Combining PGT-A with PGT-M risks trying to do too much.

The primary objective of preimplantation genetic testing for monogenic disorders (PGT-M) is to avoid having a child with a serious monogenic disease. Combining testing for unrelated sporadic chromosomal abnormalities (PGT-A) and excluding embryos with chromosomally abnormal results from transfer proffers the chance to mitigate the risk of miscarriage and to reduce the number of embryo transfers, but also risks excluding healthy embryos from transfer due to abnormal test results that do not reflect the true potential of the embryo. The theoretical utility of combining PGT-M with PGT-A is explored in this communication. It is concluded that PGT-M without PGT-A is preferred to achieve an unaffected live birth. Since PGT-M is mostly undertaken by couples where the female partner is younger than 35 years, PGT-A is likely to marginally mitigate the risk of miscarriage. Experimental non-selection studies are needed to assess the potential detrimental effect of combining PGT-M with PGT-A in a clinical setting.

Carrier screening and PGT for an autosomal recessive monogenic disorder: insights from virtual trials.

PURPOSE: To assess the costs and benefits of carrier screening and preimplantation genetic testing (PGT) for recessive autosomal monogenic disorders for couples attempting assisted conception. METHODS: A simulated first full cycle for women less than 35 years transferring embryos one at a time. The effect of testing on pregnancy outcomes was evaluated for different reporting scenarios. A Monte Carlo method utilising 1000 trials for 10,000 couples, testing 4, 16 and 38 genes, was used to assess the numbers likely to be at high risk and to estimate the incremental cost of screening and PGT to avoid an affected child. RESULTS: PGT for high-risk couples: testing embryos only for the monogenic condition avoided 1 affected pregnancy for 4 cycles started. Combined with testing for chromosomal aneuploidy: ranking test results avoided 1 adverse pregnancy (affected, biochemical, clinical miscarriage) from 3 cycles started; 1 in 2 when excluding from transfer all embryos with an abnormal test result, within 1 in 25 fewer women achieving an unaffected live birth. Carrier screening for 4, 16 and 38 gene scenarios, where 1:250, 1:196 and 1:29 couples were at high risk: the incremental cost to prevent 1 affected live birth was estimated to be less than GBP 1,150,000 (US $1,587,000), < 836,642 (1,154,566) and < 137,794 (190,156), respectively, in 95% of trials. CONCLUSIONS: Carrier screening combined with PGT, with and without testing for unrelated chromosomal abnormalities, for couples attempting assisted conception is complex but likely to be effective and also expensive.

Elucidating the PGT-A paradox: marginalising the detriment relegates the benefit.

The hypothetical analysis presented offers insight into the effect of maternal age and protocol on the cost-effectiveness of PGT-A to reduce the risk of clinical miscarriage without materially jeopardising the chance of a first live birth when attempting to transfer every suitable embryo one at a time. Reflecting current practices, the diagnostic accuracy of PGT-A is sensitive to the prevalence of embryos with chromosome aneuploidy which increases with advancing maternal age, and the power of the test to discern a non-viable embryo is higher for older women and sensitive to protocol. PGT-A is effective to mitigate (reduce not eliminate) the risk of clinical miscarriage; however, excluding embryos with intermediate copy number results from transfer is detrimental to accomplishing a first live birth from a full cycle. Paradoxically, the number of blastocysts needed to marginalise the detriment is achieved only for some younger women (≤ 40 years) who are less likely to benefit by avoiding pregnancy loss; this also makes PGT-A an expensive adjuvant. The paradox can be avoided by excluding from transfer only embryos with a 'uniform' aneuploid test result, which mitigates the risk of miscarriage for all women with the potential to be cost-effective for those > 40 years.

PGT-SR (reciprocal translocation) using trophectoderm sampling and next-generation sequencing: insights from a virtual trial.

PURPOSE: The objective of this virtual study was to simulate a full cycle and assess the costs and benefits to a couple with a reciprocal translocation, using current techniques for preimplantation genetic testing and comparing reporting every chromosome with only reporting those involved in the rearrangement. METHODS: A simulation was constructed for women under the age of 35 years, where vitrified-warmed embryos were transferred one at a time in a first full cycle following preimplantation genetic testing using next-generation sequencing and sampling the trophectoderm at the blastocyst stage. The effect on pregnancy outcomes (live birth, clinical miscarriage and biochemical pregnancy) was evaluated for different reporting strategies for embryo transfer to (i) report only the rearranged chromosomes and transfer embryos with a normal or balanced test result for the translocation (targeted), or (ii) report every chromosome and exclude from transfer all embryos with an abnormal test result (exclusion), or (iii) exclude only those consistent with an unbalanced translocation and/or unrelated non-mosaic whole-chromosome aneuploidy and assign those with samples consistent with a normal or balanced translocation complement and unrelated mosaic aneuploidy or segmental imbalance (embryos of uncertain reproductive potential) a lower transfer priority (ranking). The number of individual women whom might benefit by avoiding an adverse pregnancy outcome (biochemical pregnancy, clinical miscarriage) or be disadvantaged by not achieving a live birth was evaluated. The financial cost of the different reporting strategies and the time taken to complete a cycle were also considered. RESULTS: The simulation showed that compared to only reporting the translocation chromosomes (targeted reporting), testing every chromosome and ranking embryos by test result for transfer was a cost-effective strategy to avoid an adverse pregnancy without compromising the chance of a live birth. Excluding from transfer embryos with a test result consistent with a normal or balanced translocation complement of uncertain reproductive potential was an inferior strategy because it resulted in fewer live births from a full cycle. Reporting only the translocation chromosomes was an inferior strategy because it was less effective than ranked reporting of every chromosome to avoid an adverse pregnancy. Compared to targeted reporting, the ranked and exclusion strategies marginally reduced the overall cost and time taken to complete a full cycle. The ranking strategy avoided 1 adverse pregnancy for 12 cycles started, compared to 1 in 10 for the exclusion strategy which also resulted in 1 in 22 fewer women achieving a live birth. A minority (< 10%) of couples benefited by avoiding at least 1 adverse pregnancy whilst also reducing the time and cost for a complete cycle; most (> 70% ) couples received no benefit additional to targeted reporting and had the same outcome for pregnancy, time and cost. CONCLUSIONS: The primary objective of PGT-SR for couples with a reciprocal translocation is to avoid a pregnancy with a chromosomally unbalanced product of the translocation and to reduce the risk of miscarriage, at least to that expected for couples with normal karyotypes. Trophectoderm sampling at the blastocyst stage with testing using NGS is an effective approach; however, ranking and excluding from transfer embryos with abnormal test results for unrelated chromosomes is problematical and is likely to be detrimental to achieving a live birth. Targeted reporting, where only the results of the chromosomes involved in the translocation are known, should be preferred to achieve a live birth. A best effort should be made to follow up and investigate all pregnancies following PGT-SR. Once the reproductive outcome is known (biochemical pregnancy, clinical pregnancy, live birth), the chromosomes unrelated to the rearrangement can be analysed as an experimental study. The risk/benefit of avoiding an adverse pregnancy vs reducing the chance of a healthy delivery should be a decision for each individual couple and informed by appropriate genetic counselling for their specific translocation and history.

A tale of two studies: now is no longer the best of times for preimplantation genetic testing for aneuploidy (PGT-A).

Preimplantation genetic testing for aneuploidy (PGT-A) does not create normal embryos, but selecting a viable embryo for a fresh transfer has the potential to deliver an extra effect for live birth from a stimulated cycle by evading the attrition associated with embryo cryopreservation. Improved genetic tests are now available for selecting viable embryos; however, current embryo cryopreservation techniques also have a superior survival rate, which means it is now possible to transfer most morphologically suitable embryos from a stimulated cycle one at a time. The cumulative live birth rate from a stimulated cycle is now unlikely to be superior compared with morphological assessment alone, with any benefit likely to be associated with a reduction in the risk of miscarriage and the time to pregnancy. This communication offers a perspective on the likely benefit and disbenefit of PGT-A based on the outcome of modern-day clinical studies. Caution should be advised regarding offering PGT-A to every woman. Quantifying the likely miscarriage benefit and live birth disbenefit for an appropriate patient group may help to better inform couples who might be considering adding aneuploidy screening to their treatment cycle.

Squaring the circle of recurrent pregnancy loss (RPL).

The recent publication of a study into the contribution of embryo chromosomal abnormalities in recurrent pregnancy loss (RPL) affords the opportunity to revisit the hypothesis that women with an aneuploid pregnancy loss have a better chance of a successful pregnancy next time than women with a chromosomally normal loss. A previous miscarriage with an abnormal karyotype (unrelated to a parental chromosome rearrangement) should not be viewed as a marker of an increased likelihood of aneuploidy in a subsequent pregnancy; it is (counterintuitively) likely to be indicative of a reduced risk of clinical miscarriage (with a higher proportion of aneuploid products) and an excellent chance for the live birth of the next pregnancy. Each couple should be treated on their own merits and with appropriate investigations performed where indicated; caution should be advised regarding offering preimplantation genetic testing for aneuploidy (PGT-A).

Towards a better understanding of preimplantation genetic screening for aneuploidy: insights from a virtual trial for women under the age of 40 when transferring embryos one at a time.

BACKGROUND: The aim of this theoretical study is to explore the cost-effectiveness of aneuploidy screening in a UK setting for every woman aged under the age of 40 years when fresh and vitrified-warmed embryos are transferred one at a time in a first full cycle of assisted conception. METHODS: It is envisaged that a 24-chromosome genetic test for aneuploidy could be used to exclude embryos with an abnormal test result from transfer, or used only to rank embryos with the highest potential to be viable; the effect on cumulative outcome is assessed. The cost associated with one additional live birth event and one clinical miscarriage avoided is estimated, and the time taken to complete a cycle considered. The numbers of individual woman for whom testing is likely to be beneficial or detrimental is also evaluated. RESULTS: Adding aneuploidy screening to a first treatment cycle is unlikely to result in a higher chance of a live birth event, and can be detrimental for some women. Premature termination of a clinical trial is likely to be biased in favour of genetic testing. Testing is likely to be an expensive way of reducing the chance of clinical miscarriage and shortening treatment time without a substantial reduction in the cost of testing, and is likely to benefit a minority of women. Selecting out embryos is likely to reduce the treatment time for women whether or not they have a baby, whilst ranking embryos only to reduce the time for those that have a child and not for those who need another stimulated cycle. CONCLUSIONS: Adding aneuploidy screening to IVF treatment for women under the age of 40 years is unlikely to be beneficial for most women. To achieve an unbiased assessment of the cost-effectiveness of genetic testing for aneuploidy, clinical trials need to take account of women who still have embryos available for transfer at the end of the study period. Specifying the proportions of women for whom testing is likely to be beneficial and detrimental may help better inform couples who might be considering adding aneuploidy screening to their treatment cycle.

Successful PGD cycles for mosaic Robertsonian translocation carriers provide insights into the mechanism of formation of the derivative chromosomes.

Preimplantation genetic diagnosis (PGD) has been carried out for two couples with different mosaic Robertsonian translocations. Two PGD cycles for a mosaic 13;13 homologous Robertsonian translocation carrier resulted in the birth of a healthy child in each cycle, illustrating the importance of scanning G-banded preparations from homologous Robertsonian carriers for the presence of a normal cell line. One couple was referred for PGD because the male partner carried a mosaic 14;15 Robertsonian translocation with a normal cell line. A single PGD cycle resulted in the birth of a healthy child. Follow-up studies and extended FISH analysis of the carrier's lymphocytes detected three cell lines, two carrying different 14;15 Robertsonian chromosomes and one normal cell line. The two 14;15 Robertsonian chromosomes had different breakpoints in the proximal short arm regions. We suggest that the presence of the D15Z1 polymorphism on the short arm of one chromosome 14 mediated the post-zygotic formation of the two different Robertsonian chromosomes.

Embryo selection in IVF: is polar body array comparative genomic hybridization accurate enough?

The emergence of the array comparative genomic hybridization technique (aCGH) is considered an advance in preimplantation genetic testing. Analysis of the recently published pilot study using polar body aCGH indicates that the test accuracy compares favourably with the fluorescence in situ hybridization technique although a substantial number of euploid zygotes are still likely to be excluded incorrectly. A sound argument against selection in principle has recently been published, based on accumulating evidence that potentially all embryos can now be cryopreserved and transferred in subsequent frozen replacement cycles without impairing pregnancy rates. We suggest that vitrification and serial transfer without testing are likely to give patients the best chance for a successful pregnancy, and avoid the use of an expensive technology.

Fluorescence in situ hybridization on single cells. (Sex determination and chromosome rearrangements).

Fluorescence in situ hybridization (FISH) is the technique of choice for preimplantation genetic diagnosis (PGD) selection of female embryos in families with X-linked disease, for which there is no mutation-specific test. FISH with target-specific DNA probes is also the primary technique used for PGD detection of chromosome imbalance associated with Robertsonian translocations, reciprocal translocations, inversions, and other chromosome rearrangements, because the DNA probes, labeled with different fluorochromes or haptens, detect the copy number of their target loci. The methods described outline strategies for PGD for sex determination and chromosome rearrangements. These methods are assessment of reproductive risks, the selection of suitable probes for interphase FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring using directly labeled and indirectly labeled probes.

Preimplantation genetic diagnosis--an overview.

Since the early 1990s, preimplantation genetic diagnosis (PGD) has been expanding in scope and applications. Selection of female embryos to avoid X-linked disease was carried out first by polymerase chain reaction, then by fluorescence in situ hybridization (FISH), and an ever-increasing number of tests for monogenic diseases have been developed. Couples with chromosome rearrangements such as Robertsonian and reciprocal translocations form a large referral group for most PGD centers and present a special challenge, due to the large number of genetically unbalanced embryos generated by meiotic segregation. Early protocols used blastomeres biopsied from cleavage-stage embryos; testing of first and second polar bodies is now a routine alternative, and blastocyst biopsy can also be used. More recently, the technology has been harnessed to provide PGD-AS, or aneuploidy screening. FISH probes specific for chromosomes commonly found to be aneuploid in early pregnancy loss are used to test blastomeres for aneuploidy, with the aim of replacing euploid embryos and increasing pregnancy rates in groups of women who have poor IVF success rates. More recent application of PGD to areas such as HLA typing and social sex selection have stoked public controversy and concern, while provoking interesting ethical debates and keeping PGD firmly in the public eye.

Meiotic outcomes in reciprocal translocation carriers ascertained in 3-day human embryos.

Chromosomes involved in reciprocal translocations form quadrivalents at meiosis. These quadrivalents segregate, with or without recombination, to give 32 different meiotic outcomes, only two of which are normal or balanced. This paper presents data collected from 25 cycles of preimplantation genetic diagnosis for 18 couples carrying 15 different reciprocal translocations. Embryos were tested using fluorescence in situ hybridisation with probes for the translocated and centric segments. Overall, 47.7% (71 out of 149) of embryos tested showed signal patterns consistent with alternate segregation, 24.8% adjacent-1 segregation, 10.1% adjacent-2 segregation, 15.4% 3 : 1 segregation and 2% 4 : 0 segregation. For most translocations, alternate segregation was apparently the most frequent mode. Alternate and adjacent-1 frequencies were similar in male and female carriers; however, 5.7% of embryos from female translocation carriers showed adjacent-2 segregation and 20.0% showed 3 : 1 segregation, whilst the corresponding figures for male carriers were 20.5 and 4.5%. Overall, 2.8% of embryos were mosaic and 2.3% of embryos showed chaotic constitutions for the chromosomes tested. The pregnancy success rate for these 25 cycles was 38.8% per embryo transfer and also 38.8% per couple.

Meiotic outcomes of three-way translocations ascertained in cleavage-stage embryos: refinement of reproductive risks and implications for PGD.

Our study provides an analysis of the outcome of meiotic segregation of three-way translocations in cleavage-stage embryos and the accuracy and limitations of preimplantation genetic diagnosis (PGD) using the fluorescence in situ hybridization technique. We propose a general model for estimating reproductive risks for carriers of this class of complex chromosome rearrangement. The data presented describe six cycles for four couples where one partner has a three-way translocation. For male heterozygotes, 27.6% of embryos were consistent with 3:3 alternate segregation resulting in a normal or balanced translocation chromosome complement; 41.4% were consistent with 3:3 adjacent segregation of the translocations, comprising 6.9% reflecting adjacent-1 and 34.5% adjacent-2 segregation; 24.1% were consistent with 4:2 nondisjunction; none showed 5:1 or 6:0 segregation; the probable mode could not be ascertained for 6.9% of embryos due to complex mosaicism or nucleus fragmentation. The test accuracy for male heterozygotes was estimated to be 93.1% with 100% sensitivity and 75% specificity. With 72.4% prevalence, the predictive value was estimated to be 91.3% for an abnormal test result and 100% for a normal test result. Two of four couples had a healthy baby following PGD. The proportion of normal/balanced embryo could be significantly less for female heterozygotes, and our model indicates that this could be detrimental to the effectiveness of PGD. A 20% risk of live-born offspring with an unbalanced translocation is generally accepted, largely based on the obstetric history of female heterozygotes; we suggest that a 3% risk may be more appropriate for male carriers.

Benefits and drawbacks of preimplantation genetic diagnosis (PGD) for reciprocal translocations: lessons from a prospective cohort study.

Preimplantation genetic diagnosis (PGD) using fluorescence in situ hybridisation probes was carried out for 59 couples carrying reciprocal translocations. Before treatment, 85% of pregnancies had resulted in spontaneous miscarriage and five couples had achieved a healthy live-birth delivery. Following treatment, 33% of pregnancies failed and 21 of 59 couples had a healthy live-born child. The accuracy of diagnosis was 92% (8% false abnormal and 0% false normal results). The overall incidence of 2:2 alternate segregation products was 44%; however, products consistent with 2:2 adjacent segregation were ~twice as likely from male heterozygotes, and those with 3:1 disjunction were three times more likely from female heterozygotes. Our results indicate that up to three stimulation cycles per couple would give an ~50% chance of a successful live birth, with the risk of miscarriage reduced to the level found in the general population. In our study, 87% of all normal/balanced embryos available were identified as being suitable for transfer. We conclude that PGD provides benefit for couples with high-risk translocations by reducing the risk of miscarriage and avoiding a pregnancy with an unbalanced form of the translocation; however, for fertile carriers of translocations with a low risk of conceiving a chromosomally unbalanced offspring, natural conception may be a more viable option.

FISH for pre-implantation genetic diagnosis.

Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a cohort generated by assisted reproduction technology (ART). This selection may be required because of familial monogenic disease (e.g. cystic fibrosis), or because one partner carries a chromosome rearrangement (e.g. a two-way reciprocal translocation). PGD is available for couples who have had previous affected children, and/or in the case of chromosome rearrangements, recurrent miscarriages, or infertility. Oocytes aspirated following ovarian stimulation are fertilized by in vitro immersion in semen (IVF) or by intracytoplasmic injection of an individual spermatozoon (ICSI). Pre-implantation cleavage-stage embryos are biopsied, usually by the removal of a single cell on day 3 post-fertilization, and the biopsied cell is tested to establish the genetic status of the embryo. Fluorescence in situ hybridization (FISH) on the fixed nuclei of biopsied cells with target-specific DNA probes is the technique of choice to detect chromosome imbalance associated with chromosome rearrangements, and to select female embryos in families with X-linked disease for which there is no mutation-specific test. FISH has also been used to screen embryos for spontaneous chromosome aneuploidy (also known as PGS or PGD-AS) in order to try and improve the efficiency of assisted reproduction; however, the predictive value of this test using the spreading and FISH technique described here is likely to be unacceptably low in most people's hands and it is not recommended for routine clinical use. We describe the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to PGD in a clinical setting.