Session 5 Flashcards
What % of oocyte and sperm and chromosomally abnormal?
Oocyte (20-25% chromosomally abnormal)
Sperm (~10% chromosomally abnormal)
What is an occult abortion?
Failure to implant (~30% of conceptus) or transient implantation (~30% of conceptus) with little disruption of menstrual cycle
What is most common single trisomy?
Trisomy 16 - present in ~1% of conceptions.
Incompatible with life if full trisomy and represents ~10% of miscarriages
What trisomies are compatible with a live birth?
Trisomy 13 - Patau
Trisomy 18 - Edwards
Trisomy 21 - Down
What is most common error that produces a trisomy?
maternal meiosis I non-disjunction (Trisomy 18 is exception and usually occurs at maternal meiosis II)
What factor is most associated with increased risk of de novo aneuploidy?
Maternal age - reduced recombination, spindle errors, reduce immunological competence of an older mother
What abnormalities are seen in Second trimester pregnancy loss?
The more ‘viable’ trisomies: +21 (63%), +18 (23%), +13 (13%)
Structural rearrangements
What abnormalities are seen in third trimester pregnancy loss?
The more ‘viable’ trisomies: +21 (32%), +18 (51%), +13 (17%).
Structural rearrangements
What two types of Triploidy exist?
2/3 shown to have two paternal chromosome sets =diandry , usually arising from dispermy
1/3 shown to have two maternal chromosome sets = digyny , due to a diploid egg
What occurs in cases of diandric triploidy?
Most abort in the first or early second trimester, presenting as hydatidiform mole.
What occurs in cases of digynic triploidy?
Nonmolar and mostly abort early (mean 10 weeks). Surviving dygynic triploids develop as a severely growth retarded fetus with macrocephaly and an abnormally small and nonmolar placenta.
What is a complete mole?
Diandric diploidy - two sperm enter an ‘empty’ ovum. No embryo. High risk of choriocarcinoma.
What is an ovarian teratoma?
Dygynic diploidy - 46 chromosomes maternal in origin due to abnormal development of primary oocyte. Fatal. May contain fully differentiated tissue e.g. hair, nail.
What can occur due to chromosome abnormalities in the placenta?
Risk of misdiagnosis when testing CVS due to CPM
Pregnancy loss and intrauterine growth retardation due to incorrect placenta development
What defines recurrent miscarriage?
Three or more miscarriages before 24 weeks post-menstruation
Why can cell culture of POC be difficult?
- Maternal cell contamination
- Higher risk of infection
- Culture failure to due to lack of viable cells
- Mosaicism (biological may lead to overgrowth of normal cell line)
When is testing of POC recommended?
- Pregnancy loss or termination with significant fetal malformations
- Pregnancy loss >24 weeks
- Miscarriages (<24 weeks) for 3rd and subsequent miscarriages
What are all women in the UK offered for T21 screening?
o Information to help them decide if they want screening or not
o A screening test for Down syndrome (DS) that meets national standards
o An ultrasound scan (week 18-24+6) to check for any physical abnormalities in the fetus
What is the screening risk cut off for T21 to be offered invasive?
> 1 in 150
What is Nuchal Translucency?
Measured between 11+2 and 14+1 weeks - the NT is maximum thickness of the subcutaneous translucency between the fetal skin and the soft tissue overlying the cervical spine
What is regarded as an increased nuchal Translucency and what does it indicate?
> 3.5mm
Strongly associated with aneuploidy. Or if normal K’type of structural malformations (often causing Cardiac failure) and genetic syndromes.
What different markers are measured by maternal serum screening approaches?
Multiples of the Median for different markers :
PAPP-A - pregnancy-associated plasma protein-A is released by fetus and lower levels indicate possible genetic defect
AFP - alpha-fetoprotein (AFP) released by fetus and lower levels indicate possible genetic defect
β-hCG - Human chorionic gonadotropin is released by placenta and high levels indicate possible genetic defect (low for T13 and T18)
uE3 - Unconjugated estriol is released by placenta and low levels indicate possible genetic defect
Inhibin A is released by placenta and high levels indicate possible genetic defect
What are the two recommended Serum screening strategies and what differentiates them?
Combined Screening - β-hCG, PAPP-A+ NT - performed in 1st trimester (11+2 ideally)
Quadruple Screening – AFP, β-hCG, uE3+ Inhibin A
- second trimester (14+2 - 20)
What factors can effect serum screening?
- marker levels tend to be decreased in heavier women, and increased in lighter women
- Serum marker levels tend to be higher in Afro-Caribbean women than in Caucasian women
- IVF pregnancy (egg donor age in calculation)
- Diabetic mothers
- Smokers
- Twin pregnancies
When is the fetal analomy scan performed?
18+0 and 20+6 weeks
When was cell free fetal DNA first introduced?
In 1997, Lo et al showed that cffDNA from the Y chromosome of male fetuses was present in maternal blood
When is cffDNA detectable?
from 4-5 weeks but reliably from 7 weeks
Why is cffDNA specific to this pregnancy and not previous ones?
cffDNA is rapidly cleared from circulation within an hour after delivery (mean half-life of 16-17 minutes)
Where does cffDNA originate and what size fragments are found?
Death of placental cells - breaking up the chromosomes into short fragments = average 166bp (size range is 200 – 300 bp)
What are technical challenges of using cffDNA?
- Low concentration
- Need to use STECK tubes to stop nucleated cells that are collected releasing DNA into plasma at high levels
- Much more maternal cfDNA
- Half of the cffDNA is maternally inherited so hard to distinguish
What are the main clinical applications of NIPD?
- Fetal sex determination in pregnancies at risk of a sex-linked disease.
- Diagnosis of certain single gene disorder (bespoke NIPD)
- Detection of fetal aneuploidy
- Diagnosis of fetal blood type
- Detection of the high risk allele using RHDO (relative haplotype dosage analysis)
How is cffDNA used for fetal sex determination?
What are the two main? indications?
cfDNA extracted from maternal plasma and RT-PCR be used to look for presence of SRY
- Known X-linked condition (if female no invasive)
- congenital adrenal hyperplasia (CAH) (if female give dexamethasone to avoid virilisation)
What methods are used for confirmation of cffDNA in a sample?
Presence of paternal specific SNPs
Presence of universal fetal epigenetic markers (best are markers which can targeted by restriction enzyme to reudce
maternal cfDNA levels)
What methods can be used to enrich cffDNA?
Epigenetic (immunoprecipitation of fetal methylation pattern or restriction enzymes cutting of maternal)
Size (<50% of aternal fragments are <300bp)
Outline NIPT by MPS WGS
Plasma isolation of cfDNA
Barcoding and multiplexing
Sequencing
Distribution of reads across chomosomes to look for aneuploidy compared to normal
Can use fragment size to estimate fetal fraction and look for fetal specific markers too
How does RHDO for X-linked conditions work?
SNPs that are heterozygous in Mum to determine whether fetus has inherited same X chromosome as reference sample
Look at which SNPs are over represented to determine
How does RHDO for AR conditions work?
Requires proband to be previous child of the couple
Divides SNP genotypes in categories of usefulness:
1: Mum AA and Dad BB - used for fetal fraction
2: Mum AA and Dad AB - Fetal inheritance of paternal haplotype
3: Mum AB and Dad AA - Fetal inheritance of maternal haplotype
Presence of category 3 paternal SNPs from mutated allele = inheritance of that allele by fetus
More complex for the maternal=
1. If Mutant maternal allele SNPs matches paternal allele (alpha mat) then overrepresentation of category 4 maternal SNPs from mutated allele = inheritance of that allele by fetus
- If normal maternal allele matches paternal allele (beta mat) then will be equal category 4 maternal SNPs from mutated allele = inheritance of that allele by fetus
What complicated RHDO NIPD?
Multiple pregnancy effects SNP alleles
Low fetal fraction
Consanguinity reduced number informative SNPs
Only testing for haplotype not variant - risk of de novo
What other method can be used for NIPD for AR conditions? What does it require?
Paternal exclusion - requires parents to have different variants and then only excludes presence of paternal variant
For which two genes is NIPD for de novo AD variants available in the UK? When?
FGFR2 and FGFR3
Based on specific ultrasound findings
What occurs during oogenesis?
During female embryo development, diploid primordial germ cells migrate into the embryonic ovary and undergo rapid proliferation (mitosis) – lasts from 15th week of gestation until 7th month – gives rise to ~7 million oogonia.
Most oogonia die, but remaining (the primary oocytes) enter meiosis and pause at diplotene stage until puberty.
Oocytes then begin to move through meiosis ready for ovulation. Meiosis 1 produces a secondary oocyte and a polar body (haploid nucleus but no cytoplasm - will die).
Secondary oocytes then freeze at metaphase of meiosis 2 until fertilised.
What occurs during spermatogenesis?
Diploid primordial germ cells migrate into the embryonic testis and undergo to create spermatogonia
Some stop proliferating and differentiate into primary spermatocytes ready for meoises. In meiosis I – division is symmetrical (generating identical diploid secondary spermatocytes); in meiosis II, four equally-sized haploid spermatids are produced (24 hours).
Spermatids then differentiate into sperm (5 days)
When does an embryo become classed as a fetus?
8 weeks gestation
What occurs during fertilisation?
In the ampulla of the fallopian tube the sperm attaches to the outer layer of follicle cells of the oocyte and then reaches the zona pellucida (ZP). A cell-surface receptor on the sperm head binds to a ZP glycoprotein (PDILT gene). Acrosome (the head of the sperm) bursts – releasing enzymes that digest the ZP and allow sperm nucleus to entire oocyte.
The cortical reaction occurs - cortical granules inside the oocyte fuse with the plasma membrane of the cell and enzymes inside expell to the zone pellucida – glycoproteins in the zona pellucida to crosslink and become impermable to sperm.
Oocyte undergoes its second meiotic division
Sperm’s tail and mitochondria degenerate
Haploid oocyte and sperm nuclei form the pronuclei
and undergo first mitotic division occurs i.e. the first mitosis of the union of sperm and oocyte is the actual fusion of their chromosomes
Fertilised oocyte = totipotent zygote
What stops implantation until the embryo reaches the uterus?
What occurs if implantation happens early?
The zona pellucida stops implantation until disregarded in the uterus
Ectopic pregnancy
What occurs in the first week of embryogenesis?
Cleavage: first plane is vertical; in the second round of division, one of the cells cleaves vertically and the other horizontally (rotational cleavage) - they can divide at different times to produce embryos with odd numbers of cells.
Compaction: Loosely-associated blastomeres of the eight-cell embryo – flatten against each other and form a tightly-packed morula.
Formation of a hollow ball of cell with a fluid filled cavity - the blastocele.
Becomes the blastocyst - Outer layer of cells (trophoblast) – gives rise to the chorion (an extraembryonic membrane which provides the fetal component of the placenta). Inner cell mass (ICM) at one end of blastocele.
The zona pellucida is degraded and the blastocyst implants into the uterus
What is gastrulation?
Process where the orientation of the body is established; the embryo is converted from a bilaminar structure into a structure of three layers – ectoderm, endoderm and mesoderm
What tissues arise from each germ layer?
Endoderm = Lung, Liver, pancreas, thymus,
endocrine glands
Mesoderm = Blood, vessels, muscular tissue, connective tissue
Ectoderm = Skin and its derivatives, nervous system
At what three times points can PGD be undertaken?
o Biopsy of the polar bodies just prior to conception (first polar body) and after fertilisation (second polar body).
o Day 3 cleavage stage (5–8 cell embryonic stage) biopsy of 1-2 blastomeres.
o Trophectoderm (blastocyst) biopsy is performed on day 5–6 (or rarely day 7) embryos that consist of ~120 cells; 5-10 cells are removed from the trophectoderm as it “hatches” through the zona pellucida. Blastocyst stage biopsy is advantageous for testing compared with biopsy at the cleavage stage because more cells are biopsied and tested
What can lead to both normal and abnormal allele patterns can be obtained for a single chromosome on QF-PCR?
Somatic Microsatellite Mutations (SMMs)
Polymorphic Submicroscopic Duplications/Submicroscopic microsatellite duplications (SMDs)
Partial Chromosome Imbalance
Copy Number Variant (CNV)
Primer Binding Site Polymorphisms
What are the common ultrasound findings for T21?
nuchal oedema
cardiac defects (mainly AVSDs)
duodenal atresia (30% of cases have T21)
echogenic bowel
sandal gap
clinodactyly of the fifth finger
What are the common ultrasound findings for T18?
Strawberry shaped head
absent corpus callosum
nuchal oedema
heart defects
horseshoe kidney
diaphragmatic hernia
exomphalos
IUGR
overlapping fingers and talipes or rocker bottom feet
Decreased placental volume
What are the common ultrasound findings for T13?
Holoprosencephaly with associated facial abnormalities (cleft lip and palate)
IUGR
microcephaly
Cardiac (mostly ASD or VSD) and renal abnormalities
Often large and echogenic kidneys
exomphalos
What are the common ultrasound findings for Turner syndrome?
Lymphoedema
renal anomalies
What are the common ultrasound findings for triploidy?
Severe IUGR
Hydrops
CNS abnormalities
What is a complete mole?
Diploid (46 chromosomes) androgenetic pregnancy where both sets of chromosomes are paternally derived
No formation of a fetus or embryo
Presence of excess paternal material leads to trophoblastic hyperplasia with no formation of a fetus due to the absence of maternal chromosomal DNA
Placenta has swollen villi
Generally evacuated before 12 weeks gestation
How do complete moles arise?
Most common haploid sperm fertilises empty egg and doubles up to be 46,XX
Rarer di-spermic fertilisation of empty egg
What genes are associated with Familial recurrent hydatidiform moles (FRHM)?
NLRP7 at 19q13.42 (OMIM: 609661) and KHDC3L at 6q13 (OMIM: 611687)
Both thought to have roles in maintaining the maternal imprint within the ovum
What blood marker is associated with complete mole?
Raised hCG
What can arise from a molar pregnancy?
Invasive moles and Gestational choriocarcinomas
What are the disadvantages of mircoarray for prenatal?
Does not detect balanced rearrangements
Lower level mosaicism may be missed
Triploidy will be missed or mis-classified
Difficult interpretation of CNVs with limited phenotype info
Incidental findings
Postnatal findings (e.g. dev delay reduced penetrance)
What is a dizygotic twin?
Two eggs each fertilised by different sperm cells with their own placenta and sac (always dichorionic and diamniotic)
What is associated with increased rate of dizygotic twins?
increased incidence of multiple follicle growth and multiple ovulation rate
What is a monozygotic twin?
Develop from same egg, which splits and forms two embryos
What are the subtypes of monozygotic twins?
- 33% dichorionic/diamniotic (DC/DA)- division occurs during or before the morula stage (~ 4 days post fertilisation, 10 - 30 cells, before blastocyst stage)
-66% monochorionic/diamniotic (MC/DA)- division occurs at the blastocyst stage (after morula stage, formation of the blastocoel cavity) involving division of the inner cell mass.
- rare monochorionic/monoamniotic (MC/MA) embryos divide after amnion formed at day 9. These twins are occasionally conjoined
What is Twin to twin transfusion syndrome (TTTS)?
Affects 4-35% of MC/DA pregnancies
Artery from one twin joined to the vein of the other allowing unidirectional blood flow from a donor twin to a recipient twin, resulting in asymmetrical fetal growth and fetal mortality in 80% or more of untreated cases.
What is Twin reversed arterial perfusion (TRAP)?
In monochorionic placentas with artery-artery & vein-vein anastomoses one twin is acardiac & is perfused in a reverse direction by the ‘pump’ co-twin.
What is a vanishing twin?
What can it cause for NIPD?
One twin suffers early fetal death and isn’t aborted - may be represented by areas of degenerate villi or amorphous debris in free membranes but is often difficult to detect
CffDNA from vanished twin persists after its demise and can cause false positives for NIPD
