In Vitro Maturation (IVM) in a clinical setting
Introduction
IVM is defined as the primary intention to collect immature oocytes from hormonally unstimulated or minimally primed follicles to achieve a live birth.
The basic concept of IVM is that immature oocytes are collected from small antral follicles before spontaneous ovulation, and the oocytes are then allowed to mature in vitro after which routine in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) and embryo transfer can be performed. The immature eggs are then matured in the laboratory for 24-48 hours using culture medium with added small quantities of hormones. The procedure is based on the observations of Pincus and Enzmann in 1935 and later on by Edwards in 1965 that oocytes undergo spontaneous nuclear maturation when removed from the follicular milieu. IVM has been practiced in a clinical setting for over a decade, but only recently information have been accumulated on the clinical outcomes. The procedure is simple, lack of hormonal stimulation and the risk of ovarian hyperstimulation syndrome (OHSS) theoretically does not exist. IVM may also be less costly as there is no need for the drugs to stimulate the ovaries.
Since Cha et al. (1991) reported about the first pregnancy from in vitro matured oocytes, derived from a caesarean section donor, fertilization, embryo development and pregnancy.
Patient selection
IVM was primarily developed to make IVF safer and simpler for women with polycystic ovaries and high risk of OHSS using conventional stimulation protocols. Recently, the indications for IVM have been extended to other causes of infertility such as avoidance of the side effects resulting from gonadotropin stimulation including the risk of OHSS
• Being less costly and safer
• Simplified treatment
• Alternative to IVF for younger women with normal menstrual cycles.
• Women who have a poor response to stimulation
• Unexpected poor quality embryos
• Previous failed IVF cycles
• Fertility preservation in young cancer, with hormone-sensitive tumors, who are going to receive chemotherapy or radiotherapy
• Reserving fertility capability (Oocytes retrieved can be vitrified, or embryos created can be cryopreserved)
• Male factor infertility when the woman does not require fertility drugs
However, PCOS is the most widely used indication for IVM. It is the antral follicle count rather than the diagnosis of PCOS itself which is an important predictive factor of the number of immature oocytes obtained from an unstimulated cycle and should be used as a main criterion to select patients for IVM. Women who will benefit most from in vitro maturation are women aged 35 and younger and have antral follicle count of 10 or more.
The use of IVM in women with regular cycles and normal ovaries is more controversial. Although IVM has been applied to IVF poor responders it has mostly been used in younger patients with none or a few previous IVF attempts. The most important prognostic criteria for IVM seems to be the number of antral follicles in a baseline ultrasound scan. If the antral follicle count is less than five per ovary, IVM is not recommended. Thus, low ovarian reserve can be considered as a contraindications for IVM. The clinical experience thus far suggests that IVM is a first line treatment rather than the last resort.
Hormonal priming
Although only a single follicle usually grows to the preovulatory stage and releases its oocyte for potential fertilization, many small follicles also develop during the same follicular phase of the menstrual cycle. It seems that approximately 20 antral follicles are selected and continue to grow through to the preovulatory stages of development during each menstrual cycle.
The initial purpose of IVM was to obtain immature oocytes from antral follicles without any prior hormone stimulation. However, as the pregnancy rates initially were low there was a need to try to increase the number of good quality oocytes available for IVM and subsequent IVF-ET. One approach was to use mild ovarian stimulation with gonadotrophins. Theoretically early follicular phase FSH/HMG priming of the follicles could enhance oocyte maturation, increase the oocyte yield and make the collection of the oocytes easier. As a result, various priming protocols have been studied both in women with irregular cycles and PCOS, and regular cycles and normal ovaries. In a study by Mikkelsen et al. they used FSH 150 IU on cycle days 3-5, whereas Lin et al used both FSH (75 IU for 6 days) and HCG priming.
The main reason to use hCG priming in women with PCOS has been the finding that in vivo administered hCG enhanced the nuclear maturation of the oocytes. In non-PCO women with regular cycles, hCG priming has not been shown to have any beneficial effect.
Immature Oocyte Immediately after retrieval Mature Oocyte After 36 hours of Retrieval
During COC sliding, it is possible to observe clearly whether or not the oocyte cytoplasm contains a germinal vesicle
(GV) (A and B) or the oocyte has extruded a first polar body (1PB) into perivitelline space (PVS) (D). If neither GV is
seen in the oocyte cytoplasm nor 1PB found in PVS, then the oocyte is defined as germinal vesicle breakdown
(GVBD) or metaphase-I stage (M-I) (C).
Cycle monitoring and timing of oocyte collection
The management of an IVM cycle depends on whether it is an ovulatory or an anovulatory cycle. Ultrasonography remains the best method to monitor the cycle for the timing of immature oocyte collection. In women with PCOS the cycles are usually anovulatory without dominant follicle development in which case the monitoring of the cycle and the timing of the oocyte pick-up are more flexible than in an ovulatory cycle. The criteria for oocyte pick-up in anovulatory patients is the thickness of the endometrium which should be greater than 6 mm, although successful pregnancies have been reported with an endometrium as thin as 3 mm. Thus, the day of the cycle does not play a role in the timing of the oocyte pick-up in anovulatory cycles of PCOS women allowing more flexibility in scheduling considering the IVF laboratory and the clinic.
Based on the published literature the size of the leading follicle with successful outcome varies between less than 10 mm to 14 mm. When the leading follicle has reached 13 mm in diameter, significantly less oocytes were collected, matured and fertilized and fewer embryos transferred than in cycles with a leading follicle smaller than 13 mm, whereas others have recommended cancellation of the cycle if the leading follicle is larger than 10 mm .
Oocyte collection
The oocyte retrieval is done under ultrasound guidance with a single-lumen aspiration needle. The aspiration pressure is reduced to 7.5 kPa. The follicular fluid is collected in culture tubes containing 0.9% saline with 2 U of heparin. Because immature oocytes are enclosed in tightly packed cumulus cells, curettage of the follicle wall will dislodge the cumulus oocyte complex. The aspirates are usually blood laden and the tightly compacted oocyte complexes are difficult to identify; therefore, filters can be used to increase oocyte yield. As mentioned, multiple needle punctures are needed because lower aspiration pressures are used and bloody aspirates may block the thin needle lumen. The needle is therefore withdrawn from the vagina after aspirating a few follicles to flush and clear any block and the procedure is repeated until all follicles are aspirated.
The immature oocyte collection technique differs from the aspiration of large mature follicles. This new technology needs:
• A lower aspiration pressure
• Immobilisation of the ovary by pressure or special holding needle
• Filtering of the aspirate to recover the immature oocytes
The aspiration pressure for IVM should be lower than that used in the conventional oocyte retrieval. The range for optimal aspiration pressure is probably wide and depends on the equipment and the type of needle used. Immobilisation of the ovary by external pressure may be necessary, because the unstimulated ovary is smaller and potentially more mobile than the stimulated ovary. Alternatively a special double lumen holding needle can be used. As the immature oocytes do not have a large, expanded cumulus cell complex they are more difficult to see in the aspirate which contains more blood than the follicular fluid from large follicles. Therefore, the aspirate is usually filtered after which the immature oocytes can easily be recovered. This means that there is a longer time interval between the oocyte collection and the time the total number of retrieved oocytes is known compared to the oocyte pick-up procedure in conventional IVF. 
Follicular aspirates collected into tubes (10 ml, Falcon)
containing approximately 2-3 ml of heparinized warm (37ºC) flushing medium
In vitro maturation followed by insemination or microinjection
The culture medium for the collected immature oocytes has been supplemented with a range of gonadotropins, growth factors, steroids, serum and proteins from various sources. Many laboratories have used their in-house developed culture medias an co-culture systems and a few commercial IVM culture medias are currently available. The in vitro maturation times used have varied between 28 and 52 hours. The oocytes which mature within 30 hours after retrieval have been shown to be developmentally more potent than the ones maturing later. It is difficult to draw definite conclusions on the optimal maturation time from these studies because of the variations in culture conditions and study endpoints. For practical purposes a maturation time between 30 - 36 hours is mostly used. The quality of the oocytes at the time of collection seems to be the most important singular factor affecting the maturation rate. Oocytes surrounded by intact cumulus cells show better maturation rates than oocytes with scanty or no cumulus cells.
Intracytoplasmic sperm injection (ICSI) has been thought to be necessary for fertilization of in vitro matured oocytes even in conditions where sperm parameters are not impaired. Another reason for using ICSI instead of insemination after IVM is that the assessment of oocyte maturity is more difficult with intact cumulus cells. However, insemination may be a good alternative after IVM when the sperm is suitable for IVF. It can be speculated that the intact cumulus cells somehow enhance cytoplasmic maturation and developmental competence of the oocytes.
Embryo transfer and endometrial preparation
One of the challenges of IVM is to prepare the uterus for implantation in only a few days between the oocyte retrieval and embryo transfer. Because immature oocytes are usually retrieved before the dominant follicle develops, the endometrium is exposed to relatively low levels of estradiol by the time of oocyte pick-up. As a result, there is a dyssynchrony between the phase of the endometrium and the cleavage stage embryo. Therefore, adequate preparation of the endometrium is of crucial importance in an IVM cycle because of the absent LH surge at the time of oocyte collection. The most commonly used protocol for endometrial preparation consists of estradiol valerate 2-4 mg daily from the day of immature oocyte collection and progesterone intravaginal started 48 hours later at the time of microinjection or insemination.. In case of pregnancy, it is important to continue the hormone supplementation long enough as there is no endogenous pituitary or corpus luteum support.
Clinical outcome and follow-up of children
The clinical outcomes of IVM have continued to improve after the modest results of the early studies. The average pregnancy rates in women with PCOS have been reported between 22 and 30%, and in women with normal ovaries between 18 and 30%, Recently a pregnancy rate per embryo transfer as high as 52% and implantation rate of 27% was reported after IVM and blastocyst transfers in highly selected cases.
It has been estimated that approximately 1300 IVM babies have been born worldwide, but the birth and perinatal outcome of only some 400 babies have been reported. Reports on obstetric and perinatal outcome of IVM pregnancies show very few complications. Prematurity occurs in 4-13 % of the pregnancies which is no different from spontaneous pregnancies. This is thought to be associated to fewer multiple pregnancies compared to standard IVF treatments.
