Assisted conception. II—In vitro fertilisation and intracytoplasmic sperm injection

Introduction

In vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI) are two of the main types of assisted conception that take place in the laboratory. This article covers these two techniques in detail and looks at their safety and success.

Fertilisation: (left) each egg is surrounded by a complex of cumulus cells (purple) that the sperm need to disperse to reach the zona pellucida, the protective outer coating of the egg; (middle) capacitated sperm first bind to the zona pellucida (1) and release enzymes from the acrosome (2), which digest a pathway through the zona pellucida (3); (right) the sperm is able to fuse with the plasma membrane of the egg and becomes incorporated within the egg

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In vitro fertilisation

In IVF, oocytes (obtained surgically from ovarian follicles in superovulated cycles) and prepared sperm are brought together in a dish in the laboratory. Fertilisation takes place outside the body (in vitro = in glass). Cleavage stage embryos derived from these fertilised oocytes are placed in the uterus (embryo transfer) for pregnancy to occur.

The process

Superovulation

Patients receive superovulation treatment with gonadotrophins, usually preceded by pituitary suppression with gonadotrophin releasing hormone analogues (see last week's article). A careful balance is needed to maximise safely the number of oocytes retrieved. Ideally, there should be a choice of embryos for transfer, and some embryos should be available for cryopreservation. However, the risk of ovarian hyperstimulation syndrome also needs to be minimised. Ultrasonography of the ovaries and in some cases monitoring the rise in plasma estradiol concentration are used to check the effect of superovulation. Administration of human chorionic gonadotrophin is scheduled when the leading follicles are≥18 mm in diameter, and given 34-38 hours before planned egg retrieval. About 10% of cycles are cancelled before the planned egg collection because the response to superovulation is excessive and the risk of ovarian hyperstimulation syndrome is substantial, or, more usually, because the response to ovarian stimulation is poor.

Egg retrieval guided by ultrasonography. Patient is under sedation. A tube carrying follicular fluid from the pierced follicle to the collecting vessel can be seen

Egg collection

In the past, eggs were collected laparoscopically under general anaesthesia, but now transvaginal follicle aspiration guided by ultrasonography is the method of choice. It can be performed under intravenous sedation and allows access to ovaries that previously were not visible laparoscopically because of severe pelvic disease and adhesions. Most women are able to leave the clinic a few hours after transvaginal egg collection, and the procedure has minimal analgesic requirements.

Each follicle is aspirated in turn, usually through a single vaginal needle puncture for each ovary. The follicular fluid collected from each follicle is examined immediately under a microscope for the presence of a cumulus mass that may contain an oocyte. Once the oocytes have been collected, they are immediately placed in a culture medium containing the essential nutrients and electrolytes required for fertilisation and maintenance of embryo growth. The culture is then kept at 37°C in an incubator usually gassed with 5% carbon dioxide to maintain the appropriate pH.

Left: Aspiration of an ovarian follicle during egg collection for IVF (arrow shows needle track). Right: mature oocyte retrieved from a follicle

Insemination

Various systems are used for successful IVF and culture including test tubes, Petri dishes, multiwell dishes, and central well organ culture dishes.

Tubes and dishes used in IVF

Each oocyte is inseminated with 50 000-100 000 motile, morphologically normal sperm. Fertilisation can be detected 12-20 hours after insemination by the presence of two pronuclei formed in the cytoplasm of the egg around the maternal and paternal chromatids, and by the presence of two polar bodies in the perivitelline space. Fertilisation rates of over 60% per egg collected should be expected, although complete failure of fertilisation can occur because of previously undetected sperm or oocyte abnormalities.

Around 24 hours after insemination, the pronuclear membranes dissolve, allowing combination of the maternal and paternal chromatids (syngamy), which is followed by the first cleavage division to a two-cell embryo. Further cleavages occur at around 24 hour intervals.

Embryo with two pronuclei on day 1 after IVF

Embryo transfer

Generally, embryos are transferred to the uterus on the second or third day after insemination, by which time they have usually divided into four cells or into six to eight cells respectively. The further on in cleavage that transfer occurs, the more opportunity there is for selection of those embryos that have competence to continue with cleavage both in vitro and in vivo. Although allowing embryos to develop to the blastocyst stage (day 5) may confer this advantage, there are still unsettled concerns about the safety of long term in vitro culture.

The top photographs from left to right show human embryos in vitro at the 2-cell stage (day 1); 4-cell stage (day 2); and 8-cell stage (day 3). The bottom row shows a compacted morula (day 4); a blastocyst (day 5); and a hatching blastocyst (day 6)

Usually two, or occasionally three embryos are transferred together in a tiny drop (< 20 μl) of culture fluid using a variety of soft plastic embryo transfer catheters. Transabdominal ultrasonography can facilitiate the transfer procedure because the full bladder needed for an ultrasound scan tends to reduce anteversion of the uterus. It is also reassuring to patient and clinician to see the fluid containing the embryos placed correctly in the endometrial cavity. The procedure should be painless and the patient may be discharged shortly after transfer. Embryos of good morphological grade in excess of those transferred may be cryopreserved for future use.

Ultrasound picture of uterus during embryo transfer. The bright spot (arrow) is the small drops of fluid in which embryos were placed

Luteal support

Although in natural cycles the ovary produces progesterone after ovulation, there is evidence of premature luteolysis in some superovulatory regimens. Most IVF centres administer progesterone supplementation via vaginal pessaries, suppositories, intramuscular injections, or oral micronised progesterone tablets until menses occur or the woman has a positive pregnancy test. Alternatively, human chorionic gonadotrophin may be given two to three times a week, but it can promote ovarian hyperstimulation syndome in susceptible or heavily stimulated patients.

Intracytoplasmic sperm injection

As semen quality reduces, the proportion of oocytes fertilised by in vitro insemination decreases. In cases of multiple defects of sperm (concentration, motility, and morphology), IVF rates may be severely compromised, increasing the risk of the fertilisation failing. ICSI is a highly specialised variant of IVF treatment, in which fertilisation is achieved by the injection of a single sperm directly into the cytoplasm of the egg.

Micromanipulation equipment required to undertake ICSI procedures. The embryologist guides the needle into the egg using joystick directed servomotors

Only mature eggs (those that have extruded the first polar body and hence are at the second metaphase of meiosis) are suitable for injection with prepared sperm. As ICSI is usually used when sperm quality is extremely poor, each sperm can be examined and selected for normality of its morphology before being picked up individually with a fine glass needle and inserted directly into the cytoplasm of the egg. Sperm do not have to be motile but should show evidence of viability. Sperm suitable for ICSI may be obtained from the ejaculate even when few are present. In azoospermic men sperm can be retrieved surgically from the epididymis (percutaneous epididymal sperm aspiration) or from the testis itself (testicular sperm aspiration or extraction).

Before ICSI, the cumulus cells surrounding each oocyte are removed, allowing the assessment of egg maturity. The removal of the cumulus and corona complexes also allows the precise injection of the oocytes, which is required for successful fertilisation. Fertilisation rates are usually 60-70% per injected oocyte when ejaculated sperm are used, but rates may be lower when epididymal or testicular sperm are used

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Success of IVF and ICSI

Pregnancy outcome or success rates of IVF may be presented per cycle started, per egg collection procedure, or per embryo transfer. A pregnancy may be defined in various ways: a biochemical pregnancy (a transient rise in β human chorionic gonadotrophin concentration), a clinical pregnancy (a gestational sac and fetal heart beat present), or a live birth. The live birth rate per cycle started is sometimes called the “take home baby” rate, and it is the statistic that all clinics are obliged to make available to their patients by the Human Fertilisation and Embryology Authority (HFEA). All clinical pregnancies and their outcomes have to be reported to the HFEA. The major determinant of success in IVF is the age of the woman. A decreasing ovarian reserve and hence the number and quality of oocytes retrieved continues to decline from age 35 and especially as the woman passes 40. Success rates should be viewed according to defined age groups.

In the United Kingdom, the HFEA publishes an annual guide to IVF clinics, in which success rates are presented according to type of treatment, the overall rate, and the rate when the woman is under 38 years. Success rates in terms of live birth per cycle started, per retrieval, and per embryo transfer are also given.

Success with IVF and ICSI declines with age. Data from HFEA's guide to IVF clinics, 2000

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Safety of IVF and ICSI

An important aspect of the introduction of ICSI into clinical practice is the concern of genetic and congenital abnormalities in children born after the transfer of ICSI embryos. This is particularly true when epididymal, testicular, or sperm from men whose infertility may have some form of genetic basis, has been used. Available data show that there is a small but definite increased risk to children born of chromosomal abnormality (1.6%), especially if that abnormality involves sex chromosomes. As some of these abnormalities may be inherited from the parents (and the likelihood increases with decreasing sperm quality), it is advisable that men with sperm counts below 5 million/ml (severe oligozoospermia and azoospermia) are karyotyped before they have ICSI. Of more concern and debate is the malformation rate. It seems that the use of IVF or ICSI may increase the risk of a congenital malformation by 1-2%. It is unclear how much of this increased malformation rate is associated with the substantial multiple pregnancy rate and, hence, prematurity rate. However, it seems that even if this is taken into account, IVF babies are 2.6 times more likely to be underweight than those naturally conceived. Further work and monitoring are needed, but the unease emphasises the need for these techniques to be used with caution and only with appropriate indications.

The ABC of subfertility is edited by Peter Braude, professor and head of department of women's health, Guy's, King's, and St Thomas's School of Medicine, London, and Alison Taylor, consultant in reproductive medicine and director of the Guy's and St Thomas's assisted conception unit. The series will be published as a book in the winter.