Lectures 17&18 - Assisted reproduction Flashcards

Question

what are the causes of tubal problems

Answer 1

- Infection – most common cause ● Previous pelvic infection ● Sexually transmitted diseases eg chlamydia and gonorrhoea ● Intrauterine contraceptive device - can lead to uterine infection, which spreads to Fallopian tubes ● appendicitis, bowel infection ● after termination of pregnancy, miscarriage or delivery -Previous ectopic pregnancy - Congenital abnormality - Hydrosalpinx – blocked, dilated, fluid filled Fallopian tube usually caused by previous pelvic infection - Endometriosis

Answer 2

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 up to 10% of women of childbearing age

Answer 3

Pain relief with NSAIDs Prevent fluctuation in the woman’s hormone levels to remove the stimulation for growth of the endometriosis eg the oral contraceptive pill

Answer 4

generally no problems with getting pregnant, tend to miscarry - incidence for this is 10%

Answer 5

Fibroids - Benign growths arising from smooth muscle of the uterus Many are fertile and have no problems maintaining the pregnancy unless fibroids distort the uterine cavity and may prevent implantation Uterine Polyps - Small grows 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

Answer 6

Reproductive function declines as a woman ages, particularly after 35 Women delay starting a family due to career demands, education, financial stability, suitable partner etc However, women have a finite number of eggs Egg quality decreases with age Chromosomal abnormalities increase in late 30s Ageing also affects hormone production and ovulation Higher incidence of miscarriage in women in their late 30s

Answer 7

Poor sperm quantity (oligospermia) or quality - ~90% of male infertility. Azoospermia - 3-4% of male infertility Sperm dysfunction – 3-6% of male infertility Ejaculation disorders – 4-6% of male infertility

Answer 8

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

Answer 9

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

Answer 10

Normal semen analysis but sperm have defective fertilising capacity Defective acrosome abnormal sperm movement inability of sperm to bind to the zona pellucida

Answer 11

retrograde ejaculation – semen ejaculated backwards into the bladder impotency

Answer 12

Anti-sperm antibodies can be present in either or both partners Present in either the blood or in the genital tract secretions eg cervical mucus and ejaculate Incidence 1-2% 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 transportand fertilisation. Unknown cause but may be due to genital infection or vasectomy.

Answer 13

Failure to conceive after one year of unprotected intercourse and despite thorough investigations no cause of infertility could be determined Incidence ~25% Causes: None, even after thorough investigations of both partners

Answer 14

Regulated by the Human Fertilisation and Embryology Authority (HFEA) 1.3 million IVF treatment cycles in UK since 1991 Overall live birth rate per embryo transferred 24% in 2018 Average multiple birth rate decreased from 24% in 2008 to 10% 2017 to 6% in 2019 Single embryo transfer now common practice. In 2019 one embryo transferred in 75% of IVF cycles compared to just 13% in 1991

Answer 15

IVF-ET (In vitro Fertilisation and Embryo transfer) process: * Preliminary testing * Downregulation * Ovarian stimulation * Monitoring growth of follicles * Egg collection guided by transvaginal ultrasound * Sperm sample provided same day as egg collection * Fertilisation and embryo culture * Embryo transfer * Pregnancy testing/monitoring

Answer 16

Standard IVF – oocytes incubated with sperm over night 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

Answer 17

To stop the body’s normal control mechanisms for ovulation Gonadotrophin releasing hormone agonist (GnRH agonist) eg as a nasal spray (Nafarelin) Agonists do not dissociate quickly from the GnRH receptor - causes Flare Effect - An initial increase of pituitary hormones but after ~10 days FSH and LH levels decrease Downregulation confirmed with a blood test for oestradiol levels Side effects: hot flushes, vaginal dryness, headaches, mood swings

Answer 18

To increase the number of oocytes produced Stimulate ovaries with daily injections of FSH eg Gonal F Continue to take GnRH agonist nasal spray Monitoring response of drugs; - blood tests (oestradiol levels) - 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

Answer 19

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

Answer 20

Semen sample obtained, washed and concentrated20,000 - 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 (or occasionally 3 in women over the age of 40 and using embryos created from their own eggs) 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

Answer 21

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

Answer 22

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

Answer 23

Embryos that cleave to the 2-cell stage by 24-25h post-insemination are more viable than pronuclear embryos. On day 2 (44-48h post-insemination) embryos should be at 4-cell stage, transfer of 2-cell embryos given a lower priority backed up by Van montfoort et al. (2004) showed that pregnancy rate (%) when no early cleavage lower than when there was early cleavage

Answer 24

Grade I: Even blastomeres No fragmentation Grade II: Even blastomeres <10% fragmentation Grade II.5: Even blastomeres <30% fragmentation Grade III: Uneven blastomeres >30% fragmentation Grade IV: Only one viable blastomere Grade V: No viable blastomeres

Answer 25

Grade 1: Early blastocyst: blastocoel being less than half the volume of the embryo Grade 2: Blastocyst: blastocoel 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 4A - ICM: Tightly packed many cells, TE: Many cells forming cohesive epithelium 4B - ICML: Loosly grouped several cells, TE: Few cells forming loose epithelium 4C - ICM: Very few cells, TE: Very few large cells Made by Gardner & Schoolcraft (1999)

Answer 26

Transfer of embryo to uterus Assess embryo viability prior to transfer Increase time between cleavage stage biopsy and transfer

Answer 27

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

Answer 28

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

Answer 29

Give an image throughout development computer software will give an automated tracking of the blastomeres from this a schematic can be made found that successful development to the blastocyst stage can be predicted as early as the 4 cell stage - this is based of first 3 mitotic divisions Embryos that develop into the blastocyst stage undergo cytokinesis at the two cell stage within 15 mins and complete the second mitotic devision to the three cell stage within 11 hours and reach the fourth cell stage within an hour of the three cell stage

Answer 30

Test performed to analyse the DNA of embryos for human leukocyte antigens (HLA) typing or for determining genetic abnormalities patients undergo IVF to produce preimplantation embryos in vitro. Embryos biopsied at either the 8-cell stage (1 cell removed), or blastocyst stage (5-10 trophectoderm cells removed) and tested

Answer 31

Detects monogenic disorders. Also used for HLA testing with or without concurrent testing for monogenic disorder. Sometimes referred to in literature as PG-D (pre-implantation genertic diagnosis)

Answer 32

Detects abnormal number of chromosomes in embryo biopsies Sometimes called PGS (Pre-inmplantation genertic screening)

Answer 33

Lasers generally used to make a hole in the zona pellucida biopsy pippete can come in and aspirate off one cell, leaving a 7 cell embryo and one cell to be sent off for biopsy generally done using a calcium and magnesium free medium and this dirsrupts the adhereance junction (which is calcium dependent) allowing the removal of a cell only embryos found to be unaffected by the genetic disorder are transferred to the uterus

Answer 34

Blastocyst held with a holding pipette very close to the inner cell mass (ICM) laser is used to put a hole in the zona pellucida Biopsy pipette comes in and aspirates out 5-10 cells of the TE, and then the laser cuts this off left with the collapsed blastocyst on the holding pipette, this will re-expand 5-10 TE cells sent off for testing

Answer 35

Embryo biopsy on day 3 (cleavage stage) allows 2-3 days for PGT analysis before fresh embryo transfer at blastocyst stage. No affect on development if embryo of good quality but mosaicism thought to be higher at early cleavage stage Blastocyst biopsy removes 5-10 trophectoderm cells at blastocyst stage (day 5 or 6). Not removing cells that will form embryo. Not all zygotes will develop to blastocyst – fewer embryos to biopsy

Answer 36

Early form of prenatal diagnosis for patients at risk of transmitting a monogenic disorder to their offspring First used in 1990 to determine embryo sex allowing the transfer of unaffected females in families carrying X-linked disease

Answer 37

More than 600 monogenic disorders in the UK including: Recessive: Cystic fibrosis b-thalassaemia Sickle cell anaemia Dominant: Huntington’s Charcot-Marie-Tooth disease Myotonic dystrophy Sex linked: Fragile X syndrome Duchenne muscular dystrophy Cystic fibrosis & fragile X syndromeFanconi’s anaemia & human leucocyte antigen typing

Answer 38

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

Answer 39

Multiplex-PCR: Involves the simultaneous amplification of several DNA fragments in a single reaction. Usually a DNA fragment encompassing the site of the causative mutation is amplified along with one or more closely linked informative polymorphisms. Diagnosis can be based on the presence of the mutation itself or a particular polymorphic allele that is inherited along with it. (Allele dropout may still affect either of the amplified fragments but the probability of it affecting both in the same reaction is low.)

Answer 40

employment of whole genome amplification of biopsied cells

Answer 41

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

Answer 42

Fluorescence in situ hybridization (FISH) Original method for analysis of up to 10 chromosomes in a blastomere.Individual blastomeres are spread on a glass slide and DNA probes labelled with fluorochromes specific for the chromosomes of interest are applied. DNA denatured, hybridised, label and end up with a fluorescent spot Imaging is normal if two spots of each coloyur (coloyur represents the number chromosome e.g. 13) if more or less than two, something is incorrect

Answer 43

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

Answer 44

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

Answer 45

Step 1-3: Patient and control DNA are labelled with fluorescent dyes and applied to a microarray Step 4: Patient and control DNA compete to attach, or hybridize, to the microarray Step 5: The microarray scanner measures the flourecent signal Step 6: Computer software analyses the data and generates a plot dosage loss indicates loss of that chromosome

Answer 46

Morphology and state experiment by Yang et al. (2012) selected both and then just morphology alone, when selecting using both, ongoing pregnancy rate was 69%, whereas it was only 42% in morphology alone

Answer 47

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.

Answer 48

Cannot detect balanced translocations, or inversions or single base pair mutations

Answer 49

- Involves embryo biopsy posing a risk of harm to the embryo - Only analyses 5-10 cells, which may or 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 The use of PGT-A is a much debated in reproductive medicine with advocates and opponents Some evidence PGT-A may be beneficial for certain categories of women Counselling required to ensure patients understand the risks, benefits and limitations

Answer 50

1) Couples whose children are at increased risk for a 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 who 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 risk but whose embryos are screened for chromosomal aneuploidies to enhance their chance of an ongoing pregnancy

Answer 51

Is PGT morally 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 a limited chance of success - need for counselling and informed choice Slippery slope argument - potential for would-be parents to 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.

Answer 52

HFEA - Human Fertilisation and Embryology Authority.