This document summarizes assisted reproductive technologies (ART) such as in vitro fertilization (IVF). It discusses the history and development of IVF, including key events like the birth of the first "test-tube baby" in 1978. It provides details on the IVF process including ovarian stimulation, oocyte retrieval, fertilization, embryo transfer, and luteal phase support. It also discusses other ART procedures and their indications, as well as factors that can influence ART outcomes.

1. By – Dr Tripti Markam
Under guidance of Dr. Ishita Priyam Singh
MKCG MEDICAL COLLEGE,ODISHA

2.  The ART encompasses all the procedure that involve
manipulation of gametes & embryos outside the body
for the treatment of infertility.
 The first & still most common form of ART is in vitro
fertilization.
 IVF involve a sequence of highly coordinated steps
beginning with controlled ovarian hyperstimulation with
exogenous gonadotrophins ,followed by retrieval of
oocytes from the ovaries under the guidance of
transvaginal sonography,fertilization in the laboratory &
transcervical transfer of embryos into the uterus

3. Louise Joy Brown
World's 1st Test Tube baby
Born on
28th July 1978

5. The second birth of a test-tube baby was in
Calcutta(Kolkata), delivered by Dr Subhas Mukherjee.
Named Durga, the child was born just 67 days after the
birth of Louis Brown in the UK but the government refused
to recognise Dr Subhas’s groundbreaking medical
achievement

6. the birth of Harsha on August 6, 1986, in
Mumbai’s KEM Hospital, delivered by Dr
Indira Hinduja through IVF process has been
considered the first scientifically documented
test-tube baby in India

7. Rajo Devi and her husband
An Indian woman has become the world's
oldest mother after giving birth to her
first child at the age of 70, who married
50 years ago gave birth to a baby girl on
November 28 after receiving fertility
treatment.

8.  The first pregnancy from IVF was reported in 1976 &
was ectopic.
 The first child resulting from IVF was born in 1978.
 ART includes method of assisted fertilization by ICSI
using sperm isolate from the ejaculate or obtained by
MESA or TESE ,assisted embyo hatching & pre-
implantation genetic diagnosis.
 Other forms of ART include tubal transfer of oocytes &
sperm (GIFT),zygote(ZIFT) or embryos(tubal embryo
transfer,TET) via laparoscopy.

9.  Tubal factor infertility
 Endometriosis
 Male factor infertility
 Failed ovulation induction
 Ovulatory dysfunction
 Unexplained infertility
 Ovarian failure
 Women with normal ovaries but no functional uterus
 Women with genetic risk

10.  Age < 35 years
 Presence of ovarian reserve(D-3 sr. FSH<10IU/L)
 Husband- normal seminogram
 Couple must be screened negative for HIV &
hepatitis
 Normal uterine cavity as evaluated by
hysteroscopy/ sonohysterography.

11.  Down regulation using GnRH agonist
 Controlled ovarian stimulation(COS)
 Monitoring of follicular growth
 Oocyte retrieval
 Fertilization in vitro(IVF,ICSI,GIFT)
 Transfer of gametes or embryos
 Luteal support with progesterone

12.  GnRH agonist therapy used for downregulation of
pituitary to prevent premature LH surge.
 GnRH antagonist also used along with gonadotropin
stimulation to prevent premature LH surge or
premature ovulation.
 Different schedule for GnRH agonist are available.
 Long follicular downregulation-when therapy is started
in the follicular phase of previous cycle.
 Long luteal down regulation-it begins on D-21 of
previous cycle. Gonadotropin stimulation is started
following the menses.

14.  Short flare protocol-therapy is started in the follicular
phase(0-1) along with gonadotropin stimulation,this is
also called flare protocol,as gonadotropin can work over
the stimulatory effect of GnRH agonist.
 In short protocol,GnRH agonist (leuprolide acetate 1mg
daily)Is given on cycle day 2-4,continuing thereafter as a
reduced dose(0.5mg daily)
 Gonadotropin stimulation begins on cycle D3.

15.  GnRH antagonist are administered as a small
daily dose(cetrorelix or ganirelix) 0.25 mg by
s/c injections.ususlly starting on cycle day 6-
8 or when the lead follicle reaches 14mm in
diameter, OR as a single large dose
(cetrorelix 3mg s/c which has 4 day duration
of action) on approximately cycle day 8.
 This cycle has less risk of ovarian
hyperstimulation.

16.  Natural cycle
 Clomiphene citrate
 Clomiphene citrate & low dose exogenous
gonadotrophin
 High dose exogenous gonadotropin alone or in
combination with GnRH agonist or antagonist

17.  The 1st birth resulting from IVF derived from a
single oocyte collected in a natural ovulatory cycle.
 It was done by Steptoe & Edwards,1978.
 Advantages- it requires no medication, decrease
cost by 75-80%, minimize complication of multiple
pregnancy & ovarian hyperstimulation syndrome.
 Disadvantages- High cycle cancelation rates due to
premature LH surge. Low success rate (7%).

18.  Clomiphene citrate was the 1st method of ovarian
stimulation used in IVF.
 Clomiphene (100mg daily) is administered for 5-8
days,beginning on cycle D-3 & induce development of
two or more follicle in most normally ovulating women.
 Cycle cancelation rate are lower than natural cycle .
 Number of oocyte retrieved,embryo transferred &
pregnancy rate are greater.
 Clomiphene with modest dose of exogenous
gonadotropin(150-225 IU daily beginning on the last day
of clomiphene treatment) stimulate multifollicular
development more effectively than treatment with
clomiphene alone.

19.  The follicular growth response is monitored by cervical
mucus study, sonographic measurment of the follicle &
serum estradiol estimation commencing on the 8th day
of treatment cycle.
 The endometrial thickness is >8-9mm(trilaminar) is
optimum.
 When the cohort of ovarian follicles reaches
maturity(17-18mm in diameter),hCG(5000-10000 IU) is
administered to stimulate final stages of follicular
development.(equivalent dose –rhCG 250µg)
 Oocyte is retrieved 36 hrs after the hCG is given,hCG
induces oocyte maturation.

20.  Approximately 7-18% of stimulation cycle are cancelled
before oocyte retrieval, most for lack of adequate
response & some for excessive response.
 High responders:-when ovaries became grossly
enlarged, containing large no. of follicle of all sizes &
serum estradiol conc. are markedly
elevated(>5000pg/ml),risk of OHSS increases.
 Poor responders:- women who develope few follicle(<3-
5) despite high dose of gonadotropin stimulation, low
peak estradiol level(<500-1000pg/ml), prognosis is poor.

21. 1. Cycle cancellation
2. Coasting- To continue GnRH agonist, No gonadotropin
stimulation, To give hCG when estradiol is within
normal range.
3. Oocyte retrieval & fertilization-freezing all embryos
& no transfer to avoid OHSS.
4. To delay embryos transfer until the symptoms
subsides.

22.  To use higher dose of gonadotropin stimulation
 To decrease the dose of GnRH agonist
 To use GnRH antagonist instead of long acting agonist.

23.  Oocyte retrieval is performed approximately 34-36 hrs
after hCG administration.
 Oocyte retrieval was performed via laproscopy,transvaginal
aspiration guided by ultrasonography under intravenous
seadation is now the standard technique.
 Deep sedation is most common by propofol.
 Most women tolerate the procedure very well with consious
sedation using short acting narcotics(fentanyl) & BZPs
(midazolam).
 Prophylactic antibiotic treatment(doxycyclin 100mg or
cefoxitin 2gm) I/V 30-60 minutes before retrieval.
 Antiseptic (povidine iodine) are toxic for oocyte,thorough
irrigation with sterile saline should follow.
 A vaginal probe (5-7MHz),16-17 gauge needle, vaccum
pressure 100mmHg is used.
 Follicles within the ovaries >10mm dia. Are aspirated.

25.  Human oocyte reach full size(100-200µm) during the
early antral stage of follicular development.
 Immature oocytes collected from small antral follicles
can mature with time in culture(within 46-48hrs).
 Newer Methods for IVM-1. FSH treatment for 3-6 days
followed by retrieval on cycle day 9-10.(follicular
priming)
2.A single inj. Of hCG (10000 IU) administered
when largest follicle riches 10-12 mm in size & 36 hrs
before retrieval.
3.Combination of two technique , involving sequential
treatment with FSH & hCG before oocyte retrieval.

26.  The sperm use for insemination in vitro is prepared by
the wash & swim-up or density gradient centrifugation
technique.
 Approx. 50000-100000 capacited sperm are placed into
culture media containing the oocyte wthin 4-6 hrs of
retrieval.
 Oocyte are evaluated for evidence of fertilization 18
hrs after insemination.
 Fertilized oocyte exhibits two distinct pronuclei (one
derived from oocyte & other from the sperm & two
polar body in perivitelline space).

27.  Sperm recovery in male with retrograde ejaculation -
sympathomimetics directed at control of internal
sphincter(imipramine 25 mg twice daily),when medical
treatment prove unsuccessful than sperm can be
directly recoverd from the bladder after masturbation.
 Vibratory stimulation & electroejaculation –
psychogenic ejaculatory failure or spinal cord injury.
Men who fail vibratory stimulation Rectal probe
electrical stimulation(Electroejaculation) is
recommended.
 Epidydimal sperm aspiration – sperm is obtain by
MESA(microsurgical epididymal sperm aspiration) or
PESA(percutaneneous epididymal sperm aspiration), at
the time of vasoepidyidymostomy or in case of CBAVD.

29. Testicular Sperm Extraction & Aspiration:-
 It is done in those patient in whom epididymal sperm
aspiration technique failed & present with non
obstructive azoospermia.
Intracytoplasmic Sperm Injection(ICSI):-
It was 1st described by Van Streirteghem & colleagues in
Belgium,1992.
Indication of ICSI:- 1.severe oligospermia
2.Asthenospermia,teratospermia
3.presence of sperm antibody
4.obstruction of efferent duct system(male)
5.Congenital absence of vas(bilateral)
6.failure of fertilization in IVF
7.fertilization of crypreserved oocyte

30.  Technique:-A single selected sperm is 1st immobilize
by compressing the sperm tail with an injection
pipette(inner dia 5-7µm),then drawn into pipette.
 The oocyte is stabilized usually with polar body at 6 or
12 o’clock position & entered at the 3 o’clock
position.The pipette pierce the zona & oolemma & the
sperm is injected directly into the ooplasm.
 Result- fertilization rate- 60-70%
pregnancy rate-20-40%

31. Component for culture system:-
 carbon dioxide concentration(4-7%)
 Incubation volume-(10-50µl)
 Embryo group size(1-4)
 Type of protein supplement-human serum albumin,
recombinant albumin, synthetic serum substitute

33. Essential requierment of successful pregnancy
outcomes:-
1.successful ovum donation & IVF
2.Embryo-endometrial synchronization
3.Exogenous hormonal support until luteal placental
shift.
Indication:-
 women with premature ovarian failure
 Women with removed ovaries
 Older women(poor oocyte quality)
 Poor ovarian reserve
 Women with repeated failure with ART cycle
 Genetic disease

34. Oocyte are collected from:-
 Sister or a friend(age between 21-34year)
 Those for IVF candidates, excess oocyte following
retrieval & cryopreservation
 One undergoing laparoscopic sterilization
 Oocyte donor must be screened for infection &
genetic disease.

35.  Procedure involving the removal of one or more nuclei
from polar bodies(oocytes) or cells(blastomere,
trophoectoderm)
 Preimplantation Genetic Diagnosis- Testing for a
known genetic abnormalities carried by one or both
parents determine whether it has been transmitted to
the oocyte or embryo.
 Preimplantation Genetic Screening- Testing for
oocyte or embryo aneuploidy when parents are
normal.

36.  PGD can be performed on polar bodies removed
from oocyte before fertilization or on blastomeres
or trophoectoderm removed from embryos before
transfer.
 After creating an opening in the zona pellucida
using a laser or acid tyrode’s solution, the polar
body or blastomere are extracted for genetic
analysis.
 Specific gene mutation identified by PCR.
 Numerical & structural chromosomal abnormalities
detected by FISH technique.

37.  Embryo transfer is carried out 48-50hrs after oocyte
recovery, that is 46-48 hrs after insemination. A fine
flexible soft catheter is used for embryo transfer,
which is flushed with Earle’s medium.
 2-3 embryo suspended in 15-25µl of fluid are
transferred 1cm from the fundus i.e. about 5-6 cm
from the external os,& the catheter checked to insure
that the embryo have been transfered. The patient is
made to lie down for half an hour.
 Factor affecting embryo transfer:-1.cervical mucus-
it may plug the catheter tip leading to retention of
embryo.
2.Irritation of uterus & initiation of uterine
contraction.

39.  Luteal support:- For luteal support exogenous
progesterone treatment should begins on the day of
oocyte retrieval or at the time of embryo transfer &
continued until 10 weeks of gestation. Orally 300-
800mg daily or intramuscular injection 25-50 mg
daily ,vaginally as a bioadhesive gel 8% gel 90mg
daily.
 hCG is given in supplemented dose 1500-2500 IU
every 3rd day.

40. Embryo Transfer

41.  Embryo cryopreservation method has two distinct stage
freezing & thawing. Freezing avoid ice crystallization
of intracellular water & prevent cellular damage.
 Two basic method for embryo cryopreservation:-
1.Slow freeze technique
2.Vitrification
In both method cellular water is gradually replaced with
cryoprotactant(dimethyl sulfoxide,propanadiol glycerol)
via osmosis by passage through increasing conc. of
cryopreservatives.

43.  In slow freeze- method embryo is sealed in ampules
or vial, cooled to temp. between -30 & -110 oC in
programmed two step process & then stored in
liquid nitrogen.
 First phase of freezing process is rapid, it prevent
crystal formation & second phase is more gradual.
 In the vitrification method, embryos are flash
frozen by immersion into liquid nitrogen ,creating a
solid glass like state.

44.  Gestational surrogacy involve transfer of embryo to
the uterus of a woman willing to carry a pregnancy on
behalf of an infertile couple.
 Indications:- 1.when female partner has no
uterus(cong., hystrectomy),
2. an irreparably damaged uterus(congenital
malformation, severe intrauterine adhesion),
3. medical condition for which pregnancy may cause life
threatening risk.

45.  The host carrier may be a relative, a friend or
someone with no attachment to the couple who
may or may not be compansated for her service.
 Carrier should have previously given birth.
 A formal legal contract is required to formalize
agreements between the infertile couple & the
surrogate.

46. Gamete Intrafallopian Transfer:-
 GIFT was 1st described by Asch & colleagues in 1984.
 It is more invasive procedure than IVF, but results
seems better than IVF.
 In this procedure, both the sperm & the unfertilized
oocyte are transferred into the fallopian tubes,
fertilization is than achieved in vivo.
 Pregnancy rate is 27-30%.
.

47. Zygote Intrafallopian Transfer:-
ZIFT was first described by Devroey et al.(1986)
The placement of the zygote (following one day of in vitro fertilization) into the
fallopian tube can be done either through the abdominal ostium by laparoscope or
through the uterine ostium under ultrasonic guidance

48.  Birth defect:- most of the ART procedures are not
associated with any increased risk of fetal congenital
malformation or birth defect.
 Increased miscarriage, multiple pregnancy & ectopic &
heterotrophic pregnancy have been observed.
 Perinatal mortality & morbidity are high.
 Ovarian hyperstimulation syndrome .
 Psychological stress & anxiety of the couple are severe.