Preimplantation diagnosis for aneuploidies in assisted reproduction

At least one half of oocytes and preimplantation embryos are aneuploid and have to be avoided from transfer in in vitro fertilization (IVF) patients of advanced reproductive age. This can now be done by preimplantation genetic diagnosis, which has recently become an integral part of assisted reproduction technologies and was shown to improve implantation rate and reduce spontaneous abortions after implantation. The experience of approximately 5000 preimplantation genetic diagnosis (PGD) cycles performed for poor prognosis IVF patients have already resulted in birth of approximately 1000 apparently healthy children, suggesting that this novel technique is safe and reliable, and may in future replace the current IVF practice of preselection of embryos for transfer based on morphological parameters.     

Over a decade of experience with preimplantation genetic diagnosis

The three respondents provide additional support for preimplantation genetic diagnosis (PGD) having the pivotal place it now has in prenatal genetic diagnosis: chromosomal abnormalities (e.g., unbalanced translocations), Mendelian disorders, and HLA typing for transfer of compatible, genetically normal, embryos. Transferring euploid embryos has decreased the clinical abortion rate and increased the implantation rate in assisted reproductive technologies (ART), but it has not necessarily improved the live-birth rate. Safer embryo biopsy, more extensive diagnostic efforts (i.e., microarray analysis), and more refined patient selection may be required before shifting from preselection of embryos based solely on morphological parameters to transfer of only aneuploidy-free embryos.     

Should every embryo undergo preimplantation genetic testing for aneuploidy? A review of the modern approach to in vitro fertilization

Aneuploid conceptions constitute the majority of pregnancy failures in women of advanced maternal age. The best way to combat age-related decline in fertility is through preimplantation genetic testing for aneuploidy (PGT-A). PGT-A allows for better embryo selection, which improves implantation rates with single embryo transfer and reduces miscarriage rates. Single embryo transfers decrease multiple gestations and adverse pregnancy outcomes such as preterm or low birth weight infants. Advancements in extended embryo culture, blastocyst biopsy techniques, and 24-chromosome aneuploidy screening platforms have made PGT-A safe and accessible for all patients who undergo in vitro fertilization. Improved genomic coverage of new sequencing platforms, such as next-generation sequencing, has increased the identification and diagnosis of mosaicism and partial aneuploidies in preimplantation embryos. Mosaic embryos have decreased viability compared to euploid embryos when transferred, but some mosaic embryos result in normal live births. Whole genome amplification artifacts may contribute to a misdiagnosis of mosaicism, or some mosaic embryos may self-correct to euploid after implantation. For this reason, patients without euploid embryos should be given the option of transferring mosaic embryos after genetic counseling. Further research is needed to characterize which mosaic embryos may be viable.     

Prevention of Age-Related Aneuploidies by Polar Body Testing of Oocytes

Purpose: We previously demonstrated that aneuploidy-free oocytes may be preselected by testing the first and second polar bodies removed from oocytes following their maturation and fertilization. The present paper describes the results of the application of the method in 659 in vitro fertilization cycles from patients of advanced maternal age. Methods: Using micromanipulation techniques, 3943 oocytes were tested by polar body sampling and fluorescent in situ hybridization analysis using specific probes for chromosomes 13, 18, and 21. Results: Fluorescent in situ hybridization results were available for 3217 (81.6%) of 3943 oocytes studied, of which 1388 (43.1%) had aneuploidies; 35.7% of the aneuploidies were of first meiotic division origin, and 26.1% of second meiotic division origin. Most errors in the first meiotic division were represented by chromatid malsegregation. The transfer of embryos deriving from 1558 of 1829 aneuploidy-free oocytes in 614 treatment cycles resulted in 131 clinical pregnancies and 88 healthy children born after confirmation of the polar body diagnosis. Conclusions: Polar body testing of oocytes provides an accurate and reliable approach for prevention of age-related aneuploidies in in vitro fertilization patients of advanced maternal age.     

Pregnancies from single normal embryo transfer in women older than 40 years

The aim of this retrospective study was to determine the pregnancy rate from the transfer of single genetically normal embryos in patients of advanced reproductive age. The study group included 23 patients (mean age 42.2 +/- 1.3 years) who underwent 27 in-vitro fertilization (IVF) cycles in which preimplantation genetic diagnosis (PGD) was carried out on single blastomeres from day 3 embryos. The control group included 54 patients (mean age 43.3 +/-1.9 years) who underwent 69 cycles of IVF without PGD. Ovarian stimulation in all patients consisted of follicular phase leuprolide acetate administration, followed by ovulation induction with gonadotrophins. The mean number of biopsied embryos was 5.6 +/- 0.5. No embryo transfer occurred in six patients (10 cycles) because all embryos biopsied were abnormal. Seventeen patients (17 cycles) each had one genetically normal embryo transferred resulting in six on-going clinical pregnancies (35% per embryo transfer cycle). The mean number of embryos transferred in the control group was 4.0 +/- 0.8. Nineteen clinical pregnancies were obtained in 69 transfer cycles in the control group (28% per embryo transfer cycle). The transfer of a single normal embryo in patients of advanced reproductive age can lead to acceptable pregnancy rates. Aneuploidy appears to be a major cause of reproductive failure in this group of patients.     

The role of preimplantation diagnosis for aneuploidies

The clinical application of preimplantation genetic diagnosis (PGD) for aneuploidy has confirmed the hypothesis that implantation failure and spontaneous abortions are frequently due to aneuploidy. Following PGD, a higher implantation rate and a lower incidence of spontaneous abortions are obtained in patient categories where aneuploidy is the main cause of reproductive failure: women in advanced reproductive age, patients with an altered karyotype due to translocations or gonosomal mosaicism, and patients with recurrent spontaneous abortions. In these cases, the transfer of euploid embryos overcomes the poor prognosis condition in these couples. As expected, aneuploidy increases proportionally with female age; however, not all the chromosomes studied show this trend, suggesting that segregation errors could occur at different rates for each chromosome in relation to maternal age. Furthermore, the retrospective analysis of the results obtained in patients who repeated at least twice a PGD cycle permitted to estimate their chances of reproducing the same pattern of chromosomal abnormalities and consequently evaluating their possibility of a pregnancy: when no euploid embryos are detected at the first attempt, the chance of on-term pregnancy is below 10%; however, this chance is approximately 30% for couples with at least two euploid embryos in the first cycle.     

Preimplantation HLA typing with aneuploidy testing

Preimplantation HLA typing has been introduced for the treatment of affected siblings, requiring HLA-identical stem cell transplantation. This was applied either in combination with preimplantation genetic diagnosis (PGD) to ensure that the preselected HLA-matched embryos were also free of the genetic disorder, or without PGD, with the only purpose of selecting and transferring the HLA-matched embryos. Because patients requesting preimplantation HLA typing are usually of advanced reproductive age, aneuploidy testing allows not only the avoidance of the birth of children with chromosomal disorders, but also improvement of the reproductive outcome, which is still not sufficiently high in preimplantation HLA typing at the present time. This study presents the results of the first experience of preimplantation HLA typing combined with aneuploidy testing, demonstrating feasibility and impact of aneuploidy testing on the accuracy and outcome of preimplantation HLA typing. Of a total of 138 cycles performed, 87 were combined with PGD and 52 without testing for the causative gene, of which aneuploidy testing was performed in 27 cycles, allowing the preselection and transfer of only those HLA-matched embryos that were also euploid. Although the euploid HLA-identical embryos were available for transfer in only half of these cycles, pregnancy and birth of unaffected HLA-identical children were observed in approximately half of these cycles, suggesting the potential usefulness of incorporating aneuploidy testing into preimplantation HLA typing.     

Preimplantation genetic diagnosis for aneuploidy screening in early human embryos: a review

Embryonic aneuploidies may be responsible for pregnancy failure in many IVF patients. In recent years, fluorescent in situ hybridisation (FISH) for multiple chromosomes has been used to document a high frequency of chromosomal errors and aneuploidy in human preimplantation embryos and, after embryo biopsy, to select embryos that are more likely to implant. Such studies suggest that women with recurrent miscarriage and advanced maternal age may benefit most from preimplantation genetic diagnosis with aneuploidy screening (PGD-AS). The success of PGD-AS is likely to be enhanced by new technologies, such as comparative genomic hybridisation, which enable full karyotyping of single cells.     

The role for preimplantation genetic diagnosis in balanced translocation carriers

OBJECTIVE: Preimplantation genetic diagnosis is an established technique that provides an alternative to prenatal diagnosis for patients who are at risk of transmitting a serious genetic disorder to their offspring. Preimplantation genetic diagnosis has been used for couples who have been at risk for having offspring with single gene or X-linked disorders and for screening for common age-related aneuploidy and in couples who themselves carry balanced chromosomal rearrangements. The aim of this study was to summarize our experience using preimplantation genetic diagnosis after the identification of a parental balanced translocation, specifically as it relates to the number of embryos that are suitable for transfer after preimplantation genetic diagnosis for a known translocation and aneuploidy screening. STUDY DESIGN: This is a retrospective review of data from a single center that involved 6 couples that initiated the process of preimplantation genetic diagnosis for translocation and aneuploidy screening by fluorescent in situ hybridization. RESULTS: A total of 65 embryos were obtained, of which 56 embryos (86%) were suitable for fluorescent in situ hybridization analysis. After fluorescent in situ hybridization, 1 embryo was diagnosed as normal or balanced (1.7%). Forty-three embryos (76.8%) were unbalanced for the translocation; 8 embryos (14.3%) were aneuploid, and 4 embryos (7.1%) were uninformative. There were no clinical pregnancies. CONCLUSION: In our experience, there are very few embryos that are available for transfer from these patients after translocation and aneuploidy screening because of multiple unbalanced segregation products and a high rate of aneuploidy. Factors that contributed to this may be related to which parent carries the translocation, methods that were used for in vitro fertilization, and advanced maternal age. Although preimplantation genetic diagnosis for translocation carriers theoretically can enhance the pregnancy rate for a couple, there are limitations. This information should be shared with couples who are contemplating preimplantation genetic diagnosis for translocation, and the options of sperm or egg donor should be considered.     

Impact of chromosomal abnormalities on preimplantation embryo development

OBJECTIVES: To evaluate the influence of numerical chromosomal abnormalities on preimplantation embryo development. METHODS: This study includes 6936 embryos from 1245 women undergoing preimplantation genetic diagnosis (PGD). Indications for aneuploidy screening were: recurrent miscarriages, implantation failure, severe male factor, advanced maternal age, and mixed causes. Embryo biopsy was performed on day 3, and embryos were co-cultured until day 5, when embryo transfer was performed. RESULTS: In the aneuploidy screening regimen, normal euploid embryos showed significantly higher blastocyst rates (68.2%) compared to chromosomally abnormal (42.8%, p < 0.0001) and mosaic (53.7%, p < 0.0001) embryos. Among aneuploid embryos for autosomes, higher blastocyst rates were observed in trisomies than monosomies, although only statistically significant in patients over 36 years of age (50.8 vs 38.9%; p < 0.0001). In contrast, in embryos with sex chromosomes aneuploidy, similar blastocyst rates were observed between trisomies and monosomy X. CONCLUSION: Embryos with certain types of chromosomal abnormalities were negatively selected during preimplantation embryo development. Despite this selection, a remarkable percentage of chromosomally abnormal embryos can develop normally to blastocyst stage with high probability of implantation and pregnancy.     

Preimplantation diagnosis: a realistic option for assisted reproduction and genetic practice

PURPOSE OF REVIEW: Preimplantation genetic diagnosis (PGD) allows genetically disadvantaged couples to reproduce, while avoiding the birth of children with targeted genetic disorders. By ensuring unaffected pregnancies, PGD circumvents the possible need and therefore risks of pregnancy termination. This review will describe the current progress of PGD for Mendelian and chromosomal disorders and its impact on reproductive medicine. RECENT FINDINGS: Indications for PGD have expanded beyond those used in prenatal diagnosis, which has also resulted in improved access to HLA-compatible stem-cell transplantation for siblings through preimplantation HLA typing. More than 1000 apparently healthy, unaffected children have been born after PGD, suggesting its accuracy, reliability and safety. PGD is currently the only hope for carriers of balanced translocations. It also appears to be of special value for avoiding age-related aneuploidies in in-vitro fertilization patients who have a particularly poor prognosis for a successful pregnancy; the accumulated experience of thousands of PGD cycles strongly suggests that PGD can improve clinical outcome for such patients. SUMMARY: PGD would particularly benefit poor prognosis in-vitro fertilization patients and other at-risk couples by improving reproductive outcomes and avoiding the birth of affected offspring.     

Preimplantation diagnosis: a patient perspective.

A new dimension in the prevention of birth defects will be achieved when genetic diseases can be routinely diagnosed in embryos prior to implantation. The impressions and attitudes towards preimplantation diagnosis were studied in prospective patients, women at high reproductive risk for a genetic disease. Their perspective highlighted not only the advantages and disadvantages of this new approach, but also those changes necessary in order for preimplantation diagnosis to become a useful and practical technique. The data presented are based on information obtained by a mailed questionnaire answered by 58 women. The main benefit of preimplantation diagnosis for these high-risk women would be the ability to undertake a pregnancy without having to be subjected to the physical and/or emotional trauma of elective termination. Their major concerns related to possible damage to the embryo following biopsy, the cost of the procedure, and the low success rate of completed pregnancies. Other issues to be addressed before preimplantation diagnosis could begin to compare favourably with existing forms of prenatal testing were that the methods of obtaining oocytes or embryos should be simple, well tolerated, highly efficient, and low in maternal risk, and that the genetic analysis of embryonic or extraembryonic cells should be unequivocally accurate.     

The beneficial effects of preimplantation genetic diagnosis for aneuploidy support extensive clinical application

The aim of this study was to evaluate the clinical impact of preimplantation genetic diagnosis (PGD) for aneuploidy on 193 patients who subsequently achieved 208 clinical pregnancies, in relation to their reproductive history. The 208 clinical pregnancies included in the study resulted from 1029 assisted conception cycles in combination with PGD for aneuploidy in 740 couples with a history of poor reproductive performance. According to the reproductive history of the 193 patients, 61 had previously experienced 112 pregnancies with 105 abortions and seven deliveries, corresponding to 3.6% take-home baby rate and 10.9% implantation rate. During the PGD cycle, preimplantation embryos were analysed for 5-9 chromosomes. The transfer of euploid embryos was performed in 699 cycles (68% of oocyte retrievals), generating 171 term pregnancies with 210 infants born, whereas 34 aborted spontaneously and three were ectopic, giving a take-home baby rate per pregnant patient of 88.6% and an ongoing implantation rate per pregnant patient of 53.2%. According to these data, selection made in preimplantation embryos against chromosomal abnormalities is associated with a significantly higher (P < 0.001) take-home baby rate when compared with the previous reproductive history of the parents.     

Preimplantation Diagnosis of Common Aneuploidies by the First- and Second-Polar Body FISH Analysis

Purpose: A low pregnancy rate in in vitro fertilization (IVF) patients of advanced maternal age may be caused by aneuploidies originating from non disjunction in the first or second meiotic divisions. We introduced genetic testing of oocytes by sampling and fluorescent in situ hybridization (FISH) analysis of the first and second polar bodies, to avoid fertilization and transfer of aneuploid oocytes in IVF patients of advanced maternal age. Methods: Three hundred and sixty-three IVF patients 34 years and older participated in the study. Using micromanipulation procedures, the first and second polar bodies were removed following their extrusion from the oocytes and studied by FISH, using probes specific for chromosomes 13, 18, and 21 to detect oocytes with common aneuploidies. Results: Of a total of 538 IVF cycles, 3250 oocytes were available for FISH analysis, with conclusive FISH results in 2742 oocytes (84.3%). As many as 1102 (40%) of oocytes were predicted to be aneuploid and not transferred. Of 1640 embryos predicted to be normal, 1145 were transferred in 467 treatment cycles, resulting in 107 pregnancies (23%), from which 67 healthy children have been born, 32 pregnancies spontaneously aborted, and 15pregnancies are ongoing after being confirmed normal by prenatal diagnosis. Conclusions: Preimplantation diagnosis by first- and second-polar body FISH analysis allows us to avoid the age-related risk of common aneuploidies in IVF patients of advanced maternal age.     

Preimplantation genetic diagnosis

Preimplantation genetic diagnosis is essentially an alternative to prenatal diagnosis, in which genetic testing is performed on embryos before a clinical pregnancy is established. Preimplantation genetic diagnosis has been applied to patients carrying chromosomal rearrangements, such as translocations, in which it has been proven to decrease the number of spontaneous abortions and prevent the birth of children affected with chromosome imbalance. Preimplantation genetic diagnosis techniques have also been applied to increase implantation rates, reduce the incidence of spontaneous abortion and prevent trisomic offspring in women of advanced maternal age undergoing fertility treatment. A third group of patients receiving preimplantation genetic diagnosis are those at risk of transmitting a single gene disorder to their children. The number of monogenic disorders that have been diagnosed in preimplantation embryos has increased each year. Recent protocols have tended to be more complex and more reliable than previous methods, making greater use of multiplex polymerase chain reaction. As well as an expansion in the variety of disorders for which preimplantation genetic diagnosis is offered, new indications have been reported including the use of human leukocyte antigen histocompatibility typing and the application of preimplantation genetic diagnosis to late onset diseases.     

Can preimplantation genetic diagnosis overcome recurrent pregnancy failure?

PURPOSE OF REVIEW: Preimplantation genetic diagnosis is widely used for the detection of embryo aneuploidy before implantation, with the aim of avoiding miscarriage or pregnancy termination of an aneuploid fetus. The majority of first trimester miscarriages occur due to chromosomal imbalances. The aim of this review is to assess whether preimplantation genetic diagnosis can help women who suffer from recurrent pregnancy loss. RECENT FINDINGS: Several in-vitro fertilization clinics have employed preimplantation genetic diagnosis in women with recurrent pregnancy loss. Patients were classified into groups according to their age. Preimplantation genetic diagnosis was very successful in treating couples where one of the parents was a carrier of a balanced chromosomal abnormality such as a translocation. Similarly, recurrent pregnancy loss rate was reduced in women more than 35 years in age with a normal karyotype. On the other hand, in younger patients the beneficial effect of this procedure is debatable. In general, women with recurrent pregnancy loss produced more abnormal embryos than control groups. SUMMARY: Preimplantation genetic diagnosis can be beneficial for three major subgroups of patients with recurrent pregnancy loss: couples carrying chromosomal translocations; women more than 35 years of age; women of any age whose previous miscarriages were due to fetal aneuploidy. It is likely that the rate of miscarriage will be further reduced with the new advances in methods of performing preimplantation genetic diagnosis for more chromosomes.     

Preimplantation diagnosis for aneuploidies in patients undergoing in vitro fertilization with a poor prognosis: identification of the categories for which it should be proposed

OBJECTIVE: To verify whether advantages can derive from the implementation of preimplantation genetic diagnosis for aneuploidy in patients with a poor prognosis of full-term pregnancy, compared with conventional treatment procedures. DESIGN: A randomized, controlled study. SETTING: Reproductive Medicine Unit of the Società Italiana Studi Medicina della Riproduzione, Bologna, Italy. PATIENT(S): In a total of 262 stimulated cycles, women presented with the following poor-prognosis indications: maternal age of > or =36 years (n = 157), > or =3 previous IVF failures (n = 54), and an altered karyotype (n = 51). After giving consent, 127 patients underwent preimplantation genetic diagnosis for aneuploidy, whereas 135 controls underwent assisted zona hatching. INTERVENTION(S): Analysis of chromosomes XY, 13, 14, 15, 16, 18, 21, and 22 was carried out with the fluorescence in situ hybridization technique in a blastomere biopsied from day 3 embryos. Assisted zona hatching was performed on day 3 embryos from the control group. MAIN OUTCOME MEASURE(S): Embryo morphology and chromosomal status, number of transferred embryos, clinical pregnancies, implantation rates, and abortions. RESULT(S): In the study group, 717 embryos were analyzed by fluorescence in situ hybridization, and 60% were chromosomally abnormal. A mean of 2.3+/-0.9 euploid embryos were transferred in 99 cycles, resulting in 37 clinical pregnancies (37%) and a 22.5% ongoing implantation rate. In the control group, 126 cycles were performed with 3.2+/-1.3 embryos transferred, yielding 34 clinical pregnancies (27%) and a 10.2% ongoing implantation rate. CONCLUSION(S): The advantage of selecting embryos with a normal chromosome complement has an immediate impact on the ongoing implantation rate, especially in patients aged > or =38 years and carriers of an altered karyotype.     

Impact of preimplantation genetic diagnosis for chromosomal disorders on reproductive outcome

Despite recent controversy, existing experience suggests that aneuploidy testing has had a significant impact on the reproductive outcome of poor prognosis IVF patients, which is based on the experience of over 20,000 cases of PGD for chromosomal disorders. The clinical impact was demonstrated in the improved implantation and pregnancy rates, reduction of spontaneous abortions and improved take-home baby rate in patients of advanced reproductive age, those with repeated IVF failures and recurrent spontaneous abortions. The lack of positive effect of aneuploidy testing in a few smaller series may be due to potential detrimental effect of two blastomere removal, reducing the implantation potential of the biopsied embryos, or exclusion from testing a few key chromosomes and poor outcome of aneuploidy testing, affecting the appropriate pre-selection of embryos for transfer. Despite the need for randomized controlled studies to quantify in more detail the clinical impact of the pre-selection of aneuploidy-free zygotes, the positive impact of PGD is particularly obvious from the comparison of reproductive outcome in the same patients with and without PGD, revealing the actual benefits of PGD.     

Improved implantation after preimplantation genetic diagnosis of aneuploidy

The objective of this study was to assess the improvement in implantation rates after preimplantation genetic diagnosis (PGD) of numerical abnormalities for the sole indication of advanced maternal age when compared with a control group. Each PGD patient was matched to a control patient according to several parameters prior to obtaining pregnancy results. The diagnosis was based on the analysis of chromosomes X, Y, 13, 15, 16, 18, 21 and 22 plus a ninth probe (1, 7, 14 or 17) on a single cell per embryo. The results were also analysed in relation to the previous number of IVF cycles and the number of dipronucleated zygotes obtained, when replacing presumptively chromosomally normal embryos on day 4 of development. It was found that women of advanced reproductive age (average age 40 years) had a higher implantation rate (18%) than their matched controls treated with standard IVF (11%) (P < 0.05). This increase was not observed in patients with two or more previous IVF cycles or patients with fewer than eight zygotes. Patients with eight or more 2PN zygotes and one or no previous cycles showed the greatest improvement in implantation rate, from 8.8% in controls to 19.2% in the PGD group (average age 40 years) (P < 0.025).     

Effect of PGD on implantation and ongoing pregnancy rates in cases with predominantly macrocephalic spermatozoa

Although its occurrence is rare, the presence of large headed or macrocephalic spermatozoa and increased chromosomal abnormality has recently been reported by several groups. Moreover, when intracytoplasmic sperm injection (ICSI) was performed with samples containing macrocephalic spermatozoa, lower fertilization and implantation rates result in poor clinical outcome. In order to evaluate the impact of preimplantation genetic diagnosis (PGD) on implantation and ongoing pregnancy rates in these couples, the results of 23 PGD cycles were compared with non-PGD cycles (n = 60) as well as cycles with absolute teratozoospermia (having zero normal morphology) with (n = 14) or without PGD (n = 66). Out of 82 embryos biopsied in the macrocephalic sperm group, abnormalities were detected in 46.4% of the embryos analysed. Most of the abnormalities were trisomies (37.0%) and complex aneuploidies (51.9%). A 33.3% pregnancy rate was achieved by selectively transferring euploid embryos after PGD with the statistically higher implantation rate of 25.0% compared with non-PGD cycles (IR: 12.3%, P < 0.01). Moreover, only one missed abortion (14.3%) was observed in the PGD group, whereas seven of the 15 pregnancies resulted in abortion in the non-PGD group (46.7%). Preliminary results indicate that patients should be counselled for increased chromosomal abnormality and a possible beneficial effect of eliminating chromosomally abnormal embryos with PGD on a bortion rates.