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Addressing the risks of multiple gestation, a new method of cryopreservation of embryos, and the unique value of anti-Müllerian hormone as a marker of ovarian reserve

February 2012 · Vol. 24, No. 2


Dr. Adamson reports that he receives research grants from LabCorp and Auxogyn, and is the founder and CEO of Advanced Reproductive Care. Dr. Abusief reports no financial relationships relevant to this article.

The field of reproductive endocrinology has advanced at warp speed over the past few decades—and shows no sign of stopping any time soon. In this article, we outline noteworthy developments of the past year:

  • publication of two important Committee Opinions from the American Society for Reproductive Medicine (ASRM)—one of them on the need to reduce the rate of multiple gestation among women undergoing treatment for infertility and the other focusing on a method of achieving this goal: elective single embryo transfer
  • two studies of vitrification for cryopreservation of embryos and oocytes
  • a trio of investigations into the utility of anti-Müllerian hormone as a means of assessing ovarian reserve and reproductive potential.

Goal of non-ART infertility therapy should be to produce a single child

Practice Committee of the American Society for Reproductive Medicine. Multiple gestation associated with infertility therapy: an American Society for Reproductive Medicine Practice Committee opinion [published online ahead of print December 20, 2011]. Fertil Steril. doi:10.1016/j.fertnstert.2011.11.048.

The goal of infertility treatment is for each patient to have one healthy child at a time, according to a new Practice Committee Opinion from the American Society for Reproductive Medicine (ASRM).

In women who experience oligo-ovulation or anovulation, ovulation induction is typically offered. For ovulatory women who have unexplained or age-related infertility, the treatment often is controlled ovarian stimulation. Either intervention can lead to ovulation from multiple follicles and, ultimately, increase the risk of multiple gestation.

Multiple gestation increases maternal morbidity and both fetal and neonatal morbidity and mortality. Most of the poor perinatal outcomes relate directly to preterm birth. Treatment of women who have infertility, therefore, requires achieving a balance between two competing needs:

  • maximizing the probability of pregnancy
  • minimizing the risk of multiple (two fetuses or more) or high-order multiple (more than two fetuses) gestation.

Many multiple births are iatrogenic

Approximately 60% of twin births result from natural conception, 30% from ovulation induction and controlled ovarian stimulation, and 10% from assisted reproductive technologies (ART). For high-order multiple gestation, the figures are 20% for natural conception, 50% for ovulation induction and controlled ovarian stimulation, and 30% for ART. These statistics reveal that a very large percentage of multiple births are iatrogenic, with fertility treatment increasing the risk of twins by a factor of approximately 20 and the risk of high-order multiples by a factor of more than 100. The risk of monozygotic twinning also increases by a factor of 2 or 3 after ovulation induction, compared with natural conception.

Triplets should be a rarity

Three-dimensional sonogram of triplets.

Multiple gestation is expensive

The economic costs associated with excess perinatal and maternal morbidity are substantial. They include the immediate costs associated with maternal hospitalization and neonatal intensive care and lifetime costs associated with care for chronic illness, rehabilitation, and special education. Although these costs might be offset by the productivity of individuals, the overall benefit to society is clearly greater when a singleton is born. Personal and familial nonfinancial costs of morbidity and mortality can also be significant.

A sense of urgency on the part of the patient may contribute to an increased risk of multiple gestation by prompting more aggressive treatment. Other contributors include limited health coverage, which creates a personal financial burden, and inadequate patient education about the risks of multiple gestation.

Strategies for limiting the risk of multiple gestation

Appropriate treatment goals are the foundation of risk-reducing strategies. For example, ovulation induction in women who have oligo-ovulation or anovulation should aim toward producing a single oocyte. These women tend to respond to lower dosages of ovarian-stimulation drugs than are typically given. Therefore, women undergoing ovulation induction should receive a lower dosage of gonadotropins and be monitored very carefully for the number of developing follicles and ovarian hyperstimulation syndrome.

In contrast, the goal of controlled ovarian stimulation in ovulatory women who have unexplained or age-related subfertility is to stimulate the development and ovulation of more than one mature follicle to increase cycle fecundity.

Regrettably, efforts have failed to identify estradiol levels and the specific size and number of follicles that prevent multiple gestation. The most likely reason is that follicular size cannot accurately predict the maturity of the oocyte within—follicles as small as 10 mm sometimes yield mature and fertilizable oocytes. Moreover, the population that undergoes ovulation induction or controlled ovarian stimulation is very heterogenous. Therefore, it is not possible to propose valid guidelines to reduce the rate of multiple gestation.

Nevertheless, multiple gestation is sufficiently problematic that we recommend some strategies to reduce its incidence:

  • Use low-dosage gonadotropin stimulation with careful monitoring, and limit the number of follicles that are roughly 15 mm or larger to two in patients 37 years of age or younger; three in patient 38 to 40 years old; and more in patients older than 40
  • Develop specific cancellation criteria, which should be explained to and accepted by patients undergoing controlled ovarian stimulation. Gonadotropin-releasing hormone (GnRH) antagonists may be of benefit.1
  • When clomiphene citrate stimulates the development of two or more mature follicles, outcomes do not differ from those obtained with controlled ovarian stimulation using gonadotropins and intrauterine insemination (IUI).2 Therefore, a reasonable strategy in many patients is to consider initiating treatment with clomiphene citrate and IUI and to proceed directly to in vitro fertilization (IVF) when treatment fails, thereby avoiding controlled ovarian stimulation altogether.3
  • Pre-ovulatory ultrasonography-guided aspiration of excess follicles to reduce the risk of multiple gestation has potential benefit but needs further study.

Overall, regardless of the medication or regimen employed, it may not be possible to entirely eliminate the risk of multiple gestation associated with ovulation induction or controlled ovarian stimulation.

When to consider gestation reduction

High-order multifetal gestation reduction has been utilized as a strategy to reduce complications associated with ovulation induction and controlled ovarian stimulation, but use of this technology must be regarded as an adverse outcome of infertility treatment. Overall, data suggest that multifetal gestation reduction is associated with a reduced risk of prematurity, although its true benefit is difficult to elucidate due to potential bias in the interpretation of data. A small percentage of patients lose the entire pregnancy, and the procedure can present patients with a profound ethical dilemma and psychological trauma. Thorough counseling is imperative.

Despite feelings of loss and guilt that persist for a year or longer, most patients report that they would make the decision to undergo gestation reduction again if a similar situation arose in the future.4

The procedure should be performed only in a specialized center by an experienced practitioner.


When performing ovulation induction and controlled ovarian stimulation, use the lowest dose of drug necessary to obtain a single mature follicle in anovulatory women, two follicles in young ovulatory women, and three follicles in women 38 to 40 years old. Because of the high risk of multiple gestation associated with controlled ovarian stimulation followed by IUI, consider moving directly to IVF after use of clomiphene citrate and IUI.

Elective single embryo transfer can reduce the multiple-gestation rate in women who have a good prognosis

Practice Committee of the Society for Assisted Reproductive Technology and Practice Committee of the American Society for Reproductive Medicine. Elective single-embryo transfer [published online ahead of print December 22, 2011]. Fertil Steril. doi:10.1016/j.fertnstert.2011.11.050.

As IVF implantation rates have improved, the practice of transferring multiple embryos has resulted in a much-increased pregnancy rate but also a high percentage of multiple gestations. Elective single embryo transfer (eSET) has been advocated as the only effective means to avoid multiple pregnancy in IVF cycles, but there is significant concern that it might ultimately reduce the pregnancy rate.

ASRM recently published a Practice Committee Opinion that offers guidance for patient selection and describes barriers to eSET. Patient selection is critical.

Utilization of eSET in the United States has increased over the past decade but still lags behind other countries. Use of double embryo transfer (DET) has increased, significantly reducing the likelihood of high-order multiple pregnancies associated with ART but producing no change in the twin pregnancy rate (FIGURE). Randomized, controlled trials and other studies have demonstrated that the cumulative pregnancy rate per retrieval is no different for eSET followed by frozen embryo transfer than it is for DET in properly selected patients.

Most transfers involve two embryos

Percentage of transfer of one, two, three, or four or more embryos among all in vitro fertilization cycles performed in the United States, 1999–2008.
SOURCE: ASRM. Reproduced with permission.
eSET is most appropriate for women who have a good prognosis:

  • age younger than 35 years
  • >1 top-quality embryo available for transfer
  • first or second treatment cycle
  • prior successful IVF
  • recipients of embryos from donated eggs.

Women 35 to 40 years old can be considered for eSET if they have top-quality, blastocyst-stage embryos available for transfer.

Barriers to eSET include a lack of provider and patient education about it, financial considerations, embryo selection, and successful cryopreservation. When insurance coverage or refund guarantees are available, patient acceptance of eSET increases.


Elective single embryo transfer is the only ART embryo transfer strategy that will reduce the twin pregnancy rate. However, it is not a good approach for all patients and must be carefully utilized in selected patients who have a good prognosis.

Vitrification for cryopreservation of embryos appears to be superior to slow freezing

Leibo S, Pool T. The principal variables of cryopreservation: solutions, temperatures, and rate changes. Fertil Steril. 2011;96(2):269–276.

Cobo A, Diaz C. Clinical application of oocyte vitrification: a systematic review and meta-analysis of randomized controlled trials. Fertil Steril. 2011;96(2):277–285.

Cryopreservation is a method by which cells are suspended in a solution of salts and low-molecular-weight organic compound, cooled to subzero temperatures (approximately –196°C) in liquid nitrogen, stored, and then rewarmed. Cryopreservation has become a major component of the practice of assisted reproduction, with more than 37,000 pregnancies produced from cryopreserved embryos from 2005 through 2009 in the United States alone.5,6

Standard (slow) freezing methods for embryo cryopreservation involve suspension of the embryos in a 10% solution of propylene glycol supplemented with 3.4% sucrose, cooling them to –35°C at a rate of 0.3°C/min, submerging them in liquid nitrogen for storage, and rewarming the frozen embryos at a rate of approximately 300°C/min to thaw them.5

A major advance in the science of cryopreservation is the use of vitrification, a method of freezing in which the embryos are equilibrated with a 10% or 15% solution of cryoprotectant and then exposed briefly (30–60 seconds) to a 20% to 40% solution of cryoprotectant to achieve relative cellular dehydration. The embryos are then placed in a storage container and submerged in liquid nitrogen. During vitrification, embryos can be cooled at a rate exceeding 1,000°C/min. Vitrified embryos are stored at approximately –196°C and thawed in ultra-rapid fashion.

The development of vitrification methods has significantly advanced the technology of oocyte cryopreservation, which has been utilized for:

  • preservation of fertility in cancer patients
  • social reasons (e.g., lack of a partner)
  • egg-donation programs
  • minimization of the risk of ovarian hyperstimulation syndrome
  • storage of surplus eggs when embryo cryopreservation is not feasible.

Cobo and Diaz recently conducted a systematic review and meta-analysis of randomized, controlled trials of oocyte vitrification. They found that the potential for fertilization, embryogenesis, and pregnancy from oocytes that had undergone vitrification and warming was not significantly different from the potential for fresh oocytes and was better than the potential for oocytes that had undergone freezing and thawing from standard freezing cycles.

Although the findings of the meta-analysis were limited by the small number of studies and possible selection bias, an increasing body of evidence supports the use of vitrification for cryopreservation of oocytes. Large-scale controlled trials are needed. Until they are performed, the findings of the meta-analysis should be interpreted with caution.

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