate pelvic and abdominal pain (during or after).
• Injury to organs near the ovaries, such as the bladder, bowel, or blood vessels. Very rarely, bowel or blood vessel injury can require emergency surgery and, occasionally, blood transfusions.
• Pelvic infection (mild to severe) following egg retrieval or embryo transfer are uncommon because antibiotic medicines are usually given at the time of egg collection. Severe infection may require hospitalization and/or treatment with intravenous antibiotics.
•. Women who have had pelvic infections or endometriosis involving the ovaries are more likely to get IVF-related infections.
What are the risks associated with the embryo transfer?
A catheter containing the embryos is used to gently place them into the uterus. Women may feel mild cramping when the catheter is inserted through the cervix or they may have vaginal spotting afterward. Very rarely, an infection may develop, which can usually be treated with antibiotics.
Is there an increased risk of miscarriage and ectopic pregnancy after IVF/ICSI?
The rate of miscarriage after IVF is similar to the rate following natural conception, with the risk going up with the mother’s age. There is a small risk (1%) of an tubal ectopic pregnancy with IVF; however, this rate is similar to women with a history of infertility. If an ectopic pregnancy occurs, a woman may be given medicines to end the pregnancy or surgery to remove it. If you are pregnant and experience a sharp, stabbing pain; vaginal spotting or bleeding; dizziness or fainting; lower back pain; or low blood pressure and have not had an ultrasound confirming that the pregnancy is in the uterus, call your doctor immediately. These are all signs of a possible ectopic pregnancy. There is a 1% risk for a heterotopic pregnancy after IVF. This is when an embryo implants and grows in the uterus while another embryo implants in the tube, leading to a simultaneous ectopic pregnancy. Heterotopic pregnancies usually require surgery to remove the ectopic pregnancy so that the intrauterine pregnancy can be salvaged and grow safely to term.
Will IVF increase the risk of my child having a birth defect?
The risk of a child with a birth defect in the general fertile population is roughly 3–5 percent. After an IVF cycle, the birth defect rate increases by approximately 1 percent. This is mostly linked to delayed conception and the underlying cause of infertility. There may be a slight increased risk of sex chromosome (X or Y chromosome) abnormalities with ICSI. However, it is uncertain if these risks are due to the ICSI procedure itself or to problems with the sperm themselves. Men with sperm defects are more likely to have chromosomal abnormalities, which can be transmitted to their children. Rare genetic syndromes called imprinting disorders may be slightly increased wtih IVF.
Cryopreservation of embryos
Optimal ART requires embryo selection, which requires multiple oocytes/embryos and one cannot transfer multiple embryos and so surplus embryos need to be frozen. Freezing of embryos is also required in certain conditions like when there is risk of the ovaries overresponding to prevent complications of ovarian hyperstimulation syndrome, pre-implantation genetic testing is done, for fertility preservation or if the women wants to postpone pregnancy beyond the age of 35 years.
The embryo can be frozen at any of the following stages
Only good quality embryos are frozen, and the other bad quality embryos are usually discarded as if frozen and thawed the survival of these embryos is not good and may not result in pregnancy. This happens due to the biological consequences of embryo cryopreservation which can result in cell loss, arrested/compromised development , altered function/metabolism and prevent further cleavage and blastocyst expansion/formation.
Results of cryopreservation of human embryos either cleavage or blastocyst depend both on
Embryo quality before freezing and after thawing
Procedure used for cryopreservation – slow freezing or vitrification
Vitrification offers better protection than slow freezing
Frozen Embryo Transfer
Frozen Embryo Transfer(FET) is a cycle in which the frozen or cryopreserved embryos from a previous fresh IVF or donor egg cycle are thawed and then transferred back into the woman’s uterus. It has gained popularity in the last decade because of better quality of embryos being obtained after thawing when vitrification process is used for freezing with optimal pregnancy and live birth rate and safety both for mother and child with the use of these embryos. The chance of becoming pregnant using frozen thawed embryos is about 50%.
Elective FET is indicated in women who are at risk of hyper- response and OHSS, have an elevated progesterone levels on the day of ovulation trigger, when undergoing fertility preservation or pre-implantation testing or ERA, when random start or double stimulation protocol is used or there is a thin endometrium on the day of embryo transfer and when blastocyst develop on day 6 instead of Day 5 in afresh cycle.
FET is also done from the surplus cryopreserved embryos from a fresh cycle if the fresh cycle is unsuccessful or when you desire a second pregnancy when first cycle has succeeded.
Optimal endometrial preparation ensures the best outcome from FET. FET can be done in a natural or modified natural cycle where HCG is given for trigger. FET can also be done in a stimulated cycle where we can induce ovulation using Letrozole or gonadotropin. The day of ovulation is considered as day 0. Embryo transfer in natural, modified natural cycle or stimulated cycle is done as follows
• Cleavage stage - ET 2/3 days after ovulation or 3/4 days after LH peak
• Blastocyst stage - 5 days after ovulation or 6 days after LH peak
Luteal phase support is started on the day of ovulation.
FET can also be done in a hormone replacement treatment (HRT) cycle, where estrogen and progesterone are given to prepare the inner lining of the uterus for embryos to get attached. Estrogen is given from day 2 till the endometrial thickness reached 8 – 9 mm. Progesterone is then started. Timing of embryo transfer is stringent after initiation of P4 administration. First day of progesterone administration should be considered as Day 0. Embryo transfer is done as follows.
• P4 + 2 for 4 cell embryo
• P4 + 3 for 8 cell embryo
• P4 + 5 for blastocyst
Luteal phase support with estrogen and progesterone is continued till 12 weeks of pregnancy .
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