InVitro Fertilization

The fertilization of mammalian eggs in vitro has a history that goes back to the early 20th century. Analyses on short-term cultured mouse embryos opened a new perspective of research on human embryos in vitro.

This phase of research in 1930 allowed Pincus and his colleagues, Enzmann and Saunders, to initially retrieve undeveloped rabbit eggs from their follicles in culture and discovered that it took 12 hours for their maturation. They also studied human eggs and described similar results regarding the timing. This data misled other researchers who inseminated the eggs after 12 hours in culture and failed to achieve fertilization.

A new development in research began in the early 1950s when Edwards completed his PhD on genetic development in mice. His studies on oocyte maturation and in vitro fertilization were supported by identifying diakinesis and metaphase-2 as major indicators for the continuation of egg maturation. Mouse and other mammal eggs needed 12 hours in culture, but human eggs were seen to require more time.

At this time, Edwards also worked with John Paul and Robin Cole in Glasgow and created the world's first embryonic stem cells, using rabbit embryos from the 2-cell stage to the blastocyst. The inner cell mass was divided indefinitely in vitro for 200 or more generations. When the blastocyst was kept intact, the trophoectoderm forms a thin floor that allows a surface for the inner mass cells to differentiate into any body tissue.

Fascinated by the therapeutic prospect of these stem cells, Edwards focused on maturing human oocytes in vitro to fertilize them in vitro and to obtain human embryos for various purposes. Eventually, it was seen that human oocytes required 37 hours in culture to fully mature, indicating that insemination should be done after 35-40 hours to achieve fertilization (Edwards, 1965).

Using strict culture conditions based on earlier studies by Whitten, Biggers, and Ham, and working mostly with PhD students in Cambridge, human eggs were fertilized, in collaboration with Barry Bavister, without the need for sperm capacitation in 1969 (Edwards et al., 1969).

PGD (pre-implantation genetic diagnosis) was finally performed on rabbits in 1968, working with Richard Gardner who also announced that a single stem cell taken from the inner cell mass, injected into the recipient blastocyst, could colonize all tissues in the resulting 'chimeric' embryos. Theories on the origin of chromosomal anomalies in embryos were also formulated during this period.

Data on the timing of human oocytes in vitro showed that women ovulated 37 hours post-HCG. Edwards was already driven by multiple factors including the discovery of a very high incidence of human infertility, the endless therapeutic prospect of human stem cells, the possibility of PGD, and a determination to bring science, medicine, and ethics to human conception.

Fortunately, reading about Patrick Steptoe's work in laparoscopy revealed easy access to ovarian follicles to aspirate their oocytes. Initially, the onset of follicle rupture and ovulation in women assisted with HMG and HCG was predicted for 37 hours in culture, and human oocytes were aspirated earlier from their follicles. Ethical objections to growing human embryos in culture were numerous, and concepts of IVF, PGD, and stem cells were met with disdain; at such a stage Edwards had only three or four supporters in Cambridge.

Compelled by clinical necessity, objections were overcome as they prepared to culture human embryos in vitro. Remarkably, all stages of human division up to the 9-day post-fertilization blastocyst were achieved at Oldham and District General Hospital, while human embryology in vitro quickly surpassed results for all other mammal species. Ethics for human IVF were discussed in Nature 1971, working with Dave Sharpe, an American lawyer (Edwards and Sharpe, 1971). Edwards called upon teratologists of the world to consider the risks of IVF and in vitro culture for children conceived in this way.

Encouraged by them to continue, and impressed by the lack of anomalies in animal pups coming from embryos grown in culture and in many cases made in vitro, blastocysts were transferred to their infertile mothers treated with HMG and HCG to stimulate the maturation of several follicles and oocytes. Numerous endocrine problems were seen, particularly a very short luteal phase during stimulated cycles. Overcoming this luteal weakness with the use of progestagen, Primulot, resulted in complete failure of implantation.

Only when Ken Bagshawe in London made it possible to measure HCG in the blood of patients in Oldham around the time of implantation, did Edwards and Steptoe discover that many of their patients had short-term pregnancies, now called biochemical pregnancy, indicating that the transferred embryos did not survive in a deficient uterine environment.

Primulot was no longer used and data on early pregnancy support were described including clomiphene/HMG, bromcryptine/HMG, oocyte donation, cryopreservation of oocytes and human embryos, the first intra-Fallopian gamete transfer (GIFT), injecting HCG in the middle of the cycle, and finally measuring the LH peak in urine during a normal menstrual cycle. A single matured egg could be aspirated and fertilized in vitro. These efforts resulted in the world's first IVF child, Louise Brown, born in the UK on July 25, 1978, as a result of transferring an 8-cell embryo (Edwards et al., 1980). There was no space to complete the subsequent years at Bourn Hall.

These years achieved successes previously thought impossible with the conception of 1000 births in 8 years, more than half of the babies from IVF during that period. Among the new horizons, male infertility, detailed endocrine analyses, detailed analyses on the incidence of implantation, abortion, births, and the normality of children from IVF, the ethics of human IVF, and the first international meeting on IVF were all reported from Bourn Hall during this period. Jean Purdy and Patrick Steptoe each died at critical moments in the history of IVF.

Many of the ongoing improvements in the IVF clinic began in the early 1980s. Indicative examples include the development of ovarian stimulation protocols using different components during the follicular, mesocycle, and luteal phases, improvement of embryonic culture conditions, development of transvaginal echo (which allows monitoring of ovarian response, collection of oocytes without general anesthesia, and also transfer of embryos into the uterine cavity), cryopreservation of surplus embryos leading to additional chances for pregnancy, oocyte and embryo donation, and improved embryo transfer techniques.

In the early 1990s, the ICSI (intracytoplasmic sperm injection) technique was developed, pioneered by Devroey and van Steirteghem in Brussels, Belgium. Another step forward in the same period was made by diagnosing specific disease mutations in embryos before implantation after DNA amplification (Handyside et al., 1990).

Despite strong initial resistance from the medical community and society, IVF has now established its place in the clinical management of infertility. Indications for performing IVF have expanded from Fallopian tube occlusion to male factor, from unexplained subfertility to severe male problem requiring ICSI. Many Western societies now report that between 1 and 3% of all born children are from IVF (Fauser et al., 2005).

No one can dispute the claim that IVF now represents the main treatment for infertility, resulting in a significant contribution to births in the Western world. However, we cannot close our eyes to the side effects of today's IVF practice, the high rate of multiple pregnancies, cost and complexity of treatment, and the low use of IVF in non-Western countries.

For almost 10 years, this procedure has also been carried out in Tirana and in 2011 the opening of one of the most modern IVF centers in the Balkans, the American Hospital, was completed, achieving higher success rates than those averages published by the European Society of Human Embryology. Soon these data will also be reflected in the upcoming congress of this society where many debates are expected to open but placing Albania as one of the most attractive countries for this procedure.

References

1. Devroey P and van Steirteghem A (2004) A review of ten years experience of ICSI. Hum Reprod Update 10,19–28.

2. Edwards RG (1965) Maturation in vitro of human ovarian oocytes. Lancet 2,926–929.

3. Edwards RG and Sharpe J (1971) Social values and research in human embryology. Nature, 231,87–91.

4. Edwards RG, Bavister BD and Steptoe PC (1969) Early stages of fertilization in vitro of human oocytes matured in vitro. Nature 221,632–635.

5. Edwards RG, Steptoe PC and Purdy JM (1980) Establishing full-term human pregnancies using cleaving embryos grown in vitro. Br J Obstet Gynaecol 87,737–756.

6. Fauser BC and Macklon NS (2004) Medical approaches to ovarian stimulation for infertility. In Strauss JF and Barbieri RL (eds) Yen and Jaffe’s Reproductive Endocrinology, 5th edn. Elsevier, Philadelphia, pp. 965–1012.

7. Fauser BC, Devroey P and Macklon NS (2005) Multiple birth resulting from ovarian stimulation for subfertility treatment. Lancet 365,1807–1816.

8. Handyside AH, Kontogianni EH, Hardy K and Winston RM (1990) Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification. Nature 344,768–770.