In vitro maturation

Authors

  • Wendy Vitek MD,

    Assistant Professor, Corresponding author
    1. Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, University of Rochester Medical Center, Strong Fertility Center, Rochester, NY, USA
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  • Jared C Robins MD

    Associate Professor
    1. Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, The Warren Alpert School of Medicine at Brown University, Women and Infants Hospital of Rhode Island Providence, USA
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Abstract

Key content

  • In vitro maturation is an alternative to in vitro fertilisation for young women with polycystic ovary syndrome and young ovulatory women.
  • In vitro maturation accomplishes the maturation of immature oocytes in the laboratory, thus minimising or avoiding gonadotrophin stimulation and the associated cost, side-effects and risks.
  • Pregnancy rates are lower with in vitro maturation than with in vitro fertilisation.

Learning objectives

  • To define appropriate candidates for in vitro maturation.
  • To review variations in protocols and laboratory aspects of in vitro maturation and the rationale for these variations.
  • To highlight outcome data from in vitro maturation cycles.

Ethical issues

  • Infertile couples must consider both the risks and benefits of in vitro maturation and make an informed decision regarding their treatment options.
  • Further long-term outcome data regarding the health of children born through in vitro maturation is necessary.

Introduction

In vitro maturation (IVM) involves the retrieval of immature oocytes from antral follicles with minimal or no gonadotrophin stimulation followed by maturation and fertilisation in the laboratory. IVM offers select infertile couples a safe, convenient and less costly alternative to conventional in vitro fertilisation (IVF) by avoiding or minimising gonadotrophins. Eligible couples must weigh these benefits against the reduced pregnancy rate that is observed with IVM in comparison with IVF. Since the first reported IVM birth, research has led to improved protocols and optimised cultured conditions, yet many unanswered questions remain. The objective of this article is to discuss the definition of IVM, eligible candidates, IVM protocols, laboratory aspects and outcomes.

What is IVM?

Dr Robert Edwards first observed spontaneous in vitro maturation of immature human oocytes in 1965.[1] His subsequent association with Dr Patrick Steptoe led to the first IVF birth resulting from a single in vivo maturated oocyte retrieved prior to ovulation.[2] Subsequently, controlled ovarian hyperstimulation via gonadotrophin injections was adopted to improve the efficiency of IVF, but also increased the cost, side-effects and risks.[3] In 1983, Veeck et al.[4] first reported rescue IVM, which attempts to mature immature oocytes retrieved during gonadotrophin-stimulated IVF cycles. In rescue IVM, oocytes are stripped of their cumulus cells at the time of retrieval in order to assess for nuclear maturation and are subsequently cultured in gonadotrophin-supplemented media to promote maturation. There are concerns that oocytes that do not mature in vivo despite exposure to supraphysiological levels of gonadotrophins are dysmature, have limited developmental potential, and produce a high rate of aneuploid embryos.[3] In addition, culturing intact cumulus oocyte-complexes may be optimal given that the cumulus cells promote the cytoplasmic and nuclear maturation of the oocyte by providing the physical, nutritional and endocrine microenvironment necessary for development.[5] Current IVM techniques involve the retrieval of immature or germinal vesicle-stage oocytes that have been exposed to minimal or no gonadotrophin stimulation, followed by the culture of intact cumulus-oocyte complexes in gonadotrophin-supplemented media to accomplish in vitro maturation. In contrast, the controlled ovarian hyperstimulation utilised in IVF protocols results in the retrieval of mature or metaphase II-stage oocytes, which have resumed meiosis in vivo. In 1991, Cha et al.[6] reported the first IVM births from unstimulated immature oocytes that were cultured as cumulus-oocyte complexes. To date, there have been over 1000 reported births using current IVM techniques.

Appropriate candidates

Conventional IVF is the ‘gold standard’ treatment for infertility given excellent pregnancy rates. However, controlled ovarian hyperstimulation can result in mild ovarian hyperstimulation syndrome (OHSS) in more than 20% of IVF cycles and severe OHSS in 0.1–2% of IVF cycles.[7] Severe OHSS can have significant consequences including respiratory compromise, renal failure and stroke.[8] Therefore, the response to controlled ovarian hyperstimulation is closely monitored through serial follicular ultrasounds and serum estradiol levels. Patients at high risk of OHSS, such as young women, women with polycystic ovary syndrome (PCOS) or women with previous episodes of OHSS, may require cautious administration of gonadotrophins and daily monitoring visits while in cycle. Strategies to prevent OHSS in at-risk patients aim to minimise the impact of human chorionic gonadotrophin (hCG) by reducing the dose of the hCG used to trigger the resumption of meiosis, delaying hCG administration after withholding follicle-stimulating hormone (FSH) until lower estradiol levels are observed or substituting gonadotrophin-releasing hormone agonist (GnRHa) in place of an hCG trigger. In addition, endogenous hCG production can be avoided by cryopreserving embryos or cancelling the cycle. Though some IVM protocols incorporate an hCG trigger, the risk of OHSS and need for close monitoring is greatly reduced given that minimal or no FSH stimulation is employed.

Potential IVM candidates include young women (<38 years of age) with PCOS, high antral follicle counts and/or a history of ovarian hyper-response to gonadotrophin stimulation. Performing IVM in women with low antral follicle counts is controversial. Although fewer oocytes are retrieved from women with normal appearing ovaries, the developmental capacity of these oocytes may be superior when compared with oocytes from women with PCOS.[3] However, given that fewer oocytes are obtained, there is less opportunity to select the best embryos for transfer. Women at risk for an estrogen-associated thrombus may also benefit from IVM given the ability to retrieve oocytes without stimulating granulosa cell estrogen production with gonadotrophins. In this population, anticoagulation could be initiated with pregnancy, avoiding the risk of anticoagulation at the time of oocyte retrieval. Finally, women facing gonadotoxic therapy who desire fertility preservation may benefit from the accelerated time course of IVM in order to cryobank oocytes or embryos and avoid treatment delays.[9]

IVM protocols

The optimal timing of oocyte retrieval for IVM has yet to be established because of controversy regarding the role of dominant follicle selection. The dominant follicle is selected when the largest follicle reaches 10 mm and exceeds the diameter of the next largest follicle by at least 2 mm.[10] Some data suggest that the selection of the dominant follicle may lead to atresia of non-dominant follicles.[11] Therefore, some investigators propose cancelling an IVM cycle if the lead follicle reaches 10 mm given the possible detrimental effects of selection. However, others have observed competent embryos retrieved from small follicles in cycles where the dominant follicle size is ≥14 mm.[12] Son et al.[12] retrospectively reviewed the outcomes of IVM cycles in women with PCOS where the dominant follicle diameter was ≤10 mm, between 10 and 14 mm and >14 mm at the time of hCG priming. While the rates of maturation, fertilisation and embryo development were similar in the sibling immature oocytes retrieved in the three groups, the implantation rates and pregnancy rates were higher from the oocytes retrieved when the dominant follicle diameter was ≤14 mm. In the absence of definitive data, oocyte retrieval is commonly performed when the lead follicle diameter is less than 12–14 mm.

The necessity of follicular priming with a short course of low-dose gonadotrophins or a single dose of hCG is debated. FSH priming has been proposed in order to increase the number of oocytes retrieved, enhance oocyte maturation and aid in endometrial development by stimulating higher estradiol levels. The data regarding the benefits of this intervention are conflicting possibly because of difference in populations, timing of oocyte retrieval and culture conditions. While most of the data examining this question are retrospective, a prospective, randomised trial comparing follicular priming and an unstimulated protocol in ovulatory women found no difference in outcomes.[13] The same group found that women with PCOS benefited from FSH priming with improved pregnancy rates compared with the non-primed group.[14]

Many IVM protocols incorporate a single injection of hCG 36 hours prior to oocyte retrieval. HCG priming has been proposed to enhance oocyte maturation and may play an important role in endometrial receptivity.[3] In a prospective trial comparing FSH and hCG priming with hCG priming alone,[15] the pregnancy rates in the hCG priming alone group were 34% and there was no apparent benefit to the addition of FSH priming. Further high-quality research is necessary to determine which subpopulations of IVM candidates benefit from priming.

Given controversies regarding timing of oocyte retrieval and benefit of priming, various IVM protocols have been developed (Table 1). Natural cycle IVM typically involves a baseline transvaginal ultrasound performed after the onset of menses to ensure ovarian quiescence. Serial ultrasounds are performed on cycle days (CD) 9–11 in order to plan oocyte retrieval when the lead follicle is 12–14 mm. hCG may or may not be given prior to the retrieval. Estradiol is initiated at the time of retrieval and the dose may be determined by the thickness of the endometrium. Progesterone is started 1–2 days after retrieval. Low-stimulation IVM protocols typically initiate FSH injections at a dose of 37.5 IU/day in the early follicular phase and may escalate the dose to 150 IU/day in the late follicular phase. An ultrasound is performed on CD10 to assess follicular development and endometrial stripe. Again, hCG is typically administered with a lead follicle of 12–14 mm and progesterone is initiated 1–2 days after retrieval.

Table 1. In vitro maturation protocols
 Natural cycle IVMLow-stimulation IVMEstrogen-suppressed IVM
  1. CD = cycle day; FSH = follicle-stimulating hormone; hCG = human chorionic gonadotrophin; IVM = in vitro maturation; US = ultrasound

MonitoringCD3 – baseline USCD9 – start serial monitoring USCD3 – baseline USCD10 – start serial monitoring USNone
RetrievalTiming variesTiming variesScheduled for CD14–16
MedicationshCG may or may not be given prior to retrieval. Estradiol initiated at time of retrieval. Progesterone initiated 1–2 days after retrievalLow-dose FSH initiated after baseline US. hCG given with lead follicle of 12–14 mm. Progesterone initiated 1–2 days after retrievalEstradiol initiated with menses. hCG given on CD14. Progesterone initiated evening after retrieval

At the Center for Reproduction and Infertility at Women & Infants Hospital of Rhode Island, estrogen-suppressed IVM was developed to allow for advanced scheduling of the date of oocyte retrieval, minimal monitoring and reduced cost while maintaining excellent pregnancy rates. At the onset of menses patients begin 6 mg daily estradiol in order to inhibit development of a dominant follicle and to prepare the endometrium for implantation. hCG is administered on CD12–14 and retrieval is performed on CD14–16. Progesterone support is initiated the evening after oocyte retrieval. Oral contraceptive pills may be used to manipulate the onset of the menses for scheduling flexibility and to induce a withdrawal bleed in anovulatory patients. By avoiding gonadotrophin injections, frequent blood draws and ultrasound monitoring, patients experience less discomfort, spend less time in the office, ensue less cost and are afforded the convenience of advanced scheduling of the oocyte retrieval date.

Oocyte retrieval

An IVM oocyte retrieval is similar to a conventional IVF retrieval. However, a smaller-diameter needle and lower aspiration pressure are employed in order to recover intact cumulus-oocyte complexes from small follicles. Available needles are between 19 and 21 gauge. Because the needle is thin, some physicians will pass the needle through a 17 gauge needle to stabilise the ovary. The aspiration pressure is reduced to 80 mmHg in order to avoid denuding the oocytes of the cumulus complex. In addition, curetting small follicles may increase the oocyte yield by 22%.[16]

Laboratory aspects of IVM

Immature oocytes enclosed in cumulus cells are cultured in IVM medium for 24–48 hours. IVM medium is supplemented with gonadotrophins, such as FSH and luteinising hormone or hCG. At 24 hours, the oocytes are minimally stripped and evaluated for nuclear maturation. Mature oocytes are inseminated at this time, while immature oocytes are cultured in IVM medium for an additional 24 hours and are reassessed for maturation at 48 hours post-retrieval. Oocytes that are mature after 24 hours in culture are more developmentally competent than oocytes that mature after 48 hours in culture.[17] Insemination is commonly achieved through intracytoplasmic sperm injection (ICSI) given a theoretical concern of zona pellucida changes from IVM. While ICSI may result in higher fertilisation rates than standard insemination, the embryonic development, implantation rates and pregnancy rates are similar regard-less of insemination method.[18] Embryo transfer is typically performed at the cleavage stage. Given the lower implantation rates observed with IVM embryos, consideration can be given to transferring one additional embryo than typically recommended for conventional IVF.

IVM outcomes

Outcomes of IVM cycles by protocol are listed in Table 2. The average number of oocytes retrieved is similar between protocols, but women with PCOS tend to have more oocytes retrieved than women with normal ovarian morphology. Maturation rates are similar between protocols and are reported from 50% to 85%. Fertilisation rates with IVM/ICSI are similar to those observed with conventional IVF/ICSI. Implantation rates range from 5% to 22% and clinical pregnancy rates range from 8% to 40%, which are lower than observed in IVF patients with similar clinical characteristics. The lower implantation and pregnancy rates may reflect the increased rate of aneuploidy embryos observed with IVM.[28] Spontaneous abortion rates may be higher in women with PCOS, although it is unclear if this is related to the underlying cause of infertility or IVM.

Table 2. IVM outcomes by protocol
 PrimingAverage # oocytes retrieved% mature% fertilisedAverage # embryos transferredImplantation rate%Pregnancy rate%% SAB
  1. FSH = follicle-stimulating hormone; hCG = human chorionic gonadotrophin; ICSI = intracytoplasmic sperm injection; IVF = in vitro fertilisation; IVM = in vitro maturation; NA = not available; SAB = spontaneous abortion

Natural cycle IVM
 Mikkelsen et al. (1999)[13]None3.77662 (ICSI)1.818.833.320
 Cha et al. (2000)[19]None13.662.268 (ICSI)4.96.927.120
 Yoon et al. (2001)[20]None9.05672.6 (IVF and ICSI)3.66.517.633.3
 Cha et al. (2005)[21]None15.5NANA5.05.521.937
 Chian et al. (2000)[22]None vs hCG

7.4

7.8

68

85.2

83.9 (ICSI)

90.7 (ICSI)

2.5

2.8

14.8

16.6

27.3

38.5

0

40

 Soderstrom-Anttila et al. (2005)[23]None7.658.251.2 (IVF and ICSI)1.618.526.626.5
 Le Du et al. (2005)[24]hCG11.46370.1 (ICSI)2.510.922.540
 Lim et al. (2009)[25]hCG12.166.681.9 (ICSI)3.117.840.427.9
 Fadini et al. (2009)[26]None vs hCG

5.3

5.3

48.4

57.9

77.6 (ICSI)

71.5 (ICSI)

1.8

1.9

9.2

4.0

15.3

7.6

18.2

0

 Xu et al. (2010)[27]hCG10.847.986.3 (ICSI)3.0 14.935.928
Low-stimulation IVM
 Mikkelsen et al. (1999)[13]FSH (3d to 6d)2.4–4.271–8561–65 (ICSI)1.1–1.90–14.30–22.20
 Mikkelsen and Lindenberg (2001)[14]FSH6.55970 (ICSI)1.821.63362.5
 Lin et al. (2003)[15] FSH and hCG21.976.575.8 (ICSI)3.89.731.413
 Fadini et al. (2009)[26]FSH vs FSH and hCG

4.8

5.4

50.8

77.4

73 (ICSI)

73 (ICSI)

1.7

1.9

10.6

16.4

17.3

29.9

15.4

16.4

Estrogen-suppressed IVM
 Robins (unpublished)hCG18.65170 (ICSI)2.722.24010

It has been estimated that more than 400 children have been born worldwide from IVM.[3] While the prolonged cultured conditions necessary for IVM have raised concerns regarding the risk of imprinting disorders, there is no apparent increase in congenital anomalies in children conceived born from IVM compared with children born from IVF and IVF/ICSI.[29] In a small case series of children born through IVM, 8 of 43 were noted to have minor developmental deficits at 1 year of age, but neuropsychological development was within the normal range at 2 years of age.[30] Further long-term follow-up data are necessary.

Conclusion

IVM is an effective alternative to conventional IVF in young women with PCOS, high antral follicle counts or history of prior OHSS episodes and possibly young ovulatory women. While early enthusiasm for IVM may have been diminished by the lower pregnancy rates and the need for additional clinical and laboratory training, the benefits of reduced risk, cost and time commitment are leading to renewed interest in IVM.[31] Further research is necessary to optimise the protocols and laboratory aspects of IVM in order to achieve pregnancy rates similar to IVF. Long-term outcomes data of the health of children born through this technology are needed. While IVF remains the standard of care, IVM offers an attractive alternative to infertile couples who accept a lower pregnancy rate per cycle in order to benefit from the convenience, reduced cost and safety that IVM offers.

Disclosure of interests

None to declare.

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