project proposal

1. To determine the incidence and profile of
abnormal karyotype among fetuses with
anomalies detected by second trimester
anomaly ultrasonography scanning
Dr. Ramlingappa Antartani
Dept of Obstetrics and gynecology
KIMS, Hubballi
Co-Principal Investigators
Dr. Sahaja Kittur
Department of OBG
KIMS, Hubballi
Dr. Muragendraswami Astagimath
Department of Biochemistry
KIMS, Hubballi
Principal Investigator:

2. Introduction
The incidence rate of chromosomal anomalies in India is estimated to be
around 4-6 per 1000 live births, which translates to around 500,000-
600,000 children born with chromosomal disorders every year.
most common chromosomal anomalies in India are
• Down syndrome (Trisomy 21) (15-20%)
• Klinefelter syndrome (47, XXY) (10%)
• Turner syndrome (45, X) – (5%)
• Trisomy 13 and Trisomy 18 – (5-10% )
• Sex chromosome aneuploidies like 47,XXX; 47,XYY; 45,X/46,XX – (5-10%)

3. The major causes of chromosomal anomalies in India are advanced maternal age,
advanced paternal age, consanguineous marriages, and familial inheritance.
Compared to Western countries, the incidence of chromosomal anomalies is higher
in India, mainly due to high birth rate, high rate of consanguineous marriages, and
lack of access to prenatal screening and diagnosis.
About 94% of severe chromosomal anomalies occur in low- and middle-income
countries, where there is often limited access to prenatal screening and diagnosis.
Chromosomal anomalies are a significant contributor to infant mortality and
childhood disabilities in India.

4. Prenatal diagnosis allows for pregnancy termination,
planning for delivery management, and early therapeutic
interventions. It is a key component of prenatal care.
Prenatal diagnosis of chromosomal anomalies allows
• Pregnancy termination following diagnosis, where legal
• Planning for delivery management, including cesarean section where
indicated
• Referral to appropriate specialists for conditions like congenital heart
disease
• Early therapeutic interventions in the neonatal period and beyond

5. Karyotyping is the process of examining the number and structure of
chromosomes from a cell sample. It involves arresting cells in metaphase, staining
the condensed chromosomes to create a characteristic banding pattern, then
analyzing the chromosomes under a microscope and arranging them into a
standard karyogram to detect anomalies 1.
In prenatal diagnosis, karyotyping of fetal cells obtained through amniocentesis or
chorionic villus sampling is a key technique for detecting chromosomal
abnormalities when fetal structural anomalies are detected on ultrasound 2.
This study will examine the role and effectiveness of karyotyping specifically in
pregnancies with multiple fetal structural anomalies.

6. Karyotyping Process and Chromosomal Anomaly Detection
Karyotyping begins with culturing cells from the amniotic fluid or chorionic villi sample to stimulate
cell division and arrest the cells in metaphase when chromosomes are most condensed.
The cells are then fixed and stained, usually with Giemsa dye, to create a banding pattern unique to
each chromosome.
The chromosomes are examined under a microscope, photographed, and arranged into a
karyogram displaying the chromosome number and structure 1.
Karyotyping can detect major numerical chromosomal abnormalities like trisomies and
monosomies, where there are extra or missing chromosomes.
It can also detect large structural rearrangements such as deletions, duplications, translocations
and inversions 1.
However, it has limited resolution for detecting smaller chromosomal abnormalities below 5 Mb 3.

7. Common Fetal Structural Anomalies
Fetal structural anomalies refer to abnormalities in the development of the
fetus' bodily structure and organs 4.
Common anomalies detected on prenatal ultrasound that may warrant
karyotyping include
• Heart defects: abnormal development of the heart and major blood vessels
• Neural tube defects: incomplete closure of the brain and spinal cord like spina bifida
• Facial clefts: cleft lip and cleft palate
• Limb abnormalities: missing or malformed limbs and hands/feet
• Kidney anomalies: improper kidney formation, size or position
• Growth abnormalities: significantly larger or smaller than expected
• Brain anomalies: neural tube defects, enlarged ventricles Multiple structural anomalies in the
same pregnancy raises suspicion for an underlying genetic or chromosomal cause.

8. Role of Karyotyping in Prenatal Diagnosis of Multiple Structural Anomalies
When multiple fetal structural anomalies are detected on ultrasound, karyotyping of fetal cells is an
important part of the diagnostic workup 5.
Chromosomal anomalies are found in around 6-10% of pregnancies with multiple structural
anomalies, compared to 0.65% in the general obstetric population 6
Studies show karyotyping detects clinically significant chromosomal findings in 5.8-10.3% of fetuses
with multiple anomalies [[8],[9]].
The overall detection rate of chromosomal abnormalities in a study using conventional cytogenetic
analysis was 14.8%, the majority (72%) being associated with structural malformations, 20% with
non-immune hydrops and 4% with soft markers. Abnormal karyotypes were seen in 12.7% of
fetuses with structural malformations.7

9. The most common abnormalities were trisomy 13, 18 and 21. Karyotyping also
provides prognostic information, as chromosomal anomalies are associated with
higher risks of perinatal mortality and postnatal disabilities 8
However, karyotyping has limitations in resolution. Smaller chromosomal deletions or
duplications below 5 Mb may not be detected but could still have clinical
consequences.
However, normal karyotype results do not rule out all genetic conditions. Additional
testing and genetic counseling is still important for pregnancies with multiple
anomalies even if karyotyping is normal.
Therefore, additional genetic testing like chromosomal microarray analysis is
sometimes recommended following a normal karyotype result.

10. Aim of the study
To determine the incidence of abnormal karyotype among fetuses
with anomalies detected by detailed second trimester anomaly
ultrasonography scanning
Objectives
• 1. Screening of major chromosomal abnormalities by ultrasonography conducted in the second
trimester anomaly scan.
• 2. Analyze the chromosomal profiles of fetuses with structural anomalies using Karyotyping.
• 3. Correlate the findings with the severity and type of structural anomalies.

11. METHODOLOGY
Study Design : Prospective study
Place : Karnataka Institute of Medical Sciences, HUbballi
Study Population: mothers attending ANC in 2nd trimester of gestational period or referred to KIM’S
OBGYN department with multiple fetal structural anomalies.
Study duration: January 2024 to December 2024.
Consent is taken after explaining about the study.
Demographic information is collected
Detailed Obstetric history
Detailed data is collected on the ultrasound findings, including the type and severity of fetal
anomalies detected during the second-trimester anomaly ultrasound scan.
Anomalies are confirmed by radiologist at department of radiology KIMS,

12. After confirmation of the anomalies fetal cells from either amniocentesis,
chorionic villous sampling, or direct fetal tissue is collected and sent to MRU
for karyotyping.
Analyze the karyotyping results to determine the presence of chromosomal
anomalies in fetuses with structural anomalies detected by ultrasound.
Calculate the incidence of abnormal karyotypes among the study population.

13. Limitations:
The sample size may be small, which could limit the statistical power of the
study.
The data may be incomplete, as not all participants may have undergone
further testing.
The results of the study may not be generalizable to other populations.
The study could be limited to a specific population or location

14. Potential outcomes
Incidence of Abnormal Karyotypes:
• The study may provide an estimate of the incidence of abnormal karyotypes among fetuses with structural anomalies
detected by second-trimester ultrasound.
Types of Chromosomal Abnormalities:
• The study may identify the specific types of chromosomal abnormalities found in the study population. These could
include numerical abnormalities (e.g., trisomies) and structural abnormalities (e.g., translocations or deletions).
Prevalence of Specific Syndromes:
• The study may reveal the prevalence of specific chromosomal syndromes among fetuses with anomalies, such as Down
syndrome (Trisomy 21), Edwards syndrome (Trisomy 18), or others.
Association with Severity of Anomalies:
• The study might investigate whether there is a correlation between the severity or type of fetal structural anomalies
and the likelihood of having an abnormal karyotype.

15. Clinical Implications:
• Depending on the findings, this may include recommendations for further diagnostic testing, prenatal
counseling, and potential pregnancy management options.
Ethical Considerations: Counselling
• The study might address ethical considerations related to prenatal diagnosis, including the challenges of
balancing the desire for information with the potential emotional impact on parents.
Clinical Guidelines:
• Depending on the study's findings, it may contribute to the development or refinement of clinical guidelines for
the management of pregnancies with detected fetal anomalies.
These findings can inform healthcare providers, genetic counselors, and parents about the
importance of karyotyping in prenatal diagnosis and its implications for clinical practice.

16. References
1. O'Connor, C. (2008) Karyotyping for chromosomal abnormalities. Nature Education 1(1):27
2. https://mercy.net/service/fetal-anomaly
3. Daniel A. Queremel Milani; Prasanna Tadi. Genetics, Chromosome Abnormalities. StatPearls
Publishing; 2023 Jan-. PMID: 32491623
4. Overview of Chromosomal Anomalies By Nina N. Powell-Hamilton , MD, Sidney Kimmel Medical
College at Thomas Jefferson University.
• https://merckmanuals.com/professional/pediatrics/chromosome-and-gene-anomalies/overview-of-
chromosomal-anomalies
5. Lichtenbelt KD, Knoers NV, Schuring-Blom GH. From karyotyping to array-CGH in prenatal
diagnosis. Cytogenet Genome Res. 2011;135(3-4):241-50. doi: 10.1159/000334065. Epub 2011
Nov 12. PMID: 22086062.
6. Karyotyping: MedlinePlus Medical
Encyclopedia. https://medlineplus.gov/ency/article/003935.htm
7. The Journal of Obstetrics and Gynecology of India (July–August 2022) 72 (S1):S209–S216
• https://doi.org/10.1007/s13224-022-01626-x

17. Thank You

18. Steps of Karyotyping
• Sample Collection:
• Obtain a biological sample that contains cells with chromosomes. Common sources include amniotic fluid,
chorionic villus samples, peripheral blood, or tissue samples.
• Cell Culturing:
• If the sample contains a limited number of dividing cells, such as amniocytes or chorionic villus cells, these
cells are cultured in a special growth medium to stimulate cell division. Culturing allows for the generation of
more cells for analysis.
• Harvesting Cells:
• After cell culturing, the cells are collected and prepared for chromosome analysis. Cells are usually treated
with a mitotic spindle inhibitor (e.g., colcemid) to arrest cell division during metaphase when chromosomes
are most condensed and visible.
• Cell Fixation:
• The harvested cells are then treated with a fixative (typically methanol and acetic acid) to preserve the cell
structure and chromosomes.
• Slide Preparation:
• A small number of fixed cells are dropped onto glass slides and spread evenly to create a cell monolayer.
These slides are then heat-fixed to attach the cells firmly to the slide surface.

19. • Staining:
• The prepared slides are stained with a special dye called Giemsa or a similar chromosomal stain. This staining helps to reveal
the distinct banding patterns on chromosomes, which are essential for identifying structural abnormalities and arranging the
chromosomes in pairs.
• Microscopy:
• An experienced cytogeneticist or technologist examines the stained chromosomes under a high-powered microscope. This
involves identifying individual chromosomes, pairing them, and assessing their size, shape, and banding patterns.
• Photography and Imaging:
• Images of the chromosomes are captured using specialized imaging equipment. These images are essential for
documentation, analysis, and reporting.
• Karyogram Construction:
• The cytogeneticist or technologist creates a karyogram, which is a visual representation of the patient's chromosomes
arranged in pairs based on size and banding patterns. This step helps in identifying numerical abnormalities, such as
trisomies or monosomies.
• Analysis and Interpretation:
• The cytogeneticist analyzes the karyogram for any abnormalities, including numerical anomalies, structural rearrangements
(such as translocations, deletions, or duplications), and other chromosomal aberrations. The findings are documented in a
report.