Preimplantation genetics and other reproductive options in Huntington disease

Abstract

Preimplantation genetic diagnosis (PGD) is a form of prenatal diagnosis applied to potential parents with known carrier status of a genetic disease, such as Huntington disease. It employs the use of polymerase chain reaction to amplify single cells from early embryos obtained with in vitro fertilization (IVF) techniques. PGD allows the couple the chance to have a pregnancy and livebirth child without Huntington disease, although there are some risks and expenses related to the procedures. Success of the procedure may be greater than standard IVF because the patients are not infertility patients, but are undergoing the procedure to avoid passing a highly deleterious disease gene to offspring. Recent advances in sequencing may allow for higher success rates as the chromosomally abnormal embryos will be identified more easily and the embryos with the highest chance of survival will be transferred.

INTRODUCTION

Huntington disease (HD) affects males and females equally and each offspring of a person with an HD gene mutation has a 50% chance of inheriting the disease mutation. If the mutation is not inherited, then it cannot be transmitted to offspring. DNA testing of the HD gene for symptomatic and presymptomatic individuals from peripheral blood sampling is technically straightforward and reviewed elsewhere in this volume. In the event of a positive HD gene test result or in cases where individuals at risk of HD may not themselves wish to know their HD gene status, preimplantation genetic diagnosis (PGD) provides an option for families who wish to prevent having offspring with HD.

The first PGD analyses were performed in the United Kingdom in the early 1990s and were initially aimed at determinations of X-linked disease genes in “at-risk” embryos using single-cell polymerase chain reaction (PCR) methods (HFEA, 2014). Preliminary work in this area had been reported prior to that time (Schulman et al., 1996). Single-cell PCR has been improved and enhanced over the years and along with molecular cytogenetic techniques has allowed the diagnosis of many unique sequence gene disorder genes, chromosomal aneuploidies, and chromosomal translocations (Verlinsky et al., 2004a).

PGD is a procedure that may be useful for conception of children without the HD gene and can be used when either the prospective mother or father has tested positive for the gene mutation. This procedure is based on an initial in vitro fertilization (IVF) treatment, with the additional steps of genetic analysis of cells from each of the early embryos for detection of the disease gene. The unaffected embryo is transferred to the potential mother only if it does not have the mutated HD gene. The procedure is also useful for couples where one partner has a 50% risk of having the HD gene because of family history and has refused presymptomatic testing. In these cases, the physician does not reveal the HD mutation status of the embryos to the couple. Only unaffected embryos would be transferred if any do, in fact, have the HD gene mutation. This provides the opportunity for people who are at risk of HD because of family history to have children with no HD without disclosing their HD status. This has been referred to as an exclusion or nondisclosure test (Moutou et al., 2004).

The steps of the procedure are as follows:

1. The couple at risk for HD will meet with a certified physician from an advanced reproductive technology medical practice.

2. The HD familial mutation lab studies will be required for verification of mutation risk in the couple.

3. The female will have a typical IVF treatment to collect eggs and this will include ovarian hyperstimulation.

4. The eggs will be fertilized in the laboratory and grown for either 3 days (in the case of blastomere biopsy) or 5–6 days in the case of trophectoderm biopsy.

5. The embryos will undergo assisted hatching on day 3, as shown in Figure 9.1.

6. In the case of blastomere biopsy, on day 3, one or two of the cells will be removed from the embryo by an embryologist using a biopsy technique as shown in Figure 9.2. The embryos themselves will continue to be cultured in the lab until DNA results are obtained. In the case of trophectoderm biopsy, as shown in Figure 9.3, the embryos are cultured to the blastocyst stage, which is 5–6 days post fertilization, and the herniated trophectoderm is excised and the cells sent for DNA analysis. The trophectoderm biopsy is thought to be less harmful to the embryo than blastomere biopsy.

7. The cells which are representative of the embryo of origin are tested by PCR to determine if they contain the HD mutation. Embryos without the HD mutation can be subsequently transferred to the uterus using a catheter.

8. Additional unaffected embryos can be frozen and transferred in a future cycle. Affected embryos are allowed to perish. Those embryos that are affected by the condition are allowed to expire or, with the patients’ consent, can be used for research.

Fig. 9.1.

Assisted hatching: a small hole is made in the zona pellucida of the embryo to assist in implantation after blastocyst stage. (Courtesy of Columbia Fertility Associates.)

Fig. 9.2.

Embryo biopsy. (A) An opening in the zona pellucida is made to allow access to the blastomere. (B) First blastomere is removed from embryo. (C) Blastomere is placed in medium. (D) Second blastomere is removed (Sermon et al., 2004).

Fig. 9.3.

Trophectoderm biopsy. The embryo is in the expanded blastocyst stage at the time of cell biopsy, which is day 5–6 post fertilization. (Courtesy of Columbia Fertility Associates.)

If the embryos are grown in vitro for 5–6 days, instead of 2–3 days, there will be over 100 cells available for analysis. The cells in this later-stage embryo are divided into trophectoderm cells which comprise the placenta and inner cell mass cells which will develop into the fetus. More than one cell can be safely removed without compromising embryo viability at this stage and may allow for more accurate test results. This method is called a trophectoderm biopsy (HFEA, 2014).

The latest stage for embryo biopsy is the blastocyst stage. Many more cells can be available for PCR analysis at this stage than at earlier developmental stages. However, biopsy this late in development creates issues in rapid performance of the PCR analysis because the transfer of the normal embryos to the uterus must be performed before day 6 of in vitro embryo growth. Recently, studies have shown a higher success rate when cryopreserved or vitrified embryos are utilized to alleviated the issue of rapid laboratory turnaround and this has led to a shift from fresh embryo transfers to use of cryopreserved embryos. In these cases, embryo transfer is performed at a subsequent ovulatory cycle. Additionally, approximately one-third of embryos grow in vitro to this stage, therefore fewer embryos are available for analysis. This has improved the efficiency of the genetic testing process.

It has also been demonstrated that polar bodies, the small cells that are extruded after the first meiosis at ovulation and the second meiosis at fertilization, can be analyzed in order to determine the status of the egg prior to insemination. These cells do not develop into the embryo proper. The polar bodies represent only the female genome, and can be safely removed from embryos with little effect on IVF rates afterward. An opening can be produced in the zona pellucida, the glycoprotein layer of the egg that surrounds the plasma membrane, through the use of laser or a needle. The polar body can be carefully removed for genetic analysis using a specialized pipette.

Verlinsky and colleagues (2004b) perfected the PGD approach on the first polar body and then later determined that the second polar body should be tested for validation purposes (Evers-Kiebooms et al., 2002; Moutou et al., 2004). In a series of first and second polar body analyses performed for mutation detection for single-gene disorders, the laboratory was able to identify correctly the presence of a mutation in 98% (157 of 160) of oocytes tested (Moutou et al., 2004).

Once the polar bodies, blastomeres, or trophectoderm samples are obtained, PCR is used to amplify gene-specific DNA for confirmation of the mutation in the representative embryo (HDSA, 2015). The cells are placed in a solution that lyses the cell and releases the DNA. A PCR reaction mix is added; this contains primers which are specific for the gene of interest. Due to the possibility of contamination with unrelated DNA, scrupulous laboratory procedures and standards, such as the use of intracytoplasmic sperm injection, have been adopted (Sermon, 2001). One possible issue with the PCR technique is allele dropout, in which only one and not both of the genes in the diploid cell is amplified. This can lead to misdiagnosis of disease and the transfer of embryos containing the disease gene (Ly et al., 2011; Piyamongkol et al., 2015). More recent techniques for evaluation of the PCR fragments, such as automated sequencing, mini-sequencing, and real-time PCR, have assisted in maintaining the sensitivity of the PCR studies (Thornhill and Snow, 2002).

Families interested in learning more about PGD should request a referral to a trusted center from their family HD clinic or their gynecologist. They will be required to have a copy of the DNA test results from the index case in the family to validate the diagnosis. In cases where potential parents do not wish to undergo HD DNA testing, then the DNA result from the index case in the family can be used. A complete family history should be taken to assure that the potential HD-free offspring has no other genetic disease. Carrier testing for genetic diseases or preconception screening of over 100 genes can be performed economically and quickly due to advances in sequencing technology. Testing may be offered sequentially for recessive diseases where, if one partner tests positive for any condition, the other partner would then be offered screening for that condition.

The potential parents will review the risks and benefits of the procedure during and after a consultation with the physician. In general, it is thought that the medical risks of PGD are similar to those from conventional IVF and include issues related to superovulation, multiple births, and rare birth defects (NHS, 2015). However, with PGD there is a risk that some embryos become compromised by the process of biopsy, vitrification, and warming, which may impact the success rate. There is also the risk, as with any laboratory test, that the DNA testing is not 100% reliable (HFEA, 2014).

PGD is a complex collection of medical and genetic procedures and is expensive. One cycle of IVF costs about US$9,000 (RHTP, 2015). The addition of PGD increases the cost by US$4,000–7,500 dollars for each IVF procedure. Successful cycles resulting in a healthy live birth are affected by many factors, including the age of the female partner and fertility history. Depending on the particular case, it may be necessary to undergo multiple IVF attempts to achieve an unaffected embryo or viable pregnancy. A successful pregnancy is highly costly and may be prohibitive to HD and other prospective parents with risks of transmitting genetic diseases. The cost of PGD is likely to decrease over time as the procedures for gene analysis become more applicable to PGD. It is also possible that some insurance coverage is provided to the family for the procedure.

In order to improve the efficiency of the IVF/PGD process, often the embryos are subsequently tested for aneuploidy using inexpensive techniques such as array comparative genomic hybridization (CGH). If an embryo is numerically chromosomally normal, it has a much higher chance of implantation and development. This also encourages the transfer of only one embryo, reducing the risk of multiple gestations, which can affect the health of the baby.

It is possible that, after the PGD, embryos in excess of those used for an embryo transfer may be cryopreserved for use in a subsequent cycle. The survival rate of these embryos is excellent with the newer vitrification techniques. Vitrification allows for rapid freezing of cells without the production of ice crystallization which can affect cell viability when thawing.

Cryogenically frozen embryos can be analyzed with CGH for the detection of aneuploidy. This may become the standard in the future, especially as parents with HD are not usually undergoing the IVF process due to fertility problems; therefore they may have more successful results.

It is difficult to assess success rates for PGD because there are currently few data available. Most potential parents who undergo PGD do not have fertility problems and may have more successful results. The issues which may impact the success rate negatively are that not enough eggs are produced or fertilized, the removal of the polar body or blastomere damages the embryo, which is less significant with trophectoderm biopsy, and many or all of the embryos contain the mutations (Moutou et al., 2004). It was reported that, in 2010, 311 women received 383 cycles of PGD. These cycles resulted in 121 live births (live birth rate of 31.6% per cycle started) (HFEA, 2014).

A 2012 study reviewed 13 years of experience of PGD for HD at three European PGD practice groups in Brussels, Maastricht, and Strasbourg (Van Rij et al., 2012). Combined, 331 PGD cases specifically presented with HD family histories. Out of 331 couples, 68% requested direct testing and 32% exclusion testing. Of the total couples, 39% of the women had experienced previous pregnancies. Pregnancy termination following standard prenatal diagnosis for HD was more frequent in the direct testing group (25%) than in the group that requested exclusion testing (10%; p =.0027). A total of 389 cycles continued to the oocyte retrieval step of the process. The delivery rates of unaffected offspring per retrieval were 19.8%, and per embryo transfer 24.8%, resulting in 77 deliveries and the birth of 90 children from this cohort. There is no comparable publication that reviews the collective experience for PGD in HD from groups in Canada or the United States.

The professional guidelines for the practice of PGD are limited. The American Society for Reproductive Medicine (ASRM) issued a practice committee opinion stating that:

PGD appears to be a viable alternative to post-conception diagnosis and pregnancy termination, acknowledging the possibility of diagnostic errors and unknown long-term consequences on the fetus. ASRM also has issued an ethics committee opinion cautioning against the use of PGD for sex selection in the absence of a serious sex-linked diseases.

Two other groups, the International Society of Preimplantation Genetic Diagnosis (PGDIS) and the European Society for Human Reproduction and Embryology (ESHRE), have developed guidelines. PGDIS has developed Guidelines for Good Practice in PGD. The document outlines establishing a PGD program, IVF and PGD clinical and laboratory protocols, embryo transfer, spare embryos, follow-up of pregnancy, and quality control and assurance. ESHRE tracks PGD outcomes on a voluntary basis, but captures primarily European data (RHTP, 2015).

CONCLUSIONS

PGD provides benefits to individuals at risk for HD or positive for the gene. It allows them to prevent having offspring with HD without abortion of a fetus, and PGD can also spare “at-risk” individuals the knowledge of their gene status. PGD analyses were developed with the intention of determining X-linked disease genes in “at-risk” embryos. Single-cell PCR methods were used, and have since been enhanced and complemented by other molecular techniques which can identify chromosomally abnormal embryos. The PGD procedure for prospective parents with or at risk of HD involves IVF treatment, additional genetic analysis of one cell from each embryo, and the transfer of HD gene-free embryos to mothers. In the case of parents who are at risk of passing the HD gene on due to family history, but do not wish to know their status, exclusion or nondisclosure PGD can be performed. The physician transfers unaffected embryos, but does not reveal HD mutation status.

PGD may be financially costly and out of reach for some families, but as techniques become more efficient and successful it is likely that costs will be reduced. The procedure has variable success rates per cycle depending upon fertility of the couple. Cryogenically frozen embryos can be analyzed with CGH for the detection of aneuploidy. This may become the standard in the future, especially as parents with HD are not necessarily undergoing the IVF process because of fertility problems and may achieve better results as HD has not been associated with reproductive abnormalities, unlike other genetic diseases such as dystrophia myotonica 1 or myotonic dystrophy. Additional dimensions to PGD may continue to develop in the future, as more molecular techniques develop to enhance satisfactory results, more success rates are obtained, and professional guidelines for the practice of PGD become more uniform.