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In-vitro fertilisation
Review
In-vitro fertilisation
essential as Steptoe was able to collect the oocytes by transabdominal needle aspiration of mature follicles under laparoscopic view and Edwards had the necessary laboratory skills to achieve the growth of embryos after fertilisation in vitro. The importance of collaboration between disciplines remains as true today as it was in the 1970s. The birth of Louise Brown was soon followed by the birth of further in-vitro fertilisation (IVF) babies in Australia and the United States (US). At this time, there was considerable disquiet among both the public and the medical profession concerning the morality, and even legality, of the process of IVF. This debate led to the establishment first of a Voluntary Licensing Authority, and later the Human Fertilisation and Embryology Authority (HFEA), by the UK government. The role and powers of the HFEA continue to change as the umbrella of IVF applications continues to grow; and the organisation has been emulated in many countries over the past two decades.
Carol Coughlan William L Ledger
Abstract Infertility is not an identifiable physical disease yet its psychological impact on affected couples can be severe. One in seven couples in the UK suffers from subfertility. Of these, approximately half will end up undertaking some form of assisted conception treatment. In-vitro fertilisation (IVF) can be viewed both as a test of reproductive potential, allowing detailed assessment of oocytes, oocyte sperm interaction and embryo quality, as well as an effective treatment for most forms of subfertility. The dramatic improvements in pregnancy rates seen with IVF treatment since its inception some 30 years ago have occurred due to close multidisciplinary collaboration and the practical application of scientific advances in embryology and pharmacology. There have been several important landmarks including: the use of drugs for superovulation and pituitary downregulation; the introduction of transvaginal ultrasound for monitoring of follicle growth and oocyte retrieval; developments in embryo culture; oocyte donation; and the introduction of intracytoplasmic sperm injection for the treatment of severe forms of male infertility. In the past decade, we have seen the development of new technologies, including pre-implantation genetic diagnosis, the in-vitro culture of immature oocytes to viability, and the cryopreservation of oocytes, thereby widening the scope of clinical problems that can be addressed by IVF-associated technologies. Despite this progress, the majority of IVF cycles still do not produce a viable pregnancy. The psychological stresses imposed upon couples by assisted conception treatment need to be managed carefully and sympathetically. IVF practice continues to require support from appropriately trained and skilled counsellors.
How IVF works Although ‘human’ IVF did not come of age until 1978, most of the technological steps needed for Louise Brown’s conception had been developed years earlier in veterinary medicine. The principle of IVF is simple: collect mature oocytes (eggs) from the ovaries, fertilise them in cell culture in the laboratory and replace embryo(s) into the uterine cavity at such a time in the cycle to ensure a receptive endometrium. However, each step in this model has been refined by decades of experimentation, leading to the quasi- industrial IVF of today.
Stimulation of multiple follicle development One of Steptoe and Edwards greatest problems was a shortage of oocytes. They were able to harvest only one or two oocytes in the natural cycle, and frequently, by the time that laparoscopy had been arranged, general anaesthesia induced and surgery undertaken, the follicle had ovulated and the oocyte had been lost in the pelvis. Success rates were, therefore, low. In order to improve the chances of having embryos for transfer, the embryologist needed more oocytes and the clinicians needed to be certain that premature ovulation would not occur.
Keywords Gn RH analogues; in-vitro fertilisation (IVF); intracytoplasmic sperm injection(ICSI); pre-implantation genetic diagnosis (PGD)
Gonadotrophins Gonadotrophins synthesised and secreted by the pituitary gland play a central role in regulating the ovarian cycle of follicular recruitment and maturation, oocyte release and corpus luteum development and regression. Exogenous gonadotrophins have been used for many years to induce ovulation in anovulatory patients. Gonadotrophins were first isolated from the urine of pregnant women by Aschheim and Zondeck in 1927. Three years later, Zondeck reported their presence in the urine of menopausal women. However, the first use of gonadotrophins for inducing multiple ovulations utilised extracts of human pituitary tissue collected at post-mortem. Many pituitaries were needed to obtain sufficient extract to treat a single patient. In the 1980s, pituitary derived gonadotrophins were found to have transmitted Creuzfeld–Jakob infection in a small number of cases. It was not until 1947 that human menopausal gonadotrophins were first extracted from urine in pharmacological amounts, creating the possibility of a
Introduction In July 1978, Louise Brown, the world’s first baby to be conceived outside the human body, was born in Britain. The historic event was the result of years of research and collaboration between Robert Edwards, a scientist from Cambridge and Patrick Steptoe, a gynaecologist in Lancashire. This collaboration was
Carol Coughlan MRCOG MRCPI is Clinical Research Fellow at the Department of Obstetrics and Gynaecology, Sheffield Teaching Hospital NHS Foundation Trust, Sheffield, UK William L Ledger bm bch ma dphil frcog is Professor at the Department of Obstetrics and Gynaecology, Sheffield Teaching Hospital NHS Foundation Trust, Sheffield, UK.
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readily available pharmaceutical source for widespread clinical use. More recently, recombinant follicle-stimulating hormone (FSH) and luteinising hormone (LH) have been made available following transfection of the genes for the alpha and beta chains of human FSH or LH into Chinese hamster oocyte cell lines, producing large amounts of glycosylated FSH and LH without the need for collection and extraction from human urine. Treatment with gonadotrophins was initially by intramuscular injection of an impure fraction containing both LH and FSH. In time, purified urinary FSH and later recombinant FSH became available for subcutaneous injection. These injections can be selfadministered and are tolerated well by patients. The current era of fertility therapy focuses on simplifying treatment regimens by minimising clinic attendances, and reducing the pain and stress of treatment, thereby improving compliance. Gonadotrophin injection encourages the development of a cohort of mature follicles in a single stimulation cycle, overriding the physiological processes of follicle selection and dominance. Egg collection could yield anything up to 40 oocytes after 10–14 days of daily injection. In the early days of superovulation, ultrasound had not developed sufficiently for the assessment of follicle growth, and the only means by which clinicians could obtain an approximate measurement of ovarian response to treatment was by the daily measurement of urinary oestrogens, a process that was time-consuming and inaccurate. In addition, the rapid rises in circulating oestrogen levels were likely to trigger a premature LH surge, leading to ovulation before egg collection. Having established a means of obtaining multiple oocytes, the next development was to prevent the premature LH surge.
GnRH analogues with antagonist properties An alternative to GnRH agonists are GnRH antagonists such as Cetrorelix or Ganirelix. GnRH antagonists directly block the GnRH receptor, isolating it from the action of native GnRH, without an initial stimulation, thereby avoiding the ‘flare’ effect. This has a number of potential benefits. Since the flare is avoided, duration of treatment for controlled superovulation may decrease. The total dose of FSH required for treatment may be reduced. Use of GnRH antagonists may reduce the incidence and severity of ovarian hyperstimulation states associated with superovulation. Unless GnRH antagonists are administered for a prolonged period of time, pituitary downregulation does not occur, making them rapidly reversible. The ‘antagonist’ IVF cycle begins with the injection of FSH from day 1 or 2 of the menstrual period, with an addition of GnRH antagonist a few days later to prevent an LH surge. FSH injection is continued until hCG is given to mimic a timed LH surge. The use of GnRH antagonists allows a more rapid IVF treatment cycle without the need for preliminary downregulation, avoiding the troublesome menopausal side-effects of downregulation. Ovarian cyst formation that is seen in up to 10% of ‘long protocol’ cycles due to the ‘flare’ effect of raised FSH on the ovary are avoided by the use of GnRH antagonists.
Monitoring of follicle growth Accurate monitoring of follicle growth during ovarian stimulation is essential both for the correct timing of hCG administration (a follicle contains a mature oocyte when it reaches 17–18 mm diameter) and to avoid ovarian hyperstimulation by discontinuing FSH injection if too many follicles begin to develop. In older women, monitoring can also predict ‘poor response’, failure to develop growing follicles despite FSH injection. Such patients can again be advised to stop treatment or can at least be warned that pregnancy is unlikely if they continue stimulation and go ahead with egg collection. The growth of ovarian follicles can be monitored either directly by visualisation with ultrasound or by measuring their output of oestradiol into the circulation. The widespread availability of transvaginal ultrasound has facilitated follicle monitoring considerably – the procedure is painless and gives accurate assessment of follicle diameters and endometrial thickness and integrity. In practice, both ultrasound and blood tests for oestradiol are used in most centres. In women at high risk of developing ovarian hyperstimulation syndrome (OHSS), the use of both ultrasound scanning and serial oestradiol assays can be used to select patients suitable for prophylactic cycle cancellation or cryopreservation of all the embryos with the avoidance of fresh embryo transfer, thereby avoiding pregnancy and reducing the risk of severe pregnancy-associated OHSS. During the period of superovulation by FSH injection, follicles will grow by 1–2 mm per day, requiring 9–11 days of treatment before hCG administration. Modern ‘low-dose’ FSH regimens rarely produce more than 10 mature follicles – the reduction in number of oocytes is more than compensated for by the reduction in risk of OHSS.
Gonadotrophin-releasing hormone agonists Gonadotrophin-releasing hormone (GnRH) agonists have acquired an important place in IVF treatment. The decapeptide structure of GnRH was described in 1971. It was initially thought that agonists would be used as strong sustained stimulators of gonadotrophin secretion. It soon became evident that administration of these agents, after an initial bolus release of gonadotrophins, suppresses LH and FSH levels by pituitary receptor downregulation and desensitisation. In IVF treatment, this prevents the occurrence of a premature LH peak in response to rising oestradiol levels from the growing follicles, preventing spontaneous ovulation and allowing planned oocyte retrieval. However, since the effect of ovarian quiescence is produced at the pituitary, injections of FSH hormone would still stimulate the ovary to resume folliculogenesis. Hence, from the mid-1980s, it became common-place to begin an IVF treatment cycle with ‘pituitary downregulation’, followed by ovarian stimulation with gonadotrophin injection after downregulation had been confirmed by the measurement of low levels of oestradiol and LH in the circulation, and endometrial shedding confirmed by the ultrasound demonstration of a thin endometrium. This so-called ‘long protocol’ was followed by a number of variations on the theme. All share the same application of GnRH agonist to avoid a premature LH surge. In each protocol, a timed LH surge is created by a single injection of human chorionic gonadotrophin (hCG). This is essential to induce the oocyte to re-enter meiosis and extrude the first polar body and to begin the process of luteinisation of the follicles, which prepares the endometrium for implantation.
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Egg collection The early days of IVF used laparoscopy for egg collection. Although the quality of the laparoscopic image improved dramatically with 301
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higher than would be expected following spontaneous conception. Compared with singletons, twins have a seven-fold increase in neonatal mortality and six-fold increase in the risk of cerebral palsy. Maternal complications include both morbidity and mortality. These complications result in increased costs to the health service. As a result, multiple pregnancies are now considered the single most important risk of IVF treatment. In March 2004, the UK HFEA advocated a universal maximum of two embryos per transfer, regardless of the procedure used. In addition, the National Institute for Clinical Excellence (NICE) in the UK guideline ‘Fertility: assessment and treatment for people with fertility problems’, published in February 2004, provides recommendations for good practice. The guideline includes the recommendation that ‘no more than two embryos should be transferred during any one cycle of IVF treatment. It is now recognised that the only effective method to reduce the multiple pregnancy rate is to adopt a policy of single embryo transfer. An expert panel commissioned by the HFEA in 2005 to review multiple births and assisted conception proposed that IVF clinics should develop criteria for selecting groups of women who should be offered single embryo transfer in an effort to reduce our present multiple pregnancy rate of 24%.
fibreoptic technology, better light sources and cameras, egg collection still required general anaesthesia and carried surgical risk. Laparoscopic egg collection was also time-consuming and costly. Improvements in the quality of transvaginal ultrasound allowed the safe transvaginal ultrasound-guided needle puncture of ovarian follicles and cysts from the early 1990s onwards. This could be done with sedation and local anaesthesia and was both safer for patients and more convenient for units. Laparoscopic egg collection was quickly superseded, except for rare cases in which endometriosis, adhesions, or prior infection made the ovaries inaccessible by the transvaginal route. Egg collection is carried out some 34–36 hours after the initiation of oocyte maturation with hCG injection. Delay beyond this interval carries the risk of ovulation, and earlier egg collection may result in a higher number of immature oocytes. The procedure is generally performed as a ‘day case’ with the male partner present. Conscious sedation with analgesia is induced with intravenous opiate (pethidine or fentanyl) and tranquilliser (temazepam or diazepam). Each follicle is punctured in turn under ultrasound control. The follicular fluid is aspirated into a warmed sterile tube and immediately transferred to the adjacent embryology laboratory. The embryologist identifies the oocyte within its envelopment of cumulus cells, removes the oocyte-cumulus mass from the follicular fluid with a pipette and places it into culture medium in an incubator.
Embryo transfer Embryo transfer is performed using a flexible transfer catheter into which the embryos are loaded, suspended in 50 μl of culture medium. Transcervical transfer requires insertion of a Cusco’s speculum, cleansing of the cervix to remove excess mucus and gentle insertion of the transfer catheter to about 1 cm below the fundus. Abdominal ultrasound is increasingly used to monitor catheter placement and may result in improvement in pregnancy rates compared with the clinical touch method. Once the catheter is in place, the fluid containing the embryos is injected into the uterine cavity. The chances of pregnancy are improved by an ‘easy’ embryo transfer. If, for example, it is necessary to apply a tenaculum to the anterior lip of the cervix to allow the catheter to enter the uterine cavity, the chances of implantation fall. Further trauma, particularly if there is bleeding from the endometrium, further jeopardises the outcome. For this reason, many units perform a ‘dummy transfer’ before starting IVF, with recourse to cervical dilatation in theatre if problems are encountered. Rarely, it may be necessary surgically to dilate the cervix before the start of an IVF cycle. A number of outer sheaths, stiffeners or malleable stylettes are available to facilitate transfer in difficult cases, with recourse to a transmyometrial embryo transfer under ultrasound guidance only in cases of insurmountable cervical stenosis. Several studies have shown that trained specialist nurses can perform most embryo transfers with pregnancy rates equivalent to those of medical staff. Embryo transfer is a highly charged moment for the patient and her partner and many patients prefer their familiar nurse to undertake the procedure. Successful transfer demands a gentle and unhurried approach to the procedure.
Sperm, eggs and embryos A semen sample is provided by the male partner on the same day as the egg collection, unless previously frozen sperm are to be used. An azoospermic male may require further evaluation before IVF in order to determine the best method of obtaining spermatozoa for IVF. In non-obstructive azoospermia, the semen should be spun down to a pellet and examined, as not infrequently a sufficient number of live spermatozoa are recoverable for intracytoplasmic sperm injection (ICSI). If azoospermia is associated with a very low volume of ejaculate, retrograde ejaculation must be considered and can be diagnosed on microscopic examination of a post-ejaculatory urine sample. In obstructive and other cases of azoospermia, surgical sperm retrieval may be indicated. Retrieval methods include percutaneous epididymal sperm aspiration, simple percutaneous testicular sperm needle aspiration and percutaneous or open testicular sperm (tissue biopsy) extraction. In some centres, epididymal aspiration is performed microsurgically. The sperm sample is processed in the laboratory to isolate motile sperm from the ejaculate. Each oocyte is individually stripped of the outer cumulus layer by gentle repeated aspiration into a pipette, and is then incubated with an aliquot containing approximately 50,000 sperms or, for ICSI, injected with a single sperm. The oocytes are inspected after 24 hours in culture. If fertilisation has occurred, two pronuclei (male and female) will be visible at this stage. Culture is continued for a further 24–48 hours. Embryos will have divided up to the 4–8 cell stage and can be graded for quality by microscopic assessment of their morphology, shown in Figure 1. The best one or two embryos will generally be selected for transfer. Other ‘spare’ embryos may be cryopreserved in liquid nitrogen for later use. Due to the practice of transferring more than one embryo, the incidence of multiple pregnancies following IVF treatment has risen to over 25%. This is practically 20 times
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Luteal support Pituitary downregulation by GnRH agonists (or receptor blockage by antagonists) produces a prolonged inhibition of pituitary LH 302
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In vitro maturation Immature oocyte
Mature oocyte
In vitro fertilisation
In vitro production
1-cell
2-cell
4-cell
Expanded blastocyst
Early/expanding blastocyst
8-cell
Inner cell mass Blastocell Trophectoderm Morula
Compacting
Figure 1 Embryo development.
severe male factor infertility or those with previous failed fertilisation during IVF attempts. ICSI can be used not only for men with profound oligozoospermia or asthenoteratozoospermia but also for those with obstructive azoospermia, following microsurgical or direct aspiration of sperm from either the epididymis or the testis. It involves the injection of a single spermatozoon directly into the cytoplasm of the oocyte. The spermatozoon is immobilised before ICSI, usually by breaking the tail, as flagellation within the ooplasm is undesirable and only the genetic material of the sperm head is required. ICSI, pioneered by Van Steirteghem and his team in Brussels, has revolutionised the management of male infertility and has provided the possibility of a pregnancy for men who previously would have required their partners to undergo donor insemination. The use of ICSI is increasing worldwide and is readily available to couples based on clinical indication. There is evidence from randomised clinical trials that ICSI is not beneficial for couples with non-male infertility unless there has been a previous failed or poor fertilisation rate (NICE guideline, 2004).
secretion with a possible deleterious effect on the function of the corpus luteum. Embryo transfer is, therefore, always followed by treatment to support the luteal phase. This can be done either by stimulation of the function of the corpora lutea by repeated injection of hCG to mimic LH, or by the direct administration of progesterone or a synthetic progestogen. Progestogens can be administered via the oral or vaginal route or by intramuscular injection. Injection of hCG carries the risk of promoting ovarian hyperstimulation by its action on the corpus luteum, and has not been shown to produce better pregnancy rates than injected progestogen supplementation in meta-analysis of randomised trials. However, the optimum management of luteal support, and the related problem of implantation failure, remains controversial. Progress will come from basic research on endometrial function, receptivity and implantation in ‘in-vitro’ models of embryo–endometrial interaction.
When should IVF be offered? Conventional IVF is performed to achieve pregnancy for patients with tubal disease, anovulation, unexplained infertility, advanced age and previous failed treatment using other methods. IVF should generally be regarded as a ‘last resort’ if other simpler and cheaper approaches are available (Table 1). It is amenable to almost all causes of infertility; apart from situations in which gametes cannot be collected, such as after premature menopause or in azoospermia without sperm in testicular biopsy material. Increasing attention is being paid to the concept of ‘preparation for IVF’ including pre-IVF surgery for endometriosis or hydrosalpinx, collection and cryopreservation of epididymal or testicular sperm or prolonged pituitary downregulation in severe endometriosis.
Pregnancy rates It was recognised early in the development of IVF that the most meaningful statistic for couples is the live-birth rate per cycle started – their chance of having a live baby (or babies) if they decide to begin treatment with a centre. This statistic is published for each licensed IVF centre on a yearly basis by the HFEA, although the data is inevitably out of date by the time of publication. The success rate ‘league table’ also mitigates against commercial clinics promoting single rather than multiple embryo transfer in order to maintain a higher position. In general, a good quality IVF centre should achieve a live birth rate per cycle started of over 30% for couples in which the woman is under 36 years of age. The most powerful predictor of success in IVF is female age (v). The background health of the population
ICSI ICSI is a micromanipulation technique for in-vitro insemination, aimed at improving the fertilisation rate in couples with
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its treatment place significant psychological pressures on some couples, which can lead to the breakdown of relationships and clinical depression. From its inception, the UK HFEA has emphasised the importance of providing counselling and support for couples undergoing IVF treatment.
Alternatives to in-vitro fertilisation Cause of infertility
Alternative approach Justification
Male factor (mild/ moderate) Male factor (severe)
Intrauterine insemination Donor insemination
Anovulation (PCOS)
Clomiphene, metformin ovarian diathermy, FSH injection Dopamine agonist
Cheaper, more acceptable Cheaper, more acceptable More specific, easier, cheaper
Safety The possible effects of IVF treatment on the resultant child has been a focus of attention since the earliest days of IVF. The UK Medical Research Council sponsored the earliest significant follow-up study of IVF offspring in the 1980s, and its findings have been largely confirmed by subsequent work. The major threat to the health of IVF children is multiparity. Compared with singleton pregnancy, twins are four times and triplets eight times more likely to suffer perinatal death, with a concomitant increased risk of long-term handicap. This is the direct result of extremely premature birth rather than being an effect of fertilisation in vitro. The practice of transferring two or more embryos during IVF treatment led to a 20-fold increased risk of twins and a 400-fold increased risk of higher order multiples. An expert group report on multiple births in IVF, commissioned by the HFEA, ‘One child at a time: reducing multiple births after IVF’ was published in October 2006. This report called for success after assisted reproductive technology to be redefined to promote treatment that results in ‘full term singletons with a normal birth weight’. It outlined the rationale for elective single embryo transfer (eSET) for good prognosis IVF patients. eSET plus subsequent frozen embryo replacement is as effective as double embryo transfer in carefully selected patients. However, patient education is essential to maximise acceptance of an eSET strategy. Children born following IVF treatment resulting in a single pregnancy might have a slightly low birth weight but there do not appear to be other significant differences in the duration of pregnancy, mean birth weight, or rates of congenital anomalies and perinatal mortality between singletons conceived naturally or by IVF. The possibility of an increased incidence of genetic, chromosomal and major congenital malformations after ICSI has also been explored in a number of cohort studies. These suggest that ICSI does not increase the risks significantly. There also do not appear to be significant differences in the developmental and psychological scores of children born from ICSI compared with those conceived naturally or by IVF. There does, however, appear to be an increase in incidence of imprinting disorders in offspring conceived by ICSI, although these cases are encountered rarely in clinical practice. Genomic imprinting is the mechanism that determines the expression or repression of genes from maternal or paternal chromosomes. ICSI has been associated with imprinting disorders whereby genes from either of the parents express themselves erroneously. There is evidence that several syndromes are caused by imprinting disorders, such as Prader–Willi, Angelman’s and Beckwith–Wiedemann syndrome. OHSS is an iatrogenic and potentially life-threatening complication of superovulation. The severe form of the condition occurs in 0.5–2% of IVF cycles. The severe form of OHSS is a condition of massive ovarian enlargement with ascites and pleural effusions and haemoconcentration; in the critical form, it may be complicated by arterial and venous thromboembolism, respiratory distress syndrome and myocardial infarction. Clinics
↓ multiple pregnancy rate, cheaper Tubal disease (mild) Laparoscopic surgery Better pregnancy rates ↓ multiple pregnancy rates Endometriosis Laparoscopic surgery ↓ multiple (mild/moderate) pregnancy rates Unexplained infertility Intrauterine Cheaper, more insemination acceptable Anovulation (↑ prolactin)
PCOS, polycystic ovary syndrome; FSH, follicle-stimulating hormone.
Table 1
(percentage of smokers, prevalence of obesity, poor diet, occupational hazards) also has a significant impact on IVF outcome. Other statistics that are commonly quoted include the implantation rate (i.e. total number of gestation sacs divided by the total number of embryos transferred) and the clinical pregnancy rate (i.e. the presence of foetal heart activity on early transvaginal ultrasound per embryo transfer procedure). These measures are useful for auditing laboratory and clinical practice but can be misleading when presented to the public. The chances of a live birth following a single cycle of IVF treatment in the UK have gradually improved over the past decade, and the most recent data show a live birth rate of approximately 28% per cycle for women up to 35 years of age, falling to less than 5% per cycle for women aged 43 years and over (HFEA, 2005). Infertility and
Effect of female age on IVF success rates
Treatment cycles started (IVF and ICSI) Egg collection Embryo transfer IVF only (per ET) ICSI only (per ET) FER (per ET)
<38 years (%)
All ages (%)
22.1
19.5
23.7 25.6 25.6 25.6 14.3
21.1 22.9 22.7 23.1 13.4
IVF, in-vitro fertilisation; ICSI, intracytoplasmic sperm injection; ET, embryo transfer; FER frozen embryo replacement.
Table 2
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that provide ovarian stimulation should have protocols in place for the prevention, diagnosis and management of OHSS.
IVF for the non-infertile Pre-implantation genetic diagnosis IVF gives access to blastomere biopsy from a day 3 embryo for molecular analysis using the polymerase chain reaction. Preimplantation genetic diagnosis may be used to avoid the transmission of autosomal recessive genetic defects, such as cystic fibrosis and thalassaemia; autosomal dominant conditions including Huntington’s disease and myotonic dystrophy; and X-linked disorders such as haemophilia and fragile-X syndrome. Multiple attempts may, however, be needed before a pregnancy is established successfully, increasing the costs and complexity of treatment.
The HFEA The HFEA was established by an Act of Parliament in 1990, following the Warnock Report into IVF practice in the UK. The Act provides a legal framework within which IVF clinics are required to practice; for example, with regard to number of embryos transferred per IVF cycle. The HFEA organises the licensing and annual inspection of IVF units and, by law, receives detailed data on every IVF cycle attempted in the UK. This database has provided a useful picture of IVF practice over the years, revealing a gradual improvement in the national mean pregnancy rate from IVF, the rapid increase in ICSI treatment cycles from 1995 and the huge impact of advancing female age on IVF pregnancy rates. Since its inception, the HFEA has been heavily criticised by some IVF practitioners, and it has struggled to keep pace with the rapid technological advances in the field. However, many countries have produced similar institutions and those that have not are generally regarded as being on the fringes of ethical practice in IVF. Society continues to be both fascinated by and concerned with developments in this area and the existence of the Act of Parliament and the HFEA has protected IVF practice from undue criticism as much as it has restricted development. HFEA guidance underpins an ethical framework for IVF practice, which is highly developed in the UK but which is continually challenged by new circumstances. Views clearly change with successive generations – IVF itself was seen as unnatural and bizarre two decades ago, but is now widely accepted as a satisfactory solution to infertility. In microcosm, each IVF centre is required to have an ethics committee to scrutinise requests for treatment, which may be controversial and great attention is paid to the welfare of the potential child before treatment is offered. Such mechanisms are imperfect, and cannot operate within a clearly defined envelope of ‘ethical’ criteria, given the diversity of situations amongst presenting couples. In the past, HFEA has provided guidance, for example on the lack of evidence of safety of spermatid ICSI and the inadvisability of IVF sex selection without clear medical justification but this is currently under review.
Pre-implantation aneuploidy screening Aneuploidy is an abnormality in the number of chromosomes in an individual’s cells. Certain chromosomes (13, 15, 16, 18, 21 and 22) are particularly prone to aneuploidy. Women over 35 years old undergoing IVF have a high number of aneuploid embryos, particularly with aneuploidies of chromosomes X, Y, 13, 14, 15, 16, 18, 21 and 22. This phenomenon has been associated with recurrent implantation failures at IVF. The pre-implantation testing of blastomeres using multiplex fluorescent in-situ hybridisation before selecting normal blastocysts for transfer has not been shown to improve live birth rates in older patients when tested in randomised controlled trials, possibly due to the small number of oocytes available in this group and the presence of abnormalities in nucleus or cytoplasmic organelles that are not detectable with this technology. Pre-implantation tissue typing (‘saviour sibling’) This technique involves the human leukocyte antigen typing of IVF embryos followed by the transfer of embryos that are human leukocyte antigen-matched to a seriously ill sibling. Following the birth of the baby, umbilical cord or bone marrow stem cells are collected and stored for later transplantation to the sibling.
IVF as a source of human embryonic stem cells Stem cells are unspecialised resting progenitor cells that have the capacity to reproduce indefinitely and the ability to differentiate into various specialised cell types. The best source of naturally occurring, apparently totipotent human stem cells is the early embryo, which has the capacity to develop into any of the cells required for full human development, including the extra-embryonic tissues that form the umbilical cord and placenta. Stem cell research promises many improvements in the prevention and treatment of many diseases. Although the ethical justification for the use of ‘spare’ human embryos for stem cell derivation remains controversial, the procedure has been legalised by UK Parliament, and rapid progress has been made in the derivation, culture and storage of human embryonic stem cells.
Who pays? Less than 25% of the IVF treatments carried out in the UK are paid for by the NHS. Rightly or wrongly, most couples wishing this treatment have to pay from earned income, which creates a unique model of healthcare provision in Britain. Some IVF centres operate as entirely private institutions, are run for profit and are answerable to shareholders. Others operate within a university or NHS environment, either at cost or with a ‘research levy’ added to treatment costs. The cost of IVF in Britain remains lower than in the US, but a single cycle of treatment frequently costs £3000 or more, with added costs for ICSI, embryo cryopreservation, etc. The newly developed concept of clinical governance may have wide-reaching effects on the regulation of IVF practice, with acceptance of continuing responsibility for clinical care and, possibly, a requirement for the private sector to pay the true cost of medical care for its patients when admitted to NHS hospitals with treatment complications.
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Oocyte cryopreservation Technological advances in cryobiology involving rapid freezing of cells by vitrification have been successfully applied to human oocyte cryopreservation. The human oocyte is a large cell that is prone to damage by intracellular ice formation in conventional slow freezing. Vitrification has improved the chances of a human 305
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oocyte surviving the freeze-thaw process, and several hundred live births from frozen oocytes have now been reported worldwide. Oocyte cryopreservation is useful for young women who are to have gonadotoxic chemotherapy or radiotherapy. Those in a stable relationship may prefer the greater security of embryo cryopreservation, but this is only an option for women with long-term partners. Oocyte cryopreservation allows ‘unattached’ patients to store fertility potential until they have been successfully treated and later meet the right partner. More controversially, oocyte cryopreservation is being advocated by some as an option for healthy women who plan to put-off starting their family until an age when natural fertility is on the decline. The need for the healthy person to undergo superovulation and transvaginal oocyte collection imparts small but significant risks, and the small number of live births recorded makes it impossible to give a guarantee of success later, at which point natural conception may not be an option. As usual, media ‘hype’ exceeds reality, and recent reports describing the ‘success’ of oocyte vitrification describe only survival of eggs after thawing rather than high rates of live birth.
Further reading Edwards RG. The bumpy road to human in vitro fertilisation. Nat Med 2001; 7: 1091–1094. Fauser BC, Devroey P, Yen SS, et al. Minimal ovarian stimulation for IVF, appraisal for potential for benefits and drawbacks. Hum Reprod 1999; 14: 2681–2686. Felberbaum RE, Ludwig M, Diedrich K. Clinical application of GnRH –antagonists. Mol Cell Endocrinol 2000; 166: 9–14. Gianaroli L, Magli MC, Ferraretti AP, Munne S. Preimplantation diagnosis for aneuploidies in patients undergoing in vitro fertilization with a poor prognosis: identification of the categories for which it should be proposed. Fertil Steril 1999; 72: 837–844. Grondahl C. Oocyte maturation. Basic and clinical aspects of in vitro maturation (IVM) with special emphasis of the role of FF-MAS. Dan Med Bull 2008; 55: 1–16. Human Fertilisation and Embryology Authority. Code of practice, 6th ed. London: HFEA, 2004. Human Fertilisation and Embryology Authority. Guide to infertility and directory of clinics, 2005–6. London: HFEA, 2005. Mathur R, Jenkins J. Ovarian hyperstimulation syndrome: an endocrinopathy? Curr Opin Obstet Gynecol 2001; 13: 329–333. National Institute for Clinical Excellence. Guidelines on management of infertility. London: NICE, 2004. Ozturk O, Templeton A. In vitro fertilisation and risk of multiple pregnancies. Lancet 2000; 359: 232. Ponjaert-Kristoffersen I, Tjus T, Nekkebroeck J, et al. Collaborative study of Brussels, Goteborg and New York. Psychological follow-up study of 5-year-old ICSI children. Hum Reprod 2004; 19: 2791–2797. Pritts EA, Atwood AK. Luteal phase support in infertility treatment: a meta-analysis of the randomized trials. Hum Reprod 2002; 17: 2287–2299. Reya T, Morrison S, Clarke M, Weissman I. Stem cells, cancer and cancer stem cells. Nature 2001; 414: 105–111. Salhar O, Balen AH. New concepts in superovulation strategies for assisted conception treatments. Curr Opin Obstet Gynecol 2000; 12: 201–206. Sallam HN, Sadek SS. Ultrasound-guided embryo transfer: a metaanalysis of randomized controlled trials. Fertil Steril 2003; 80: 1042–1046. Wolner-Hanssen P, Rydhstroem H. Cost-effectiveness analysis of in-vitro fertilisation: estimated costs per successful pregnancy after transfer of one or two embryos. Hum Reprod 1998; 13: 88–94.
In-vitro maturation The technique of in-vitro maturation (IVM) involves collecting immature oocytes by transvaginal needle aspiration after minimal or no ovarian stimulation. The oocytes are then matured in culture in the IVF laboratory, fertilised by ICSI and resultant embryos are replaced. IVM significantly reduces risk of OHSS and hence may be used for women with severe polycystic ovary syndrome who are at high risk of OHSS. Pregnancy and live birth rates are currently lower than after conventional IVF and further research is needed before the technique is widely applied.
Conclusion Infertility treatment has seen many changes since the birth of Louise Brown. The experience of IVF is much less challenging to patients and is no longer a mysterious journey into the unknown. The process has become much simpler, safer and effective over the past 30 years. In the future, the whole concept of a ‘treatment cycle’ may change – monthly replacement of a single frozenthawed embryo from a pool of six or seven embryos may come to be the ‘norm’ in IVF practice, avoiding risk of multiple pregnancy and creating a more accurate facsimile of natural conception. As society’s knowledge of assisted conception, genetics and cell biology becomes more sophisticated, we can only hope that a ‘regulated yet permissive’ environment for research and treatment continues. Ethical and legal constraints must continue to develop in parallel with new science, and simplistic methods of communication – such as the HFEA ‘league table’ – may need to be superseded if they begin to do more harm than good.
Practice points • IVF is a commonly used treatment for many forms of infertility • Female age is the most important determinant of outcome • Complications, although rare, can be life-threatening • Multiple pregnancy is the major cause of poor obstetric outcome in IVF pregnancies • NHS funding for assisted conception treatment in the UK is extremely limited
Useful websites • Human Fertilisation and Embryology Authority: http://www. hfea.gov.uk • One at a time: http://www.oneatatime.org.uk • Infertility Network UK: http://www.infertilitynetworkuk. com ◆ OBSTETRICS, GYNAECOLOGY AND REPRODUCTIVE MEDICINE 18:11
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essential as Steptoe was able to collect the oocytes by transabdominal needle aspiration of mature follicles under laparoscopic view and Edwards had the necessary laboratory skills to achieve the growth of embryos after fertilisation in vitro. The importance of collaboration between disciplines remains as true today as it was in the 1970s. The birth of Louise Brown was soon followed by the birth of further in-vitro fertilisation (IVF) babies in Australia and the United States (US). At this time, there was considerable disquiet among both the public and the medical profession concerning the morality, and even legality, of the process of IVF. This debate led to the establishment first of a Voluntary Licensing Authority, and later the Human Fertilisation and Embryology Authority (HFEA), by the UK government. The role and powers of the HFEA continue to change as the umbrella of IVF applications continues to grow; and the organisation has been emulated in many countries over the past two decades.
Carol Coughlan William L Ledger
Abstract Infertility is not an identifiable physical disease yet its psychological impact on affected couples can be severe. One in seven couples in the UK suffers from subfertility. Of these, approximately half will end up undertaking some form of assisted conception treatment. In-vitro fertilisation (IVF) can be viewed both as a test of reproductive potential, allowing detailed assessment of oocytes, oocyte sperm interaction and embryo quality, as well as an effective treatment for most forms of subfertility. The dramatic improvements in pregnancy rates seen with IVF treatment since its inception some 30 years ago have occurred due to close multidisciplinary collaboration and the practical application of scientific advances in embryology and pharmacology. There have been several important landmarks including: the use of drugs for superovulation and pituitary downregulation; the introduction of transvaginal ultrasound for monitoring of follicle growth and oocyte retrieval; developments in embryo culture; oocyte donation; and the introduction of intracytoplasmic sperm injection for the treatment of severe forms of male infertility. In the past decade, we have seen the development of new technologies, including pre-implantation genetic diagnosis, the in-vitro culture of immature oocytes to viability, and the cryopreservation of oocytes, thereby widening the scope of clinical problems that can be addressed by IVF-associated technologies. Despite this progress, the majority of IVF cycles still do not produce a viable pregnancy. The psychological stresses imposed upon couples by assisted conception treatment need to be managed carefully and sympathetically. IVF practice continues to require support from appropriately trained and skilled counsellors.
How IVF works Although ‘human’ IVF did not come of age until 1978, most of the technological steps needed for Louise Brown’s conception had been developed years earlier in veterinary medicine. The principle of IVF is simple: collect mature oocytes (eggs) from the ovaries, fertilise them in cell culture in the laboratory and replace embryo(s) into the uterine cavity at such a time in the cycle to ensure a receptive endometrium. However, each step in this model has been refined by decades of experimentation, leading to the quasi- industrial IVF of today.
Stimulation of multiple follicle development One of Steptoe and Edwards greatest problems was a shortage of oocytes. They were able to harvest only one or two oocytes in the natural cycle, and frequently, by the time that laparoscopy had been arranged, general anaesthesia induced and surgery undertaken, the follicle had ovulated and the oocyte had been lost in the pelvis. Success rates were, therefore, low. In order to improve the chances of having embryos for transfer, the embryologist needed more oocytes and the clinicians needed to be certain that premature ovulation would not occur.
Keywords Gn RH analogues; in-vitro fertilisation (IVF); intracytoplasmic sperm injection(ICSI); pre-implantation genetic diagnosis (PGD)
Gonadotrophins Gonadotrophins synthesised and secreted by the pituitary gland play a central role in regulating the ovarian cycle of follicular recruitment and maturation, oocyte release and corpus luteum development and regression. Exogenous gonadotrophins have been used for many years to induce ovulation in anovulatory patients. Gonadotrophins were first isolated from the urine of pregnant women by Aschheim and Zondeck in 1927. Three years later, Zondeck reported their presence in the urine of menopausal women. However, the first use of gonadotrophins for inducing multiple ovulations utilised extracts of human pituitary tissue collected at post-mortem. Many pituitaries were needed to obtain sufficient extract to treat a single patient. In the 1980s, pituitary derived gonadotrophins were found to have transmitted Creuzfeld–Jakob infection in a small number of cases. It was not until 1947 that human menopausal gonadotrophins were first extracted from urine in pharmacological amounts, creating the possibility of a
Introduction In July 1978, Louise Brown, the world’s first baby to be conceived outside the human body, was born in Britain. The historic event was the result of years of research and collaboration between Robert Edwards, a scientist from Cambridge and Patrick Steptoe, a gynaecologist in Lancashire. This collaboration was
Carol Coughlan MRCOG MRCPI is Clinical Research Fellow at the Department of Obstetrics and Gynaecology, Sheffield Teaching Hospital NHS Foundation Trust, Sheffield, UK William L Ledger bm bch ma dphil frcog is Professor at the Department of Obstetrics and Gynaecology, Sheffield Teaching Hospital NHS Foundation Trust, Sheffield, UK.
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readily available pharmaceutical source for widespread clinical use. More recently, recombinant follicle-stimulating hormone (FSH) and luteinising hormone (LH) have been made available following transfection of the genes for the alpha and beta chains of human FSH or LH into Chinese hamster oocyte cell lines, producing large amounts of glycosylated FSH and LH without the need for collection and extraction from human urine. Treatment with gonadotrophins was initially by intramuscular injection of an impure fraction containing both LH and FSH. In time, purified urinary FSH and later recombinant FSH became available for subcutaneous injection. These injections can be selfadministered and are tolerated well by patients. The current era of fertility therapy focuses on simplifying treatment regimens by minimising clinic attendances, and reducing the pain and stress of treatment, thereby improving compliance. Gonadotrophin injection encourages the development of a cohort of mature follicles in a single stimulation cycle, overriding the physiological processes of follicle selection and dominance. Egg collection could yield anything up to 40 oocytes after 10–14 days of daily injection. In the early days of superovulation, ultrasound had not developed sufficiently for the assessment of follicle growth, and the only means by which clinicians could obtain an approximate measurement of ovarian response to treatment was by the daily measurement of urinary oestrogens, a process that was time-consuming and inaccurate. In addition, the rapid rises in circulating oestrogen levels were likely to trigger a premature LH surge, leading to ovulation before egg collection. Having established a means of obtaining multiple oocytes, the next development was to prevent the premature LH surge.
GnRH analogues with antagonist properties An alternative to GnRH agonists are GnRH antagonists such as Cetrorelix or Ganirelix. GnRH antagonists directly block the GnRH receptor, isolating it from the action of native GnRH, without an initial stimulation, thereby avoiding the ‘flare’ effect. This has a number of potential benefits. Since the flare is avoided, duration of treatment for controlled superovulation may decrease. The total dose of FSH required for treatment may be reduced. Use of GnRH antagonists may reduce the incidence and severity of ovarian hyperstimulation states associated with superovulation. Unless GnRH antagonists are administered for a prolonged period of time, pituitary downregulation does not occur, making them rapidly reversible. The ‘antagonist’ IVF cycle begins with the injection of FSH from day 1 or 2 of the menstrual period, with an addition of GnRH antagonist a few days later to prevent an LH surge. FSH injection is continued until hCG is given to mimic a timed LH surge. The use of GnRH antagonists allows a more rapid IVF treatment cycle without the need for preliminary downregulation, avoiding the troublesome menopausal side-effects of downregulation. Ovarian cyst formation that is seen in up to 10% of ‘long protocol’ cycles due to the ‘flare’ effect of raised FSH on the ovary are avoided by the use of GnRH antagonists.
Monitoring of follicle growth Accurate monitoring of follicle growth during ovarian stimulation is essential both for the correct timing of hCG administration (a follicle contains a mature oocyte when it reaches 17–18 mm diameter) and to avoid ovarian hyperstimulation by discontinuing FSH injection if too many follicles begin to develop. In older women, monitoring can also predict ‘poor response’, failure to develop growing follicles despite FSH injection. Such patients can again be advised to stop treatment or can at least be warned that pregnancy is unlikely if they continue stimulation and go ahead with egg collection. The growth of ovarian follicles can be monitored either directly by visualisation with ultrasound or by measuring their output of oestradiol into the circulation. The widespread availability of transvaginal ultrasound has facilitated follicle monitoring considerably – the procedure is painless and gives accurate assessment of follicle diameters and endometrial thickness and integrity. In practice, both ultrasound and blood tests for oestradiol are used in most centres. In women at high risk of developing ovarian hyperstimulation syndrome (OHSS), the use of both ultrasound scanning and serial oestradiol assays can be used to select patients suitable for prophylactic cycle cancellation or cryopreservation of all the embryos with the avoidance of fresh embryo transfer, thereby avoiding pregnancy and reducing the risk of severe pregnancy-associated OHSS. During the period of superovulation by FSH injection, follicles will grow by 1–2 mm per day, requiring 9–11 days of treatment before hCG administration. Modern ‘low-dose’ FSH regimens rarely produce more than 10 mature follicles – the reduction in number of oocytes is more than compensated for by the reduction in risk of OHSS.
Gonadotrophin-releasing hormone agonists Gonadotrophin-releasing hormone (GnRH) agonists have acquired an important place in IVF treatment. The decapeptide structure of GnRH was described in 1971. It was initially thought that agonists would be used as strong sustained stimulators of gonadotrophin secretion. It soon became evident that administration of these agents, after an initial bolus release of gonadotrophins, suppresses LH and FSH levels by pituitary receptor downregulation and desensitisation. In IVF treatment, this prevents the occurrence of a premature LH peak in response to rising oestradiol levels from the growing follicles, preventing spontaneous ovulation and allowing planned oocyte retrieval. However, since the effect of ovarian quiescence is produced at the pituitary, injections of FSH hormone would still stimulate the ovary to resume folliculogenesis. Hence, from the mid-1980s, it became common-place to begin an IVF treatment cycle with ‘pituitary downregulation’, followed by ovarian stimulation with gonadotrophin injection after downregulation had been confirmed by the measurement of low levels of oestradiol and LH in the circulation, and endometrial shedding confirmed by the ultrasound demonstration of a thin endometrium. This so-called ‘long protocol’ was followed by a number of variations on the theme. All share the same application of GnRH agonist to avoid a premature LH surge. In each protocol, a timed LH surge is created by a single injection of human chorionic gonadotrophin (hCG). This is essential to induce the oocyte to re-enter meiosis and extrude the first polar body and to begin the process of luteinisation of the follicles, which prepares the endometrium for implantation.
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Egg collection The early days of IVF used laparoscopy for egg collection. Although the quality of the laparoscopic image improved dramatically with 301
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higher than would be expected following spontaneous conception. Compared with singletons, twins have a seven-fold increase in neonatal mortality and six-fold increase in the risk of cerebral palsy. Maternal complications include both morbidity and mortality. These complications result in increased costs to the health service. As a result, multiple pregnancies are now considered the single most important risk of IVF treatment. In March 2004, the UK HFEA advocated a universal maximum of two embryos per transfer, regardless of the procedure used. In addition, the National Institute for Clinical Excellence (NICE) in the UK guideline ‘Fertility: assessment and treatment for people with fertility problems’, published in February 2004, provides recommendations for good practice. The guideline includes the recommendation that ‘no more than two embryos should be transferred during any one cycle of IVF treatment. It is now recognised that the only effective method to reduce the multiple pregnancy rate is to adopt a policy of single embryo transfer. An expert panel commissioned by the HFEA in 2005 to review multiple births and assisted conception proposed that IVF clinics should develop criteria for selecting groups of women who should be offered single embryo transfer in an effort to reduce our present multiple pregnancy rate of 24%.
fibreoptic technology, better light sources and cameras, egg collection still required general anaesthesia and carried surgical risk. Laparoscopic egg collection was also time-consuming and costly. Improvements in the quality of transvaginal ultrasound allowed the safe transvaginal ultrasound-guided needle puncture of ovarian follicles and cysts from the early 1990s onwards. This could be done with sedation and local anaesthesia and was both safer for patients and more convenient for units. Laparoscopic egg collection was quickly superseded, except for rare cases in which endometriosis, adhesions, or prior infection made the ovaries inaccessible by the transvaginal route. Egg collection is carried out some 34–36 hours after the initiation of oocyte maturation with hCG injection. Delay beyond this interval carries the risk of ovulation, and earlier egg collection may result in a higher number of immature oocytes. The procedure is generally performed as a ‘day case’ with the male partner present. Conscious sedation with analgesia is induced with intravenous opiate (pethidine or fentanyl) and tranquilliser (temazepam or diazepam). Each follicle is punctured in turn under ultrasound control. The follicular fluid is aspirated into a warmed sterile tube and immediately transferred to the adjacent embryology laboratory. The embryologist identifies the oocyte within its envelopment of cumulus cells, removes the oocyte-cumulus mass from the follicular fluid with a pipette and places it into culture medium in an incubator.
Embryo transfer Embryo transfer is performed using a flexible transfer catheter into which the embryos are loaded, suspended in 50 μl of culture medium. Transcervical transfer requires insertion of a Cusco’s speculum, cleansing of the cervix to remove excess mucus and gentle insertion of the transfer catheter to about 1 cm below the fundus. Abdominal ultrasound is increasingly used to monitor catheter placement and may result in improvement in pregnancy rates compared with the clinical touch method. Once the catheter is in place, the fluid containing the embryos is injected into the uterine cavity. The chances of pregnancy are improved by an ‘easy’ embryo transfer. If, for example, it is necessary to apply a tenaculum to the anterior lip of the cervix to allow the catheter to enter the uterine cavity, the chances of implantation fall. Further trauma, particularly if there is bleeding from the endometrium, further jeopardises the outcome. For this reason, many units perform a ‘dummy transfer’ before starting IVF, with recourse to cervical dilatation in theatre if problems are encountered. Rarely, it may be necessary surgically to dilate the cervix before the start of an IVF cycle. A number of outer sheaths, stiffeners or malleable stylettes are available to facilitate transfer in difficult cases, with recourse to a transmyometrial embryo transfer under ultrasound guidance only in cases of insurmountable cervical stenosis. Several studies have shown that trained specialist nurses can perform most embryo transfers with pregnancy rates equivalent to those of medical staff. Embryo transfer is a highly charged moment for the patient and her partner and many patients prefer their familiar nurse to undertake the procedure. Successful transfer demands a gentle and unhurried approach to the procedure.
Sperm, eggs and embryos A semen sample is provided by the male partner on the same day as the egg collection, unless previously frozen sperm are to be used. An azoospermic male may require further evaluation before IVF in order to determine the best method of obtaining spermatozoa for IVF. In non-obstructive azoospermia, the semen should be spun down to a pellet and examined, as not infrequently a sufficient number of live spermatozoa are recoverable for intracytoplasmic sperm injection (ICSI). If azoospermia is associated with a very low volume of ejaculate, retrograde ejaculation must be considered and can be diagnosed on microscopic examination of a post-ejaculatory urine sample. In obstructive and other cases of azoospermia, surgical sperm retrieval may be indicated. Retrieval methods include percutaneous epididymal sperm aspiration, simple percutaneous testicular sperm needle aspiration and percutaneous or open testicular sperm (tissue biopsy) extraction. In some centres, epididymal aspiration is performed microsurgically. The sperm sample is processed in the laboratory to isolate motile sperm from the ejaculate. Each oocyte is individually stripped of the outer cumulus layer by gentle repeated aspiration into a pipette, and is then incubated with an aliquot containing approximately 50,000 sperms or, for ICSI, injected with a single sperm. The oocytes are inspected after 24 hours in culture. If fertilisation has occurred, two pronuclei (male and female) will be visible at this stage. Culture is continued for a further 24–48 hours. Embryos will have divided up to the 4–8 cell stage and can be graded for quality by microscopic assessment of their morphology, shown in Figure 1. The best one or two embryos will generally be selected for transfer. Other ‘spare’ embryos may be cryopreserved in liquid nitrogen for later use. Due to the practice of transferring more than one embryo, the incidence of multiple pregnancies following IVF treatment has risen to over 25%. This is practically 20 times
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Luteal support Pituitary downregulation by GnRH agonists (or receptor blockage by antagonists) produces a prolonged inhibition of pituitary LH 302
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In vitro maturation Immature oocyte
Mature oocyte
In vitro fertilisation
In vitro production
1-cell
2-cell
4-cell
Expanded blastocyst
Early/expanding blastocyst
8-cell
Inner cell mass Blastocell Trophectoderm Morula
Compacting
Figure 1 Embryo development.
severe male factor infertility or those with previous failed fertilisation during IVF attempts. ICSI can be used not only for men with profound oligozoospermia or asthenoteratozoospermia but also for those with obstructive azoospermia, following microsurgical or direct aspiration of sperm from either the epididymis or the testis. It involves the injection of a single spermatozoon directly into the cytoplasm of the oocyte. The spermatozoon is immobilised before ICSI, usually by breaking the tail, as flagellation within the ooplasm is undesirable and only the genetic material of the sperm head is required. ICSI, pioneered by Van Steirteghem and his team in Brussels, has revolutionised the management of male infertility and has provided the possibility of a pregnancy for men who previously would have required their partners to undergo donor insemination. The use of ICSI is increasing worldwide and is readily available to couples based on clinical indication. There is evidence from randomised clinical trials that ICSI is not beneficial for couples with non-male infertility unless there has been a previous failed or poor fertilisation rate (NICE guideline, 2004).
secretion with a possible deleterious effect on the function of the corpus luteum. Embryo transfer is, therefore, always followed by treatment to support the luteal phase. This can be done either by stimulation of the function of the corpora lutea by repeated injection of hCG to mimic LH, or by the direct administration of progesterone or a synthetic progestogen. Progestogens can be administered via the oral or vaginal route or by intramuscular injection. Injection of hCG carries the risk of promoting ovarian hyperstimulation by its action on the corpus luteum, and has not been shown to produce better pregnancy rates than injected progestogen supplementation in meta-analysis of randomised trials. However, the optimum management of luteal support, and the related problem of implantation failure, remains controversial. Progress will come from basic research on endometrial function, receptivity and implantation in ‘in-vitro’ models of embryo–endometrial interaction.
When should IVF be offered? Conventional IVF is performed to achieve pregnancy for patients with tubal disease, anovulation, unexplained infertility, advanced age and previous failed treatment using other methods. IVF should generally be regarded as a ‘last resort’ if other simpler and cheaper approaches are available (Table 1). It is amenable to almost all causes of infertility; apart from situations in which gametes cannot be collected, such as after premature menopause or in azoospermia without sperm in testicular biopsy material. Increasing attention is being paid to the concept of ‘preparation for IVF’ including pre-IVF surgery for endometriosis or hydrosalpinx, collection and cryopreservation of epididymal or testicular sperm or prolonged pituitary downregulation in severe endometriosis.
Pregnancy rates It was recognised early in the development of IVF that the most meaningful statistic for couples is the live-birth rate per cycle started – their chance of having a live baby (or babies) if they decide to begin treatment with a centre. This statistic is published for each licensed IVF centre on a yearly basis by the HFEA, although the data is inevitably out of date by the time of publication. The success rate ‘league table’ also mitigates against commercial clinics promoting single rather than multiple embryo transfer in order to maintain a higher position. In general, a good quality IVF centre should achieve a live birth rate per cycle started of over 30% for couples in which the woman is under 36 years of age. The most powerful predictor of success in IVF is female age (v). The background health of the population
ICSI ICSI is a micromanipulation technique for in-vitro insemination, aimed at improving the fertilisation rate in couples with
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its treatment place significant psychological pressures on some couples, which can lead to the breakdown of relationships and clinical depression. From its inception, the UK HFEA has emphasised the importance of providing counselling and support for couples undergoing IVF treatment.
Alternatives to in-vitro fertilisation Cause of infertility
Alternative approach Justification
Male factor (mild/ moderate) Male factor (severe)
Intrauterine insemination Donor insemination
Anovulation (PCOS)
Clomiphene, metformin ovarian diathermy, FSH injection Dopamine agonist
Cheaper, more acceptable Cheaper, more acceptable More specific, easier, cheaper
Safety The possible effects of IVF treatment on the resultant child has been a focus of attention since the earliest days of IVF. The UK Medical Research Council sponsored the earliest significant follow-up study of IVF offspring in the 1980s, and its findings have been largely confirmed by subsequent work. The major threat to the health of IVF children is multiparity. Compared with singleton pregnancy, twins are four times and triplets eight times more likely to suffer perinatal death, with a concomitant increased risk of long-term handicap. This is the direct result of extremely premature birth rather than being an effect of fertilisation in vitro. The practice of transferring two or more embryos during IVF treatment led to a 20-fold increased risk of twins and a 400-fold increased risk of higher order multiples. An expert group report on multiple births in IVF, commissioned by the HFEA, ‘One child at a time: reducing multiple births after IVF’ was published in October 2006. This report called for success after assisted reproductive technology to be redefined to promote treatment that results in ‘full term singletons with a normal birth weight’. It outlined the rationale for elective single embryo transfer (eSET) for good prognosis IVF patients. eSET plus subsequent frozen embryo replacement is as effective as double embryo transfer in carefully selected patients. However, patient education is essential to maximise acceptance of an eSET strategy. Children born following IVF treatment resulting in a single pregnancy might have a slightly low birth weight but there do not appear to be other significant differences in the duration of pregnancy, mean birth weight, or rates of congenital anomalies and perinatal mortality between singletons conceived naturally or by IVF. The possibility of an increased incidence of genetic, chromosomal and major congenital malformations after ICSI has also been explored in a number of cohort studies. These suggest that ICSI does not increase the risks significantly. There also do not appear to be significant differences in the developmental and psychological scores of children born from ICSI compared with those conceived naturally or by IVF. There does, however, appear to be an increase in incidence of imprinting disorders in offspring conceived by ICSI, although these cases are encountered rarely in clinical practice. Genomic imprinting is the mechanism that determines the expression or repression of genes from maternal or paternal chromosomes. ICSI has been associated with imprinting disorders whereby genes from either of the parents express themselves erroneously. There is evidence that several syndromes are caused by imprinting disorders, such as Prader–Willi, Angelman’s and Beckwith–Wiedemann syndrome. OHSS is an iatrogenic and potentially life-threatening complication of superovulation. The severe form of the condition occurs in 0.5–2% of IVF cycles. The severe form of OHSS is a condition of massive ovarian enlargement with ascites and pleural effusions and haemoconcentration; in the critical form, it may be complicated by arterial and venous thromboembolism, respiratory distress syndrome and myocardial infarction. Clinics
↓ multiple pregnancy rate, cheaper Tubal disease (mild) Laparoscopic surgery Better pregnancy rates ↓ multiple pregnancy rates Endometriosis Laparoscopic surgery ↓ multiple (mild/moderate) pregnancy rates Unexplained infertility Intrauterine Cheaper, more insemination acceptable Anovulation (↑ prolactin)
PCOS, polycystic ovary syndrome; FSH, follicle-stimulating hormone.
Table 1
(percentage of smokers, prevalence of obesity, poor diet, occupational hazards) also has a significant impact on IVF outcome. Other statistics that are commonly quoted include the implantation rate (i.e. total number of gestation sacs divided by the total number of embryos transferred) and the clinical pregnancy rate (i.e. the presence of foetal heart activity on early transvaginal ultrasound per embryo transfer procedure). These measures are useful for auditing laboratory and clinical practice but can be misleading when presented to the public. The chances of a live birth following a single cycle of IVF treatment in the UK have gradually improved over the past decade, and the most recent data show a live birth rate of approximately 28% per cycle for women up to 35 years of age, falling to less than 5% per cycle for women aged 43 years and over (HFEA, 2005). Infertility and
Effect of female age on IVF success rates
Treatment cycles started (IVF and ICSI) Egg collection Embryo transfer IVF only (per ET) ICSI only (per ET) FER (per ET)
<38 years (%)
All ages (%)
22.1
19.5
23.7 25.6 25.6 25.6 14.3
21.1 22.9 22.7 23.1 13.4
IVF, in-vitro fertilisation; ICSI, intracytoplasmic sperm injection; ET, embryo transfer; FER frozen embryo replacement.
Table 2
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that provide ovarian stimulation should have protocols in place for the prevention, diagnosis and management of OHSS.
IVF for the non-infertile Pre-implantation genetic diagnosis IVF gives access to blastomere biopsy from a day 3 embryo for molecular analysis using the polymerase chain reaction. Preimplantation genetic diagnosis may be used to avoid the transmission of autosomal recessive genetic defects, such as cystic fibrosis and thalassaemia; autosomal dominant conditions including Huntington’s disease and myotonic dystrophy; and X-linked disorders such as haemophilia and fragile-X syndrome. Multiple attempts may, however, be needed before a pregnancy is established successfully, increasing the costs and complexity of treatment.
The HFEA The HFEA was established by an Act of Parliament in 1990, following the Warnock Report into IVF practice in the UK. The Act provides a legal framework within which IVF clinics are required to practice; for example, with regard to number of embryos transferred per IVF cycle. The HFEA organises the licensing and annual inspection of IVF units and, by law, receives detailed data on every IVF cycle attempted in the UK. This database has provided a useful picture of IVF practice over the years, revealing a gradual improvement in the national mean pregnancy rate from IVF, the rapid increase in ICSI treatment cycles from 1995 and the huge impact of advancing female age on IVF pregnancy rates. Since its inception, the HFEA has been heavily criticised by some IVF practitioners, and it has struggled to keep pace with the rapid technological advances in the field. However, many countries have produced similar institutions and those that have not are generally regarded as being on the fringes of ethical practice in IVF. Society continues to be both fascinated by and concerned with developments in this area and the existence of the Act of Parliament and the HFEA has protected IVF practice from undue criticism as much as it has restricted development. HFEA guidance underpins an ethical framework for IVF practice, which is highly developed in the UK but which is continually challenged by new circumstances. Views clearly change with successive generations – IVF itself was seen as unnatural and bizarre two decades ago, but is now widely accepted as a satisfactory solution to infertility. In microcosm, each IVF centre is required to have an ethics committee to scrutinise requests for treatment, which may be controversial and great attention is paid to the welfare of the potential child before treatment is offered. Such mechanisms are imperfect, and cannot operate within a clearly defined envelope of ‘ethical’ criteria, given the diversity of situations amongst presenting couples. In the past, HFEA has provided guidance, for example on the lack of evidence of safety of spermatid ICSI and the inadvisability of IVF sex selection without clear medical justification but this is currently under review.
Pre-implantation aneuploidy screening Aneuploidy is an abnormality in the number of chromosomes in an individual’s cells. Certain chromosomes (13, 15, 16, 18, 21 and 22) are particularly prone to aneuploidy. Women over 35 years old undergoing IVF have a high number of aneuploid embryos, particularly with aneuploidies of chromosomes X, Y, 13, 14, 15, 16, 18, 21 and 22. This phenomenon has been associated with recurrent implantation failures at IVF. The pre-implantation testing of blastomeres using multiplex fluorescent in-situ hybridisation before selecting normal blastocysts for transfer has not been shown to improve live birth rates in older patients when tested in randomised controlled trials, possibly due to the small number of oocytes available in this group and the presence of abnormalities in nucleus or cytoplasmic organelles that are not detectable with this technology. Pre-implantation tissue typing (‘saviour sibling’) This technique involves the human leukocyte antigen typing of IVF embryos followed by the transfer of embryos that are human leukocyte antigen-matched to a seriously ill sibling. Following the birth of the baby, umbilical cord or bone marrow stem cells are collected and stored for later transplantation to the sibling.
IVF as a source of human embryonic stem cells Stem cells are unspecialised resting progenitor cells that have the capacity to reproduce indefinitely and the ability to differentiate into various specialised cell types. The best source of naturally occurring, apparently totipotent human stem cells is the early embryo, which has the capacity to develop into any of the cells required for full human development, including the extra-embryonic tissues that form the umbilical cord and placenta. Stem cell research promises many improvements in the prevention and treatment of many diseases. Although the ethical justification for the use of ‘spare’ human embryos for stem cell derivation remains controversial, the procedure has been legalised by UK Parliament, and rapid progress has been made in the derivation, culture and storage of human embryonic stem cells.
Who pays? Less than 25% of the IVF treatments carried out in the UK are paid for by the NHS. Rightly or wrongly, most couples wishing this treatment have to pay from earned income, which creates a unique model of healthcare provision in Britain. Some IVF centres operate as entirely private institutions, are run for profit and are answerable to shareholders. Others operate within a university or NHS environment, either at cost or with a ‘research levy’ added to treatment costs. The cost of IVF in Britain remains lower than in the US, but a single cycle of treatment frequently costs £3000 or more, with added costs for ICSI, embryo cryopreservation, etc. The newly developed concept of clinical governance may have wide-reaching effects on the regulation of IVF practice, with acceptance of continuing responsibility for clinical care and, possibly, a requirement for the private sector to pay the true cost of medical care for its patients when admitted to NHS hospitals with treatment complications.
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Oocyte cryopreservation Technological advances in cryobiology involving rapid freezing of cells by vitrification have been successfully applied to human oocyte cryopreservation. The human oocyte is a large cell that is prone to damage by intracellular ice formation in conventional slow freezing. Vitrification has improved the chances of a human 305
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Review
oocyte surviving the freeze-thaw process, and several hundred live births from frozen oocytes have now been reported worldwide. Oocyte cryopreservation is useful for young women who are to have gonadotoxic chemotherapy or radiotherapy. Those in a stable relationship may prefer the greater security of embryo cryopreservation, but this is only an option for women with long-term partners. Oocyte cryopreservation allows ‘unattached’ patients to store fertility potential until they have been successfully treated and later meet the right partner. More controversially, oocyte cryopreservation is being advocated by some as an option for healthy women who plan to put-off starting their family until an age when natural fertility is on the decline. The need for the healthy person to undergo superovulation and transvaginal oocyte collection imparts small but significant risks, and the small number of live births recorded makes it impossible to give a guarantee of success later, at which point natural conception may not be an option. As usual, media ‘hype’ exceeds reality, and recent reports describing the ‘success’ of oocyte vitrification describe only survival of eggs after thawing rather than high rates of live birth.
Further reading Edwards RG. The bumpy road to human in vitro fertilisation. Nat Med 2001; 7: 1091–1094. Fauser BC, Devroey P, Yen SS, et al. Minimal ovarian stimulation for IVF, appraisal for potential for benefits and drawbacks. Hum Reprod 1999; 14: 2681–2686. Felberbaum RE, Ludwig M, Diedrich K. Clinical application of GnRH –antagonists. Mol Cell Endocrinol 2000; 166: 9–14. Gianaroli L, Magli MC, Ferraretti AP, Munne S. Preimplantation diagnosis for aneuploidies in patients undergoing in vitro fertilization with a poor prognosis: identification of the categories for which it should be proposed. Fertil Steril 1999; 72: 837–844. Grondahl C. Oocyte maturation. Basic and clinical aspects of in vitro maturation (IVM) with special emphasis of the role of FF-MAS. Dan Med Bull 2008; 55: 1–16. Human Fertilisation and Embryology Authority. Code of practice, 6th ed. London: HFEA, 2004. Human Fertilisation and Embryology Authority. Guide to infertility and directory of clinics, 2005–6. London: HFEA, 2005. Mathur R, Jenkins J. Ovarian hyperstimulation syndrome: an endocrinopathy? Curr Opin Obstet Gynecol 2001; 13: 329–333. National Institute for Clinical Excellence. Guidelines on management of infertility. London: NICE, 2004. Ozturk O, Templeton A. In vitro fertilisation and risk of multiple pregnancies. Lancet 2000; 359: 232. Ponjaert-Kristoffersen I, Tjus T, Nekkebroeck J, et al. Collaborative study of Brussels, Goteborg and New York. Psychological follow-up study of 5-year-old ICSI children. Hum Reprod 2004; 19: 2791–2797. Pritts EA, Atwood AK. Luteal phase support in infertility treatment: a meta-analysis of the randomized trials. Hum Reprod 2002; 17: 2287–2299. Reya T, Morrison S, Clarke M, Weissman I. Stem cells, cancer and cancer stem cells. Nature 2001; 414: 105–111. Salhar O, Balen AH. New concepts in superovulation strategies for assisted conception treatments. Curr Opin Obstet Gynecol 2000; 12: 201–206. Sallam HN, Sadek SS. Ultrasound-guided embryo transfer: a metaanalysis of randomized controlled trials. Fertil Steril 2003; 80: 1042–1046. Wolner-Hanssen P, Rydhstroem H. Cost-effectiveness analysis of in-vitro fertilisation: estimated costs per successful pregnancy after transfer of one or two embryos. Hum Reprod 1998; 13: 88–94.
In-vitro maturation The technique of in-vitro maturation (IVM) involves collecting immature oocytes by transvaginal needle aspiration after minimal or no ovarian stimulation. The oocytes are then matured in culture in the IVF laboratory, fertilised by ICSI and resultant embryos are replaced. IVM significantly reduces risk of OHSS and hence may be used for women with severe polycystic ovary syndrome who are at high risk of OHSS. Pregnancy and live birth rates are currently lower than after conventional IVF and further research is needed before the technique is widely applied.
Conclusion Infertility treatment has seen many changes since the birth of Louise Brown. The experience of IVF is much less challenging to patients and is no longer a mysterious journey into the unknown. The process has become much simpler, safer and effective over the past 30 years. In the future, the whole concept of a ‘treatment cycle’ may change – monthly replacement of a single frozenthawed embryo from a pool of six or seven embryos may come to be the ‘norm’ in IVF practice, avoiding risk of multiple pregnancy and creating a more accurate facsimile of natural conception. As society’s knowledge of assisted conception, genetics and cell biology becomes more sophisticated, we can only hope that a ‘regulated yet permissive’ environment for research and treatment continues. Ethical and legal constraints must continue to develop in parallel with new science, and simplistic methods of communication – such as the HFEA ‘league table’ – may need to be superseded if they begin to do more harm than good.
Practice points • IVF is a commonly used treatment for many forms of infertility • Female age is the most important determinant of outcome • Complications, although rare, can be life-threatening • Multiple pregnancy is the major cause of poor obstetric outcome in IVF pregnancies • NHS funding for assisted conception treatment in the UK is extremely limited
Useful websites • Human Fertilisation and Embryology Authority: http://www. hfea.gov.uk • One at a time: http://www.oneatatime.org.uk • Infertility Network UK: http://www.infertilitynetworkuk. com ◆ OBSTETRICS, GYNAECOLOGY AND REPRODUCTIVE MEDICINE 18:11
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