GENETICS IN IN-VITRO FERTILIZATION TECHNIQUE

(PRE-IMPLANTATION GENETIC TESTING) AND

PRENATAL GENETIC TESTING
 IN VITRO FERTILIZATION (IVF) PROCEDURE

1. Stimulation phase
2. Egg retrieval
3. Collect sperm
4. In vitro fertilization
(Genetic Testing)
5. Embryo Transfer
6. Implantation
THREE TYPES OF PRE- IMPLANTATION GENETIC TESTING
 PGT-A
(ANEUPLOIDY SCREENING)

• PGT-A detects aneuploidy among IVF embryos Aneuploidy exists across all ages and
increases with maternal age.
• Chromosomal aneuploidy is known to be a major cause of IVF failure.

Indication for PGT-A:

1. Women of advanced maternal age (>34 yo)

2. History of recurrent early pregnancy loss
3. Repeated IVF failure
4. Severe male infertility
 PGT-A
(ANEUPLOIDY SCREENING)
PGT
FISH
Traditional genetic testing platform
(chr 13,18,21,X,Y)

ARRAY
Automated array technology
Detect 23 pairs of chromosome

NGS
Latest technology
Detect 23 pairs of chromosome
High-throughput
Easier experimental operation
 PGT-SR
(STRUCTURAL REARRANGEMENTS)
• PGT-SR, preimplantation genetic testing for structural chromosomal rearrangements, is
a genetic test performed on embryos created through IVF to screen for chromosomal
structural rearrangements normally caused by balanced translocations and inversions.

Chromosomal structural rearrangements:

• Duplications: A segment of the chromosome is repeated

• Deletions: A segment of the chromosome is missing
• Inversion: A segment of chromosome reverses its orientation
• Translocation: A segment of the chromosome changes its location. Two different types
of translocations are observed.
 PGT-SR
(STRUCTURAL REARRANGEMENTS)
 PGT-SR
(STRUCTURAL REARRANGEMENTS)

PGT-SR for 46XY,t(5;21)(q11.2;q11.2)

 PGT-SR
(STRUCTURAL REARRANGEMENTS)

PGT-SR for 46XY,t(5;21)(q11.2;q11.2)

 PGT-M
(MONOGENIC DISORDERS)

• One or both genetic parents carry a gene mutation

• Testing is performed to determine specific mutation

Indication for PGT-M:

1. With known single gene disorders

2. Autosomal dominant
3. Autosomal recessive
4. X-linked disorders
5. Carriers of mutations
6. Human Leukocyte antigen (HLA) matching
 PGT-M
(MONOGENIC DISORDERS)
PGT-M AND PGT-A PERFORMED TOGETHER
PRENATAL GENETIC TESTING
PRENATAL GENETIC TESTING
 ANEUPLOIDY

• By definition, aneuploid cells have an abnormal number of chromosomes. Because

each chromosome contains hundreds of genes, the addition or loss of even a single
chromosome disrupts the existing equilibrium in cells, and in most cases, is not
compatible with life.

• In humans, the most common aneuploidies are trisomies, which represent about
0.3% of all live births. Trisomies are characterized by the presence of one
additional chromosome, bringing the total chromosome number to 47.

• With few exceptions, trisomies do not appear to be compatible with life. In fact,
trisomies represent about 35% of spontaneous abortions
Science of identifying structural and/or functional
abnormalities-birth defects in the fetus

NON INVASIVE INVASIVE

US Biochemical Karyotyping
MRI cffDNA Chromosomal DNA
NON- INVASIVE PRENATAL TESTING (NIPT)
NON- INVASIVE PRENATAL TESTING (NIPT)
 NIPT to the rescue

• Singleton Pregnancy - test can be performed any time after 10 weeks within the first
trimester.
• Twin Pregnancy - test can be performed any time after 12 weeks within the first
trimester.
• On the basis of available data, detection rates appear to be higher.
• There is a high negative predictive value for Down syndrome. This may be important
for patients seeking to avoid the risks (e.g., fetal loss) inherent with invasive testing.
• NIPT has a lower false-positive rate, meaning fewer women will receive a “positive”
screen, necessitating fewer invasive procedures.
• Risk assessment is less dependent on gestational age
 What NIPT is not – Points to remember

• Multiple Pregnancy - This test cannot be performed in multiple pregnancies.

• NIPT does not screen for open neural tube defects. Maternal serum α-
fetoprotein testing should still be offered at 15–20 weeks gestation to screen for
open neural tube defects even when NIPT is performed.
• NIPT does not replace the utility of a first-trimester ultrasound examination,
which has been proven to be useful for accurate gestational dating,
assessment of the nuchal translucency region to identify a fetus at increased
risk for a chromosome abnormality, identification of twins and higher-order
pregnancies, placental abnormalities, and congenital anomalies.
• There is a possibility of false-positive screening results, which may be due to
confined placental mosaicism or theoretically a “vanishing twin.”
 Quadruple Marker
• Alpha-fetoprotein (AFP), a protein made by the developing fetus
• Human chorionic gonadotropin (HCG), a hormone made by the placenta
• Estriol, a hormone made by the placenta and the fetal liver
• Inhibin A, another hormone made by the placenta
• Ideally, the quad screen is done between weeks 15 and 18 of pregnancy — during the second
trimester. However, the procedure can be done up to week 22.
 sFlt1 and PIGF –New kids on the block, beyond
aneuploidies!
• sFlt1 levels are increased, and free (unbound) PlGF levels are
suppressed in the serum of women during clinical preeclampsia.
• The changes in these markers precede the onset of clinical disease
by at least 5 weeks.
• Decreased free PlGF levels are observed even before 20 weeks
gestation in women who go on to develop preeclampsia
 Screening Timeline

NIPT Penta/Double Marker Quadruple sFlt1/ PIGF

100% sensitivity and 99.9% Penta - 92% DR for T21,

specificity T21, Sensitivity is 95% for early
88% for T18 and T13 81% DR for T21, T18, NTD onset pre eclampsia
92.9% sensitivity and 100%
specificity T18 100% and abdominal wall defects Near 100% DR for pre-
Double - 86% DR for T21, eclampsia within 1 week of
sensitivity and 99.9% 80% for T18
specificity T13. testing.
60% for T13
PPVs T21 - 98.3%,
T18 -100%,
T13 - 90.0%
 CONFIRMATORY DIAGNOSIS
Karyotyping
Low Diagnostic Yield
Only 3% of cases have a
diagnosis made
Slow Time to Results
Sample to Result – 7-14 days
Miss small changes
5MB is the smallest deletion you
can detect with a microscope and
a human eye
Very specially trained people
Difficult to scale
Subjective method
Prone to human error
QFPCR
Requires DNA for analysis- No
Cell Culture Issues
Completely Automated Process
Assay is also equally accurate
and efficient if performed on
minute amounts of DNA-
performing even in slow-growing
or contaminated cytogenetic
cultures
Can Identify and Quantify MCC
100% - Sensitive and Specific
CMA
Diagnostic yield ~30%
More information per test/ fewer
retests/reflex tests
Greater number of patients benefit
from definitive diagnosis
Rapid turn round of samples
Meaningful and actionable
information to patients in a timely
Superior resolution
Whole genome / high density CN
and SNP content
Increased confidence in results
Quantitative vs subjective
Increased confidence
CONFIRMATORY DIAGNOSIS
 Products of Conception Analysis
Sporadic Pregnancy Loss Recurrent Pregnancy Loss

• 30-50% Cytogenetic Abnormality

• 50-70%-Cytogenetic Abnormality
• Trisomy -60%, • Random aneuplodies with low recurrence.
• Monosomy X -20%, • Non-Random genetic aberrations.
• Polyploidy- 20% • Influence of maternal age & meiotic errors
INFERTILI
TY
 When to Take the Test?
MALE INFERTILITY PANEL
➔ Failure to achieve a clinical pregnancy
after 12 months or more of regular
unprotected sexual intercourse
➔ Abnormal sperm analysis results—very
low sperm count, or no sperm in the
semen
➔ Physical features that suggest a genetic
cause
➔ History of recurrent miscarriages
➔ Molecular confirmation of a clinical
diagnosis3
➔ Abnormal hormone test results
FEMALE INFERTILITY PANEL
➔ Failure to achieve a clinical pregnancy
after 12 months or more of regular
unprotected sexual intercourse
➔ History of recurrent miscarriages
➔ Molecular confirmation of a clinical
diagnosis
➔ Irregular ovulation
➔ Early menopause
➔ Abnormal hormone test results
 Benefits of Infertility Screening: Male And Female
● Identification of the infertility causes
● Assessment of reproductive risks for a couple planning to conceive
● For couples who are planning to become pregnant to know their reproductive risk
● Identification of genetic diseases transmissible to the baby
● Optimization of the assisted reproductive technology (ART)

Benefit for Men Benefit for Women

● for diagnostic purposes
● during clinical decision-making to establish the
most appropriate ART strategy (for example, in
the presence of deletions of the AZFa and AZFb
regions, the possibility of sperm recovery using o early diagnosis of polycystic ovary syndrome can
testicular biopsy is extremely low), and
● for prognostic purposes (to establish the risk of
transmitting the pathology and plan a prenatal
or preimplantation diagnostic procedures).
o early diagnosis of predisposition to severe
diminished ovarian response or premature ovarian
failure may help providers to better guide patients
to gamete cryopreservation in anticipation of future
pregnancies
lead to initiation of hormonal therapy to thereby
reducing ovarian hyperstimulation syndrome
Carrier Screening
 What is Carrier Screening?
Carrier screening is a genetic method that identifies couples who are at risk of passing inherited disorders to their children.
Most people do not even know if they are a carrier for an inherited genetic disease until they have a child afflicted with the disease. GenePass
Carrier Screening Test is a comprehensive investigation, which enables couples to make informed reproductive decisions and hence, should ideally
be performed when they start planning a pregnancy This test also allows doctors if an individual could pass serious inherited health conditions on
to your child .

“The American College of Obstetricians and Gynecology recommends offering carrier screening to all pregnant women or
couples considering pregnancy”
Couple Carrier Screening:
WHY And WHO
Couples who:
● Carriers are typically healthy and do not have
symptoms.
● Most of us are carriers of at least one genetic
condition.
● For most diseases, both partners have to be
carriers for the same condition for the child
children to be at increased risk
● Each individual harbours an average of 2.8
known severe recessive mutations.#
● are currently pregnant or planning a
pregnancy
● have a family history of a genetic disorder
● would like additional information about the
reproductive risks of having a child with a
genetic disorder
● are planning to donate eggs, sperm, or
embryos
● belong to a high-risk ethnic group
● have a consanguineous partner
 CASE STUDY 1

• Couple married non- consanguineously was currently pregnant with gestational age
of 22 weeks+ 05 days. The fetus ultrasound scan reports indicated
polyhydramnios, and club foot appearance.

• No genetic screening/ testing done.

• No family history
 CASE STUDY 1

Association of club foot with genetic syndromes:

• 20% of cases, clubfoot is associated with distal arthrogryposis, congenital myotonic
dystrophy, myelomeningocele, amniotic band sequence, or other genetic syndromes
such as trisomies, chromosome 22q11 deletion syndrome.
• In rest of the cases: deformity is isolated.

• Genetic testing recommended: Chromosomal Microarray+ Clinical Exome

Sequencing.
 CASE STUDY 1

Post Test Counseling:

Counseling for Trisomy 21 (Down Syndrome), Management options, connecting with support
groups “Down Syndrome Federation of India” (DSFI). Discussion with referring doctor.
 CASE STUDY 2

The couple married non- consanguineously for 7 years presented with history of 2
MTPs:

• G1- 2020, ?Lung issues, MTP

• G2- 2022; 24 weeks; bilateral echogenic kidneys, oligohydramnios and echogenic
intracardiac focus in left ventricle. Amniocentesis (Whole Exome Sequencing) was
done and further patient decided to terminate the pregnany.
 CASE STUDY 2

A complete three-generation family history was obtained. Husband is first born to a non- consanguineous
parents. He has 2 brothers and 1 sister. Wife has 2 sisters and 4 brothers. There is no history of cancer in
the family. Following is the pedigree based on family history reported:
 CASE STUDY 2

Whole Exome Sequencing for fetus:

 CASE STUDY 2

Whole Exome Sequencing for fetus:

• Likely compound heterozygous variant in PKHD1 gene associated with Polycystic kidney disease 4 with
or without polycystic liver disease (263200). The variant c.5751G>Ap.Gln1917Gln was identified in Exon
35 and was classified as Uncertain Significance.
• Another variant, c.982C>Tp.Arg328* was identified in Exon 14 and classified as Pathogenic. This
disorder is inherited in Autosomal Recessive manner.
• The couple was counseled about the condition associated with the disorder.
• Sanger validation of identified variants in the parents recommended.
 CASE STUDY 3

• Couple married non- consanguineously presented with history of two neonatal deaths (male child).
• Genetic testing was done for second child and a variant of Uncertain significance was identified in G6PD
gene variant c.961G>A (p.Val32Met) on exon 9 in hemizygous state corresponding to G6PD deficiency.
• Presently, the wife is diagnosed to have polycystic ovarian disease and she is under treatment for the
same.
• The couple was counseled about the disorder and explained testing options which included parental
sanger analysis, Couple Clinical Exome Sequencing and Couple Whole Exome Sequencing.
• The patient opted for Couple Whole Exome Sequencing.
 CASE STUDY 3

PEDIGREE:
 CASE STUDY 3

• Couple Whole Exome Sequencing report showed carrier for pathogenic variant in the wife for G6PD
deficiency. The husband’s report was negative.
 CASE STUDY 3

• The couple was explained about the Whole Exome Sequencing report.

• Further recommendations was shared with the couple which included:

• Sanger testing in every pregnancy to check the identified variants in the fetus by performing CVS/
Amniocentesis.
• IVF pregnancy with PGT- A and PGT-M.
• The couple opted for an IVF pregnancy with PGT-A and PGT-M. Pre- PGT workup report is awaited.
 CASE STUDY 4

• Couple aged 31 years married non- consanguineously.

• Gestational age: 19 weeks.
• Their NT/ NB scan at 12 weeks 05 days was normal. Their prenatal first trimester screening reported
normal. There is no history of miscarriage.
 CASE STUDY 4

• Couple Carrier Screening panel recommended.

Husband Wife

GENETIC TESTING RESULTS CAN BE VERY SURPRISING!

 CASE STUDY 4

Follow up and recommendations:

• Sanger Sequencing of the identified variant
and QFPCR test is suggested for the fetus OR
Post delivery Newborn Screening is
recommended with Sanger Sequencing of the
identified variant.
• Pre-natal testing in every pregnancy to check
for identified variant.
• Follow up with obstetrician/gynecologist is
recommended for routine ultrasounds.
• Post test genetic counseling is highly
recommended.
 CASE STUDY 5

• Couple presented with Bad obstetric history (BOH) and having a history of two neonatal death.
• In their first pregnancy (G1), there is history of neonatal death of a male child at 28th day of life with
congenital anomalies.
• In their second pregnancy (G2), there is history of neonatal death of a male child at first few months of
life due to clinically suspected Hemophagocytic Lymphohistiocytosis (HLH).
 CASE STUDY 5

• Clinical exome sequencing report of husband revealed a likely-pathogenic variant – c.658G>C

(p.Gly220Arg) in exon 3 of the PRF1 gene (NM_001083116.3) in heterozygous form causing autosomal
recessive Familial hemophagocytic lymphohistiocytosis 2 when mutation in a homozygous or compound
heterozygous state. Same variant is not detected in the wife but another missense variant detected in
the same gene in heterozygous state.
• Clinical exome sequencing report of wife revealed a likely-pathogenic variant – c.386G>C (p.Trp129Ser)
in exon 2 of the PRF1 gene (NM_001083116.3) in heterozygous form causing autosomal recessive
Familial hemophagocytic lymphohistiocytosis 2 when mutation in a homozygous or compound
heterozygous state. Same variant is not detected in the husband but another missense variant detected
in the same gene in heterozygous state.
 CASE STUDY 5

• After detailed genetic counseling, Pre-PGT-M opted for both the variants. Father’s
mutation c.658G>C (p.Gly220Arg) in the exon 3 of the PRF1 gene and Mother’s
mutation c.386G>C (p.Trp129Ser) in the exon 2 of the PRF1 gene causative of
autosomal recessive Familial hemophagocytic lymphohistiocytosis 2.
 CASE STUDY 5

• Based on Pre-PGT-M report, PGT-M was offered to the couple to screen embryos for the mentioned two
variants causative of Autosomal recessive Familial hemophagocytic lymphohistiocytosis 2.