Assisted conception. II--in vitro fertilisation and intracytoplasmic sperm injection

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. 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. | • Severe tubal damage | | • Bilateral salpingectomy | | • Endometriosis | | • Mild male infertility | | • Idiopathic infertility | | • Immunologic infertility | 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 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. Insemination Various systems are used for successful IVF and culture including test tubes, Petri dishes, multiwell dishes, and central well organ culture dishes. 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 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. 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. 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. 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). 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. | Ejaculated sperm | | • Oligozoospermia (<20 × 106/ml) | | • Asthenozoospermia (<30% progressive motility) | | • Teratozoospermia (<15% normal forms—according to strict Kruger criteria) | | • Antisperm antibodies | | • Fertilisation failure after conventional IVF | | • Ejaculatory disorders | | • Preimplantation diagnosis using polymerase chain reaction analysis | | Epididymal sperm or testicular sperm | | • Congenital bilateral absence of vas deferens | | • Obstruction of both ejaculatory ducts | | • Azoospermia | | • Failed vasovasostomy | | • Failed epididymovasostomy | 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. 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 HFEA's guide to IVF clinics is available at www.hfea.gov.uk 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. Competing interests: None declared. The figure showing fertilisation is adapted from Primaton P, Myles DG. Science 2002:296: 5576.. The photographs of human embryos in vitro are courtesy of Dr S Pickering, Guy's Hospital, London. References - • Templeton A, Ashok P, Bhattacharya S, Gazvani R, Hamilton M, Macmillan S, et al. Management of infertility for the MRCOG and beyond. London: RCOG Press, 2000 - • Winston R, Hardy K. Are we ignoring potential dangers of in vitro fertilization and related treatments? Nat Cell Biol 2002:4; 14-8 [DOI] [PubMed] [Google Scholar] - • Ombelet W, Menkveld R, Kruger TF, Steeno O. Sperm morphology assessment: historical review in relation to fertility. Hum Reprod Update 1995;1: 543-57 [DOI] [PubMed] [Google Scholar] - • Rowe PJ, Comhaire FH, Hargreave TB, Mellows HJ. WHO manual for the standardised investigation and diagnosis of the infertile couple. Cambridge: Cambridge University Press, 1999