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In Vitro Maturation of Oocytes
INTRODUCTION
Since the first live birth resulting from in vitro fertilization (IVF) was reported 26 years ago , over two million live births have been reported as a result of IVF. IVF success rates have steadily improved over the years and in many leading IVF centers today, the live-birth rate per cycle in women younger than 35 years may approach 50%. Conventional IVF treatment requires that the ovaries be stimulated with gonadotropins, which contain follicle-stimulating hormone (FSH) and luteinizing hormone (LH), in order to increase the number of mature oocytes retrieved, the number of embryos available for transfer, and, consequently, to improve pregnancy rates. Using controlled ovarian stimulation protocols, the success rates of IVF treatment have steadily increased and the results of many leading IVF centers today exceed those of spontaneous conceptions in healthy, fertile couples. However, ovarian stimulation protocols are associated with high costs, daily injections of gonadotropins and close monitoring, and carry a considerable risk of causing ovarian hyperstimulation syndrome (OHSS). Although mild or moderate degrees of OHSS may not be very dangerous, severe OHSS may be associated with significant morbidity. Patients with polycystic ovaries (PCO) or polycystic ovarian syndrome (PCOS) are particularly prone to develop OHSS with an incidence of up to 6%. The most severe manifestation of OHSS involves massive ovarian enlargement and multiple cysts, hemoconcentration, and third-space accumulation of fluid. The syndrome may be complicated by renal failure and oliguria, hypovolemic shock, thromboembolic episodes, and adult respiratory distress syndrome which, in extreme cases, can even be fatal. Despite many years of clinical experience, no precise methods have been developed that will completely prevent severe OHSS after ovarian stimulation and the only certain method is to avoid stimulating the ovaries with exogenous FSH. Some patients may also be deterred by the suggested association between multiple repeated cycles of ovarian stimulation and potential increased incidence of malignant diseases, a worrisome but unproven association. Avoiding ovarian stimulation and collection of immature oocytes would eliminate the risk of OHSS. Indeed, research on immature oocytes and their maturation was conducted as early as the mid-1930s.
OOCYTE MATURATION IN VIVO AND IN VITRO
Follicle Development and Oocyte Maturation In Vivo The development of human oocytes is arrested at the prophase I stage of meiosis during fetal life. At birth, there are approximately one million primordial follicles in the ovaries, each of which consists of an oocyte surrounded by a few flattened pregranulosa cells enclosed by a basement membrane. Although large numbers of follicles can leave the primordial pool and begin to grow, very few will be selected to mature and to ovulate for potential fertilization. Follicles respond to rising levels of gonadotropins by growing and fully maturing, then being released into the fallopian tube by ovulation only after the onset of puberty. During a woman’s reproductive life, only about 400–500 mature oocytes will be released from the ovaries for potential fertilization. The process of follicular development within the ovary is directly influenced by gonadotropins, namely FSH and LH. From the growing cohort of antral follicles, only a portion is able to respond to the rising levels of FSH; consequently, a large number of follicles die at the early antral stage of development. During the early antral stage, the follicle has multilaminar granulosa cell layers and acquires vascularized, distinct layers of thecal cells that are separated from the granulosa cells by the basement membrane. It appears that approximately 20 antral follicles are selected and continued throughthe preovulatory stages of development during each menstrual cycle . During the later antral stage of follicular development, granulosa cells rapidly proliferate and differentiate into two populations, namely the mural granulosa cells that are adjacent to the basement membrane and the cumulus cells that surround the oocyte. Gonadotropins (FSH and LH) are necessary for follicular development in vivo, and both these hormones use the cyclic adenosine monophosphate pathway system as the intracellular second messenger. In addition, there are many other growth factors and cytokines that modulate the actions of gonadotropins; the follicle-enclosed oocyte being, as mentioned previously, arrested at the prophase stage of the first meiotic division. The resumption of the first meiotic division occurs in preovulatory follicles following the preovulatory LH surge. The nuclear membrane dissolves and the chromosomes progress from the metaphase I to the telophase I stage. The dissolution of the nuclear membrane is known as germinal vesicle breakdown. After the first meiotic division, which is characterized by the extrusion of the first polar body, the second meiotic division begins and a secondary metaphase plate (metaphase II) is formed. Therefore, oocyte maturation is defined morphologically as the reinitiation and completion of the first meiotic division from the germinal vesicle stage to the metaphase II stage with accompanying cytoplasmic maturation necessary for oocyte fertilization and early embryonic development. Oocytes that have not reached the metaphase II stage cannot be fertilized and undergo embryo cleavage. Knowledge of meiosis at the molecular level has accumulated rapidly in the last two decades. A major breakthrough was the discovery of a non-specific factor, the maturation-promoting factor, which is responsible for the G2 to the M-phase transition of the cell cycle. Molecular characterization of the maturation-promoting factor has shown that the active form is a protein dimmer composed of catalytic p34cdc serine/threonine kinase and regulatory cyclin B subunits.
In Vitro Maturation Oocytes
Although it is clear that the LH surge triggers the resumption of meiosis in vivo, cumulus–oocyte complexes can be spontaneously induced to resume meiosis when they are released from follicles into culture in vitro. Therefore, the action of endocrine factors affecting oocyte maturation in vitro may be quite different from in vivo conditions. Immature oocytes, with or without surrounding cumulus cells, can be matured to the metaphase II stage; however, the capacity of early embryonic development from the denuded oocytes is questionable. The beneficial effects of cumulus cells on early embryonic development have been reported in many species including humans. The actions of endocrine, paracrine, and autocrine factors that control oocyte maturation in vitro, either directly or indirectly, are mediated by the cumulus cells. Although FSH and LH play an important part in the development and maturation of preantral, antral, and preovulatory follicles in vivo, these gonadotropins may not play the same role in promoting oocyte maturation in vitro. Currently, most in vitro maturation (IVM) protocols supplement FSH or LH in a culture medium for oocyte maturation. However, the effects of FSH or LH on oocyte maturation and subsequent fertilization as well as early embryonic development are still controversial. The idea of supplementing these hormones in a culture medium is based on their physiological role in oocyte maturation in vivo.
The contradictory reports that FSH or LH are major hormones involved in IVM may be related to cross-contamination of FSH with LH or LH with FSH, as each preparation is derived from urinary extracts . Although it has been reported that using a combination of recombinant FSH with recombinant LH in IVM of immature oocytes resulted in significantly higher developmental competence, as evidenced by increased development to the blastocyst stage compared with recombinant FSH alone or no gonadotropins, conclusive results require further study. In addition, recently Hreinsson et al.showed that use of recombinant human chorionic gonadotropin (hCG) or recombinant LH is equally effective in promoting oocyte maturation in vitro, although there was no proper control group in their study to substantiate this conclusion. It was initially considered that FSH and LH probably act to induce oocyte maturation in in vitro conditions through an indirect action mediated by cumulus cells, because it is believed that there are no FSH or LH receptors on the oocytes. However, recent reports indicate that messenger RNA for FSH and LH receptors is present in mouse and human oocytes, zygotes, and preimplantation embryos, indicating a potential role for gonadotropins in the modulation of meiotic resumption and the completion of oocyte maturation. In addition, it has been known that culture medium supplemented with a physiological concentration of FSH or LH stimulates steroid secretions (estradiol and progesterone) from cultured granulosa and cumulus cells . Therefore, it is likely that one of the actions of gonadotropins is mediated by either estradiol or progesterone, which may control oocyte maturation in vitro. A recent report indicated that LH-receptor formation in the cumulus cells surrounding porcine oocytes plays an important role in oocyte cytoplasmic maturation. However, its importance in oocyte maturation in vitro and how this action is linked to other signal transduction (pathways) are still largely unknown.
Estradiol and progesterone are mediators of normal mammalian ovarian function. Inhibition of steroid synthesis in whole cultured follicles impairs the subsequent fertilization and developmental capacity of oocytes in sheep. The presence of estradiol in the culture medium of in vitro matured human oocytes had no effect on the progression of meiosis but improved fertilization and cleavage rates. However, it may not be necessary to add estradiol to the oocyte maturation medium when the oocytes are cultured with cumulus cells because the culture medium supplemented with gonadotropins stimulates estradiol secretion from the granulosa and cumulus cells during culture in vitro . Little information is currently available about how progesterone contained in the culture medium affects oocyte maturation. However, we have found that progesterone has a negative effect on bovine oocyte maturation in vitro, and it is well known that many growth factors are contained in follicular fluid. These growth factors must be secreted from the granulosa and cumulus cells that respond to gonadotropins and subsequently act on the oocyte via paracrine and autocrine pathways. Although a growing number of studies have indicated that growth factors produce beneficial effects on oocyte maturation, it seems that only denuded oocytes require the supplementation of growth factors in the culture medium for proper oocyte maturation. This suggests that the granulosa and cumulus cells can secrete some growth factors during culture and play some functional roles during oocyte maturation in vitro. In practice, the culture medium is also supplemented with the patient’s own serum or human serum albumin as a protein source. Both serum and human serum albumin are a rich source of growth factors. Therefore, it is not necessary to add growth factors to the IVM medium for oocyte maturation in vitro, especially when the IVM medium contains serum or human serum albumin.
IN VITRO MATURATION OF OOCYTES IN
INFERTILITY TREATMENTS
The research into maturation of immature oocytes initiated by Pincus and Enzmann and continued by Edwards et al. was not incorporated as a treatment for human infertility until 1991. Cha et al. reported that human follicular oocytes were harvested from unstimulated ovaries during gynecological surgery, matured in vitro, then fertilized, and five embryos were transferred to a woman with premature ovarian failure. The recipient subsequently delivered healthy triplet girls. Trounson et al. further suggested that immature oocyte recovery could be developed as a new method for the treatment of women with infertility due to PCO because the oocytes of these patients retain their maturational and developmental competence.
However, the initial reported IVM pregnancy rates were low. Our group demonstrated that priming with hCG 36 hours prior to immature oocyte collection significantly improved the maturation rate, and the pregnancy rate exceeded 30%. IVM was initially considered as a treatment for patients with PCOS, but the indications are now expanding to include various other fertility problems.
IVM of Oocytes from Women with PCOS
PCOS is a very heterogeneous syndrome, often first diagnosed when the patient presents complaining of infertility; approximately 75% of these women suffer infertility due to anovulation. The majority of women with anovulation or oligo ovulation due to PCOS have menstrual irregularities, usually oligo- or amenorrhea, associated with clinical and/or biochemical evidence of hyperandrogenism. In almost all these patients, ultrasonic scan of the ovaries typically reveals numerous antral follicles . Fertility treatments for women with PCOS include lifestyle management, administration of insulin-sensitizing agents, laparoscopic ovarian drilling, ovulation induction, ovarian stimulation, and IVF. As previously mentioned, this group of patients has an increased risk of severe OHSS from gonadotropin stimulation compared with women who have normal ovaries. The risk of multiple-follicle ovulation and subsequent multiple pregnancies is also of crucial importance. However, the high number of antral follicles in patients with PCO makes them prime candidates for IVM treatment, even if the appearance of PCO in the scan is not associated with an ovulation disorder. Indeed, the main determinant clinically of success rates of IVM treatment is antral follicle count. When hCG priming is used before oocyte retrieval, it has been found that immature oocytes retrieved from normal ovaries, PCO, or women with PCOS have a similarly high maturation, fertilization, and cleavage potential . However, although the implantation rate was lower, the live-birth rates were not significantly different and, as expected, the OHSS rate was significantly lower in the IVM group. These results suggested that IVM is a promising alternative to conventional IVF treatment for women with PCO or a high antral follicle count who require assisted conception.
IVM for High Responders to Gonadotropin Stimulation
When patients receiving gonadotropins hyper-respond to treatment, there are no precise methods to completely prevent severe OHSS. However, the risk can be reduced by withholding the ovulation-inducing trigger of hCG . Thus, in conventional ovarian stimulation for IVF where there has been an over-response and there is a high chance of developing OHSS, the cycle would be cancelled. Immature oocyte retrieval followed by IVM and IVF may provide an alternative to cancellation of these cycles. Initially, one live birth was reported from immature oocytes collected from a patient at substantial risk of developing OHSS . More recently, Lim et al. reported 17 patients with a high risk of developing OHSS during the course of their IVF cycles. Instead of canceling the cycles, they undertook immature oocyte collection followed by IVM. hCG was administered 36 hours before oocyte collection when the leading follicle had reached a mean diameter of 12–14mm and indeed 11.6% of the oocytes had already reached the metaphase II stage at collection. Eight out of 17 (47.1%) clinical pregnancies were achieved in this group of patients. Even though the safest method of preventing OHSS is to withhold hCG administration, no cases of OHSS were reported among these patients, who were at a high risk of developing the syndrome. To date, more than 30 healthy live births have been reported from this group of patients following oocyte retrieval and IVM treatment (personal communication). Therefore, patients who are at risk of developing OHSS during controlled ovarian hyperstimulation can resort to immature oocyte retrieval followed by IVM as an alternative to canceling the cycle.
IVM for Poor Responders
Poor response to gonadotropin stimulation occurs more often in older women but may also be present in young women, including those with normal endocrine profiles as well as those with abnormal endocrine parameters—namely, high baseline FSH and estradiol (E2) levels—known to be associated with poor response. Some poor responders appear to respond to stimulation but have a low estrogen level, whereas others have few or slow-growing follicles. Normally, these patients require prolonged stimulation and higher doses of gonadotropins. They also experience a high cancellation rate because of the smaller number or size of follicles. Many different ovarian stimulation protocols have been tried for treatment of poor responders in IVF. No single protocol seems to benefit all poor responders and treatment continues to challenge those involved in IVF programs. Although oocyte donation would be the ideal treatment for these patients, some may refuse this option because they would prefer to try using their own oocytes. In these cases, poor responders to previous gonadotropin stimulation may benefit from immature oocyte collection from unstimulated ovaries. In a study by Child et al., eight women with a previous poor response to IVF underwent oocyte collection without ovarian stimulation. hCG was administrated 36 hr before collection. An average of 2.3 immature oocytes were collected and an average of 1.7 matured in vitro. Six of the eight women underwent embryo transfer of 1–3 embryos (average of 1.7); one patient became pregnant and subsequently delivered. The number of embryos produced and available for embryo transfer was similar to that for previous IVF treatments. During ovarian stimulation, the small number and size of follicles often warrant cancellation of the cycle. As an alternative to cancellation, immature oocytes could be collected from the stimulated but unresponsive ovaries and then matured in vitro. Such pregnancies were first reported after cryopreservation of in vitro matured oocytes . Liu et al. reported eight cases of immature oocyte collection in young patients who had shown poor response to gonadotropin stimulation; three pregnancies were achieved. In another report, 41 patients were identified as being resistant to gonadotropin stimulation as the follicles did not grow despite increasing the dosage of gonadotropins. To optimize the successful pregnancy rate among these poor responders, hCG was administered and oocyte retrieval performed 36 hours later because at least some in vivo matured oocytes could be collected after hCG administration. This indicates that immature oocyte retrieval followed by IVM is a possible alternative to cancellation of the treatment cycle in women with poor response following ovarian stimulation. Based on the results of these preliminary studies, it seems that IVM is a possible option for patients with a poor ovarian response in an ongoing stimulated IVF cycle or with a history of a previous low response to gonadotropin stimulation. Although IVM does not always produce better results than conventional IVF in these cases, it will at least give comparable results without the need for prolonged stimulation with large doses of gonadotropins.
Immature Oocyte Retrieval
Oocyte retrieval is done under spinal anesthesia or intravenous sedation using fentanyl and midazolam (1–2 mg). Intravenous fentanyl is administered at intervals of 15–20 min up to a total dose of 150–200 mg. Local infiltration of bupivacaine 0.5% in the vagina reduces the discomfort of multiple needle punctures. Retrieval is performed under ultrasound guidance with a 19-G, 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 2U/mL of heparin. Because immature oocytes are enclosed in tightly packed cumulus cells, curettage of the follicle wall will dislodge the cumulus oocyte complex. In an immature oocyte collection, multiple needle punctures are needed. Because the aspiration pressure is low and a small-gauged needle is used, the bloodstained aspirate may often block the needle. Therefore, the needle is withdrawn from the vagina after aspirating a few follicles to flush and clear any blockage. The procedure is repeated until all follicles seen are aspirated.
Maturation In Vitro and Fertilization
Immature oocytes are incubated in a culture dish containing maturation medium. The maturation medium is supplemented with 75mIU/mL of FSH and LH. The oocytes are cultured at 37_C in an atmosphere of 5% carbon dioxide and 95% air with high humidity. Oocytes are checked for maturity 24 and 48 hours after culture. The oocytes are denuded of granulosa cells, and mature oocytes (detected by the presence of an extruded polar body) are fertilized by ICSI. ICSI is performed for in vitro matured oocytes because it reduces the risk of unexpected poor fertilization as compared with IVF. However, it has been demonstrated that ICSI may not always be essential for the fertilization of in vitro matured human oocytes collected from unstimulated ovaries when the sperm parameters are normal . After ICSI, the oocytes are transferred into 1mL of IVF medium in a tissue culture dish. Fertilization is assessed 18 hours after ICSI by examining the oocytes for the appearance of two distinct pronuclei and two polar bodies.
Embryo Transfer
The fertilized oocytes are further cultured up to day 2 or 3, and then embryo transfer is performed. Assisted hatching is performed to avoid reduced implantation due to a hardened zona pellucida. When a large number of embryos have been formed, alternative approaches could be either an extended culture to the blastocyst stage or a double transfer . A double transfer is performed on day 2 or 3 and a blastocyst transfer on day 5 or 6.
The embryo transfer technique is the same as that employed for conventional IVF.
IVM TREATMENT OUTCOME
Pregnancy rates with IVM are correlated with the number of immature oocytes retrieved. In women younger than 35 years from whom we retrieved more than 10 immature GV oocytes, we have achieved a clinical pregnancy rate of 38% per cycle. With an oocyte retrieval rate of more than 50% from the follicles present, women with 20 or more follicles at the baseline scan for IVM would be the best candidates for IVM. Our implantation rates are approximately 12%. As with IVF, clinical pregnancy and implantation rates decrease with increasing age. In women younger than 35 years, we have achieved a clinical pregnancy rate of 38% per oocyte retrieval and an implantation rate of 13%. In women between 36 and 40 years old, the clinical pregnancy rate is 21% per retrieval and the implantation rate 5%. Based on more than 1000 IVM cycles in four centers performing IVM cycles with hCG priming before oocyte collection, the pregnancy rates reached 30–35% and the implantation rates 10–15% . Some authors have expressed concerns regarding the safety of IVM, especially in relation to imprinting gene disorders . In various published series, no increased rates of congenital malformations have been reported with IVM. A recent analysis of the obstetrical, neonatal, and infant outcome in our IVM conceptions showed pregnancy rates of 73% singleton, 24% twin, and 2.7% triplet. The median gestation age was 39 weeks for singletons and 37 weeks for multiple pregnancies. There were only two malformations, including a ventriculo septal defect and a congenital dislocation of the hips. There was no increased relative risk of
malformations when IVM pregnancies were compared with IVF and spontaneous pregnancies. Similar reassuring results have been published by others.
Figure 1 Immature human oocyte retrieved from a follicle at 4mm in diameter. The
oocyte with several layers of compacting cumulus cells. Source: From R.C. Chian, with
permission.
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