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Cellular & Molecular Pathology > Infertility, IVF and genetic testing > Flashcards

Infertility, IVF and genetic testing Flashcards

1
Q

what fraction of couples in the UK are affected by infertility?

A

1 in 6/ 1 in 7

around 3.5 million people

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2
Q

what are the types of infertility?

what percentage do each ocntribute

5

A

25% unexplained
25% ovulatory
20% tubal
30% male factor
10% uterine

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3
Q

what are the main causes of infertility for females?

A
  • Problems with ovulation
  • Tubal problems
  • Uterine problems
  • Age
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4
Q

what are the main causes of infertility for males?

A
  • Poor sperm quality
  • azoospermia
  • sperm dysfunction
  • ejaculation disorders
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5
Q

what two causes of infertility can apply to both sexes?

A
  • unexplained
  • immunological infertility
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6
Q

what is anovulation?

A

Anovulation happens when an egg (ovum) doesn’t release from your ovary during your menstrual cycle

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7
Q

what is oligoovulation?

A

a condition that causes irregular or infrequent periods.
because eggs don’t mature or get released like they should be

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8
Q

what are the primary causes of ovulation problems?

A

 Surgical removal of ovary
 Ovaries damaged by radiotherapy/ chemotherapy
 Premature menopause (affects 1-2% <40years)
 Congenital defect
 Polycystic ovaries

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9
Q

what are the secondary causes of ovulation problems?

A

 Severe stress
 Recent large gain or loss of weight
 Tumour
 Excess prolactin
 Disturbances in thyroid and adrenal gland

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10
Q

What are luteal phase defects?

A

Defect of progesterone secretion by corpus luteum or defective response of the endometrium to hormonal stimulation.
- Results in inadequate endometrium for embryo implantation

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11
Q

what happens in luteal phase defects?
3

A
  1. Poor follicle production
  2. Premature failure of corpus luteum
  3. Failure of uterine lining to respond to progesterone
    - resulting in inadequate endometrium for embryo implantation
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12
Q

what is polycystic ovarian syndrome?

A

Most common cause of ovulation disorders in women of reproductive age.
Characterised by many minute follicles in the ovaries and an excess production of androgens. (cysts less than 1mm and rarely grow to maturity)
Associated with weight gain, excessive hair growth, irregular, infrequent or absent periods and infrequent or absent ovulation

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13
Q

what is the incidence of PCOS in those with oligomenorrhoea, amenorrhoea and anovulation?

A

90% women with oligomenorrhoea
30% women with amenorrhoea
70% women with anovulation

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14
Q

what is oligomenorrhoea?

A

irregular and inconsistent menstrual blood flow in a woman

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15
Q

what is amenorrhoea?

A

the absence of menstruation, often defined as missing one or more menstrual periods

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16
Q

what treatment options are there for people with polycystic ovarian syndrome?

A

 Lose weight if over weight
 Induce ovulation with clomiphene tablets
 Controlled ovarian stimulation with FSH and hCG
 Surgery

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17
Q

give two examples conditions causing ovulation problems?

A
  • luteal phase defect
  • polycystic ovarian syndrome
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18
Q

what are common tubal problems?
3 broad

A
  • damaged fimbriae (prevent movement of oocyte into the fallopian tube)
  • adhesions may distort the tube
  • tubal blockage (prevents the sperm from reaching the oocyte or prevents the zygote fom moving to the uterus leading to increase incidence of ectopic pregnancy)
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19
Q

what are the causes of tubal problems?

A
  • infection
  • previous ectopic pregnancy
  • congenital abnormality
  • hydrosalpinx
  • endometriosis
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20
Q

what is hydrosalpinx?

A

blocked, dialated, fluid filled fallopian tubes usually caused by previous pelvic infection

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21
Q

what is endometriosis?

what percentage of women of childbearing age does it affect?

A

Misplaced endometrial tissue outside the uterus
Commonly affects the ovaries and Fallopian tube, less commonly the bowel and bladder
If severe, may reduce fertility
Associated with mild, severe or chronic pain during menstruation and sexual intercourse may be painful

affects 10% of women of childbearing age

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21
Q

what is endometriosis?

what percentage of women of childbearing age does it affect?

A

Misplaced endometrial tissue outside the uterus
Commonly affects the ovaries and Fallopian tube, less commonly the bowel and bladder
If severe, may reduce fertility
Associated with mild, severe or chronic pain during menstruation and sexual intercourse may be painful

affects 10% of women of childbearing age

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22
Q

what treatment is available to those with endometriosis?

A
  • Pain relief with NSAIDs - non steroidal anti-inflammatory drugs
  • Prevent fluctuation in the woman’s hormone levels to remove the stimulation for growth of the endometriosis eg the oral contraceptive pill
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23
Q

what are the causes of uterine problems?

A

 Fibroids – benign growths arising from smooth muscle of the uterus.
 Uterine polyps – small growths of endometrial tissue dangling in the uterus may interfere with implantation
 Uterine adhesions – may be consequence of infection or surgery may occlude uterine cavity
 Congenital problems – absent uterus, hypoplastic uterus, double uterus, uterine septum etc

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24
Q

why is female age a cause for infertility?

5

A

reproductive function declines as a women ages, particularly after 35

women have a finite number of eggs
egg quality decreases with age
chromosomal abnormalities increase in late 30s
aging also affects hormone production and ovulations
higher incidence of miscarriage in women in their later 30s

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25
Q

what are the main causes of male infertility?
how often do they occur?

A
  • poor sperm quantity or quality (90%)
  • azoospermia (3-4%)
  • sperm dysfunction (3-6%)
  • ejacualtion disorders (4-6%)
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26
Q

what could cause poor sperm quality or quantity?

A
  • Low motility (asthenozoospermia)
  • High percentage of abnormal sperm (teratozoospermia)
  • Hormone deficiency
  • Testicular varicocele
  • infection
  • drugs eg antidepressants, antihypertensives, anabolic steroids, smoking and excessive alcohol
  • antisperm antibodies
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27
Q

what is asthenozoospermia?

A

low sperm motility

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28
Q

what is azoospermia?

A

the medical term used when there are no sperm in the ejaculate.

It can be “obstructive,” where there is a blockage preventing sperm from entering the ejaculate, or it can be “nonobstructive” when it is due to decreased sperm production by the testis.

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29
Q

what is teratozoospermia?

A

high percentage of abnormal sperm

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30
Q

what could cause azoospermia?
6

A
  • no spermatogenesis
  • blocked vas deferens, or congenitally did not develop
  • trauma to the testicles,
  • severe mumps infection after puberty
  • chemotherapy/radiotherapy
  • Klinefelter’s syndrome
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31
Q

what could cause sperm dysfunction?

normal semen analysis but sperm have defective fertilising capacity
3

A
  • Defective acrosome
  • Abnormal sperm movement
  • Inability of sperm to bind to the zona pellucida
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32
Q

give two examples of ejaculation disorders?

A
  • Retrograde ejaculation – semen ejaculated backwards into the bladder 1%
  • Impotency
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33
Q

what is immunological infertility?

A

antispem antibodies can be present in either or both partners
present in either the blood or the genital tract secretions (eg cervical mucus or ejaculate)

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34
Q

what can cause immunological infertility?

A
  • In ejaculate anti-sperm antibodies cause sperm to stick together and prevent them from being released
  • In the female anti-sperm antibodies may interfere with sperm transport and fertilisation
  • Unknown cause but may be due to genital infection or vasectomy
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35
Q

what is unexplained infertility?

A
  • Failure to conceive after one year of unprotected intercourse and despite thorough investigations no cause of infertility could be determined
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36
Q

what are the origins of human in vitro fertilisation?

A

 Pioneered in 1970s by Patrick Steptoe and Robert Edwards
 1978 first baby born by IVF

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37
Q

who regulates human in vitro fertilisation in the uk?

A

human fertilisation and embryology authority (HFEA)

38
Q

since 1991 how many IVF treatment cycles have been performed andd how many babies have been born in the uk?

A

1.3 million IVF treatment cycles
~390000 babies born

39
Q

what was the overalll birth rate per embryo transferred in 2018

A

24%

40
Q

what were the average multiple birth rates in 2008 vs 2019

A

24% in 2008
6% in 2019

41
Q

single embryo transfer has increased to what in 2019 vs 1991

A

in 2019 one embryo transferred in 75% of IVF cycles compared to just 13% in 1991

42
Q

describe the trend of fresh vs frozen birth rate between women of different ages

A

for those under 35 fresh cycles have a slightly higher success rate
If you have a frozen cycle then the older women have a better chance, because their eggs were obtained when they were younger so they were better quality so there is a higher success rate

43
Q

what are the 9 stages in in vitro fertilisation and embryo transfer (IVF-ET)

A
  1. Preliminary testing – ultrasound to check anatomy check for cysts and other things
  2. Downregulation
  3. Ovarian stimulation
  4. Monitoring growth of follicles
  5. Egg collection guided by transvaginal ultrasound
  6. Sperm sample provided same day as egg collected
  7. Fertilisation and embryo culture
  8. Embryo transfer
  9. Pregnancy testing/ monitoring
44
Q

what are two common types of IVF?

A
  • Standard IVF – oocytes incubated with sperm overnight and allowed to fertilise the oocytes
  • ICSI (Intracytoplasmic sperm injection) – single sperm injected directly into the oocyte

- In 2017 ICSI accounted for 34% of fresh IVF treatments in UK

44
Q

what are two common types of IVF?

A
  • Standard IVF – oocytes incubated with sperm overnight and allowed to fertilise the oocytes
  • ICSI (Intracytoplasmic sperm injection) – single sperm injected directly into the oocyte

- In 2017 ICSI accounted for 34% of fresh IVF treatments in UK

45
Q

what occurs in the downregulation stage of IVF treatment and why?

A

To stop body’s normal control mechanisms for ovulation
Gonadotropic releasing hormone agonist is administered sometimes via nasal spray Nafarelin - GnRH dont dissociate readily from their receptors and cause the Flare effect, an initial increase of pituitary hormones FSH and LH (which lasts 10 days)
confirmed via blood test for oestradiol levels

46
Q

what occurs at the ovarian stimulation stage of IVF treatment and why?

A

To increase the number of oocytes produced
Stimulate ovaries with daily injections of FSH (Eg Gonal F)
(Continue to take GnRH agonist nasal spray)
Monitor response of drugs
- Blood tests (so that oestradiol levels don’t rise too quickly)
- Ultrasound scans to measure the number & size of follicles
When follicles have reached right size (lead follicles ~18mm)a hCG injection (eg Profasi) given to trigger final oocyte maturation
Stop the GnRH agonist nasal spray and FSH injections

47
Q

what occurs during the oocyte retrival stage of IVF treatment?
when does it occur?

A
  • Occurs 34-36h post hCG injection
    A thin needle is passed through the vaginal wall into the ovaries guided by vaginal ultrasound.
    The follicular fluid is gently aspirated and given to embryologists to search for oocytes
    Procedure takes ~20-30mins.
    A good cycle should produce ~10 oocytes
48
Q

what occurs during insemination, fertilisation and embryo transfer?

A

Semen sample obtained, washed and concentrated
20,000 - 30,000 motile sperm/ml are incubated with the oocytes overnight in culture medium to allow fertilisation to occur.
Embryologists perform a fertilisation check to obtain the number of fertilised oocytes (zygotes) of normal morphology ie the presence of 2 pronuclei = Day 1
If embryos have developed, 1 or 2 are transferred via a catheter through the cervix into the uterus, either on day 2 or 3 (2-cell to 8-cell stage), or at the blastocyst stage
Progesterone vaginal pessaries are taken daily to support the endometrium and increase the chance of success

49
Q

when does pregnancy testing occur in IVF treatment cycle?

A

Blood test on day 14 – measures level of hCG
If positive repeated on day 16 to check for rising hCG levels
Stop taking progesterone pessaries

50
Q

where is there a dramatic drop between stage cycle reached?

A

between embryo and transfer and pregnancy

51
Q

how are embryos selected for transfer in Assisted Reproductive Technologies?

A

Predominantly based on morphological criteria
- rate of cleavage
- embryo scoring
- blastocyst scoring
More recent developments
- live cell imaging

52
Q

how are embryos selected for transfer in Assisted Reproductive Technologies?

A

Predominantly based on morphological criteria
- rate of cleavage
- embryo scoring
- blastocyst scoring
More recent developments
- live cell imaging

53
Q

embryos that cleave to the 2 cell stage between what time period are more viable than pronuclear embryos?

A

24-25hr post insemination

54
Q

on day 2 (44-48hr postinsemination) what stage should the embryos be at

to have a higher priority transfer

A

4 cell stage

55
Q

what are the morphological embryo grades?

A

Grade 1 - even blastomeres, no fragmentation
Grade 2 - even blastomeres, <10% fragmentation
Grade 2.5 - even blastomeres, <30% fragmentation
Grade 3 - eneven blastomeres, >30% fragmentation
Grade 4 - only one viable blastomere
Grade 5 - no viable blastomere

56
Q

what are the grades in blastocyst scoring?

A

Grade 1 - early blastocyst: blastocoel being less than half the volume of the embryo
Grade 2- blastocyst: blastocoeal being greater than half the volume of the embryo
Grade 3 - full blastocyst: blastocoel completely fills embryo
Grade 4 - expanded blastocyst: blastocoel volume is now larger than that of early embryo and zona is thinning
Grade 4A - ICM tight packed many cells, TE many cells forming cohesive epithelium
Grade 4B - ICM loosley group several cells, TE few cells forming loose epithelium
Grade 4C - ICM very few cells, TE very few large cells

57
Q

what are the pros and cons of blastocyst culture?

A

Pros
* Transfer of embryo to uterus - Blastocyst is usually found in the uterus where as early zygotes are usually in the fallopian tubes at the stage they are injected
* Assess embryo viability prior to transfer
* Increase time between cleavage stage biopsy and transfer

Cons
* Requires ~8 or more oocytes
* Prolonged culture in artificial environment
* Higher risk of no transfer/ culture cancellation
* Reduced number of embryos for cryopreservation

58
Q

what percentage of transfer are at the cleavage stages vs the blastocyst?

A

In UK ~75% of women have a blastocyst transfer and ~25% cleavage stage transfer

59
Q

what is live cell imaging and how is it used in assisted reproductive technologies?

A

multiple images taken of embyo thoughout development
computer software can analyse images to determine success
automated tracking of blastomeres can determine succesful development to the blastocyst stage by the 4 cell embryo

60
Q

what factor shows the importance of egg quality?

A

ESSP1 is maternally inherited DNA that is degraded as the embryo develops
ESSP2 is first transcribed on day 3 at 8 cell stage

we can predict success prior to embryonic gene activation at the 4 cell stage - so good quality egg detemines success

61
Q

what is preimplantation genetic testing (PGT)?

A

test perfromed to analyse the DNA of embryos for human leukocyte antigens (HLA) typing or for determining genetic abnormalities

62
Q

what are the two types of preimplantaion genetic testing?

A

Preimplantation genetic testing for monogenic/ single gene defects PGT-M - Detects monogenic disorders. Also used for HLA testing with or without concurrent testing for monogenic disorder.
Preimplantation genetic testing for aneuploidy PGT-A – Detects abnormal number of chromosomes in embryo biopsies

63
Q

at what two stages are the embryos biopsied for preimplantaiton genetic testing?

A

8-cell stage (1 cell removed), or blastocyst stage (5-10 trophectoderm cells removed)

64
Q

what are the pros and cons of biopsy at cleavage stage vs blastocyst?

A

Cleavage stage Pros - on day 3 so allows 2-3days for PGT analysis before fresh embryo transfer at blastocyst stage. No effect on development if embryo is good quality. Cons - mosacism thought to be higher at early cleavage stage

Blastocyst Pros - not removing cells that will form the embryo. Cons - not all zygotes will develop to blastocyst so there will be fewer embryos to biopsy

65
Q

why is PGT-M used?

A

for patients at risk of transmitting a monogenic disorder to their offspring

66
Q

when and why was PGT-M first used?

A

First used in 1990 to determine embryo sex allowing the transfer of unaffected females in families carrying X-linked disease

67
Q

PGT-M can be used to diagnosed what recessive and dominant monogenic disorders?

A

Recessive:
* Cystic fibrosis
* b-thalassaemia
* Sickle cell anaemia

Dominant:
* Huntington’s
* Charcot-Marie-Tooth disease
* Myotonic dystrophy

68
Q

PGT-M can be used to diagnosed what sex linked monogenic disorders?

A
  • Fragile X syndrome
  • Duchenne muscular dystrophy
  • Cystic fibrosis & fragile X syndrome
  • Fanconi’s anaemia & human leucocyte antigen typing
69
Q

what are the two major issues of using PCR in PGT-M?
how can they be overcome?

A

Contamination and allele dropout
Paternal contamination overcome by using intracytoplasmic sperm injection (ICSI) for all PCR-based PGT cases.
Amplification failure sometimes affects only one of the two alleles in a cell. Thus a heterozygous cell may appear to be homozygous – termed allele dropout.
Allele dropout can be overcome using multiplex-PCR

70
Q

why is preimplantation genetic testing for aneuploidy (PGT-A) used?

A

Aims to improve IVF success by selecting euploid embryos for transfer increasing the chance of implantation and decreasing spontaneous miscarriage.

71
Q

what three techniques can be used for PGT-A?

A

o Fluorescence in situ hybridization (FISH)
o Array-Comparative Genomic Hybridization (array-CGH)
o Next generation sequencing (NGS)

72
Q

how is PGT-A performed using FISH?

A

individual blastomeres are spread on a glass slide and DNA probes labelled with fluorochromes specific for the chromosomes of interest are applied

a more robust method for sex determination compared to PGT-M

72
Q

how is PGT-A performed using FISH?

A

individual blastomeres are spread on a glass slide and DNA probes labelled with fluorochromes specific for the chromosomes of interest are applied

chromosomes shown by different coloured fluorescent spots
aneuploidy shown by more than two spots of each colour

a more robust method for sex determination compared to PGT-M

73
Q

what are the limitations of using PGT-A using FISH?

A

Inability to screen simultaneously for all 24 chromosomes. Thus, some embryos diagnosed as normal are undoubtedly abnormal due to aneuploidies of chromosomes not analysed
Typically screen 7 chromosomes (13, 16, 18, 21, 22, X, Y) most frequently associated with miscarriage
Limited interrogation of chromosomes and thus prone to errors associated with chromosome extrapolation based on the presence or absence of a single locus
Technically challenging – optimise single cell lysis, chromosomal spreading and fixation difficult

74
Q

How is PGT-A performed using array-CGH

A

Array-CGH uses microarray technology to screen all chromosomes and requires whole genome amplification.
* Tests for aneuploidy and unbalanced translocations.
Array-CGH involves competitive hybridization of differentially labelled test and reference DNA samples to DNA probes affixed to a microscope slide.
Each probe specific to a different chromosomal region and occupies a discrete spot on the slide.
Chromosomal loss or gain revealed by the ratio of fluorescence intensity for the 2 colours

75
Q

what are the steps of array-CGH?

A
  1. patient and control DNA are labelled with fluorescent dyes and applied to the microarray
  2. patient and control DNA compete to attach, or hybridize, to the microarray
  3. the microarray scanner measures the fluorescent signals
  4. computer software analyzes the data and generates a plot
75
Q

what are the steps of array-CGH?

A
  1. patient and control DNA are labelled with fluorescent dyes and applied to the microarray
  2. patient and control DNA compete to attach, or hybridize, to the microarray
  3. the microarray scanner measures the fluorescent signals
  4. computer software analyzes the data and generates a plot
76
Q

what are the benefits and limitations of PGT-A using array-CGH?

A

Benefits:
* Comprehensive chromosomal analysis achieved in ~24 hours by the evaluation of multiple loci along the length of each chromosome.
* Compared to FISH does not require cell fixation onto slides.
* Amplification step produces enough DNA to detect aneuploidy via array-CGH and gene defects using PCR

Limitations:
* Cannot detect balanced translocation or inversions or single base pair mutations

77
Q

what are the risks/ downfalls of PGT-A?

A
  • Involved embryo biopsy posing isk to harm to the embryo
  • Only analyses 5-10 cells, which may not be representative of the rest of the embryo
  • Results may be inconclusive or may obtain false positives or false negatives
  • May lead to fewer embryos to use in treatment
78
Q

who might consider PGT?

A

1) Couples whose children are at increased risk of specific genetic disorder – eg carriers of monogenic disease or of chromosomal aberrations such as translocations
* Those who have opted to terminate previous pregnancy following prenatal tests

  • Those whose suffer recurrent miscarriages
  • Those with religious or moral objections to pregnancy termination
  • Those who have had previous failed attempts at IVF without explanation

2) Couples being treated with IVF who may have a low genetic ridk but whose embryos are screen for chromosomal aneploidies enhance their chance or an ongoing pregnancy
* Women over 37 undergoing IVF whose low success rate might be attributable to chromosomal aneuploidies

79
Q

what are the ethical considerations of PGT

A

Is PGT orally acceptable?
- Depends on how moral status of embryo is viewed
- Is selective transfer favourable to pregnancy termination?
Is strain on women acceptable
- IVF involves risk to women with only limited chance of success
- Need for counselling and informed choice
Slippery slope argument
- Potential for would be parents o design the ‘perfect child’

● Social sex selection - In UK sex selection only allowed for medical reasons eg to avoid giving birth to a child with a sex-linked disorder.
● Saviour sibling - Selection of an embryo for implantation to provide a treatment for a sibling with life-limiting blood disorders.

80
Q

what are the ethical considerations with PGT and saviour siblings?

A

● Saviour sibling - Selection of an embryo for implantation to provide a treatment for a sibling with life-limiting blood disorders.
A couple with a child affected with Fanconi’s anaemia can request PGT for this disease along with HLA typing. Cord blood or bone marrow from their second child can be used to cure their first – saviour sibling.
Is it ethical to select an embryo solely for the purpose of treating a child who is already alive? Potential adverse psychological and emotional effects
If cord blood transplant is unsuccessful, the second child will be required to donate bone marrow, a painful procedure with no direct benefit to the donor.

81
Q

how does downregulation help in the success of in vitro fertilisation

A

can avoid a premature luteinizing hormone (LH) surge, favor follicle development, synchronize the growth of the follicles and endometrium, and thus improve IVF success

82
Q

what is the window for implantation in the menstrual cycle of someone receiving IVF?

A

Transfers performed on the 17th and 20th days of the cycle can result in successful implantation, although the rates of implantation are highest when transfers are done on days 18 and 19.

83
Q

what causes the symptoms that are associated with PCOS?

A

an excess production of androgens

84
Q

what are fibroids?

how do they affect fertility

A

benign growths arising from smooth muscle of the uterus that can distort the uterine cavity and prevent implantation

85
Q

hrwhat are uterine polyps?

how do they affect fertility

A

small growths of endometrial tissue dangling in the uterus interfering with implantation

86
Q

what is the purpose of Nafarelin?

A

Nafarelin a nasal spray containing a gonadotropic releasing hormone agonist is administered in order to stop the bodys normal control mechanisms and cause the flare effect: rise of gonadotropin levels and short term activation of gonadal metabolism and steroid production.
It helps avoid premature lutinizing hormone surge and synchronise the growth of the follicles and endometrium thus improving IVF success.

87
Q

what size do the follicles have to be before administering the hCG injection

A

around 18mm

88
Q

why is live cell imaging better than scoring based off mrophological criteria?

A

it monitors the embryonic development continually from fertilisation to transfer and based on previous knowledge of success, determines which cells cleave and develop at the optimum rate for a successful cycle

89
Q

how do they increase chance of success after embryo transfer

A

progesterone pessaries are taken daily to support the endothelium increasing the chance of success.

90
Q

how is PGT-M carried out?

A

It uses PCR to amplify the DNA fragment containing the causative mutation
Alternatively, PGT-M now employs whole genome amplification of biopsied cells.

Cellular & Molecular Pathology (12 decks)

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