History and Development of HRT

Question 1

Discovery of spermatozoa: Anton Van Leeuwenoek

Answer 1

Father of microbiology, first microbiologist.
First to observe and describe single-celled organism
(animalcules)…microorganisms, muscle fibres, bacteria, capillaries and
spermatozoa.

Drew the first sketches of sperm

Question 2

John Hunter (Artificial Insemination)

Answer 2

Appointed as surgeon at St George’s in 1768.
He advised a patient with severe hypospadias to collect the semen which
escaped during coitus in a warmed syringe and inject the sample into the
vagina. A successful pregnancy resulted.

Question 3

Define Hypospadias

Answer 3

Hypospadias opening
of the urethra
develops abnormally,
usually on the
underside of the penis.
Abnormal closure of
the urethral fold over
the genital groove.

Question 4

Walter Heape (embryo transfer)

Answer 4

Walter Heape Cambridge University 1891
Transferred 4 cell embryos from the uterine tubes of Angora rabbits and placed them into the tubes of a recently mated Belgian hare.
2 Angora rabbits (and 4 Belgians) in the resulting litter.
General anaesthetic and the embryos transferred on the point of forceps…they were not transferred to any kind of media.
First to take pre-implantation embryos and transfer them to a gestational carrier without affecting their development.

Question 5

Oestrogens & Sex Steroids: Edward Doisy

Answer 5

Edward Doisy Harvard Medical School 1929
Extracts from sow ovaries (female pig) injected into ovarectomized mice (removed ovary), resulted in the production of cornified cells in the vagina – is a bioassay. Later isolated estradiol from pig follicular fluid.
When mice are about to ovulate, they enter into the oestrus cycle. They enter oestrus as they have a lot of estrogen, produced from the developing follicle. You can tell when a mouse has entered oestrus and it is ready to mate as there will be cornified cells around the vagina.
Doisy took the sow extract and injected into mice that had ovaries removed, he found that they stared to go into oestrus. So something in the sow extract resulted in the cornified cells in the mice.

Question 6

Oestrogens & Sex Steroids ( Adolf Butenandt)

Answer 6

Adolf Butenandt University of Göttingen 1929
Isolated estrogen from hundreds of gallons of human pregnancy urine.

Question 7

Pituitary & Gonadotrophins: Samuel Crowe

Answer 7

Samuel Crowe 1910
Partial pituitary ablation resulted in gonadal atrophy in dogs (no development of gonad/ testes) and persistence of infantilism in puppies.
If done to puppies they do not mature

Question 8

Pituitary & Gonadotrophins: Bernard Aschner

Answer 8

Bernard Aschner 1912
Postulated that pituitary function determined by higher centres in the brain (hypothalamus) after observing gonadal atrophy in patients with brain injury.
Sectioned pituitary stalk which resulted in gonadal atrophy and hypothesised that brain/pituitary extracts might affect the gonads.

Question 9

Identification of FSH & LH: Bernhard Zondek

Answer 9

Bernhard Zondek Berlin 1930

Proposed the idea that the pituitary secretes two hormones – Prolan A stimulated follicular growth (FSH) and prolan B stimulated ovulation and formation of the corpus luteum (LH).
He isolated these gonadotrophins from post menopausal blood and urine.
As after menopause there is no estrogen so with no estrogen, there is no negative feedback on the pituitary so the pituitary produces FSH and LH.

Also isolated hCG from pregnancy urine and injected into mice leading to follicular maturation and ovulation – potential pregnancy test.

Question 10

Why does removal of hypothalamus/ pituitary result in tiny testes?

Answer 10

No FSH

FSH controls the development of Sertoli Cells

Question 11

Friedman test- Bioassay of pregnancy

Answer 11

Maurice Friedman & Maxwell Lapham Pennsylvania 1931
Inject the tested woman’s urine into a female rabbit, then examined the rabbit’s ovaries a few days later… presence of corpus luteum indicated pregnancy.
Hormone responsible is hCG which binds to LH receptors causing luteinisation.
Drawback that the rabbit had to be operated on to examine ovaries.

Later development used the African clawed frog, which responds to hCG by laying eggs and so removing the need for surgery.

Question 12

Human in- vitro fertilisation

Answer 12

John Rock & Miriam Menkin Harvard 1944
Culmination of 6 years of experiments changing conditions.
Oocytes obtained from patients around 10th day of the menstrual cycle by laparotomy.
Oocytes washed in Locke’s solution and incubated for 27 hours in serum obtained from the egg donor; exposure to a sperm suspension also washed in Locke’s solution for 1 hour. This resulted in an embryo

Transferred to a serum from a post-menopausal patient and observed over the following days where they divided into 2-4 cell embryos.
There was no attempt to transfer the embryos to a recipient.

BUT, use to create babies did not happen straight after

Question 13

Fertility treatment 1950’s- 1970’s

Answer 13

1st use of hyperstimulation was in mice used crude extracts of Post menopausal serum (PMS) as it contains lots of LH and FSH, 1950’s

Hypogonadotrophic women treated with crude pituitary extracts & hCG (Gemzel, 1967).

Human menopausal gonadotrophins to treat amenorrhoeic women (Lunenfeld, 1969).

Anovulatory PCOS patients treated using clomiphene (Kistner, 1972). Blocks estrogen receptors.

Question 14

Disadvantages of fertility treatment

Answer 14

(MULTIPLE PROBLEMS, ALSO NO ULTRASOUND MONITORING):

Multiple pregnancies
Ovarian Hyperstimulation syndrome (OHSS)
Miscarriage.
Purity of preparations. – danger of transmitting viruses.
CJD…other infectious agents.
Virtually no monitoring.

Question 15

Ovarian Hyperstimulation syndrome (OHSS)

Answer 15

When a woman ovulates, there is continuous high estrogen from the developing follicle. This triggers the negative feedback to switch to positive, this causes the LH surge from the pituitary.
This LH spike acts on the ovary causing two main things to happen:
Causes resumption of meiosis 1
Triggers a series of inflammatory and tissue remodelling reactions that causes the release of the egg. One of the cytokines is vascular endothelial growth factor (VEGF), this causes increased vascular permeability this results in oedema and also more cytokines and cells to the site of inflammation.
But if you have given the woman a lot of FSH she could have 20 follicles ovulate. Then VEFG levels rise so high that they cause systemic vascular permeability. In the lungs this causes pulmonary oedema.
SO if you give FSH, you must monitor the number of follicles using ultrasound.

Question 16

Laparotomy

Answer 16

surgical incision (cut) into the abdominal cavity

Question 17

Laparoscopy

Answer 17

Patrick Steptoe - Graduated from St George’s in 1939.
Studied obstetrics and, in 1951 learned the technique of laparoscopy from one of its pioneers Raoul Palmer and promoted its usefulness.
One of the first people to use a laparoscope – can operate using just a couple of small holes.

Question 18

Robert Edwards- Fertilisation

Answer 18

Began to study human fertilisation around 1960. Optimised culture media.
Discovered when to collect eggs after hCG trigger using ‘ovulations’ from pieces of ovary and oocyte maturation in vitro, 1965.
In-vitro matured fertilised eggs did not develop, problems with timing egg maturity and sperm capacitation, 1968.

Achieved fertilisation of a human egg in the laboratory 1969.
Next problem was obtaining follicular oocytes from selected patients. To solve this clinical problem, an inspired collaboration was initiated with Patrick Steptoe.

Question 19

Steptoe & Edwards

Answer 19

Began working together
Measured urinary oestrogen in a gonadotrophin stimulated cycle.
Timed collection by laparoscopy IVF and replacement of embryos …..failure 1971.
Decided to give luteal support using Primulot which turned out to be an abortive agent. This wasted 3 years.
Switched to using a natural cycle and achieved 1st pregnancy but it was ectopic, 1977.
But was first successful in 1978 – Louise Brown

Question 20

Clomiphene

Answer 20

Oestrogen receptor blocker

First line of treatment for PCOS

Question 21

Progress in IVF technology

Answer 21

Purer urinary FSH/LH preparations, recombinant gonadotrophins
GnRH agonists/antagonists
Better ultrasound monitoring
Micromanipulation for ICSI, MESA, TESA etc
Cryopreservation of oocytes (vitrification)
Reduction in OHSS less stimulation & GnRH agonist/Kisspeptin triggers
Sequential media for blastocyst culture
Single embryo transfer
Pre-implantation diagnosis or screening
Ovarian tissue cryopreservation
Mitochondrial donation (3 parent family)
In vitro maturation of oocytes

Question 22

OHSS

Answer 22

Too much FSH

VEGF causes systemic vascular permeability, may lead to pulmonary oedema

Question 23

Preimplantation genetic testing (PGT)

Answer 23

Taking a cell from the embryo – embryo biopsy. Will then test these cells
PGT information used to decide which embryo to transfer to the mother.

PGT-A aneuploidy
PGT-SR chromosomal structural rearrangements
Screening of embryos for aneuploidy or chromosomal translocations. Often used for advanced maternal age risk of aneuploidy, know translocations or repeated implantation failure.
Eg increased risk of downsyndrome

PGT-M monogenic/single gene defects
Screening of embryos for known genetic mutations where the parents are carriers.

Screening of embryos includes fluorescence in situ-hybridisation (FISH), array-based comparative genomic hybridisation (aCGH), next-generation sequencing (NGS) and single nucleotide polymorphism (SNP) array. Also, whole genome amplification (WGA) based versions of techniques for PGT-M.
BUT you can only use it to test for genetic diseases

Question 24

Background of origin of mitochondria

Answer 24

All mitochondria comes from the mother, there are some genetic diseases of the mitochondrial genes, so it will be passed onto a mothers children.
You can get a donor to donate their mitochondria
So offspring will have mothers and fathers nuclear DNA, but a different mitochondrial DNA.

Question 25

2 ways of mitochondrial donation

Answer 25

Meiotic spindle transfer (MST)- most common

Pronuclear transfer (PNT)- less common

Question 26

MST

Answer 26

Meiotic spindle transfer (MST) – Most common
Meiotic spindle of donor oocyte removed during MII and replaced by patient spindle.
You get two oocytes that have been triggered = they have completed meiosis 1
You take the meiotic spindle out of the donor and dispose of it.
Then take the meiotic spindle out of the patient, and put it in the donor egg.
This means that you get the donor cytoplasm, but the mums nuclear DNA.
You then fertilise that egg, so in the embryo now you have the mothers and fathers normal DNA, but the donors mitochondria.

Question 27

PNT

Answer 27

Pronuclei of fertilised patient egg transferred to fertilised donor egg which has had pronuclei removed.
With this you get two embryos.
You get an embryo from the parents and embryo from the donor
You discard the pronuclei from the donor embryo and transfer the pronuclei from the patient embryo.

Question 28

Types of cloning

Answer 28

Natural
Reproductive
Therapeutic

Question 29

Natural cloning

Answer 29

Mitotic division of a cell.
Asexual reproduction…plants, invertebrates.
Identical twins.

Question 30

Reproductive cloning

Answer 30

Somatic cell nuclear transfer.
Designed to create a new ‘being’.

Question 31

Therapeutic cloning

Answer 31

Cloning to create stem cells which are compatible with a recipient.

Question 32

Human stem cells: blastocyst

Answer 32

The first thing it does (except from dividing) is to differentiate into two cell types.
So by 5 days you have:
A blastocele – fluid filled space
Trophoblast cells around the edge that will become the embryos contribution to the placenta.
Inner cell mass – which contains the pluripotent stem cells

Question 33

Blastocyst: differentiation

Answer 33

Found in embryos and to a lesser extent in some adult tissues.
In adults they differentiate into other cell types in the tissue in which they are found. Embryonic stem cells are pluripotent and can differentiate into a wide variety of tissues. Stem cells from a cloned embryo will have identical genetic make up of the patient eliminating immunological rejection issues.

Question 34

Human stem cells: therapeutic uses

Answer 34

Potential to replace cells lost due to age, damage or disease.
Brain or nerve tissue – Parkinson’s, Alzheimer’s or spinal cord injury.
Heart disease – repair ischaemic damage to cardiac muscle.
Bone marrow – restore bone marrow/blood cells in cancer patients.
Skin grafts – replace damaged skin in accident or burn victims.

Question 35

Focus of biomedical research & drug development

Answer 35

Understanding how stem cells differentiate into their target tissues adds to knowledge regarding disease processes.
Assess the effectiveness or toxicity of new drugs and treatments.

Question 36

Cloning technique overview

Answer 36

• Collect a mature oocyte and remove the haploid nucleus.
• Take an adult diploid somatic cell and transfer the nucleus into the enucleated egg.
• Fusion activation with electricity & chemical stimulus to mimic fertilisation.
• The embryo will have identical DNA to the adult from whom the nucleus came.
• Harvest inner cell mass and create therapeutic stem cells

Question 37

Human cloning is banned in most countries due to:

Answer 37

Welfare of the child.
Ethical, moral & religious objections.
Relatively low success rate at the moment.
Reproductive cloning is banned in the UK but cloning human embryos for research into stem cells is allowed under licence from the HFEA.

Question 38

Therapeutic cloning for creation of stem cells: Why are stem cells needed for ART?

Answer 38

Huge future potential for differentiating them into skin, pancreas, heart, neurones etc.

Question 39

Why do we need foetal cells to create stem cells?

Answer 39

We need the entire genome of undamaged DNA
Embryonic cells are easier to re-programme into the cell of choice.
We might create an embryonic clone in order to create cells for donation that will not be rejected. It is possible to re-programme some adult cells but it’s complex and they are not totally pluripotent.