In Vitro Fertilization – Test Tube Baby Program in Animals and Man

2020. 11. 02.

UNIVERSITY OF VETERINARY MEDICINE, BUDAPEST

In vitro fertilization – test tube baby program in animals and man

László Solti Professor emeritus

Brackett with Willy and Nilly, the first two test tube goats born in the US

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Timeline of in vitro fertilization

Embryo development (IVF-ET)

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In vitro fertilization

Large Offspring Syndrome Liver, kidney and heart can be affected Common in ruminants (dystocia → †) In vitro culture?

Major steps of IVF

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Ultrasound guided Ovum Pick Up (OPU)

Instruments and drugs used for IVF

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Hormonal Stimulation for IVF

Weekly scheme and hormone treatment for Ovum pick- Blastocyst yields from the animals subjected to OPU up (OPU). (a) The animals were not treated with without FSH (open bars) and OPU with FSH treatment hormone FSH. Transvaginal OPUs (triangle) were (solid bars). performed on Monday and Thursday of each week for consecutive two weeks, resulting in a total of four sessions (Presicce et al, Vet Med Int. 2011; 2011: 145626) (b) FSH treatment: FSH (arrow) at dosage of 80mg IM and 120 SC was simultaneously administered on Day 1 (Saturday). Ovum pick-ups were performed 48 and 96 hours post FSH injection, meaning on Day 3 and Day 5 of each week for consecutive two weeks, resulting in a total of four sessions.

Donor preparation

(Luis Ferré, INTA Rafaela) 10

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Follicle aspiration

Isolation and maturation of oocytes

(Luis Ferré, INTA Rafaela) 12

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Cumulus-Oocyte-Complex (COC)

IVF

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Cumulus-Oocyte-Complex and oocytes at the end of maturation

At the end of maturation (Katherine Hendrics)

After treatment with hyaluronidase (Firdous Khan)

In vitro fertilization and culture

(Luis Ferré, INTA Rafaela) 16

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ET and pregnancy diagnosis

(Luis Ferré, INTA Rafaela) 17

Embryo production in vitro

Human Animals (ruminants)

IVM ø + IVF + +

IVC  ++

Intermediate recipients ø 

Cocultivation , ø ++

Ultrasound guided ovum pick up (OPU) + , ø

(Human: Edwards and Steptoe 1978 (Louise Brown) Cattle: Brackett et al. 1981, goat: Brackett et al, 1983

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Sperm preparation for IVF (Swim up)

 Heparin containing medium (M 199)

Sperm preparation for IVF Percoll gradient centrifugation

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Sperm preparation for IVF Glass wool filtration

MOET vs IVF

Parameters MOET IVF

Collection/year 6a 24b

Embryos/collection 6 4.5

Annual Embryo Production 36 108

Pregnancy Rate (%) 60c 45c

TOTAL Born Animals 22 49

a Collections every 60 days b 2 OPUs/month c Fresh transfers

(Luis Ferré, INTA Rafaela)

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MOET vs IVF with sexed semen

Parameters MOET IVF (sexed semen)

Collections/year 6a 24b

Embryos/collection 6 3

Annual Embryo Production 36 60

Sex Ratio (Females:Males) 50:50 90:10

Pregnancy Rate (%) 60c 45c

TOTAL Females Born 11 29

a Collections every 60 days b 2 OPUs/month c Fresh transfers

(Luis Ferré, INTA Rafaela)

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ET vs IVF worldwide

Source: 27th Annual Report of IETS (2018)

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Production of bovine embryos in Brazil

embryos

of of Number

ET: conventional embryo transfer, IVF: embryos produced in a laboratory

Source: L.C. Cruz and A. Sarabia ([email protected])

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Current status of ET and IVF using Ovum Pick Up in cattle

Az anyák átlagos életkora első gyermekük születésekor

1990 23

1995 23,5

1998 24,2

2000 25

2005 27

2008 28

0 5 10 15 20 25 30

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In vitro production of human embryos

Brave New World is a dystopian novel written in 1931 by English author Aldous Huxley, and published in 1932. Largely set in a futuristic World State of genetically modified citizens and an intelligence-based social hierarchy, the novel anticipates huge scientific developments in reproductive technology, sleep-learning, psychological manipulation, and classical conditioning that are combined to make a utopian society that goes challenged only by a single outsider. (1931)

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"for the development of in vitro fertilization."

https://www.youtube.com/watch?v=fy61qs_tXTQ https://www.youtube.com/watch?v=1zQhwiGhzJk https://www.youtube.com/watch?v=TIRbsqvjuRs

Births and deaths in Hungary

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World population according to continents, 1950-2100 UN, World Population Prospects 2019

https://www.ksh.hu/interaktiv/grafikonok/vilag_nepessege.html

Population data

Hungary, June 2017  Live births: 73.300  Deaths: 92.200  Population decrease: 1.900

https://www.ksh.hu/docs/hun/xftp/gyor/nep/nep1706.html

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Decreasing population

Natural Population Natural Total fertility Livebirths Deaths increase (in thousands) increase index (per 1000 head)

1960 9 984 146 461 101 525 44 936 4.5 2,02

2005 10 088 97 496 135 732 -38 236 -3.8 1,32

2018 9 778 89 807 131 045 -26 900 -4.2 1,50

Population forecast until 2100

Average fertility rate in Hungary is 1.4 which cannot stop the population decline. According to UN the population decreases to approx. 6.5 m by 2100 even if the fertility rate goes up to 1.8 which is the moderate rate. Migration balance is also negative – if it would go down to zero, at moderate fertility rate the population would be below 6 m. An increasing trend to become above 10 m could be reached by a fertility rate >2.3 which is almost impossible.

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Composition of the Hungarian population

Arrow: missing age group as a consequence of WW-I

Population pyramid 1960-2014

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Patrick Steptoe Bob Edwards

• 1970-ies: millions of couples failed to get child. With oviductal patency failure (approx 20%) there was no chance for conception • Lesley and John Brown visited different physicians for 9 years before they came to Dr. Patrick Steptoe (†1988), who performed in vitro fertilization (IVF) in 1977 when it was in experimental phase • July 25, 1978: birth of Louise Brown, the first test tube baby • Since then >8 million IVF children were born worldwide. Pregnancy rates now steady at around 36 percent, according to ESHRE monitoring (Science Daily July 2018) • In spite of resistance of churches, governments and media as well as the scepticism of scientific world, Edwards and Steptoe went on working

Patrick Steptoe and Robert Edwards: Brave New Baby Doctors

„The astonishing feat brought instant celebrity to Patrick Steptoe and his partner, Robert Edwards - as well as a barrage of criticism. The Roman Catholic Church and other religious groups denounced it as playing God, and even scientists like James Watson, unraveler of DNA, were worried about tinkering with a process as sacrosanct as procreation. But the debate faded as it became clear that the brave new world of babymaking that Steptoe had ushered in was providing a desperately sought service.”

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• The history of IVF and ET dates back as early as the 1890's when Walter Heape successfully transferred embryos between rabbits, long before the applications to human fertility were even suggested • „We were called everything under the sun - immoral, unethical, dehumanising”

BBC News ( 10/4/2010) Robert Edwards, pioneer of in vitro fertilization (IVF), has won this year's Nobel Prize in medicine, and the award carries a resonance in modern science Livemint ( 10/4/2010) Very few scientists can say that four million people are alive because of their work, but Robert Edwards is one of those few. ZDNet (blog) ( 10/4/2010) A Vatican official has said the awarding of the Nobel Prize for Medicine to British IVF pioneer Robert Edwards is "completely out of order" National Geographic (10/4/2010) Vatican bioethics official: Nobel prize winner started new chapter but IVF not Hindustan Times (10/4/2010) Edwards' prize a political act. If having a family does not require sex, and all that entails, if humanity is something that can come

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2014 Annual Report, Society for ART (USA)

• Since 2007, the birth rate has been declining steadily

• 2,000 more IVF babies were born in 2012 than 2011 The average cost of one IVF treatment in the US is $12,400, and that's without the extra medicines the couple may need. Often, couples will need more than one treatment to conceive. The number of multiple embryos being transferred in IVF treatments has also declined

• IVF success rates decrease as women age The IVF success rate was significantly higher for women under the age of 35 compared with those who were older. Looking only at the IVF treatments that used fresh embryos from non- donor eggs, the number of women under the age of 35 who gave birth using IVF was about 40%, compared with only 31% for women between the ages of 35 and 37. Only 3.9% of 42-year-olds got pregnant

Severino Antinori

• Starting in veterinary medicine, he changed to human medicine at the La Sapienza University. Graduated in 1972 • First he worked in gastroenterology but after meeting with de Steptoe changed to gynecology and obstetrics • Main field of interest was infertility, in 1982 he opened his own clinic • In 1989 he extended the IVF activity to elder menopausal women • In 1994 his 63-year-old patient, Rosanna Della Cortes became the oldest pregnant woman giving birth to a sound normal child

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Sunday, 25 July 1993

• Severino Antinori, the Italian obstetrician at the centre of the storm over fertility treatments for older women, is very angry. • 'They have said that I'm unprofessional. That I'm an extortionist, a blackmailer. • Antinori denies absolutely that there is anything wrong in what he does. „Every woman has the right to have a child. These women come to me when they have nowhere else to go”. • Of course, there are ethical issues to be discussed. • But who says it's not right for a woman to have a child at 55? A man can have a child at that age, and everyone says, 'Isn't he clever'. But those same people say that a woman of 55 is a dishrag, good for throwing out on the dungheap. It's a disgrace.

Severino Antinori

• In 2006, the 62-year-old Patricia Rashbrook got pregnant as a result of treatment performed by Antinori who regarded this as the ultimate age limit for IVF • He thinks that minimum 20 years of life expectancy is needed required for IVF patients • Josephine Quintavalle (Comment on Reproductive Ethics, CORE), charged Rashbrook with selfishness because „it must be extremely difficult for a child whose mother is as old as his grandmother”

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 In 2009, when the 66-year-old british woman (Elizabeth Adeney) became pregnant, Antinori criticised, saying she is 'Mother-to-be too old at 66’. The boy delivered by Caesarean is normal  Pregancy of Adeney opened up the debate about how long for a woman is ethical to get pregnant?

Cultural Studies from Birmingham, Vol. 2 (1), (1998) ISSN 1463-9734

• Still the known liberal IVF-doctor Antinori has prejudices! His double standard is proven by his refusal for treating Martina Navratilova. • He does not accept lesbians because „children need father, too”. • But what about children with mothers over 60 who may loose one of their parents any time? (Claire Townley-Jones)

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Astonishing: Elton John became father! 2010. 12. 28., MTI

• In Christmas secretely came to world the son of Elton John, Zachary Jackson Levon Furnish-John. The agreement with the surrogate mother is not public • Elton John lives with a Canadian filmmaker David Furnish, with whom the relationship became official in 2005 • „In this special moment we felt an overwhelming happiness and joy" – said Elton John and his 48-yr-old partner

Comments from Hungary

• It is a disgrace to the world! In the good old days those people would have been fired on stake! • In fact which disturbs me is that a rich man can order a child for him and he is allowed to do so. He thinks that he survives everything and may order organs, too, for himself… • It is going to be hard for this child! He was born to make a 63-yr-old homosexual star happy – when he will be 18 his father is 81. Straight road to the drug clinic. • So many bullshit! If somebody is a world star must not have child? Because he is rich or because he is gay?

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Reflexions from abroad

• German christiandemocrat politician Volker Kauder (21/12/2010): „we should not allow gays for raising children! I do not think children would like to grow in homosexual partnership!„ • In fact twice as many lesbian pairs want to establish family by IVF as earlier • UK: in 2009, 350 lesbian pairs enrolled into IVF programs while in 2007 this was 176, in 2000 only 36. IVF lead to birth of 358 babies between 2007 and 2009, while donor insemination remained constant at about 300 per year

Robert G. Edwards dies at 87 April 10, 2013

Dr. Edwards holding Robert G. Edwards with the world’s first “test tube Louise in 1978 baby,” Louise Brown, second from right, in 2008, just before her 30th birthday. With them are her mother, Lesley, and her son, Cameron

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Obituary:

Dr. Edwards was known to have dementia and was said to have been unable to appreciate the tribute when he was awarded the Nobel Prize for Physiology or Medicine in 2010. Dr. Steptoe, who died in 1988, did not receive a share of the Nobel because the prizes are not awarded posthumously. The technique has resulted in the births of five million babies. Yet, like so many pioneers of science, Dr. Edwards and Dr. Steptoe achieved what they did in the face of a skeptical establishment and choruses of critics, some of whom found the idea of a “test tube baby” morally repugnant. Denied government support, the two men resorted to private financing. And they did their work in virtual seclusion, in a tiny, windowless laboratory at a small, out-of-the-way English hospital outside Manchester. In 1971, Dr. Edwards’s application for research support from the British government was turned down, in part because a committee reviewing his application thought it would be more prudent to perfect the method in primates before jumping to humans. Then there was Dr. Edwards’s personality. Committee members wrote that they were uncomfortable with his “tendency to seek publicity in the press, television and so on.” Finally, the committee thought that the hospital where Dr. Edwards and Dr. Steptoe worked was insufficiently equipped. The two men obtained private funds and continued their work.

In a paper published in Nature Medicine, Dr. Edwards explained that he first got the idea for human IVF when he was a Ph.D. student at Edinburgh University. Years later, he asked gynecologists if they would give him ovarian tissue that they had removed from patients for other reasons. Dr. Edwards sought to induce the eggs in the tissue to mature. Then he would fertilize them and transfer them to infertile women to produce pregnancies. One gynecologist, Dr. Molly Rose, who had delivered two of Dr. Edwards’s five daughters, said she would do it. The immature eggs, though, would not mature. Dr. Edwards had assumed it took 12 hours for the process; that was what another research group had said. He finally succeeded in 1972, when, “after two disappointing years,” he let the eggs grow for as long as 25 hours. Then, he wrote, “a joy unbounding,” the eggs matured. What followed, however, was failure after failure to achieve the steps needed for an actual pregnancy. The first hurdle was trying to get a human egg to be fertilized by human sperm in the lab. Dr. Edwards tried repeatedly for several years, to no avail. Meanwhile, Dr. Bavister, working next door to Dr. Edwards’s lab, had developed a solution to nourish hamster sperm while he fertilized hamster eggs. “One day,” Dr. Bavister recalled, “Bob said, ‘Barry, why don’t we try your hamster medium with human eggs?’ ”It worked. Dr. Bavister said he could still recall his first glimpse of that fertilized human egg in the microscope. “It was unbelievable, like ‘Eureka!” he said.

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The next step was to get the embryos to start to grow and divide in a petri dish so that they could be transferred to a woman’s uterus. Many did not develop, and those that did did not survive long. Even after Dr. Edwards and Dr. Steptoe managed to get the eggs to grow to the point where they could be transferred, no pregnancies resulted. They made over 100 attempts, but no woman got pregnant, except one, who had an ectopic pregnancy, with the embryo in her fallopian tube instead of her uterus. “Most sensible scientists would have given up,” Dr. Bavister said. “But they plugged on and plugged on.” There were only three remaining patients. Dr. Steptoe could not retrieve any eggs from the first two. One woman did not get pregnant. Another had a tubal pregnancy that could not be carried to term. Then he tried his method with Mrs. Brown. The night Mrs. Brown was to give birth to the world’s first IVF baby, the press descended on the Oldham hospital. Dr. Edwards left the hospital that afternoon and told reporters that nothing was happening yet and that they could go home. He and Dr. Steptoe sneaked in the back entrance that night, and Dr. Steptoe delivered the baby by Caesarean. It was Dr. Steptoe’s decision to do a Caesarean, “If the baby was abnormal, they sure did not want the press in the delivery room.” In fact, he said, if the baby had been abnormal, that would have spelled the end of IVF. But after the birth of Louise Brown, Dr. Edwards was triumphant. He and Dr. Steptoe founded an IVF center, Bourn Hall Clinic, in Cambridge.

The Roman Catholic Church denounced the Nobel committee for awarding Dr. Edwards his prize, arguing that human life should begin only through intercourse and not artificially. The Vatican said Dr. Edwards “bore a moral responsibility for all subsequent developments in assisted reproduction technology and for all abuses made possible by IVF.”

In 2011, Dr. Edwards was knighted by Queen Elizabeth II “for services to human reproductive biology.” But the most moving tribute to Dr. Edwards was in a message posted on the Nobel Web site after Dr. Edwards had received the prize. It was from a man who had been born through IVF. “Dr. Edwards, thank you for my life,” it said.