History of Human Assisted Reproductive TechnologyChapter 1

Trying to fertilize oocytes in vitro was unsuccessful even in animals till the 1960s. Various reasons like sperm capacitation (Austin and Chang 1969).³ In the 1950s Robert Edwards, Mervyn Runner and Ruth Fowler (Edward's wife) tried to use mare's serum (PMS) to induce ovulation and estrus in mice.^4–6

In USA at around 1950s, a lot of genetic work on identifying chromosomes was being done and also at this time Stein and Levinthal described polycystic ovarian syndrome and described “wedge” resection as a treatment.

Robert Edwards used to study these wedges, which were provided to him by gynecologist Molly Rose and he studied various stages of oocyte development.⁷

Maturation of oocytes at 36 hours after insemination was also described by Edwards here. And also stem cell development was documented by John Paul.⁸

Robert Edwards was first to classify the stages of meiosis in human oocyte and this had a clinical significance of 2nd polar body extrusion 37 hours after injection of human chorionic gonadotropin (hCG). The first attempts to fertilize oocytes removed 36 hours after injection of hCG were tried and failed at this stage (sperm capacitation and use of epididymal sperms).

The work of Prof Robert Edwards at this stage in United Kingdom raised many questions in the medical, social and religious circles on ethics, abnormal babies, sexing and cloning. Professor Robert Geoffrey Edwards, born in Leeds on the September 27, 1925, is presently the Chief Editor of Reproductive BioMedicine Online. As a young science graduate from the University of Wales, he began his PhD at the Edinburgh University in 1951. His early work was on altering chromosomal content of mouse embryos. This gave him a strong background in embryology and reproductive physiology of mice. He teamed up with a colleague (and his future wife), Ruth Fowler to study the induction of multiple ovulations in mice with pregnant mares' serum (PMS) which was rich in gonadotropins. They were able to induce oocyte maturation and meiosis in vitro as early as 1957. Edwards traveled to California to widen his horizons 2into molecular biology and genetics. He returned to London and spent two years working intensely on immunology until his curiosity retuned to oocyte maturation and fertilization.

In vivo and in vitro experiments with gonadotropins confirmed the earlier success with rabbit, hamster and rat oocytes which matured within 12 hours of administering hCG followed by insemination. However, oocytes from cows, sheep and monkeys showed no signs of maturity when inseminated at 12 hours. Similarly, human oocyte maturation was also thought to be impossible in vitro. Molly Rose, the gynecologist who had delivered two of his daughters, agreed to provide him with human ovarian tissue. He worked with oocytes harvested from slithers or wedges of human ovaries removed from patients with Stein-Leventhal syndrome. The results from the first two years of his work were disappointing. A common basis of his failure, as well of other workers who had experimented in this field, was the early work by Pincus et al which proposed that human oocytes matured at 12 hours after hCG administration.⁸ Having exhausted a number of options such as changing culture media, adding higher doses of hormones and sera, Edwards decided that there might be some difference in mammalian species regarding the time to oocyte maturation. This was a vital breakthrough and he was finally able to demonstrate meiosis between 24 and 48 hours. This was further refined and the time of ovulation for human oocytes was identified as 36 hours. Pig oocytes were closest to humans, requiring 37 hours!⁹ Edwards moved to Cambridge where he further explored and optimized incubators, media and cell line cultivation.

Patrick Christopher Steptoe was born on June 9, 1913 in Oxfordshire, England. His father was a church organist, while his mother served as a social worker. Steptoe studied medicine at the University of London's St. George Hospital Medical School. After qualifying, his medical career was interrupted by World War II. Steptoe volunteered as a naval surgeon. He was captured with his shipmates by Italian forces in 1941 after their ship sank in the Battle of Crete. Initially granted special privileges in prison because he was a physician, Steptoe was placed in solitary confinement after officials detected his efforts to help fellow prisoners escape. Following the war, Steptoe completed additional studies in obstetrics and gynecology. In 1948, he became a member of the Royal College of Obstetricians and Gynecologists and moved to Manchester to set-up a private practice. In 1951, Steptoe began working at Oldham General and District Hospital in northeast England. While at Oldham General and District Hospital, Steptoe pursued his interest in fertility problems. He was a pioneer of laparoscopy in England, having trained with Palmer in Paris and Frangenheim in Germany. In the early part of his career he had trouble convincing the profession about its utility. Five years passed before Steptoe published his first paper on laparoscopy. He also described laparoscopy at ovulation and the ability to collect oocytes from the ovaries.¹⁰ This drew attention from all quarters of the medical profession and included Robert Edwards.

The historic collaboration began with a phone call from Edwards to Steptoe and flourished at the Oldham and District General Hospital and later at the Kershaw's Hospital. They set-up a small laboratory at Oldham. Women were stimulated with purified urinary human menopausal gonadotropin (hMG) and ovulation triggered with hCG. Oocytes were aspirated 36 hours later via laparoscopy. Human embryo formation was routinely achieved. Once again, there were disappointing results in the first three years with no pregnancies. One of the mistakes that was identified was the use of Primolut depot as a form of luteal support. It was later found to be a 3luteolytic and abortifacient. They switched to hCG and progesterone thereafter. The pressures from critics, ethicists and colleagues were mounting. The patients, however, remained loyal and a steady stream ensured that work progressed. Much of the early work was funded by Edwards and Steptoe themselves. More bad news was in store. Though a clinical pregnancy was established in 1976, it turned out to be an ectopic which had to be removed at about 10 weeks.¹¹ Working against the odds, Steptoe and Edwards experimented with various stimulation protocols and natural cycle IVF. Lesley and John Brown were the second entrants in the natural IVF group. Lesley had no oviducts, having lost them to ectopic pregnancies earlier. This proved to be a valuable discussion to critics who would later claim that the pregnancy was a fraud.

After seven years of work at Oldham, the positive pregnancy test in a natural cycle was a major milestone. Louise Brown was born at 11:47 pm on July 25th 1978 at Oldham, through a planned caesarean section delivered by registrar John Webster. She weighed 5 pounds, 12 ounces (2.608 kg) at birth. Her younger sister, Natalie Brown, was also conceived through IVF, four years later, and became the world's fortieth IVF baby, and the first one to give birth herself, naturally, in 1999. Louise Brown also has a son, conceived naturally and born in 2006, with Professor Edwards in attendance. Steptoe and Edwards published their early results in 1980 with four pregnancies out of 32 embryo transfers.¹²

The Oldham team wound up the small laboratory and set-up a private clinic at Bourn Hall. Criticism and plaudits were earned in equal measure until the late 1980s when IVF became an international phenomenon and was widely accepted in clinical practice and the public view. Patrick Steptoe died from cancer in 1988. Professor Robert Edwards continues to work on the genetic aspects controlling human reproduction and the ethics of assisted reproduction.¹³

Assisted reproductive technologies (ART) have enabled millions of people in the world to have biological children who otherwise would not have been able to do so. According to the European Society for Human Reproduction and Embryology, more than three million babies have been born using ART worldwide in the last 30 years, enabling infertile women and men; single women and men; and lesbian, gay, and transgender couples to form genetically-related families.

These new technologies have transformed the way we view reproduction. While they have created new hopeful possibilities, they also require that we pay attention to issues of health, ethics, law, and policy. Key concerns include: lack of access; health effects on women and children; potential for devaluation of the lives of people with disabilities; limitations on use by lesbian, gay, bisexual, transgender, questioning, and intersex (LGBTQI) individuals and couples; dangers of selecting characteristics of children; the commercial environment surrounding ART; and the nature of regulation in the US and other countries.

Some of the major milestones which happened over the last 30 years are as follows:

In 1932, ‘Brave New World’ was published by Aldous Huxley. In this science fiction novel, Huxley realistically described the technique of IVF as we know it. Five years later in 1937, an editorial appeared in the New England Journal of Medicine (NEJM 1937, 21 October) which is note worthy.

“Conception in a watch glass: The ‘Brave New World’ of Aldous Huxley may be nearer realization. Pincus and Enzmann have started one step earlier with the rabbit, isolating an ovum, fertilizing it in a watch glass and reimplanting it in a doe other than the one which furnished the oocyte and have thus successfully inaugurated pregnancy in the unmated animal. If such an accomplishment with rabbits were to be duplicated in the human being, we should in the words of ‘flaming youth’ be ‘going places’.

In 1934, Pincus and Enzmann, from the Laboratory of General Physiology at Harvard University, published a paper in the Proceedings of the National Academy of Sciences of the USA, raising the possibility that mammalian eggs can undergo normal development in vitro. Fourteen years later, in 1948, Miriam Menken and John Rock retrieved more than 800 oocytes from women during operations for various conditions. One hundred and thirty-eight of these oocytes were exposed to spermatozoa in vitro. In 1948, they published their experiences in the American Journal of Obstetrics and Gynecology.

However, it was not until 1959 that indisputable evidence of IVF was obtained by Chang (Chang M. Nature 1959;184:466) who was the first to achieve births in a mammal (a rabbit) by IVF. The newly-ovulated eggs were fertilized, in vitro by incubation with capacitated sperm in a small Carrel flask for 4 hours, thus opening the way to assisted procreation.

Professionals in the fields of microscopy, embryology, and anatomy laid the foundations for future achievements. The recent rapid growth of IVF-ET and related techniques worldwide are further supported by the social and scientific climate which favors their continuation.5

Through the years numerous modifications have been made in the development of IVF-ET in humans: Refinement of fertilization and embryo culture media; earlier transfer of the embryo; improvements in equipment; use of a reduced number of spermatozoa in the fertilization dish and embryo biopsy among others.

The purpose of this introduction is to acknowledge those who initiated new steps in the development of the treatment protocols and techniques that we now use facilitating such simple and promising IVF-ET procedures.

Steptoe and Edwards published a report on an ectopic pregnancy following transfer of a human embryo at the late morulae /early blastocyst stage. (Steptoe and Edwards. Lancet 1976;1:880).

Introduction of Clomiphene Citrate and hMG in the IVF treatment protocol (Trounson et al. Science 1981;212:681).

The Clamart group in France developed an LH assay (LH-SIR0) which could detect the initial LH rise in plasma allowing accurate prediction of the ideal time for the retrieval of oocytes (Testart et al. Fertil Steril 1981;36:50).

The first frozen embryo twins were born in Australia.

The introduction of culture medium (Mohr and Trounson. J Reprod Fertil 1980;58:189).

The first report of the need for a delay between oocyte collection and insemination to allow oocytes collected to complete maturation (Trounson et al. J Reprod Fertil 1982;64:285).

Susan Lenz and Jörgen G Lauritsen demonstrated transabdominal transvesical oocyte aspiration using an ultrasound-guided needle (Lenz and Lauritsen. Fertil Steril 1982;38:673).8

Monash IVF team reported on the birth of the first frozen embryo baby (Embryo freezing developed in Cambridge, England on cattle – minor adaptations to adjust to humans). (Trounson and Mohr. Nature 1983;305:707).

Maturation and fertilization of morphologically immature human oocytes in a program of in vitro fertilization (Veek et al. Fertil Steril 1983;39:594).

First pregnancy after oocyte donation. (Trounson et al. Br Med J 1983;286:835).

First successful delivery following egg donation. (Buster et al. Lancet 1983;2:223).

Gleicher and his group reported the first vaginal egg retrieval using an abdominal ultrasound (Gleicher et al. Lancet 1983;2:508).

First surrogacy embryo transfer baby born in California.

First report on pregnancy following translaparoscopic gamete intrafallopian transfer (GIFT) procedure (Asch et al. Lancet 1984; 2(8410):1034).

The first report on pregnancy following IVF and egg donation in a woman with primary ovarian failure (Lutien et al. Nature 1984;307(5947):174).

Introduction of GnRH agonists to the IVF treatment protocol (Porter et al. Lancet 1984;2:1284).

A report of pregnancy following transfer of intact frozen-thawed embryos (Zeilmaker et al. Fertil Steril 1984;42:293).

An unusual report of the possibility that abnormal spermatozoa could be enriched and give rise to healthy babies (Cohen et al. Lancet 1984; 1(8388):1239-40).

Casper and his colleagues were the first to described the use of low dose hCG for support of the luteal phase in ART cycles (Casper et al. Lancet 1983;2:1191).

Jacqueline Mandelbaumm with Dan Szollosi described the microstructures of the human oocyte, which became known as ‘oocyte dysmorphia’ (Szollosi et al. J In Vitro Fertil Embryo Trans 1986;3:232).

Matts Wikland in Gothenburg, Sweden, described the possibility of using a vaginal sector scanner (transvaginal technique) for oocyte aspiration (Wikland et al. Ann NY Acad Sci 1985; 442:182).

First report of the use of abdominal ultrasound guidance for embryo transfer (Strickler et al. Fertil Steril 1985;43:54).

The first reported birth after replacement of hatching blastocyst cryopreserved at expanded blastocyst stage (Cohen et al. Lancet 1985;1(8429): 647).

First report about the Canadian IVF baby by the group of Victor Gomel at the University of British Columbia (Young S Moon et al. Gamete Research 1985;11:289).

In 1985, Quinn and Warnes published a formula entitled Human Tubal Fluid (HTF) which mimics the in vivo environment to which the embryo is exposed (Quinn P, Kerin JF, Warnes GM. Fertil Steril 1985;1985:493).

First description of transvaginal sector scan sonography for needle-guided transvaginal follicle aspiration (Fiechtinger and Kemeter. Fertil Steril 1986;45:722).

First pregnancy, following IVF donated oocytes, in a non-ovarian failure patient (Rosenwaks et al. Fertil Steril 1986;45:417).

Navot et al. reported the possibility to artificially induced endometrial cycles and establishment of pregnancies in the absence of ovaries (Navot et al. NEJM 1986;314:806).

First report on pregnancy after translaparoscopic zygote intrafallopian transfer (Devroey et al. Lancet 1986;1(8493):1329).

First two babies born after epididymal sperm aspiration for men with congenital absence of the vas deferens and naming of the technique MESA (Patrizio et al. Lancet 1988;2(8624):1364).

Australia's first IVF surrogate birth.

The first baby birth using subzonal sperm injection at National University of Singapore (Ng et al. Lancet 1988;2:790).

Pregnancy was obtained from micromanipulation using zona drilling or mechanical partial zona dissection (Cohen et al. Lancet 1988;2(8603):162).

The death of Dr Patrick Steptoe—March 21 1988.12

First report of the use of laser techniques in the field of assisted reproduction for application in gametes or embryos (Tadir et al. Fertil Steril 1989;52:870).

Gonen and her colleagues in Toronto pioneered the use of ultrasound for endometrial quality (thickness and pattern) related to IVF pregnancy. (Gonen et al. Fertil Steril 1989;52:446).

Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification (Handyside et al. Nature 1990; 344:768).

First report of assisted hatching in human embryos (Cohen et al. Human Reprod 1990;5:7).

The first report on polar body biopsy, transfer of the embryo and achieving pregnancy (Verlinsky et al. Hum Reprod 1990;7:826).

Gonen et al proposed the use of GnRH agonist in place of hCG as a means to trigger the gonadotropic surge for IVF (Gonen et al. J Clin Endocrinol Metab 1990;71:918).

Gonen, Jacobson and Casper pioneered the use of combined oral contraceptives for follicle synchronization and cycle scheduling in IVF (Gonen et al. Fertil Steril 1990;53:282).

1991 In Vitro Maturation (IVM) in unstimulated cycle resulted in pregnancy in a donor oocyte program (Cha et al. Fertil Steril 1991;55:109).

The first report suggesting the use of GnRH antagonist, Nal-Glue, to prevent premature LH rise and progesterone in controlled ovarian hyperstimulation treatment (Frydman et al. Fertil Steril 1991;56:932).

The first report on the use of a laser for Zona Pelucida drilling (Palanker et al. Laser Surg Med 1991;11:589).

Navot et al confirmed that the age-related decline in female fertility is attributable to oocyte quality (Navot et al. Lancet 1991;337(8754):137).

Assisted zona hatching was introduced in IVF programs to breach the zona pellucida and promote the natural process of hatching (Cohen et al. Hum Reprod 1992;7:685-91).

Discovery that men with congenital absence of the vas deferens have a mild form of cystic fibrosis: Two papers: (Anguiano et al. JAMA 1992;267(13): 1794) (Patrizio and Asch. Annals Acad Med Singapore 1992;21:533).15

Report of the first pregnancy after intracytoplasmic sperm injection (ICSI) by the group in Brussels (Palermo et al. Lancet 1992;2:17).

Pregnancy after embryo biopsy and coamplification of DNA from X and Y chromosomes (Gilbert et al. JAMA 1992;268:727).

Ovulation induction by endogenous LH released by the administration of an LHRH agonist after follicular stimulation for in vitro fertilization (Emperaire et al. J Gynecol Obstet Biol Reprod (Paris) 1992;21:489).

First report of cumulative conception and live-birth rates after IVF in relation to patient age and cause of infertility (Tan et al. Lancet 1992; 339:1390).

Confirmation that men with congenital absence of the vas defference, have cystic fibrosis mutations which can be transmitted to the offspring (Patrizio P et al. Hum Reprod 1983;8:215).

First report on the use of testicular sperm extraction (TESE) and ICSI (Silber et al. Hum Reprod 1994).

First two births from replacement of frozen embryos produced with epididymal sperm (Patrizio et al. Hum Reprod 1992;7:652).

The first reported birth resulting a frozen-thawed oocyte and ICSI (Chen Lancet 1986;1:884).

Birth after blastocyst development from IVM oocyte plus ICSI plus Assisted Hatching (Barnes et al. Hum Reprod 1995;10:3243) The first report of aneuploidy testing (Munné et al. Am J Obstet Gynecol 1995;172:1191).

Zenzes from the group of Casper in Toronto demonstrated adverse effects of cigarette smoking on nuclear spindle (Zenzes et al. Hum Reprod 1995;10:3213).

Discovery that some men with severe oligoasthenospermia have deletions in the Y-chromosome (Rejio et al. Lancet 1996;347(9011): 1290).

Jurisicova from the group of Casper was the first to recognize that preimplantation human embryo fragmentation involved programmed cell death. (Jurisicova et al. Mol Hum Reprod 1996;2:93).

Casper and his colleagues were the first to demonstrate and introduce the use of the hypo-osmotic swelling test for selection of immotile sperm for ICSI (Casper et al. Fertil Steril 1996; 65:972).

First report on Cytoplasmic transfer (Cohen et al. Lancet 1996;350:186).

Gardner introduced sequential media and blastocyst transfer which is now greatly assisting in the move to single embryo transfer (Gardner et al. Human Reproduction 1998;13:3434).

Births after intracytoplasmic injection of sperm obtained by testicular extraction from men with nonmosaic Klinefelter's sydrome (Palermo et al. N Engl J Med 1998;338:588).

First report on cytoplasmic transfer (Cohen et al. Lancet 1996;350(9072):186).

Birth following vitrification of human oocyte (Kuleshova et al. Hum Reprod 1999;14:3077).

Chian et al demonstrated that hCG priming prior to immature oocyte retrieval in women with PCO increases the maturation rate and produces high pregnancy rates of 40 percent per IVM cycle started (Chian et al. N Eng J Med 1999;341:1624).

World's first IVF/ICSI pregnancy and livebirth after successful air transport of oocytes reported by the McGill Reproductive Center, enabling the creation of an airtransport IVF program in large countries with scattered populations or in areas remote from infertility centers (Buckett WM et al. Fertil Steril 1999;71:753).

Embryo transfer catheter is visualized by vaginal ultrasound.

Birth of an infant from cryopreserved embryos (zygotes) produced by IVM oocytes derived from an unstimulated patient with polycysticovary syndrome (PCOS) (Chian et al. Hum Reprod 2001;16:1700).

Ongoing twin pregnancy after ICSI of PESA-retrieved spermatozoa into in vitro matured oocytes reported by the McGill group. (Ahmad Kamal Abdul-Jalil et al. Hum Reprod 2001;16:1424).

Livebirth after sperm retrieval from a moribund man (Belker et al. Fertil Steril 2001;76:841).

First clinical application of comparative genomic hybridization and polar body testing for preimplantation genetic diagnosis of aneuploidy. (Wells et al. Fertil Steril 2002;78:543).

First livebirth after ovarian stimulation using a chimeric long-acting human recombinant follicle-stimulating hormone (FSH) agonist (recFSH-CTP) for in vitro fertilization (Beckers et al. Fertil Steril 2003;79:621).

The first reported liveborn following preimplantation genetic diagnosis for Retinoblastoma (Xu et al. Am J Opthalmol 2003;18) Implantation of the human embryo is the limiting factor in the success of IVF.

Jaques Donnez reporting about the first Livebirth after orthotopic transplantation of cryopreserved ovarian tissue (Donnez et al. Lancet 2004;364(9443):1405).

Gardner and colleagues performed the world's first prospective single blastocyst trial, which showed the feasibility of SBT and in keeping high pregnancy rates (Gardner et al. Fertil Steril 2004; 81:551).

The first preimplantation HLA matching for stem cell transplantation to affected sibling (Verlinsky et al. JAMA 2004;291:2079).

First report of fertility preservation for cancer patients using IVM and oocyte vitrification (Rao et al. Lancet 2004;363:1829). First report on natural cycle in IVF combined with IVM as a potential approach to infertility treatment (Chian et al. Fertil and Steril 2004;82:1675).

First birth in Israel from thawed ovarian cortex re-transplants (Meirow et al. N Engl J Med 2005; 535(3):318).

Cryopreservation of intact human ovary with its vascular pedicle (Bedaiwy et al. Hum Reprod 2006;21:3258).

First successful pregnancy after preimplantation genetic diagnosis for aneuploidy screening in embryos generated from natural-cycle IVF combined with IVM achieved at the McGill Reproductive Centre (Ao et al. Fertil Steril 2006;85: 1510).

Introducing the concept of mild treatment strategy for IVF (Heijnen et al. Lancet 2007;369 (9563):743).

A novel multi-gradient freezing technique for the cryopreservation of the whole ovary. Thawing the ovary resulted in normal ovarian architecture and no damage to the vascular wall or intima (Arav and Pasquale. Yale Practice 2007;12:2).

First IVM egg donation pregnancy in North America achieved by McGill Reproductive Centre (Holzer et al. Fertil Steril 2007;88:62).