In vitro maturation
In vitro maturation is the technique of letting the contents of ovarian follicles and the oocytes inside mature in vitro. It can be offered to women with infertility problems, combined with in vitro fertilization, offering women pregnancy without ovarian stimulation.
History
In 1935, Pincus & Enzmann did the first experiment on immature rabbit oocyte, showing in vitro spontaneous maturation and fertilization. They showed maturation occurs in isolation from normal follicular environment. In 1965 Edwards then continued IVM studies in mouse, sheep, cow, pig, rhesus monkey and human. By 1991, the first human pregnancy was recorded using IVM followed by IVF, and in 1994 the first birth using IVM oocytes from polycystic ovarian syndrome patients was recorded, confirming that their oocytes are capable of maturation.Background
takes place during embryonic life, in which primordial germ cells undergo mitosis until a few weeks prior to birth, forming oogonia. These then begin meiosis to form the oocyte within the primordial follicle. This follicle consists of the oocyte surrounded by flattened pregranulosa cells. Babies are born with 1-2 million primordial follicles, and by puberty have around 300,000. Of these primordial follicles, only around 400 mature oocytes are released and could be potentially fertilised, with the rest undergoing atresia.'Maturation' of an oocyte is the process by which an 'oocyte attains the competence to be fertilised and undergo embryogenesis'.
Folliculogenesis is the mechanism by which the ovarian follicles mature. This can take many months in vivo and involves primordial follicle growth and differentiation.
Primordial follicles containing the primary oocyte, arrested at prophase of meiosis I, develop into primary follicle containing cuboidal granulosa cells. A secondary follicle is formed with a few granulosa cell layers, as well as a theca layer. Finally before ovulation, a tertiary follicle is formed containing a follicular-fluid filled antrum. Of these small antral follicles, 1 will become dominant and ovulate. During ovulation, the primary oocyte will resume meiosis in response to signals, arresting in metaphase meiosis II, ready for fertilization. The dominant follicle contains the mature oocyte. Follicular development is directly under gonadotropins control, LH and FSH. These use cAMP as an intracellular second messenger, with growth factors and cytokines also influencing their development in vivo.
Through in vitro maturation, folliculogenesis and latter parts of oogenesis are being mimicked outside of the ovaries- trying to recreate the conditions for these processes.
Techniques
If a follicle has reached the early tertiary or antral stage, IVM can be carried out.Firstly, the oocytes need to be obtained from the subject. The timing of this is dependent on the stage of the cycle the subject is in, which is usually monitored using ultrasonography. If without the use of priming, oocytes are obtained when the largest follicles are around 10mm in size.
In humans, this can be done with an aspiration needle, using ultrasound to allow accuracy. Depending on whether you are aspirating mature or immature follicles, the protocol differs slightly. In both procedures the aspiration pressure is reduced, but to varying degrees. Additionally, it is more important that the aspirate is filtered when retrieving immature follicles, as the follicles are much smaller and harder to see in the fluid extracted.
Priming is the process by which the oocytes are primed with follicle-stimulating hormone or human chorionic gonadotrophin before retrieval. hCG is important in women with polycystic ovarian syndrome. This results in an expanding or dispersed pattern of the cumulus oophorus around the egg cell, facilitating its identification within follicular fluid. This leads to improved maturation and quality of the oocytes. However, the evidence of a clinical effect of hCG priming is still lacking. When IVM was initially introduced, successful pregnancies were low, leading to the use of ovary priming.
This technique is also used in sheep, pigs and other animals. See In animals.
Oocytes classification
Oocytes are classified depending on their condition, such as number of cumulus cells. The best oocytes are chosen to be matured in the hope of then being implanted using in vitro fertilisation techniques.Cultured in media
The oocytes are then cultured in media containing nutrients important for oocyte survival, such as gonadotrophins, growth factors and steroids. These vary between clinics and research laboratories. McLaughlin et al. biopsied human ovarian tissue and achieved a 10% rate of maturation from unilaminar follicles into metaphase II by a multi-step culture system:- 8 days of culture in a serum-free medium
- 8 days of culture in a serum-free medium with activin A
- 4 days of culture on membranes with activin A and follicle-stimulating hormone.
In vitro fertilisation
A few live births have already been made by taking small early tertiary follicles, letting them mature in vitro and subsequently fertilizing them.
However, for follicles that haven't reached the early tertiary stage, IVM is still under development. There are a lot of cellular changes in the oocyte and the rest of the cells in the follicle, which makes it very susceptible. Nevertheless, it is possible to let a primordial follicle mature to a secondary follicle outside the body by growing it in a slice of ovarian tissue. The subsequent maturity from secondary to early tertiary stage can then be supported in test-tubes. It has been suggested that photoirradiation of granulosa cells and oocytes may facilitate IVM.
Clinical applications
In vitro maturation is an assistive reproductive technique typically used in patients with fertility issues including polycystic ovary syndrome, high antral follicle counts and ovarian hyper-responsiveness. However, more recently IVM has also become widely utilised in areas such as fertility preservation in cancer patient who have undergone treatment involving gonadotoxic therapies. There have been over 1000 live births recorded from mothers using IVM.Polycystic ovary syndrome
PCOS is a common disorder involving dysfunction of the endocrine system associated with female reproduction. PCOS involves discrepancies in the Hyphophyseal-pituitary-gonadal endocrine axis which can result in hormonal dysfunction, excess androgens and frequent anovulatory menstrual cycles. Therefore, it is common for women suffering from PCOS to require assistance in order to conceive. In these patients IVM can be used to mature oocytes and aid conception. Few studies shows that substituting IVM in PCOS patients eliminates the risk of OHSS and lowers the cost of treatment. The same group conducted a retrospective analysis study to compare the treatment outcome of IVM with IVF in patients with PCOS.They have further concluded there was significant increase pregnancy rates, implantation rates and number of embryos transferred in IVM group.Alternative to ovarian hyperstimulation
The use of in vitro maturation in assisted reproduction has advantages over standard ART procedures. In typical IVF practice, controlled ovarian hyperstimulation is performed, which is where supraphysiological levels of gonadotropins are administered to the patient in order to hyperstimulate the antral follicles and hence induce oocyte maturation to metaphase II at a rate that is above normal physiological capabilities. This practice can be disadvantageous in several ways: It is very costly, can become complicated and may also predispose to several undesirable side effects, such as ovarian hyperstimulation syndrome. Ovarian hyperstimulation can cause severe OHSS in up to 2% of cases. OHSS can have serious consequences, including respiratory problems, renal impairment and even stroke. Patients with PCOS and younger women are at an increased risk of OHSS. In these women, it may be even more beneficial to employ IVM rather than conventional IVF treatment.In IVM, immature oocytes are removed from the antral follicle of a woman and then are matured in vitro in a culture rich in gonadotrophins. This hence negates the need for gonadotrophin stimulation.
IVM is not an entirely perfected technique. Pregnancy rates are lower in IVM than in standard IVF. There is also research required into whether or not babies born to mothers who have undergone IVM have any health concerns later in life.
Women with a personal or family history of an oestrogen associated thrombus, or of severe cardiovascular disease, may also benefit from IVM. This is because conventional IVF, with its hyperstimulation of the ovaries, has the potential to stimulate mass synthesis of oestrogen via the stimulation of granulosa cell oestrogen production.