No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Pre-implantation genetic diagnosis and screening: Now and the future
Gynecological Endocrinology
Abstract
Since 1989, the year of the first pre-implantation genetic diagnosis (PGD), many developments occurred both in assisted reproduction techniques and in molecular tools. While PGD is a well-established and documented application, pre-implantation genetic screening (PGS) for the detection of aneuploid embryos is still debated due to the presence of mosaicism in the embryo, but especially to the knowledge of the limits that label an embryo as healthy or as appropriate to the life. The aim of this review is to present the state-of-the-art in the field of PGD and PGS, illustrating its benefits and limitations, along with biopsy techniques and the use of new high-throughput technologies.
... Polar body biopsy has more limitations than advantages; genetic information provided is only maternal and therefore cannot inform about parental or mitotic division abnormalities. 30 Furthermore, data show it is highly expensive, 31 and at this developmental stage, meiotic errors are common, 32 and a high risk of aneuploidy (32.5%-67%) exists. [33][34][35] Polar body biopsy has been poorly studied in comparison to other types of biopsies. ...
... One limitation of day 3 biopsies is the requirement to remove 1 to 2 cells, or up to 33% of the embryo, to obtain the necessary genetic material for PGT-A. 30 Two-cell biopsy may increase accuracy but compromise development, implantation, and overall viability, 43,44 whereas one-cell biopsy can result in misleading or incorrect analysis results. 45,46 Studies have examined the differences between 1-cell and 2-cell biopsy at this stage of development. ...
... 54 Five-to-ten TE cells are removed during biopsy from the 100-to 150-cell embryo, comprising a lower proportion of cell loss (<10%) than cleavage-stage biopsy. 17 Although day 5 biopsies are technically more difficult, 55 they allow the obtainment and analysis of additional genetic material than day 3 biopsies 30 and have increased cost-effectiveness as fewer embryos are tested 56,57 due to natural attrition of poor quality embryos before reaching the blastocyst stage. They also ensure that the biopsy can target the TE cells, leaving the ICM intact. ...
Importance:
Preimplantation genetic testing for aneuploidy (PGT-A) has undergone many technical developments over recent years, including changes in biopsy timings, methodology, and genetic analysis techniques. The evidence surrounding the efficaciousness of PGT-A is sporadic and inconsistent; as such, significant doubt and concern remain regarding its widespread implementation.
Objective:
This review seeks to describe the historical development of PGT-A and to analyze and summarize the current published literature.
Conclusions:
At times during its infancy, PGT-A failed to display conclusive improvements in results; with newer technologies, PGT-A appears to yield superior outcomes, including reductions in miscarriages and multiple gestations. Clinicians and patients should assess the use of PGT-A on a case-by-case basis, with laboratories encouraged to utilize blastocyst biopsy and next-generation sequencing when conducting PGT-A. Further studies providing cumulative live birth rates and time to live birth are required if PGT-A is to be proven as producing superior outcomes.
Relevance:
PGT-A has the potential ability to impact in vitro fertilization success rates, and as it is increasingly adopted worldwide, it is crucial that clinicians are aware of the evidence for its continued use.
... For the last 2 decades, screening strategies to select euploid embryos have emerged as a major interest. A preimplantation genetic screening (PGS) to select euploid embryo to transfer in couples who do not have specific disease is widely used as a good screening tool before going through IVF-embryo transfer (ET) [5]. Indication for PGS varies depending on center. ...
... A preimplantation genetic diagnosis (PGD) is the method to avoid the transmission of genetic disease such as recessive monogenic disorders, dominant monogenic disorder, sexlinked disorders, chromosomal disorders or human leukocyte antigen matching [5,7]. Also, diagnosis of embryos for chromosome abnormalities including aneuploidy screening has been invigorated by introduction of microarray-based testing methods, which allowed analysis of 24 chromosomes [8]. ...
... Among this, array CGH is widely used around the world as it is known to be reliable, accurate, and relatively rapid for whole chromosome analysis [11]. aCGH is genomic hybridization method which requires labelled DNA from both test and control sample which are hybridized to DNA microarray [5]. ...
Objective:
Indications for preimplantation genetic diagnosis (PGD)/preimplantation genetic screening (PGS) cycles and clinical outcomes were evaluated at CHA Gangnam Medical Center.
Methods:
This is retrospective cohort study. All patients (n=336) who went through in vitro fertilization (IVF)-PGD/PGS cycles (n=486) between January 2014 and December 2015 were included in Fertility Center of CHA Gangnam Medical Center. Patients underwent IVF-PGD/PGS with 24-chromosome screening. Patients with euploid embryos had transfer of one or 2 embryos in a fresh cycle with any subsequent frozen embryo transfer (ET) cycle. Compared implantation, clinical pregnancy, ongoing pregnancy, and early abortion rates were the main outcome measures.
Results:
The most common indication for PGD/PGS was recurrent spontaneous abortion (n=160). The chromosome rearrangement cases (n=116) included 24 Robertsonian translocations, 60 reciprocal translocations, 3 inversions, 2 deletions, 4 additions, and 23 mosaicisms. PGS cases rather than the PGD cases showed higher implantation rates (26.4% vs. 20.3%), ongoing pregnancy rates (19.5% vs. 16.4%), and clinical pregnancy rates (28.6% vs. 23.3%). Implantation rates (30.3% vs. 23.7%), clinical pregnancy rates (39.2% vs. 25.2%), and ongoing pregnancy rates (25.7% vs. 17.5%) were significant higher in the blastocyst evaluation group than cleavage stage evaluation group.
Conclusion:
This was the largest study of PGD/PGS for 2 years at a single center in Korea. The pregnancy outcomes of PGD cases are slightly lower than PGS cases. It was confirmed again that success rate of PGD/PGS is higher if biopsy was done at blastocyst than cleavage stage.
... This feature allows the parental identification of the analyzed allele. For characterization of haplotypes, analyses of Short Tandem Repeats (STR) are performed [17][18][19]. ...
... (71) were of paternal origin, and 52.9% (81) were of maternal origin (Table 3). When alterations were classified, 87.5% (133) displayed monosomy, and only 12.5% (19) presented trisomy (Table 4). However, as we mentioned earlier, due to methodology limitations, we detected only a minority of trisomy proportions. ...
Introduction
Analyses of miscarriage products indicate that the majority of aneuploidies in early developing embryos derive from errors occurring during maternal meiosis and the paternal contribution is less than 10%. Our aim was to assess the aneuploidy (mainly monosmies) frequencies at the earliest stages of embryo development, 3 days following fertilization during In vitro fertilization (IVF) treatments and to elucidate their parental origin. Later, we compared monosomies rates of day 3 to those of day 5 as demonstrated from Preimplantation Genetic Testing for Structural chromosomal Rearrangement (PGT-SR) results.
Methods
For a retrospective study, we collected data of 210 Preimplantation Genetic Testing for Monogenic Disorder (PGT-M) cycles performed between years 2008 and 2019.This study includes 2083 embryos, of 113 couples. It also included 432 embryos from 90 PGT-SR cycles of other 45 patients, carriers of balanced translocations. Defining the parental origin of aneuploidy in cleavage stage embryos was based on haplotypes analysis of at least six informative markers flanking the analyzed gene. For comprehensive chromosomal screening (CCS), chromosomal microarray (CMA) and next generation sequencing (NGS) was used.
Results
We inspected haplotype data of 40 genomic regions, flanking analyzed genes located on 9 different chromosomes.151 (7.2%) embryos presented numerical alterations in the tested chromosomes. We found similar paternal and maternal contribution to monosomy at cleavage stage. We demonstrated paternal origin in 51.5% of the monosomy, and maternal origin in 48.5% of the monosomies cases.
Conclusion
In our study, we found equal parental contribution to monosomies in cleavage-stage embryos. Comparison to CCS analyses of PGT-SR patients revealed a lower rate of monosomy per chromosome in embryos at day 5 of development. This is in contrast to the maternal dominancy described in studies of early miscarriage. Mitotic errors and paternal involvement in chemical pregnancies and IVF failure should be re-evaluated. Our results show monosomies are relatively common and may play a role in early development of ART embryos.
... Preimplantation genetic diagnosis (PGD) is a very early form of prenatal diagnosis before the implantation of the embryo in the uterus and involves testing to avoid the transmission of specific genetic or chromosomal birth defects. 38 It was performed for the first time by Handyside in 1989 in the UK. 39 This technique also aims to bypass the obvious issue of voluntary TOP because it allows to select and transfer in the maternal uterus only the healthy embryos obtained in vitro by assisted reproductive techniques (IVF-ICSI). ...
... At present time, the most accurate analysis is the wholegenome amplification and genome-wide technologies. 38 The success rate of analysis is high even if in a few cases a diagnosis may not be available due to DNA amplification problems or contamination. The success of PGD is strongly influenced by Preimplantation genetic diagnosis can also be employed for HLA compatibility by human leukocyte antigen matching in case of bone marrow transplantation after birth even if these techniques raise some ethical problems and controversies. ...
... Generally, TE biopsies are performed five days (D5) after fertilization, with three to ten trophoblast cells obtained for further genetic testing. This can improve the accuracy and validity of PGD [3][4][5]. Although these three PGD methods have achieved respectable results with embryos biopsied at either the zygote, cleavage, or blastocyst stages, zona drilling is required for each technique. ...
... Additionally, they identified a 97.4% ploidy concordance between BF-DNA and TE cells. Palini et al. (2015) showed that genomic DNA was present in approximately 90% of blastocoel fluid samples harvested during vitrification procedures [14]. ...
Preimplantation genetic diagnosis (PGD) has become a widely accepted and routine technique for testing chromosomal integrity during in vitro fertilization (IVF) procedures, and has been successfully applied to high-risk couples for more than two decades [1,2]. During the development of PGD, polar bodies, blastomeres, and trophectoderms (TE) have been used as biopsy material for analysis. The polar body is expelled from oocytes during the first meiotic division, and therefore only contains maternal genetic information. Blastomere biopsies are typically performed on third day (D3) embryos that contain at least six cells, and one or two blastomeres are used for subsequent genetic analysis, including chromosome examination, identifying chromosomal structural abnormalities, molecular diagnosis of single gene diseases, and several other tests. The main disadvantage of these D3 biopsies of blastomeres is that any resultant analysis cannot completely rule out the impact of mosaics, affecting detection accuracy. Generally, TE biopsies are performed five days (D5) after fertilization, with three to ten trophoblast cells obtained for further genetic testing. This can improve the accuracy and validity of PGD [3-5]. Although these three PGD methods have achieved respectable results with embryos biopsied at either the zygote, cleavage, or blastocyst stages, zona drilling is required for each technique. This is accompanied by lesions to the embryo, the extent of which depends on the technique. However, it is not clear whether this damage affects the development of the embryo or fetus.
... Since embryonic aneuploidy is one of the main causes of RIF, preimplantation genetic testing for aneuploidy (PGT-A) is applied to select euploid embryos for successful implantation [14]. Through examination of numeral and structural changes of chromosomes of embryos before transfer, the chances of delivering a healthy baby could be maximized [15]. Next-generation sequencing (NGS) techniques are the latest and most popular technique adopted for testing embryos by massively parallel genome sequencing [16]. ...
Purpose
Recurrent implantation failure (RIF) is one of the most common conditions affecting In Vitro Fertilization (IVF)/Intracytoplasmic sperm injection (ICSI) outcomes. Aneuploidy embryos, one of the main types of embryos-related factors, was reported to be a major contributor to RIF. The present study aimed to examine the association between sperm DNA fragmentation index (DFI) and outcomes of next-generation sequencing (NGS)-based preimplantation genetic testing for aneuploidy (PGT-A) in unexplained RIF patients.
Methods
This study analyzed 119 couples with unexplained RIF who underwent 119 PGT-A cycles between January, 2017 and March, 2022. The 119 males were divided into 3 groups according to their sperm DFI levels: Group1 (low, DFI ≤ 15%, n = 50), Group2 (medium, 15% < DFI < 30%, n = 41) and Group3 (high, DFI ≥ 30%, n = 28). Sperm DFI was measured by sperm chromatin structure analysis (SCSA) technique. Trophectoderm biopsy on day 5 or 6 were performed with NGS technique. The following outcomes of PGT-A were analyzed and compared: fertilization, good-quality embryos, aneuploidy rate, miscarriage, live birth and newborn defects.
Results
The component of aneuploidy embryos was significantly higher in high DFI group (42.71%) than that of medium group (28.39%) and low group (27.80%). The miscarriage rate of high DFI group (27.27%) and medium group (14.29%) is significantly higher than that of low group (0.00%). No significant differences were found regarding fertility, good-quality embryo rate, pregnancy rate, live birth rate or newborn defects among three groups.
Conclusion
The sperm DNA damage is associated with blastocyst aneuploidy and miscarriage rate in unexplained RIF cases. Embryo selection by PGT-A and efforts to decrease sperm DFI before IVF/ICSI treatments should be considered for those male patients with high DFI.
... 30 In fact, PGD is used to detect chromosomal abnormalities or specific genetic diseases in the embryos of IVF patients such as recessive monogenic disorders, dominant monogenic disorder, sexlinked disorders, chromosomal disorders or human leukocyte antigen matching and consequently avoid its transmission. 31,32 Pehlivan et al. revealed that aneuploidies, with other chromosomal anomalies, presented higher rates in the case of RIF and when normal embryos after genetic screening were selected for transfer the implantation rate was similar to of young fertile controls. 33 Age also have an important impact on chromosomal abnormalities. ...
With more than three decades of Assisted Reproductive Technology (ART) practice worldwide. There are nearly 10 to 15% of patients suffering from unexplained Recurrent Implantation Failures (RIF) and Repeated Pregnancy Loss (RPL). The literature reported different causes of RIF-RPL, mainly multifactorial, endometrial and idiopathic. RIF remain as a dark box because of the complicate categorization and causes of this physio-pathological dysregulation of implantation and pregnancy process after ovarian stimulation. Many options were suggested as solutions to treat RIF/RPL with controversial results on their usefulness. In this article, we reviewed different possible causes of RIF and therapeutics options to improve implantation rates and clinical outcomes.
... Preimplantation genetic testing (PGT) is an in vitro fertilization (IVF) technique with the aim of assisting couples with heritable genetic disorders to avoid transmitting the genetic disorder to their offspring [11]. PGT encompasses testing for monogenic disorders (PGT-M), for structural rearrangements, and for aneuploidy [12]. ...
Background:
Alport syndrome (AS) is a hereditary renal basement membrane disease that can lead to end-stage renal disease in young adults. It can be diagnosed by genetic analysis, being mostly caused by mutations in COL4A3, COL-4A4, and COL4A5. To date, there is no radical cure for this disease.
Objectives:
The aim of this study was to avoid the transmission of AS within an affected family by selecting healthy embryos for uterine transfer. The embryos were identified by preimplantation genetic testing for monogenic disorders (PGT-M).
Methods:
We used next-generation sequencing (NGS) to identify mutations in the proband and his parents. The results of NGS were confirmed by Sanger sequencing. Targeted NGS combined with targeted single-nucleotide polymorphism haplotyping was used for the in vitro identification of COL4A5 mutations in human embryos to prevent their intergenerational transmission.
Results:
The c.349_359delGGACCTCAAGG and c.360_361insTGC mutations in COL4A5 were identified in a family affected by X-linked AS. Whole-genome sequencing by NGS with targeted haplotyping was performed on biopsied trophectoderm cells. A healthy baby was born after transfer of a single freeze-thawed blastocyst.
Conclusions:
The use of targeted NGS for identifying diagnostic markers combined with targeted haplotyping is an easy and efficient PGT-M method for preventing intergenerational transmission of AS.
... In this study, we implemented NGS-based PGT for translocation testing as a valuable alternative for FISHbased testing aiming to increase the sensitivity and specificity of PGT and to improve pregnancy outcomes in couples with balanced translocations. Blastocyst trophectoderm biopsy provids more genetic material for subsequent analysis, and cryopreservation of biopsied blastocysts allows for enough time for genetic testing and a more acceptable embryo-endometrial synchronization cycle for transfer [Palini et al., 2015]. Fifty-four normal/balanced blastocysts were transferred, and the overall clinical pregnancy rate was 64.8% in our study. ...
The purpose of this study was to evaluate the effectiveness of next-generation sequencing (NGS)-based preimplantation genetic testing (PGT) for balanced translocation carriers to identify normal/balanced blastocysts and to measure pregnancy outcomes following euploid embryo transfer. We enrolled 75 couples with a balanced translocation who underwent 83 PGT cycles (58 cycles for carriers with reciprocal translocations and 25 cycles for carriers with Robertsonian translocations) and 388 blastocysts were diagnosed. Moreover, we transferred single euploid blastocysts through frozen embryo transfer and calculated the biochemical pregnancy, clinical pregnancy, miscarriage, and ongoing pregnancy rates per embryo transfer cycle. Despite a mean maternal age of 29.8 years and mean of 4.34 embryos biopsied, there was a 32.8% chance of recording no chromosomally normal/balanced embryos for reciprocal translocation carriers. The proportion of normal/balanced embryos was significantly higher (44.1 vs. 27.8%) in Robertsonian translocation carriers than in reciprocal translocation carriers. Female carriers had a significantly lower (23.3 vs. 42.4%, 34.7 vs. 54.7%, respectively) percentage of normal/balanced embryos than male carriers, regardless of the translocation. After transfering single blastocysts, we obtained a 64.4% clinical pregnancy rate per transfer, and the clinical miscarriage rate was 5.7%. Amniocentesis results showed that all karyotypes of the fetuses were consistent with PGT results. The clinical outcomes are probably not influenced by the type of translocation, maternal age, and blastocyst morphology following the transfer of euploid blastocysts. Therefore, we conclude that NGS-based PGT is an efficient method for analyzing balanced translocation carriers, and aneuploidy screening had good clinical outcomes.
... 4 PGD or preimplantation genetic testing (PGT) is a very early form of prenatal diagnosis performed for the first time by Handyside in 1989 for cystic fibrosis. 26 This technique aims to avoid the transmission of specific genetic or chromosomal birth defects 27 and to bypass the obvious issue of TOP. Thus, only the healthy embryos obtained in vitro by in vitro fertilization and intracytoplasmic sperm injection get to to be transferred. ...
The genetic screening and prenatal diagnosis panorama have been profoundly modified in the last 30 years. This study analyzes the changes on international level and shares the experience of our maternal-fetal-perinatal medicine center at the Microcitemico Hospital, Cagliari. We observed the evolution of screening tests for fetal aneuploidies. There was an overall reduction of invasive prenatal procedures, probably due to denatality, the innovations in ultrasound imaging technology, and the introduction of noninvasive prenatal testing. Furthermore, we reported the decrease of amniocentesis as compared to chorionic villous sampling (CVS) and the decline of fetal loss rates following both of these procedures. The demand for training fellows in invasive prenatal procedures and especially in CVS is continuously increasing.
... Since then, this technology has been used as an alternative to prenatal diagnosis to avoid transmitting a genetic or chromosomal abnormality to offspring (2). Preimplantation genetic testing enables the identification of embryos with specific disease-causing mutations, such as recessive monogenic disorders, dominant monogenic disorders, and sexlinked disorders, or with chromosomal disorders (3,4). With the evolution of molecular genetics, an increasing number of monogenic diseases have been identified, which has promoted the rapid development of PGT for monogenic diseases (PGT-M) (5). ...
Objective:
To investigate whether aneuploidy screening in preimplantation genetic testing (PGT) for monogenic diseases improves the ongoing pregnancy/live birth rate of single frozen/thawed embryo transfer (FET) cycles in young women.
Design:
Retrospective cohort study.
Setting:
Single university-based fertility center.
Patient(s):
From January 2016 to December 2017, 569 FET cycles were selected for analysis. The aneuploidy screening (AS) group included 131 FET cycles from 105 oocyte retrieval cycles in 98 patients who underwent PGT for monogenic diseases with aneuploidy screening, and the non-AS group included 438 FET cycles from 280 oocyte retrieval cycles in 266 patients who underwent PGT for monogenic diseases without aneuploidy screening.
Intervention(s):
The patient population was all under the age of 35 years and underwent PGT for monogenic diseases with and without AS.
Main outcome measure(s):
Ongoing pregnancy/live birth rate, live birth rate, implantation rate, and miscarriage rate.
Result(s):
Aneuploidy screening significantly improved the ongoing pregnancy/live birth rate (61.22% vs. 43.98%), implantation rate (64.29% vs. 50.38%), and live birth rate (53.06% vs. 36.09%) of young women carrying monogenic diseases in the first FET cycles. When adjusted for the parity, number of previous miscarriages, and percentage of infertility, the likelihood of implantation was 1.874 times higher (95% confidence interval 1.126-3.119), and an ongoing pregnancy/live birth was 2.139 times more likely (95% confidence interval 1.295-3.534). In addition, the miscarriage rate was significantly decreased (3.17% vs. 11.94%). In the cumulative pregnancy outcomes, the cumulative ongoing pregnancy/live birth rate both per transfer and per patient were significantly higher in the AS group (62.24% vs. 50.38% and 79.59% vs. 68.80%), but no difference existed after adjusting for the parity, number of previous miscarriage, and percentage of infertility. Nevertheless, aneuploidy screening reduced the time interval from the first ET to the achievement a pregnancy.
Conclusion(s):
Aneuploidy screening in PGT significantly improved the ongoing pregnancy/live birth rate of young women carrying monogenic diseases in the first FET cycles.
... Embryonic aneuploidy is the main cause of miscarriage and failure of assisted reproductive technology (ART) [3]. Aneuploidy screening of embryos originating from IVF patients is termed as preimplantation genetic testing for aneuploidy (PGT-A); it enables the examination of numeral and structural chromosomes of embryos before transfer [4]. This technique has been applied to treat patients with an increased risk of aneuploid embryos, such as those with advanced maternal age (AMA) [5e7], repeated implantation failure (RIF) [8e10], and recurrent miscarriage (RM) [11e13]. ...
OBJECTIVE:
The primary objective of this study was to investigate whether preimplantation genetic testing for aneuploidy (PGT-A) of blastocysts through array comparative genomic hybridization (aCGH) improves live birth rates (LBR) in IVF cycles for patients with high prevalence of aneuploidy.
MATERIALS AND METHODS:
This study included 1389 blastocysts with aCGH results derived from 296 PGT-A cycles in IVF patients with advanced maternal age (AMA) (n = 87, group A), those with repeated implantation failure (RIF) (n = 82, group B), those with recurrent miscarriage (RM) (n = 82, group C), and oocyte donors (OD) (n = 45, young age, as a control group). Another 61 AMA patients without PGT-A procedures were used as a control group for group A. Vitrification was performed after blastocyst biopsy, and thawed euploid embryos were transferred in a nonstimulated cycle.
RESULTS:
For the AMA group, a significant increase in LBRs was found in the PGT-A group compared with the non-PGT-A group (54.1% vs. 32.8%, p = 0.018). Consistent LBRs (54.1%, 51.6%, 55.9%, and 57.1%, respectively, in group A, B, C, and young age group) were obtained for all the indications.
CONCLUSIONS:
LBRs can be improved using PGT-A of blastocysts with aCGH in IVF cycles for patients with a high rate of aneuploidy, especially for patients with AMA.
... Embryonic aneuploidy is the main cause of miscarriage and failure of assisted reproductive technology (ART) [3]. Aneuploidy screening of embryos originating from IVF patients is termed as preimplantation genetic testing for aneuploidy (PGT-A); it enables the examination of numeral and structural chromosomes of embryos before transfer [4]. This technique has been applied to treat patients with an increased risk of aneuploid embryos, such as those with advanced maternal age (AMA) [5e7], repeated implantation failure (RIF) [8e10], and recurrent miscarriage (RM) [11e13]. ...
Objective:
To investigate the effects of growth hormone (GH) cotreatment in ovarian stimulation in infertile women of advanced age, poor responders, and patients with one or more previous IVF treatment failures.
Materials and methods:
We conducted a retrospective observational study of 436 patients undergoing GH cotreatment in ovarian stimulation. The first arm included 134 infertile women of advanced age. The second arm included 236 patients with one or more IVF previous treatment failures, and the third arm included 66 younger poor responders. Main outcome measures were the number of oocytes and embryos, quality of embryos, and implantation and pregnancy rates.
Results:
In infertile women of advanced age, GH plus ovarian stimulation yielded no statistical differences in the numbers of oocytes and embryos, quality of embryo, and rates of implantation and pregnancy. In the second arm, the mature oocyte number (8.2 vs. 6.8), implantation rate (16.1% vs. 0%), and pregnancy rate (33.9% vs. 0%) in the GH cotreatment group differed significantly from those in the control group; the rate of good-quality embryos in the GH cotreatment group improved from 35.5% ± 31.1%-41.4% ± 30.6% in this arm. Similar results were observed in the third arm; in this arm, the clinical pregnancy rate was 30.3% in the GH cotreatment group and 6.1% in the control group.
Conclusion:
No significant differences were observed in infertile women of advanced age, which may be due to the low GH dose. The GH adjuvant therapy for patients with one or more previous IVF treatment failures and for poor responders significantly improved the oocyte and embryo numbers as well as implantation and pregnancy rates.
... Therefore, testing women that are undergoing in vitro fertilization (IVF) for aneuploidies or for genetic disorders can improve Assisted Reproductive Technology outcomes. By selecting chromosomally integral embryos for transfer, patients with advanced maternal age can improve their chances for implantation, reduce pregnancy loss, and considerably reduce the risk of delivering a child with genetic abnormalities [3][4][5][6][7][8]. There is currently enough evidence provided by a series of randomized controlled trials to support preimplantation genetic screening (PGS) as a reliable tool to improve clinical outcomes in IVF [9][10][11]. ...
Background
Preimplantation genetic screening (PGS) is an important procedure for in vitro fertilization (IVF). A key step of PGS, blastomere removal, is abundant with many technical issues. The aim of this study was to compare a more simple procedure based on the Stipper Micropipetter, named S-biopsy, to the conventional aspiration method. Methods
On Day 3, 368 high-quality embryos (>7 cells on Day3 with <10% fragmentation) were collected from 38 women. For each patient, their embryos were equally separated between the conventional method (n = 188) and S-biopsy method (n = 180). The conventional method was performed using a standardized protocol. For the S-biopsy method, a laser was used to remove a significantly smaller portion of the zona pellucida. Afterwards, the complete embryo was aspirated with a Stripper Micropipetter, forcing the removal of the blastomere. Selected blastomeres went to PGS using CGH microarrays. Embryo integrity and blastocyst formation were assessed on Day 5. Differences between groups were assessed by either the Mann-Whitney test or Fisher Exact test. ResultsBoth methods resulted in the removal of only one blastomere. The S-biopsy and the conventional method did not differ in terms of affecting embryo integrity (95.0% vs. 95.7%) or blastocyst formation (72.7% vs. 70.7%). PGS analysis indicated that aneuploidy rate were similar between the two methods (63.1% vs. 65.2%). However, the time required to perform the S-biopsy method (179.2 ± 17.5 s) was significantly shorter (5-fold) than the conventional method. Conclusion
The S-biopsy method is comparable to the conventional method that is used to remove a blastomere for PGS, but requires less time. Furthermore, due to the simplicity of the S-biopsy technique, this method is more ideal for IVF laboratories.
... The exponential growth of the diagnostic technologies in genetics in the last decade has enabled state-of-the-art evaluation of newborns and infants. Now, we have the technology to provide genetic testing on a single cell in an embryo before implantation, 1,2 and to extract fetal DNA from maternal plasma to test for significant genomic imbalances. 3,4 With the growing awareness of the role of genetics in human diseases, susceptibility to complex diseases and innate responsiveness to environmental triggers such as drugs, toxins, and infectious agents, it is crucial for physicians to implement individualized medicine in clinical practice and apply the best diagnostic and therapeutic tools for pediatric care. ...
In summary, the phenomenal advancement of molecular technologies in the diagnosis of genetic disease in recent years has dramatically changed the landscape of neonatal medicine. While the utility of whole genome sequencing is being deliberated currently for its inclusion in the newborn screening program,
⁵²
• King J.S.
• Smith M.E.
Whole-genome screening of newborns? The constitutional boundaries of state newborn screening programs.Pediatrics. 2016; 137: S8-S15
• Crossref
• PubMed
• Scopus (10)
• Google Scholar
the implementation of this technique in the clinical evaluation of critically ill neonates is imminent. It is vital for pediatric healthcare providers to be cognizant of the available diagnostic tools for providing the best clinical care to their patients with genetic disorders.
β-thalassemia is an autosomal recessive disease with the reduction or absence in the production of β-globin chain in the hemoglobin, which is caused by mutations in the Hemoglobin subunit beta ( HBB ) gene. In Vietnam, the number of β-thalassemia carriers range from 1.5 to 25.0%, depending on ethnic and geographical areas, which is much higher than WHO’s data worldwide (1.5%). Hence, preimplantation genetic diagnosis (PGD) plays a crucial role in reducing the rate of β-thalassemia affected patients/carriers. In this research, we report the feasibility and reliability of conducting PGD in combination with the use of short tandem repeat (STR) markers in facilitating the birth of healthy children. Six STRs, which were reported to closely linked with the HBB gene, were used on 15 couples of β-thalassemia carriers. With 231 embryos, 168 blastocysts were formed (formation rate of 72.73%), and 88 were biopsied and examined with STRs haplotyping and pedigree analysis. Thus, the results were verified by Sanger sequencing, as a definitive diagnosis. Consequently, 11 over 15 couples have achieved pregnancy of healthy or at least asymptomatic offspring. Only three couples failed to detect any signs of pregnancy such as increased Human Chorionic Gonadotropin (HCG) level, foetal sac, or heart; and one couple has not reached embryo transfer as they were proposed to continue with HLA-matching to screen for a potential umbilical cord blood donor sibling. Thus, these results have indicated that the combination of PGD with STRs analysis confirmed by Sanger sequencing has demonstrated to be a well-grounded and practical clinical strategy to improve the detection of β-thalassemia in the pregnancies of couples at-risk before embryo transfer, thus reducing β-thalassemia rate in the population.
Objective:
To review the effect of comprehensive chromosome screening-based preimplantation genetic testing for aneuploidy (PGT-A) in women undergoing in vitro fertilization (IVF) treatment, we conducted this meta-analysis to compare pregnancy outcomes of women who did and did not undergo such testing.
Data sources:
We searched Medline, EMBASE, the Cochrane Central Register of Controlled Trials, and ClinicalTrials.gov from their inception until February 28, 2022, for randomized controlled trials focusing on PGT-A treatment without any language restrictions.
Methods of study selection:
Randomized controlled trials involving women undergoing IVF with or without PGT-A and comprehensive chromosome testing. Pooled relative risks (RRs) with 95% CIs were calculated for the primary outcome using a random-effects model with the Mantel-Haenszel method.
Results:
A total of nine trials with 3,334 participants were included. Overall, PGT-A was not associated with an increased live-birth rate (RR 1.13, 95% CI 0.96-1.34, I2=79%). However, PGT-A raised the live-birth rate in women of advanced maternal age (RR 1.34, 95% CI 1.02-1.77, I2=50%) but not in women of nonadvanced age (RR 0.94, 95% CI 0.89-0.99, I2=0%).
Conclusion:
Preimplantation genetic testing for aneuploidy increases the live-birth rate in women of advanced maternal age.
Systematic review registration:
PROSPERO, CRD42022311540.
Research question:
Can next-generation sequencing (NGS) based on copy number variation sequencing (CNV-Seq) identify normal/balanced embryos in balanced reciprocal translocation carriers and what are their reproductive outcomes?
Design:
One hundred couples with balanced reciprocal translocation who underwent a total of 134 preimplantation genetic testing (PGT) cycles between January 2015 and October 2017 were evaluated. Trophectoderm cells of blastocysts were biopsied for CNV-Seq-based NGS. All the balanced/normal blastocysts were vitrified and cryopreserved. Single balanced/normal blastocysts were warmed and transferred in the subsequent frozen embryo transfer (FET) cycle.
Results:
During the study period, 400 blastocysts were analysed by NGS-PGT, of which 109 (27.25%) were balanced and euploid. A total of 52 blastocysts were transferred in the FET cycle. Clinical pregnancy was confirmed in 34 women (65.38%), with a miscarriage rate of 2.94%; 26 healthy term babies were born, including 24 singletons and one set of twins, while eight couples had ongoing pregnancies. Amniocentesis revealed a fetal chromosome status that was consistent with the NGS-PGT results. Female carriers had a significantly higher blastocyst rate than did the male carriers (37.01% versus 31.27%, P = 0.04). The transferable blastocyst rate was higher in couples treated with gonadotrophin-releasing hormone (GnRH) antagonist than in those treated with GnRH agonist (38.20% versus 24.37%, P = 0.01). However, neither carrier sex nor ovarian stimulation protocol influenced the clinical pregnancy rate.
Conclusions:
CNV-Seq-based NGS is an efficient and reliable PGT method for balanced reciprocal translocation.
Practical Problems in Assisted Conception - edited by Ying Cheong September 2018
Cambridge Core - Obstetrics and Gynecology, Reproductive Medicine - - edited by Ying Cheong
Background:
In in vitro fertilization (IVF) treatment, preimplantation genetic diagnosis/screening (PGD/S) attempts to detect chromosomal abnormalities in embryos before implantation. Using the meta-analytic and qualitative review approaches, this study aims to evaluate the effect of PGD/S on clinical pregnancy, live births, and childhood outcomes.
Methods:
We conducted a literature search using 1) PubMed and other search engines, and 2) an ancestry search by tracking references cited in prior work. After screening the studies, we extracted information pertinent to the meta-analysis. We calculated the effect sizes for clinical pregnancy and live birth rates, and performed a moderation analysis by maternal age, type of genetic screening, and timing of the biopsy. For childhood outcomes, we conducted a systematic review of studies reporting the anthropometric, psychomotor, cognitive, behavioral, and family functioning of PGD/S children.
Results:
We included 26 studies for clinical pregnancy and live births, and 18 studies for childhood outcomes. Results indicated that women who underwent comprehensive chromosome screening-based PGD/S had significantly higher clinical pregnancy rates (rr 1.207, 95% CI 1.017-1.431) and live birth rates (rr 1.362, 95% CI 1.057-1.755) than those whose IVF treatment did not include PGD/S. Early childhood outcomes of PGD/S children did not differ from those of non-PGD/S children.
Conclusions:
Comprehensive chromosome screening-based PGD/S can improve clinical pregnancy and live birth rates without adversely affecting functioning in childhood at least up to age 9. Results are discussed in the context of bioethical, financial, legal, and psychological issues surrounding PGD/S.
Objective:
To determine the efficiency of pre-implantation genetic screening (PGS) among women scheduled to undergo intracytoplasmic sperm injection who had experienced recurrent in vitro fertilization (IVF) failure.
Methods:
The present retrospective cohort study reviewed the medical records of consecutive women who had experienced recurrent IVF failure and had presented at a private IVF facility in Trabzon Province, Turkey, to undergo intracytoplasmic sperm injection between May 1, 2012, and December 31, 2014. Patient data and perinatal outcomes were compared between patients who underwent PGS and those who did not.
Results:
There were 88 patients included in the study; 43 patients had undergone PGS and 45 had declined to do so. No differences were detected in the clinical pregnancy rate (P=0.846), spontaneous abortion rate (P=0.416), number of perinatal deaths (P=0.162), and the number of live deliveries (P=0.188) between the groups of patients. The pregnancies included in the study resulted in 25 neonates being delivered; 24 had normal karyotypes, and one neonate from the control group had a karyotype of 46, XX, 9ph. Among the 19 embryos that were not transferred, the most frequently encountered chromosomal anomalies were diploidy, monosomy X, and 2N/N/4N mosaicism, detected in 7 (37%), 2 (11%), and 2 (11%) embryos, respectively.
Conclusion:
PGS had no effect on perinatal outcomes among women experiencing recurrent IVF failure. This article is protected by copyright. All rights reserved.
Although healthy infants have developed from non- and mono-pronuclear zygotes, the transfer of embryos from non- and mono-pronuclear zygotes is not recommended because there are no proper selection criteria. In the present study, we discuss how to select non- and mono-pronuclear embryos with the highest developmental potential at 19-20 hours post-insemination. We found that the percentage of blastocysts with normal chromosome constitution in non-pronuclear zygotes was slightly higher than in mono-pronuclear zygotes. Non- and mono-pronuclear embryos that were at the 4-cell stage on D2 and/or at the 6- to 8-cell stage on D3 had higher incidence rates of blastocysts with normal chromosome constitutions. We also found higher incidences of blastocysts with normal chromosome constitution on D6 than on D5. The results suggest that if high quality non- and mono-pronuclear zygotes develop to the 4-cell stage on D2 and the 6-to 8- cell stages on D3, along with high quality D6 blastocysts, the incidence of blastocysts with normal chromosome constitution is higher.
Background
Preimplantation genetic diagnosis (PGD) currently relies on biopsy of one or few embryo cells. Our aim was to evaluate the embryo extracellular matrices (spent medium and blastocoele fluid) as source of DNA for embryo genotyping.
Results/methodology
We first evaluated the amplifiability and the amount of genomic DNA in spent embryo culture media from day 3 (n = 32) and day 5/6 (n = 54). Secondly, we evaluated the possibility to genotype the MTHFR polymorphism C677T from media at day 5/6 (n = 8) and blastocoele fluids (n = 9) by direct sequencing. The C677T polymorphism detection rate was 62.5 and 44.4% in medium and fluid, respectively.
Conclusion
A noninvasive approach for embryo genotyping was possible, but still with limitations due to low detection rate and possible allele dropout.
To develop a noninvasive medium-based preimplantation genetic diagnosis (PGD) test for α-thalassemias-SEA.
The embryos of α-thalassemia-SEA carriers undergoing in vitro fertilization (IVF) were cultured. Single cells were biopsied from blastomeres and subjected to fluorescent gap polymerase chain reaction (PCR) analysis; the spent culture media that contained embryo genomic DNA and corresponding blastocysts as verification were subjected to quantitative-PCR (Q-PCR) detection of α-thalassemia-SEA. The diagnosis efficiency and allele dropout (ADO) ratio were calculated, and the cell-free DNA concentration was quantitatively assessed in the culture medium.
The diagnosis efficiency of medium-based α-thalassemias–SEA detection significantly increased compared with that of biopsy-based fluorescent gap PCR analysis (88.6% vs 82.1%, P < 0.05). There is no significant difference regarding ADO ratio between them. The optimal time for medium-based α-thalassemias–SEA detection is Day 5 (D5) following IVF.
Medium-based α-thalassemias–SEA detection could represent a novel, quick, and noninvasive approach for carriers to undergo IVF and PGD.
Unlabelled:
The study evaluated the proportion of patients whose pituitary glands respond with a sharp decrease in luteinizing hormone (LH) levels when exposed to a conventional dose of 0.25 mg gonadotropin releasing hormone (GnRH) antagonist in a prospective, single-center, non-randomized, proof-of-concept study. Fifty women eligible for in vitro fertilization (IVF) received recFSH (Gonal-F) from day 2 or 3 of menstrual period. Basal estradiol, progesterone, and LH were measured on the same day and 4-5 days later-immediately before GnRH antagonist 0.25 mg administration, and 24 hours after its administration. Responders were defined as "normal" if 24 hours after the first GnRH antagonist injection, LH level was ≥50% of the pre-injection level and as "over-suppressed" if it was <50% of the pre-injection level. Twelve patients (26% of the total) were "over-suppressed" with a mean LH level of 37% of the level 24 hours earlier. These patients also demonstrated a significant decrease in estradiol rise during the first 24 hours after initial antagonist administration. This effect was reversed for the rest of the stimulation period during which recLH (Luveris, 150 IU/day) was added to the "over-suppressed." If proven advantageous in terms of pregnancy rate, this approach to individualized treatment would be easy to implement.
Trial registration:
ClinicalTrials. gov Identifier: NCT01936077.
In assisted reproduction technology, embryo competence is routinely evaluated on morphological criteria. Over the last decade, efforts in improving non-invasive embryo assessment have looked into the secretome of embryos. Human embryos release genomic (gDNA) and mitochondrial DNA (mtDNA) into the culture medium, and the mtDNA/gDNA ratio is significantly correlated with embryo fragmentation. Here we investigate whether mtDNA/gDNA ratio in embryo spent medium is correlated with blastulation potential and implantation. The mtDNA/gDNA ratio was assessed in 699 day 3 culture media by quantitative PCR to investigate its correlation with embryo morphology, blastocyst development, and implantation. A logistic regression model evaluated whether mtDNA/gDNA ratio in the secretome may improve prediction of blastulation. We found that embryos that successfully developed into blastocysts exhibited a significantly higher mtDNA/gDNA ratio in the culture medium compared to those that arrest (P=0.0251), and mtDNA/gDNA, combined with morphological grading, has the potential to predict blastulation better than morphology alone (P=0.02). Moreover, mtDNA/gDNA ratio was higher in the media from good quality embryos that reached the full blastocyst stage on day 5 compared with embryos that developed more slowly (P<0.0001). With respect to blastocyst morphology, higher trophectoderm quality was associated with a higher mtDNA/gDNA ratio in the culture medium. Finally, a high mtDNA/gDNA ratio in spent medium was associated with successful implantation outcome (P=0.0452) of good quality embryos. In summary, the mtDNA/gDNA ratio in the day 3 embryo secretome, in combination with morphological grading, may be a novel, non-invasive, early, biomarker to improve identification of viable embryos with high developmental potential.
BACKGROUND
Chromosomal mosaicism, the presence of two or more distinct cell lines, is prevalent throughout human pre- and post-implantation development and can lead to genetic abnormalities, miscarriages, stillbirths or live births. Due to the prevalence and significance of mosaicism in the human species, it is important to understand the origins, mechanisms and incidence of mosaicism throughout development.METHODS
Literature searches were conducted utilizing Pubmed, with emphasis on human pre- and post-implantation mosaicism.RESULTSMosaicism persists in two separate forms: general and confined. General mosaicism is routine during human embryonic growth as detected by preimplantation genetic screening at either the cleavage or blastocyst stage, leading to mosaicism within both the placenta and fetus proper. Confined mosaicism has been reported in the brain, gonads and placenta, amongst other places. Mosaicism is derived from a variety of mechanisms including chromosome non-disjunction, anaphase lagging or endoreplication. Anaphase lagging has been implicated as the main process by which mosaicism arises in the preimplantation embryo. Furthermore, mosaicism can be caused by any one of numerous factors from paternal, maternal or exogenous factors such as culture media or possibly controlled ovarian hyperstimulation during in vitro fertilization (IVF). Mosaicism has been reported in as high as 70 and 90% of cleavage- and blastocyst-stage embryos derived from IVF, respectively.CONCLUSIONS
The clinical consequences of mosaicism depend on which chromosome is involved, and when and where an error occurs. Mitotic rescue of a meiotic error or a very early mitotic error will typically lead to general mosaicism while a mitotic error at a specific cell lineage point typically leads to confined mosaicism. The clinical consequences of mosaicism are dependent on numerous aspects, with the consequences being unique for each event.
Only a few years ago the American Society of Assisted Reproductive Medicine (ASRM), the European Society for Human Reproduction and Embryology (ESHRE) and the British Fertility Society declared preimplantation genetic screening (PGS#1) ineffective in improving in vitro fertilization (IVF) pregnancy rates and in reducing miscarriage rates. A presumably upgraded form of the procedure (PGS#2) has recently been reintroduced, and is here assessed in a systematic review. PGS#2 in comparison to PGS#1 is characterized by: (i) trophectoderm biopsy on day 5/6 embryos in place of day-3 embryo biopsy; and (ii) fluorescence in-situ hybridization (FISH) of limited chromosome numbers is replaced by techniques, allowing aneuploidy assessments of all 24 chromosome pairs. Reviewing the literature, we were unable to identify properly conducted prospective clinical trials in which IVF outcomes were assessed based on "intent to treat." Whether PGS#2 improves IVF outcomes can, therefore, not be determined. Reassessments of data, alleged to support the efficacy of PGS#2, indeed, suggest the opposite. Like with PGS#1, the introduction of PGS#2 into unrestricted IVF practice again appears premature, and threatens to repeat the PGS#1 experience, when thousands of women experienced reductions in IVF pregnancy chances, while expecting improvements. PGS#2 is an unproven and still experimental procedure, which, until evidence suggests otherwise, should only be offered under study conditions, and with appropriate informed consents.
What are the analytical and clinical validity and the clinical utility of in vitro screening of embryos by whole-genome sequencing?
At present there are still many limitations in terms of analytical and clinical validity and utility and many ethical questions remain.
Whole-genome sequencing of IVF/ICSI embryos is technically possible. Many loss-of-function mutations exist in the general population without serious effects on the phenotype of the individual. Moreover, annotations of genes and the reference genome are still not 100% correct.
We used publicly available samples from the 1000 Genomes project and Complete Genomics, together with 42 samples from in-house research samples of parents from trios to investigate the presence of loss-of-function mutations in healthy individuals.
In the samples, we looked for mutations in genes that are associated with a selection of severe Mendelian disorders with a known molecular basis. We looked for mutations predicted to be damaging by PolyPhen and SIFT and for mutations annotated as disease causing in Human Genome Mutation Database (HGMD).
More than 40% of individuals who can be considered healthy have mutations that are predicted to be damaging in genes associated with severe Mendelian disorders or are annotated as disease causing.
The analysis relies on current knowledge and databases are continuously updated to reflect our increasing knowledge about the genome. In the process of our analysis several updates were already made.
At this moment it is not advisable to use whole-genome sequencing as a tool to set up health profiles to select embryos for transfer. We also raise some ethical questions that have to be addressed before this technology can be used for embryo selection.
This research was supported by: Research Council KU Leuven (Projects: GOA/10/09 MaNet, KUL PFV/10/016 SymBioSys); Flemish Government: IWT - Agency for Innovation by Science and Technology (Project: O&O ExaScience Life), Hercules Foundation (Project: Hercules III PacBio RS), iMinds Future Health Department (Projects: SBO 2013, Art&D Instance), Flemish tier-1 Supercomputer (Project: VSC Tier 1 Exome sequencing); K.H. was supported by the Centre for Society and Life Sciences (CSG, non-profit organization) (Project number: 70.1.074).
None of the authors has any conflict of interest to declare. TRIAL REGISTRATION NUMBER: N/A.
High proportions of human embryos produced by in vitro fertilization are aneuploidy and mosaic. DNA microarray is one of the most practical screening methods to select euploid embryos for transfer. However, mosaic pregnancy is still possible due to embryonic mosacism. Here we report a successful pregnancy after transfer of a mosaic blastocyst with euploid inner cell mass.
A woman with a previous trisomy 13 pregnancy pursued infertility treatment with preimplantation genetic screening by a trophectoderm biopsy and DNA microarray. NimbleGen oligonucleotide DNA microarray was applied to biopsied samples from 13 blastocysts. A euploid blastocyst was transferred to the patient and subsequent prenatal cytogenetic tests were performed by FISH and/or G banding.
Following DNA microarray, it was found that 5 blastocysts were euploid and 8 were aneuploidy. Transfer of one euploid blastocyst resulted in a clinical pregnancy. Prenatal cytogenetic tests of samples biopsied from chorionic villi sample showed both trisomy 21 (47 XX, +21) and euploid (46, XX) cells. Further prenatal cytogenetic test with a sample from amniotic fluid indicated that all cells were euploid (46, XX). The pregnancy was continued and a healthy girl was delivered after 41 weeks of gestation.
This is the first report to indicate a mosaic pregnancy after transfer of a "euploid" blastocyst that was screened by DNA microarray, and the case further confirms that mosaicism is present in human blastocysts produced by in vitro fertilization.
Study question:
Does comprehensive chromosome screening (CCS) of cells sampled from the blastocyst trophectoderm (TE) accurately predict the chromosome complement of the inner cell mass (ICM)?
Summary answer:
Comprehensive chromosome screening of a TE sample is unlikely to be confounded by mosaicism and has the potential for high diagnostic accuracy.
What is known already:
The effectiveness of chromosome aneuploidy screening is limited by the technologies available and chromosome mosaicism in the embryo. Combined with improving methods for cryopreservation and blastocyst culture, TE biopsy and CCS is considered to be a promising approach to select diploid embryos for transfer.
Study design, size, duration:
The study was performed between January 2011 and August 2011. In the first part, a new ICM isolation method was developed and tested on 20 good morphology blastocysts. In the main phase of the study, fluorescence in situ hybridization (FISH) was used to reanalyse the ICMs and TEs separated from 70 embryos obtained from 26 patients undergoing blastocyst stage array comparative genome hybridization (aCGH) PGS cycles.
Materials, setting, methods:
The isolated ICM and TE fractions were characterized by immunostaining for KRT18. Then, non-transferrable cryopreserved embryos were selected for the FISH reanalysis based on previous genetic diagnosis obtained by TE aCGH analysis. Blastocysts either diploid for chromosome copy number (20) or diagnosed as single- (40) or double aneuploid (10) were included after preparing the embryo into one ICM and three equal-sized TE sections. Accuracy of the aCGH was measured based on FISH reanalysis. Chromosomal segregations resulting in diploid/aneuploid mosaicism were classified as 'low-', 'medium-' and 'high-' grade and categorized with respect to their distribution (1TE, 2TE, 3TE, ICM or ALL embryo). Linear regression model was used to test the relationship between the distributions and the proportion of aneuploid cells across the four embryo sections. Fisher's exact test was used to test for random allocation of aneuploid cells between TE and ICM.
Main results and the role of chance:
All ICM biopsy procedures displayed ICM cells in the recovered fraction with a mean number of ICM cells of 26.2 and a mean TE cell contamination rate of 2%. By FISH reanalysis of previously aCGH-screened blastocysts, a total of 66 aneuploidies were scored, 52 (78.8%) observed in all cells and 14 (21.2%) mosaic. Overall, mosaic chromosomal errors were observed only in 11 out of 70 blastocysts (15.7%) but only 2 cases were classified as mosaic diploid/aneuploid (2.9%). Sensitivity and specificity of aCGH on TE clinical biopsies were 98.0 and 100% per embryo and 95.2 and 99.8% per chromosome, respectively. Linear regression analysis performed on the 11 mosaic diploid/aneuploid chromosomal segregations showed a significant positive correlation between the distribution and the proportion of aneuploid cells across the four-blastocyst sections (P < 0.01). In addition, regression analysis revealed that both the grade and the distribution of mosaic abnormal cells were significantly correlated with the likelihood of being diagnosed by aCGH performed on clinical TE biopsies (P = 0.019 and P < 0.01, respectively). Fisher's exact test for the 66 aneuploidies recorded showed no preferential allocation of abnormal cells between ICM and TE (P = 0.33).
Limitations, reasons for caution:
The study is limited to non-transferable embryos, reanalyzed for only nine chromosomes and excludes segmental imbalance and uniparental disomy. The prevalence of aneuploidy in the study group is likely to be higher than in the general population of clinical PGD embryos.
Wider implications of the findings:
This study showed high accuracy of diagnosis achievable during blastocyst stage PGS cycles coupled with 24-chromosomes molecular karyotyping analysis. The new ICM isolation strategy developed may open new possibilities for basic research in embryology and for clinical grade derivation of human embryonic stem cells.
Study funding/competing interest(s):
No specific funding was sought or obtained for this study.
Objective:
To determine whether performing comprehensive chromosome screening (CCS) and transferring a single euploid blastocyst can result in an ongoing pregnancy rate that is equivalent to transferring two untested blastocysts while reducing the risk of multiple gestation.
Design:
Randomized, noninferiority trial.
Setting:
Academic center for reproductive medicine.
Patient(s):
Infertile couples (n = 205) with a female partner less than 43 years old having a serum anti-Müllerian hormone level ≥ 1.2 ng/mL and day 3 FSH <12 IU/L.
Intervention(s):
Randomization occurred when at least two blastocysts were suitable for trophectoderm biopsy. The study group (n = 89) had all viable blastocysts biopsied for real-time, polymerase chain reaction-based CCS and single euploid blastocyst transfer. The control group (n = 86) had their two best-quality, untested blastocysts transferred.
Main outcome measure(s):
The ongoing pregnancy rate to ≥ 24 weeks (primary outcome) and the multiple gestation rate.
Result(s):
The ongoing pregnancy rate per randomized patient after the first ET was similar between groups (60.7% after single euploid blastocyst transfer vs. 65.1% after untested two-blastocyst transfer; relative risk [RR], 0.9; 95% confidence interval [CI], 0.7-1.2). A difference of greater than 20% in favor of two-blastocyst transfer was excluded. The risk of multiple gestation was reduced after single euploid blastocyst transfer (53.4% to 0%), and patients were nearly twice as likely to have an ongoing singleton pregnancy (60.7% vs. 33.7%; RR, 1.8; 95% CI, 1.3-2.5).
Conclusion(s):
In women ≤ 42 years old, transferring a single euploid blastocyst results in ongoing pregnancy rates that are the same as transferring two untested blastocysts while dramatically reducing the risk of twins.
We have assessed the numbers of potentially deleterious variants in the genomes of apparently healthy humans by using (1) low-coverage whole-genome sequence data from 179 individuals in the 1000 Genomes Pilot Project and (2) current predictions and databases of deleterious variants. Each individual carried 281-515 missense substitutions, 40-85 of which were homozygous, predicted to be highly damaging. They also carried 40-110 variants classified by the Human Gene Mutation Database (HGMD) as disease-causing mutations (DMs), 3-24 variants in the homozygous state, and many polymorphisms putatively associated with disease. Whereas many of these DMs are likely to represent disease-allele-annotation errors, between 0 and 8 DMs (0-1 homozygous) per individual are predicted to be highly damaging, and some of them provide information of medical relevance. These analyses emphasize the need for improved annotation of disease alleles both in mutation databases and in the primary literature; some HGMD mutation data have been recategorized on the basis of the present findings, an iterative process that is both necessary and ongoing. Our estimates of deleterious-allele numbers are likely to be subject to both overcounting and undercounting. However, our current best mean estimates of ∼400 damaging variants and ∼2 bona fide disease mutations per individual are likely to increase rather than decrease as sequencing studies ascertain rare variants more effectively and as additional disease alleles are discovered.
Single embryo transfer (SET) remains underutilized as a strategy to reduce multiple gestation risk in IVF, and its overall lower pregnancy rate underscores the need for improved techniques to select one embryo for fresh transfer. This study explored use of comprehensive chromosomal screening by array CGH (aCGH) to provide this advantage and improve pregnancy rate from SET.
First-time IVF patients with a good prognosis (age <35, no prior miscarriage) and normal karyotype seeking elective SET were prospectively randomized into two groups: In Group A, embryos were selected on the basis of morphology and comprehensive chromosomal screening via aCGH (from d5 trophectoderm biopsy) while Group B embryos were assessed by morphology only. All patients had a single fresh blastocyst transferred on d6. Laboratory parameters and clinical pregnancy rates were compared between the two groups.
For patients in Group A (n = 55), 425 blastocysts were biopsied and analyzed via aCGH (7.7 blastocysts/patient). Aneuploidy was detected in 191/425 (44.9%) of blastocysts in this group. For patients in Group B (n = 48), 389 blastocysts were microscopically examined (8.1 blastocysts/patient). Clinical pregnancy rate was significantly higher in the morphology + aCGH group compared to the morphology-only group (70.9 and 45.8%, respectively; p = 0.017); ongoing pregnancy rate for Groups A and B were 69.1 vs. 41.7%, respectively (p = 0.009). There were no twin pregnancies.
Although aCGH followed by frozen embryo transfer has been used to screen at risk embryos (e.g., known parental chromosomal translocation or history of recurrent pregnancy loss), this is the first description of aCGH fully integrated with a clinical IVF program to select single blastocysts for fresh SET in good prognosis patients. The observed aneuploidy rate (44.9%) among biopsied blastocysts highlights the inherent imprecision of SET when conventional morphology is used alone. Embryos randomized to the aCGH group implanted with greater efficiency, resulted in clinical pregnancy more often, and yielded a lower miscarriage rate than those selected without aCGH. Additional studies are needed to verify our pilot data and confirm a role for on-site, rapid aCGH for IVF patients contemplating fresh SET.
Since it was established in 1997, the ESHRE PGD Consortium has been collecting data from international preimplantation genetic diagnosis (PGD) centres. Ten papers have been published, including data from January 1997 to December 2007.
The data collection originally used a hard-copy format, then an excel database and finally a FileMaker Pro database. The indications are divided into five categories: PGD for chromosome abnormalities, sexing for X-linked disease, PGD for single gene defects, preimplantation genetic screening (PGS) and PGD for social sexing. The main end-points are pregnancy outcome and follow-up of deliveries.
In data collection I, 16 centres contributed data, which increased to 57 centres by data X (average of 39 centres per data collection). These centres contributed data on over 27 000 cycles that reached oocyte retrieval. Of these cycles, 61% were for aneuploidy screening, 17% for single gene disorders, 16% for chromosomal abnormalities, 4% for sexing of X-linked disease and 2% for social sexing. Cumulatively, 5187 clinical pregnancies gave rise to 4140 deliveries and 5135 newborns (singletons: 3182, twins: 921, triplets: 37).
In this paper, we present an overview of the first 10 years of PGD data, highlighting trends. These include the introduction of laser-assisted biopsy, an increase in polar body and trophectoderm biopsy, new strategies, methodologies and technologies for diagnosis, including recently arrays, and the more frequent use of freezing biopsied embryos. The Consortium data reports represent a valuable resource for information about the practice of PGD.
Blastomere biopsy of human embryos is performed for preimplantation genetic diagnosis (PGD). The impact on further development is largely unexplored, though studies on mice suggest an influence on the hatching process. The objective of this study was to evaluate the effect of blastomere biopsy on early human embryonic development using time-lapse analysis.
Embryos from couples undergoing PGD treatment or IVF/ICSI were included. In the PGD group, 56 human embryos had one blastomere biopsied. As controls, 53 non-biopsied IVF/ICSI embryos were selected. All embryos were cultured until 5 days after fertilization in a time-lapse incubator (EmbryoScope™). Images of embryos were acquired every 20 min. Time-points of key embryonic events were registered, and development in the two groups was compared.
Duration of the biopsied cell-stage in the PGD group was longer than in the control group (P < 0.001), causing biopsied embryos to reach subsequent embryonic stages until hatching at significantly later time-points (P(compaction) < 0.001; P(morula) < 0.001; P(earlyblast) < 0.001; P(fullblast) = 0.01), but with unchanged intervals. Embryos in the PGD group started hatching at the same time-point as the control group, but had a smaller diameter (P < 0.001), and a thicker zona pellucida (P < 0.001) when hatching. Time-lapse videos revealed that in the control group, expansion of the blastocyst caused continuous thinning of zona pellucida until the blastocyst hatched, whereas in the PGD group the blastocyst hatched through the opening in zona pellucida artificially introduced prior to the biopsy.
We find that blastomere biopsy prolongs the biopsied cell-stage, possibly caused by a delayed compaction and alters the mechanism of hatching.
Recent studies have suggested that biopsy of several trophectoderm (TE) cells from blastocysts followed by comparative genomic hybridization (CGH) analysis might represent an optimal strategy for aneuploidy detection, but few data on accuracy are available. The main question concerns the rate of mosaicism at the blastocyst stage, and to what extent this might cause misdiagnoses. We assessed blastocyst aneuploidy and mosaicism rates and evaluated the accuracy and efficiency of CGH and microarray-CGH (aCGH) for TE analysis.
A total of 52 blastocysts, from 20 couples, were biopsied and their chromosomes examined by CGH. The remaining cells were spread and tested by fluorescent in situ hybridization (FISH). Of the 52 blastocysts, 20 underwent a second TE biopsy and were tested using aCGH.
CGH and aCGH produced results for 98% of TE samples. 42.3% of blastocysts were uniformly euploid, 30% were uniformly aneuploid and 32.4% were mosaic. Of the mosaic embryos, 15.4% were found to be composed of a mixture of different aneuploid cell lines, while 17% contained both normal and aneuploid cells. Mosaic diploid-aneuploid blastocysts with >30% normal cells accounted for <6% of analysed embryos.
Comprehensive chromosome screening and follow-up assessment of large numbers of cells provided a unique insight into the cytogenetics of human blastocysts. Meiotic and post-zygotic errors leading to mosaicism were common. However, most mosaic blastocysts contained no normal cells. Hence, CGH or aCGH TE analysis is an accurate aneuploidy detection tool and may assist in identifying viable euploid embryos with higher implantation potential.
In 2005, the European Society for Human Reproduction and Embryology (ESHRE) Preimplantation Genetic Diagnosis (PGD) Consortium published a set of Guidelines for Best Practice to give information, support and guidance to potential, existing and fledgling PGD programmes (Thornhill AR, De Die-Smulders CE, Geraedts JP, Harper JC, Harton GL, Lavery SA, Moutou C, Robinson MD, Schmutzler AG, Scriven PN et al. ESHRE PGD Consortium best practice guidelines for clinical preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS). Hum Reprod 2005;20:35-48.). The subsequent years have seen the introduction of a number of new technologies as well as the evolution of current techniques. Additionally, in light of ESHRE's recent advice on how practice guidelines should be written and formulated, the Consortium believed it was timely to revise and update the PGD guidelines. Rather than one document that covers all of PGD as in the original publication, these guidelines are separated into four new documents that apply to different aspects of a PGD programme; Organization of a PGD centre, fluorescence in situ hybridization-based testing, amplification-based testing and polar body and embryo biopsy for preimplantation genetic diagnosis/screening (PGD/PGS). Here we have updated the sections that pertain to embryology (including cryopreservation) and biopsy of embryos prior to PGD or PGS. Topics covered in this guideline include uses of embryo biopsy, laboratory issues relating to biopsy, timing of biopsy, biopsy procedure and cryopreserving biopsied embryos.
The 10th report of the European Society of Human Reproduction and Embryology (ESHRE) PGD Consortium is presented, documenting
cycles collected for the calendar year 2007 and follow-up of the pregnancies and babies born until October 2008 which resulted
from these cycles. Since the beginning of the data collections there has been a steady increase in the number of cycles, pregnancies
and babies reported annually. For data collection X, 57 centres participated, reporting on 5887 cycles to oocyte retrieval
(OR), along with details of the follow-up on 1516 pregnancies and 1206 babies born. A total of 729 OR were reported for chromosomal
abnormalities, 110 OR for sexing for X-linked diseases, 1203 OR for monogenic diseases, 3753 OR for preimplantation genetic
screening and 92 OR for social sexing. Data X is compared with the cumulative data for data collections I–IX.
Aneuploidy has been well-documented in blastocyst embryos, but prior studies have been limited in scale and/or lack mechanistic data. We previously reported preclinical validation of microarray 24-chromosome preimplantation genetic screening in a 24-h protocol. The method diagnoses chromosome copy number, structural chromosome aberrations, parental source of aneuploidy and distinguishes certain meiotic from mitotic errors. In this study, our objective was to examine aneuploidy in human blastocysts and determine correspondence of karyotypes between trophectoderm (TE) and inner cell mass (ICM). We disaggregated 51 blastocysts from 17 couples into ICM and one or two TE fractions. The average maternal age was 31. Next, we ran 24-chromosome microarray molecular karyotyping on all of the samples, and then performed a retrospective analysis of the data. The average per-chromosome confidence was 99.95%. Approximately 80% of blastocysts were euploid. The majority of aneuploid embryos were simple aneuploid, i.e. one or two whole-chromosome imbalances. Structural chromosome aberrations, which are common in cleavage stage embryos, occurred in only three blastocysts (5.8%). All TE biopsies derived from the same embryos were concordant. Forty-nine of 51 (96.1%) ICM samples were concordant with TE biopsies derived from the same embryos. Discordance between TE and ICM occurred only in the two embryos with structural chromosome aberration. We conclude that TE karyotype is an excellent predictor of ICM karyotype. Discordance between TE and ICM occurred only in embryos with structural chromosome aberrations.
Although selection of chromosomally normal embryos has the potential to improve outcomes for patients undergoing IVF, the clinical impact of aneuploidy screening by fluorescence in situ hybridization (FISH) has been controversial. There are many putative explanations including sampling error due to mosaicism, negative impact of biopsy, a lack of comprehensive chromosome screening, the possibility of embryo self-correction and poor predictive value of the technology itself. Direct analysis of the negative predictive value of FISH-based aneuploidy screening for an embryo's reproductive potential has not been performed. Although previous studies have found that cleavage-stage FISH is poorly predictive of aneuploidy in morphologically normal blastocysts, putative explanations have not been investigated. The present study used a single nucleotide polymorphism (SNP) microarray-based 24 chromosome aneuploidy screening technology to re-evaluate morphologically normal blastocysts that were diagnosed as aneuploid by FISH at the cleavage stage. Mosaicism and preferential segregation of aneuploidy to the trophectoderm (TE) were evaluated by characterization of multiple sections of the blastocyst. SNP microarray technology also provided the first opportunity to evaluate self-correction mechanisms involving extrusion or duplication of aneuploid chromosomes resulting in uniparental disomy (UPD). Of all blastocysts evaluated (n = 50), 58% were euploid in all sections despite an aneuploid FISH result. Aneuploid blastocysts displayed no evidence of preferential segregation of abnormalities to the TE. In addition, extrusion or duplication of aneuploid chromosomes resulting in UPD did not occur. These findings support the conclusion that cleavage-stage FISH technology is poorly predictive of aneuploidy in morphologically normal blastocysts.
The impact of gonadotropin-releasing hormone (GnRH) agonist long compared with GnRH antagonist protocols, under in vitro fertilization conditions on endometrial receptivity, is still debated. Therefore, we compared the effect of both GnRH antagonist and agonist long protocols on the endometrial receptivity by analyzing, to our knowledge for the first time, the global gene expression profile shift during the prereceptive and receptive stages of stimulated cycles under the two GnRH analogue protocols compared with natural cycles in the same patients. For the same normal-responder patients, endometrial biopsies were collected on the day of oocyte retrieval and on the day of embryo transfer after human chorionic gonadotropin administration of a stimulated cycle with either GnRH agonist long or GnRH antagonist protocols and compared with the prereceptive and receptive stages of a natural cycle. Samples were analyzed using DNA microarrays. Gene expression profiles and biological pathways involved during the prereceptive stage to the receptive endometrial transition of stimulated and natural cycles were analyzed and compared for each patient. Both protocols affect endometrial receptivity in comparison with their natural cycle in the same patients. Major differences in endometrial chemokines and growth factors under stimulated cycles in comparison with natural cycles were observed. Such an effect has been associated with gene expression alterations of endometrial receptivity. However, the endometrial receptivity under the GnRH antagonist protocol was more similar to the natural cycle receptivity than that under the GnRH agonist protocol.
Human embryo biopsy is performed for preimplantation genetic diagnosis (PGD). The impact of 1- or 2-cell removal at cleavage-stage on future embryo development and implantation capacity is highly debated.
In order to explore this issue further, a cohort of Day 5 single embryo transfers was analysed prospectively for embryological and clinical outcome. All transferred embryos resulted from 8-cell embryos on Day 3, from which subsequently either one cell (group I, n = 182) or two cells (group II, n = 259) were removed, or on which no invasion by means of embryo biopsy was performed (group III, control group, n = 702). RESULTS Blastocyst formation was significantly better in group III compared with group II, and similar to group I. Group I and group II did not differ in Day 3 nor in Day 5 embryo development. The overall live birth rate was significantly higher in group I (37.4%, CI 29.0-47.4%) than in group II (22.4%, CI 17.0-28.9%), and comparable to the reference ICSI population (35.0%, CI 30.8-39.7%).
The clinical outcome of 1-cell biopsy was significantly better than that of 2-cell biopsy, even when adjusted for availability of genetically transferable embryos.
Embryonic aneuploidy is highly prevalent in IVF cycles and contributes to decreased implantation rates, IVF cycle failure and early pregnancy loss. Preimplantation genetic screening (PGS) selects the most competent (euploid) embryos for transfer, and has been proposed to improve IVF outcomes. Use of PGS with fluorescence-in-situ hybridization technology after day 3 embryo biopsy (PGS-v1) significantly lowers live birth rates and is not recommended for use. Comprehensive chromosome screening technology, which assesses the whole chromosome complement, can be achieved using different genetic platforms. Whether PGS using comprehensive chromosome screening after blastocyst biopsy (PGS-v2) improves IVF outcomes remains to be determined. A systematic review of randomized controlled trials was conducted on PGS-v2. Three trials met full inclusion criteria, comparing PGS-v2 and routine IVF care. PGS-v2 is associated with higher clinical implantation rates, and higher ongoing pregnancy rates when the same number of embryos is transferred in both PGS and control groups. Additionally, PGS-v2 improves embryo selection for elective single embryo, maintaining the same ongoing pregnancy rates between PGS and control groups, while sharply decreasing multiple pregnancy rates. These results stem from good-prognosis patients undergoing IVF. Whether these findings can be extrapolated to poor-prognosis patients with decreased ovarian reserve remains to be determined.
Preimplantation genetic diagnosis (PGD) for human leukocyte antigen (HLA) typing is an established procedure for conceiving a child who may donate cord blood or haematopoietic stem cells for transplantation to save an ill sibling. Haematopoietic stem cell transplantation (HSCT) from related matched donors improves overall survival compared with unrelated or non-matched donors. As IVF for PGD–HLA is unavailable for 70% of patients, it is a relevant clinical option. Recent success of HSCT after PGD–HLA, and excellent health and family support of the children born, suggests that debate over this kind of ‘designer baby’ and ‘gift of life’ should subside. Discussions about IVF for PGD–HLA should be held with families when a related matched-donor is unavailable, when HSCT can wait at least 9–12 months, within weeks of diagnosis irrespective of prognosis, and when the mother is of reproductive age. Related half-matched egg donors may also be considered. National and international collaborations should be established, and couples choosing this modality should be referred to experienced IVF and PGD centres. Clinical guidelines will improve physician and patient awareness of IVF for PGD-HLA and its role in advancing the clinical care of children in need of HSCT.
Comprehensive chromosome screening (CCS) methods are being extensively used to select chromosomally normal embryos in human assisted reproduction. Some concerns related to the stage of analysis and which aneuploidy screening method to use still remain. In this study, the reliability of blastocyst-stage aneuploidy screening and the diagnostic performance of the two mostly used CCS methods (quantitative real-time PCR (qPCR) and array comparative genome hybridization (aCGH)) has been assessed. aCGH aneuploid blastocysts were rebiopsied, blinded, and evaluated by qPCR. Discordant cases were subsequently rebiopsied, blinded, and evaluated by single-nucleotide polymorphism (SNP) array-based CCS. Although 81.7% of embryos showed the same diagnosis when comparing aCGH and qPCR-based CCS, 18.3% (22/120) of embryos gave a discordant result for at least one chromosome. SNP array reanalysis showed that a discordance was reported in ten blastocysts for aCGH, mostly due to false positives, and in four cases for qPCR. The discordant aneuploidy call rate per chromosome was significantly higher for aCGH (5.7%) compared with qPCR (0.6%; P<0.01). To corroborate these findings, 39 embryos were simultaneously biopsied for aCGH and qPCR during blastocyst-stage aneuploidy screening cycles. 35 matched including all 21 euploid embryos. Blinded SNP analysis on rebiopsies of the four embryos matched qPCR. These findings demonstrate the high reliability of diagnosis performed at the blastocyst stage with the use of different CCS methods. However, the application of aCGH can be expected to result in a higher aneuploidy rate than other contemporary methods of CCS.European Journal of Human Genetics advance online publication, 29 October 2014; doi:10.1038/ejhg.2014.222.
Objective
To investigate the presence of DNA in blastocyst fluids (BFs) and to estimate whether the chromosomal status predicted by its analysis corresponds with the ploidy condition in trophectoderm (TE) cells, the whole embryo, and that predicted by polar bodies (PBs) or blastomeres.
Design
Prospective study.
Setting
In vitro fertilization unit.
Patient(s)
Seventeen couples undergoing preimplantation genetic screening with the use of array comparative genomic hybridization on PBs (n = 12) or blastomeres (n = 5).
Intervention(s)
BFs and TE cells were retrieved from 51 blastocysts for separate chromosomal analysis.
Main Outcome Measure(s)
Presence of DNA in BFs and assessment of the corresponding chromosome condition; correlation with the results in TE cells and those predicted by the analysis done at earlier stages.
Result(s)
DNA was detected in 39 BFs (76.5%). In 38 of 39 cases (97.4%) the ploidy condition of BFs was confirmed in TE cells, and the rate of concordance per single chromosome was 96.6% (904/936). In relation to the whole embryo, the ploidy condition corresponded in all cases with a per-chromosome concordance of 98.1%. The testing of PBs and blastomeres had 93.3% and 100% prediction of BF ploidy condition with a concordance per chromosome of 93.5% and 94%, respectively.
Conclusion(s)
Blastocentesis could represent an alternative source of material for chromosomal testing, because the BF is highly predictive of the embryo ploidy condition and chromosome content. Our data confirm the relevance of the oocyte and of the early-cleavage embryo in determining the ploidy condition of the resulting blastocyst.
Objective
To validate a next-generation sequencing (NGS)–based method for 24-chromosome aneuploidy screening and to investigate its applicability to preimplantation genetic screening (PGS).
Design
Retrospective blinded study.
Setting
Reference laboratory.
Patient(s)
Karyotypically defined chromosomally abnormal single cells and whole-genome amplification (WGA) products, previously analyzed by array comparative genomic hybridization (array-CGH), selected from 68 clinical PGS cycles with embryos biopsied at cleavage stage.
Intervention(s)
None.
Main Outcome Measure(s)
Consistency of NGS-based diagnosis of aneuploidy compared with either conventional karyotyping of single cells or array-CGH diagnoses of single blastomeres.
Result(s)
Eighteen single cells and 190 WGA products from single blastomeres, were blindly evaluated with the NGS-based protocol. In total, 4,992 chromosomes were assessed, 402 of which carried a copy number imbalance. NGS specificity for aneuploidy call (consistency of chromosome copy number assignment) was 99.98% (95% confidence interval [CI] 99.88%–100%) with a sensitivity of 100% (95% CI 99.08%–100%). NGS specificity for aneuploid embryo call (24-chromosome diagnosis consistency) was 100% (95% CI 94.59%–100%) with a sensitivity of 100% (95% CI 97.39%–100%).
Conclusion(s)
This is the first study reporting extensive preclinical validation and accuracy assessment of NGS-based comprehensive aneuploidy screening on single cells. Given the high level of consistency with an established methodology, such as array-CGH, NGS has demonstrated a robust high-throughput methodology ready for clinical application in reproductive medicine, with potential advantages of reduced costs and enhanced precision.
Preimplantation Genetic Diagnosis (PGD) allows identifying genetic traits in early embryos. Since in some equine breeds, like Polo Argentino, females are preferred to males for competition, PGD can be used to determine the gender of the embryo before transfer and thus allow the production of only female pregnancies. This procedure could have a great impact on commercial embryo production programs. The present study was conducted to adapt gender selection by PGD to a large-scale equine embryo transfer program. To achieve this, we studied i) the effect on pregnancy rates of holding biopsied embryos for 7 to 10 hours in holding medium at 32°C before transfer, ii) the effect on pregnancy rates of using embryos of different sizes for biopsy, and iii) the efficiency of amplification by heating biopsies before PCR. Equine embryos were classified by size (≤300 μm, 300 to 1000 μm and >1000 μm), biopsied and transferred 1 to 2 or 7 to 10 hours after flushing. Some of the biopsy samples obtained were incubated for 10 min at 95°C and the rest remained untreated. Pregnancy rates were recorded at 25 days of gestation; fetal gender was determined by ultrasonography and compared to PGD results. Holding biopsied embryos for 7 to 10 hours before transfer produced pregnancy rates similar to those for biopsied embryos transferred within two hours (63% and 57%, respectively). These results did not differ from pregnancy rates of non-biopsied embryos undergoing the same holding times (50% for 7 to 10 hours and 63% for 1 to 2 hours). Pregnancy rates for biopsied and non-biopsied embryos did not differ between size groups or between biopsied and non-biopsied embryos within the same size group (P>0.05). Incubating biopsy samples for 10 min at 95°C before PCR significantly increased the diagnosis rate (78.5% versus 45.5% for treated and non-treated biopsy samples respectively). Gender determination using incubated biopsy samples matched the results obtained by ultrasonography in all pregnancies assessed (11 of 11, 100%); untreated biopsy samples were correctly diagnosed in 36/41 assessed pregnancies (87.8%), although the difference between treated and untreated biopsy samples was not significant. Our results demonstrated that biopsied embryos can remain in holding medium before being transferred, until gender diagnosis by PGD is complete (7 to 10 hours), without affecting pregnancy rates. This simplifies the management of an embryo transfer program willing to incorporate PGD for gender selection, by transferring only embryos of the desired sex. Embryo biopsy can be performed in a clinical setting on embryos of different sizes, without affecting their viability. Additionally, we showed that pretreating biopsy samples with a short incubation at 95°C improved the overall efficiency of embryo sex determination.
The randomized clinical trial (RCT) was developed to assess the efficacy of medical interventions by comparing a treatment group with a control group that did not receive treatment or received placebo. It has been evident that this study design is more suited to evaluation of drugs and less to complex interventions such as surgical procedures. The same can be said about assisted reproduction technology, which clearly belongs in the complex category. Yet, there have been no specific guidelines for the design of RCT in clinical embryology. Moreover, the reliability of the published data from arbitrarily designed trials has not been debated. The near absence of allocation concealment and blinding and the difficulties inherent in randomizing oocytes and embryos in addition to patients require further consideration. This work evaluates systematic reviews to assess the efficacy of interventions, including the use of different culture media, culture under reduced oxygen tension, blastocyst culture, co-culture with somatic cells, assisted hatching, preimplantation genetic screening and intracytoplasmic morphologically selected sperm injection. The overall quality of a RCT and the appropriate application of new technologies in clinical embryology may be improved if a specific set of guidelines were to be developed for such investigations.
Chromosome aneuploidy, an abnormal number of chromosomes, in human gametes and embryos is a major cause of IVF failure and miscarriage and can result in affected live births. To avoid these outcomes and improve implantation and live birth rates, preimplantation genetic screening aims to identify euploid embryos before transfer but has been restricted to analysis of a limited number of chromosomes. Over the past 15 years, various technologies have been developed that allow copy number analysis of all 23 pairs of chromosomes, 22 autosomes, and the sex chromosomes, or "24-chromosome" copy number analysis in single or small numbers of cells. Herein the pros and cons of these technologies are reviewed and evaluated for their potential as screening or diagnostic tests when used in combination with oocyte or embryo biopsy at different stages.
Is the amount of cell-free DNA released by human embryos into culture medium correlated with embryo morphological features?
The mitochondrial DNA (mtDNA) content of culture medium is significantly associated with the fragmentation rate on Days 2 and 3 of embryo development, whether the oocyte came from women ≤35 or >35 years old.
Cellular fragmentation is often utilized as one of the morphological parameters for embryo quality assessment. The amount of cellular fragments is considered to be an important morphological parameter for embryo implantation potential. It has been hypothesized that fragments are apoptotic bodies or anuclear cytoplasmatic pieces of blastomeres, although no definitive conclusion has been drawn about their pathogenesis.
Human fertilized oocytes were individually cultured from Day 1 to Days 2 and 3. A total of 800 samples (166 spent media from Day 2 and 634 from Day 3) were enrolled into the present study.
Double-stranded DNA (dsDNA) was quantified in 800 spent embryo culture media by Pico Green dye fluorescence assay. After DNA purification, genomic DNA (gDNA) and mtDNA were profiled by specific quantitative PCR. Statistical analyses defined correlations among DNA contents, embryo morphology and maternal age.
Different independent tests confirmed the presence of DNA into embryo culture medium and, for the first time, we demonstrate that both gDNA and mtDNA are detectable in the secretome. The amount of DNA is larger in embryos with bad quality cleavage compared with high-grade embryos, suggesting that the DNA profile of culture medium is an objective marker for embryo quality assessment. In particular, DNA profiles are significantly associated with fragmentation feature (total dsDNA: P = 0.0010; mtDNA; P = 0.0247) and advanced maternal age.
It is necessary to establish whether DNA profiling of spent embryo culture medium is a robust onsite test that can improve the prediction of blastulation, implantation and/or pregnancy rate.
The approach we are proposing may provide a novel, non-invasive, objective tool for embryo quality grading. The correlation between a high mtDNA concentration and the fragmentation rate of embryos is suggestive that fragments are mainly anuclear cytoplasmatic debris arising during cleavage. Therefore, blastomere shaping as an early event during in vitro development may play a homeostatic role and be related to embryo competence.
This project was funded by Merck Serono (Grant for Fertility Innovation 2011). The sponsor had no role in study design, data collection, data analysis, data interpretation and writing of the paper. Authors declare no conflicts of interest.
ClinicalTrials.gov Identifier: NCT01397136.
The mechanisms underlying the appearance of asymmetry between cells in the early embryo and consequently the specification of distinct cell lineages during mammalian development remain elusive. Recent experimental advances have revealed unexpected dynamics of and new complexity in this process. These findings can be integrated in a new unified framework that regards the early mammalian embryo as a self-organizing system.
Objective:
To determine if cleavage- or blastocyst-stage embryo biopsy affects reproductive competence.
Design:
Paired randomized clinical trial.
Setting:
Academic-assisted reproduction program.
Patient(s):
Attempting conception through IVF.
Intervention(s):
After selecting two embryos for transfer, one was randomized to biopsy and the other to control. Both were transferred within shortly thereafter. The biopsy was submitted for microarray analysis and single-nucleotide polymorphism (SNP) profiling. Buccal DNA obtained from the neonate after delivery had microarray analysis and SNP profile compared with that of the embryonic DNA. A match confirmed that the biopsied embryo implanted and developed to term, whereas a nonmatch indicated that the control embryo had led to the delivery.
Main outcome measure(s):
Paired analysis of the delivery rates of the transferred embryos. Either twin delivery or failure to deliver represents equivalent outcomes for the biopsied and control embryos. In contrast, singletons were determined to be from the biopsied or the control embryo.
Result(s):
Blastomere biopsy on day 3 of development resulted in a significant reduction in sustained implantation. Only 30% of biopsied embryos had sustained implantation and ultimately developed into live-born infants versus 50% of unbiopsied controls, a relative reduction of 39%. In contrast, sustained implantation rates were equivalent (51% vs. 54%) for biopsied and control blastocysts.
Conclusion(s):
Cleavage-stage biopsy markedly reduced embryonic reproductive potential. In contrast, trophectoderm biopsy had no measurable impact and may be used safely when embryo biopsy is indicated.
Clinical trial registration number:
NCT01219504.
Objective:
To determine whether blastocyst biopsy and rapid quantitative real-time polymerase chain reaction (qPCR)-based comprehensive chromosome screening (CCS) improves in vitro fertilization (IVF) implantation and delivery rates.
Design:
Randomized controlled trial.
Setting:
Academic reproductive medicine center.
Patient(s):
Infertile couples in whom the female partner (or oocyte donor) is between the ages of 21 and 42 years who are attempting conception through IVF.
Intervention(s):
Embryonic aneuploidy screening.
Main outcome measure(s):
Sustained implantation and delivery rates.
Result(s):
We transferred 134 blastocysts to 72 patients in the study (CCS) group and 163 blastocysts to 83 patients in the routine care (control) group. Sustained implantation rates (probability that an embryo will implant and progress to delivery) were statistically significantly higher in the CCS group (89 of 134; 66.4%) compared with those from the control group (78 of 163; 47.9%). Delivery rates per cycle were also statistically significantly higher in the CCS group. Sixty one of 72 treatment cycles using CCS led to delivery (84.7%), and 56 of 83 (67.5%) control cycles ultimately delivered. Outcomes were excellent in both groups, but use of CCS clearly improved patient outcomes.
Conclusion(s):
Blastocyst biopsy with rapid qPCR-based comprehensive chromosomal screening results in statistically significantly improved IVF outcomes, as evidenced by meaningful increases in sustained implantation and delivery rates.
Clinical trial registration number:
NCT01219283.
Genomic technologies, including next-generation sequencing (NGS) and single-nucleotide polymorphism (SNP) microarrays, have provided unprecedented opportunities to assess genomic variation among, and increasingly within, individuals. It has long been known that cancer is a mosaic genetic disorder, but mosaicism is now apparent in a diverse range of other clinical disorders, as indicated by their tissue distributions and inheritance patterns. Recent technical advances have uncovered the causative mosaic variant underlying many of these conditions and have provided insight into the pervasiveness of mosaicism in normal individuals. Here, we discuss the clinical and molecular classes of mosaicism, their detection and the biological insights gained from these studies.
IVF often requires embryo cryopreservation through vitrification. During the vitrification process, the embryos can be collapsed by withdrawing the blastocoele fluid. The metabolomic profile of blastocoele fluid has been recently investigated by high-performance liquid chromatography-electrospray ionization-mass spectrometry to provide metabolite information that can help estimations of implantation efficiency. However, the presence of embryo DNA in blastocoele fluid has not been reported to date. This study shows using real-time PCR that genomic DNA was present in about 90% of blastocoele fluid samples harvested during the vitrification procedure. Moreover, the potential for determining embryo sex directly from blastocoele fluid is demonstrated by amplifying the multicopy genes TSPY1 (on the Y chromosome) and TBC1D3 (on chromosome 17). This opens up the possibility of screening embryos from couples carrying an X-linked disorder to identify male embryos at high risk of disease. The application of whole-genome amplification technologies to fluid samples is also shown to be feasible, potentially allowing more comprehensive genetic tests. As proof of principle, microarray comparative genomic hybridization was attempted to confirm the sex of embryos as well as detect several aneuploidies. However, further studies are needed to validate this approach and confirm that the accuracy is sufficient for diagnostic purposes.
Objective:
To compare the incidence of ectopic pregnancy (EP) after fresh ET and thawed ET.
Design:
Retrospective cohort study.
Setting:
Private fertility center.
Patient(s):
This retrospective study included 2,150 blastocyst transfers, including all 1,460 fresh autologous blastocyst transfers and all 690 transfers of autologous blastocysts derived from post-thaw extended culture of thawed bipronuclear oocytes in the 8-year study period 2004-2011.
Intervention(s):
None.
Main outcome measure(s):
Visualized EP and treated persistent pregnancy of unknown location.
Result(s):
The rate of visualized EP was 1.5% in pregnancies in fresh autologous cycles, which was significantly more than the rate of 0 with autologous post-thaw extended culture. The rates of treated persistent pregnancy of unknown location were 2.5% and 0.3% in these two groups, respectively, a difference that was also statistically significant (relative risk 7.3, 95% confidence interval 1.7-31.0).
Conclusion(s):
Relative to fresh transfer, thawed ET was associated with significantly reduced incidence of EP. These findings are consistent with ovarian stimulation increasing the risk of EP.
Advances in cell culture media have led to a shift in in vitro fertilization (IVF) practice from early cleavage embryo transfer to blastocyst stage transfer. The rationale for blastocyst culture is to improve both uterine and embryonic synchronicity and enable self selection of viable embryos thus resulting in higher implantation rates.
To determine if blastocyst stage (Day 5 to 6) embryo transfers (ETs) improve live birth rate and other associated outcomes compared with cleavage stage (Day 2 to 3) ETs.
Cochrane Menstrual Disorders and Subfertility Group Specialised Register of controlled trials, Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library), MEDLINE, EMBASE and Bio extracts. The last search date was 21 February 2012.
Trials were included if they were randomised and compared the effectiveness of early cleavage versus blastocyst stage transfers.
Of the 50 trials that were identified, 23 randomised controlled trials (RCTs) met the inclusion criteria and were reviewed (five new studies were added in this update). The primary outcome was rate of live birth. Secondary outcomes were rates per couple of clinical pregnancy, cumulative clinical pregnancy, multiple pregnancy, high order pregnancy, miscarriage, failure to transfer embryos and cryopreservation. Quality assessment, data extraction and meta-analysis were performed following Cochrane guidelines.
Twelve RCTs reported live birth rates and there was evidence of a significant difference in live birth rate per couple favouring blastocyst culture (1510 women, Peto OR 1.40, 95% CI 1.13 to 1.74) (Day 2 to 3: 31%; Day 5 to 6: 38.8%, I(2) = 40%). This means that for a typical rate of 31% in clinics that use early cleavage stage cycles, the rate of live births would increase to 32% to 42% if clinics used blastocyst transfer.There was no difference in clinical pregnancy rate between early cleavage and blastocyst transfer in the 23 RCTs (Peto OR 1.14, 95% CI 0.99 to 1.32) (Day 2 to 3: 38.6%; Day 5 to 6: 41.6%) and no difference in miscarriage rate (13 RCTs, Peto OR 1.18, 95% CI 0.86 to 1.60). The four RCTs that reported cumulative pregnancy rates (266 women, Peto OR 1.58, 95% CI 1.11 to 2.25) (Day 2 to 3: 56.8%; Day 5 to 6: 46.3%) significantly favoured early cleavage. Embryo freezing rates (11 RCTs, 1729 women, Peto OR 2.88, 95% CI 2.35 to 3.51) and failure to transfer embryos (16 RCTs, 2459 women, OR 0.35, 95% CI 0.24 to 0.51) (Day 2 to 3: 3.4%; Day 5 to 6: 8.9%) favoured cleavage stage transfer.
This review provides evidence that there is a small significant difference in live birth rates in favour of blastocyst transfer (Day 5 to 6) compared to cleavage stage transfer (Day 2 to 3). However, cumulative clinical pregnancy rates from cleavage stage (derived from fresh and thaw cycles) resulted in higher clinical pregnancy rates than from blastocyst cycles. The most likely explanation for this is the higher rates of frozen embryos and lower failure to transfer rates per couple obtained from cleavage stage protocols. Future RCTs should report miscarriage, live birth and cumulative live birth rates to enable ART consumers and service providers to make well informed decisions on the best treatment option available.
To perform a systematic review and meta-analysis of obstetric and perinatal complications in singleton pregnancies after the transfer of frozen thawed and fresh embryos generated through IVF.
Systematic review.
Observational studies, comparing obstetric and perinatal outcomes in singleton pregnancies subsequent to frozen thawed ET versus fresh embryo transfer, were included from Medline, EMBASE, Cochrane Central Register of Clinical Trials, DARE, and CINAHL (1984-2012).
Women undergoing IVF/intracytoplasmic sperm injection (ICSI).
Two independent reviewers extracted data and assessed the methodological quality of the relevant studies using critical appraisal skills program scoring. Risk ratios and risk differences were calculated in Rev Man 5.1. Subgroup analysis was performed on matched cohort studies.
Antepartum hemorrhage, very preterm birth, preterm birth, small for gestational age, low birth weight, very low birth weight, cesarean section, congenital anomalies, perinatal mortality, and admission to neonatal intensive care unit.
Eleven studies met the inclusion criteria. Singleton pregnancies after the transfer of frozen thawed embryos were associated with better perinatal outcomes compared with those after fresh IVF embryos. The relative risks (RR) and 95% confidence intervals (CI) of antepartum hemorrhage (RR = 0.67, 95% CI 0.55-0.81), preterm birth (RR = 0.84, 95% CI 0.78-0.90), small for gestational age (RR = 0.45, 95% CI 0.30-0.66), low birth weight (RR = 0.69, 95% CI 0.62-0.76), and perinatal mortality (RR = 0.68, 95% CI 0.48-0.96) were lower in women who received frozen embryos.
Although fresh ET is the norm in IVF, results of this systematic review of observational studies suggest that pregnancies arising from the transfer of frozen thawed IVF embryos seem to have better obstetric and perinatal outcomes.
The extent to which birth defects after infertility treatment may be explained by underlying parental factors is uncertain.
We linked a census of treatment with assisted reproductive technology in South Australia to a registry of births and terminations with a gestation period of at least 20 weeks or a birth weight of at least 400 g and registries of birth defects (including cerebral palsy and terminations for defects at any gestational period). We compared risks of birth defects (diagnosed before a child's fifth birthday) among pregnancies in women who received treatment with assisted reproductive technology, spontaneous pregnancies (i.e., without assisted conception) in women who had a previous birth with assisted conception, pregnancies in women with a record of infertility but no treatment with assisted reproductive technology, and pregnancies in women with no record of infertility.
Of the 308,974 births, 6163 resulted from assisted conception. The unadjusted odds ratio for any birth defect in pregnancies involving assisted conception (513 defects, 8.3%) as compared with pregnancies not involving assisted conception (17,546 defects, 5.8%) was 1.47 (95% confidence interval [CI], 1.33 to 1.62); the multivariate-adjusted odds ratio was 1.28 (95% CI, 1.16 to 1.41). The corresponding odds ratios with in vitro fertilization (IVF) (165 birth defects, 7.2%) were 1.26 (95% CI, 1.07 to 1.48) and 1.07 (95% CI, 0.90 to 1.26), and the odds ratios with intracytoplasmic sperm injection (ICSI) (139 defects, 9.9%) were 1.77 (95% CI, 1.47 to 2.12) and 1.57 (95% CI, 1.30 to 1.90). A history of infertility, either with or without assisted conception, was also significantly associated with birth defects.
The increased risk of birth defects associated with IVF was no longer significant after adjustment for parental factors. The risk of birth defects associated with ICSI remained increased after multivariate adjustment, although the possibility of residual confounding cannot be excluded. (Funded by the National Health and Medical Research Council and the Australian Research Council.).
To develop and validate a quantitative real-time polymerase chain reaction (qPCR)-based method for blastocyst trophectoderm comprehensive chromosome screening (CCS) of aneuploidy.
Prospective, randomized, and blinded.
Academic center for reproductive medicine.
Nine cell lines were obtained from a commercial cell line repository, and 71 discarded human blastocysts were obtained from 24 IVF patients that underwent preimplantation genetic screening.
None.
Consistency of qPCR diagnosis of aneuploidy compared with either conventional karyotyping of cell lines or microarray-based diagnoses of human blastocysts.
Samples from nine cell lines with well characterized karyotypes were diagnosed by qPCR with 97.6% (41/42) consistency. After applying a minimum threshold for concurrence, 100% consistency was achieved. Developmentally normal blastocysts designated as aneuploid or arrested blastocysts designated as euploid by single-nucleotide polymorphism microarray analyses were assigned identical 24 chromosome diagnoses by qPCR in 98.6% of cases (70/71). Overall euploidy (n = 37) and aneuploidy (n = 34) were assigned with 100% consistency. Data was obtained for both sample types in 4 hours.
These data demonstrate the first qPCR technology capable of accurate aneuploidy screening of all 24 chromosomes in 4 hours. This methodology provides an opportunity to evaluate trophectoderm biopsies with subsequent fresh euploid blastocyst transfer. Randomized controlled trials to investigate the clinical efficacy of qPCR-based CCS are currently underway.
To examine the effect of mosaicism in the array comparative genomic hybridization result during preimplantation genetic screening after blastocyst biopsy.
Experimental study.
University laboratory. MATERIAL(S): Epithelial cell lines.
Mixing of euploid and aneuploid cells to create mosaic trophectoderm and blastocyst models.
The level of aneuploidy in samples with different ratios of aneuploid cells was measured after array comparative genomic hybridization.
A shift from normality was present when the level of aneuploid cells in the sample was >25%. Aneuploidy could be confidently called when the level of aneuploid cells was >50%.
This study determined that aneuploidy in mosaic samples can be detected by array comparative genomic hybridization and that the result may also indicate the proportion of the aneuploid cells present in the sample.
Preimplantation genetic screening (PGS) has increasingly been used in the past decade. Here we present a systematic review and meta-analysis of RCTs on the effect of PGS on the probability of live birth after IVF.
PubMed and trial registers were searched for RCTs on PGS. Trials were assessed following predetermined quality criteria. The primary outcome was live birth rate per woman, secondary outcomes were ongoing pregnancy rate, miscarriage rate, multiple pregnancy rate and pregnancy outcome.
Nine RCTs comparing IVF with and without PGS were included in our meta-analysis. Fluorescence in situ hybridization was used in all trials and cleavage stage biopsy was used in all but one trial. PGS significantly lowered live birth rate after IVF for women of advanced maternal age (risk difference: -0.08; 95% confidence interval: -0. 13 to -0.03). For a live birth rate of 26% after IVF without PGS, the rate would be between 13 and 23% using PGS. Trials where PGS was offered to women with a good prognosis and to women with repeated implantation failure suggested similar outcomes.
There is no evidence of a beneficial effect of PGS as currently applied on the live birth rate after IVF. On the contrary, for women of advanced maternal age PGS significantly lowers the live birth rate. Technical drawbacks and chromosomal mosaicism underlie this inefficacy of PGS. New approaches in the application of PGS should be evaluated carefully before their introduction into clinical practice.
To describe a method of amplifying DNA from blastocyst trophectoderm cells (two or three cells) and simultaneously performing 23-chromosome single nucleotide polymorphism microarrays and single-gene preimplantation genetic diagnosis.
Case report.
IVF clinic and preimplantation genetic diagnostic centers.
A 36-year-old woman, gravida 2, para 1011, and her husband who both were carriers of GM(1) gangliosidosis. The couple wished to proceed with microarray analysis for aneuploidy detection coupled with DNA sequencing for GM(1) gangliosidosis.
An IVF cycle was performed. Ten blastocyst-stage embryos underwent trophectoderm biopsy. Twenty-three-chromosome microarray analysis for aneuploidy and specific DNA sequencing for GM(1) gangliosidosis mutations were performed.
Viable pregnancy.
After testing, elective single embryo transfer was performed followed by an intrauterine pregnancy with documented fetal cardiac activity by ultrasound.
Twenty-three-chromosome microarray analysis for aneuploidy detection and single-gene evaluation via specific DNA sequencing and linkage analysis are used for preimplantation diagnosis for single-gene disorders and aneuploidy. Because of the minimal amount of genetic material obtained from the day 3 to 5 embryos (up to 6 pg), these modalities have been used in isolation of each other. The use of preimplantation genetic diagnosis for aneuploidy coupled with testing for single-gene disorders via trophectoderm biopsy is a novel approach to maximize pregnancy outcomes. Although further investigation is warranted, preimplantation genetic diagnosis for aneuploidy and single-gene testing seem destined to be used increasingly to optimize ultimate pregnancy success.
Genetic variants predicted to seriously disrupt the function of human protein-coding genes—so-called loss-of-function (LOF)
variants—have traditionally been viewed in the context of severe Mendelian disease. However, recent large-scale sequencing
and genotyping projects have revealed a surprisingly large number of these variants in the genomes of apparently healthy individuals—at
least 100 per genome, including more than 30 in a homozygous state—suggesting a previously unappreciated level of variation
in functional gene content between humans. These variants are mostly found at low frequency, suggesting that they are enriched
for mildly deleterious polymorphisms suppressed by negative natural selection, and thus represent an attractive set of candidate
variants for complex disease susceptibility. However, they are also enriched for sequencing and annotation artefacts, so overall
present serious challenges for clinical sequencing projects seeking to identify severe disease genes amidst the ‘noise’ of
technical error and benign genetic polymorphism. Systematic, high-quality catalogues of LOF variants present in the genomes
of healthy individuals, built from the output of large-scale sequencing studies such as the 1000 Genomes Project, will help
to distinguish between benign and disease-causing LOF variants, and will provide valuable resources for clinical genomics.
To test the hypothesis that patients with advanced maternal age (AMA) have a higher implantation rate (IR) after embryo transfer of embryos with a normal chromosomal pattern for the chromosomes studied with preimplantation genetic screening (PGS) compared with patients who had an embryo transfer without PGS.
Prospective randomized controlled trial (RCT).
Academic tertiary setting.
Patients with AMA (> or =35 years).
In an RCT, the clinical IR per embryo transferred was compared after embryo transfer on day 5 or 6 between the PGS group (analysis of chromosomes 13, 16, 18, 21, 22, X, and Y) and the Control group without PGS.
No differences were observed between the PGS group and the Control group for the clinical IR (15.1%; 14.9%; rate ratio 1.01; exact confidence interval [CI], 0.25-5.27), the ongoing IR (at 12 weeks) (9.4%; 14.9%), and the live born rate per embryo transferred (9.4%; 14.9%; rate ratio 0.63; exact CI, 0.08-3.37). Fewer embryos were transferred in the PGS group (1.6 +/- 0.6) than in the Control group (2.0 +/- 0.6). A normal diploid status was observed in 30.3% of the embryos screened by PGS.
In this RCT, the results did not confirm the hypothesis that PGS results in improved reproductive outcome in patients with AMA.