The reproducibility of trophectoderm biopsies in euploid,aneuploid, and mosaic embryos using independently verified next-generation sequencing (NGS): a pilot study

To assess the accuracy and reliability of comprehensive chromosome screening by next-generation sequencing (NGS) of human trophectoderm (TE) biopsy specimens. The reliability and accuracy of diagnoses made by preimplantation genetic testing for aneuploidy (PGT-A) from TE biopsy were tested. Repeat biopsies of TE and inner cell mass (ICM) samples were obtained from thawed blastocysts previously tested by NGS. To test for the reliability of the NGS assay, biopsy samples were compared with the original PGT-A results. Prior NGS testing classified the TE samples as euploid, aneuploid, or aneuploid-mosaic. The resulting re-biopsied samples underwent SurePlex whole genome amplification followed by NGS via the MiSeq platform, with copy number value (CNV) determined using BlueFuse Multi Software. The primary outcome measure was reliability, defined as concordance between initial TE result and the repeat biopsies. Accuracy was determined by concordance between the TE and ICM samples, and compared between three chromosome types (disomic, aneuploid, and mosaic). Re-biopsies were performed on 32 embryos with prior PGT-A showing euploidy (10 embryos), aneuploidy of one or two chromosomes (4 embryos), or aneuploid-mosaic with one aneuploid chromosome and one mosaic chromosome (18 embryos). One hundred twenty-nine biopsy samples completed NGS (90 TE and 39 ICM biopsies) and 105 biopsy results were included in the analysis. TE biopsies provide a highly accurate test of the future fetus, with the ICM disomic concordance rate of 97.6%. Clinical concordance rates indicate that TE biopsies provide a reliable test when the result is euploid (99.5%) or aneuploid (97.3%), but less reliable when the result is mosaic (35.2%). TE biopsies predict euploidy or aneuploidy in the ICM with a high degree of accuracy. PGT-A with NGS of TE biopsies is shown to be highly reliable, with clinically relevant concordance rates for aneuploidy and euploidy over 95%. TE biopsies indicating mosaicism were less reliable (35.2%), presumably because mitotic non-disjunction events are not uniformly distributed throughout the blastocyst. However, classification of TE biopsy of PGT-A with NGS results as either aneuploid or euploid provides a highly reliable test.     

Two different microarray technologies for preimplantation genetic diagnosis and screening,due to reciprocal translocation imbalances,demonstrate equivalent euploidy and clinical pregnancy rates

Purpose

To compare single nucleotide polymorphism (SNP) and comparative genomic hybridization (aCGH) microarray platforms to evaluate embryos for parental translocation imbalances and aneuploidy.

Methods

A retrospective review of preimplantation genetic diagnosis and screening (PGD/PGS) results of 498 embryos from 63 couples undergoing 75 in vitro fertilization cycles due to parental carriers of a reciprocal translocation.

Results

There was no significant difference between SNP and aCGH microarrays when comparing the prevalence of embryos that were euploid with no translocation imbalance, euploidy with a parental translocation imbalance or aneuploid with or without the parental chromosome imbalance. The clinical pregnancy rates were also equivalent for SNP (60 %) versus aCGH (65 %) microarrays. Of 498 diagnosed embryos, 45 % (226/498) were chromosomally normal without translocation errors or aneuploidy, 22 % (112/498) were euploid but had a parentally derived unbalanced chromosomal segregant, 8 % (42/498) harbored both a translocation imbalance and aneuploidy and 24 % (118/498) of embryos were genetically balanced for the parental reciprocal translocation but were aneuploid for other chromosomes. The overall clinical pregnancy rate per IVF cycle following SNP or aCGH microarray analysis was 61 % and was higher if the biopsy was done on blastocysts (65 %) versus cleavage stage embryos (59 %), although not statistically significant.

Conclusions

SNP or aCGH microarray technologies demonstrate equivalent clinical findings that maximize the pregnancy potential in patients with known parental reciprocal chromosomal translocations.     

Negligible interchromosomal effect in embryos of Robertsonian translocation carriers

It has been suggested that translocations, and perhaps other chromosome rearrangements, disturb meiotic disjunction of chromosome pairs not involved in the translocation, resulting in non-disjunction in those chromosomes (interchromosomal effect) and predisposition to trisomy offspring. Other reports have suggested an increased risk of mosaicism and chaotic embryos in translocation carriers. This study was designed to determine if such interchromosomal effects are producing significantly more chromosome abnormalities than those expected from unbalanced gametes. For that purpose, two groups of PGD patients were compared, Robertsonian translocation carriers (RBT) and carriers of X-linked diseases (XLI), of similar age. Both groups were analysed by FISH with similar DNA probes. The results indicate that overall, the higher rate of chromosome abnormalities in the RBT group was solely due to unbalanced gametes and not to an interchromosomal effect or higher incidence of mosaicism. If unbalanced and normal were combined, this proportion was 53% in XLI and 59% in RBT. However, when specific RBT translocations were studied, only a slight increase in embryos with aneuploidy for chromosome 22 was found for the t(13;14) translocation carriers, while a higher rate of post-zygotic abnormalities was observed in the more rare RBT. In conclusion, the overall rate of non-translocation related abnormalities was not increased in the RBT group compared with the control group, but a slight interchromosomal effect may exist, as some Robertsonian translocations may be more prone to produce mosaic and chaotic embryos.     

Evaluation of chromosomal abnormalities from preimplantation genetic testing to the reproductive outcomes: a comparison between three different structural rearrangements based on next-generation sequencing

PurposeThe aim of this study was to determine factors affecting the chromosome imbalance in blastocysts and reproductive outcomes by a comparison between the reciprocal translocation (REC), inversion (INV), and Robertsonian translocation (ROB) carriers.MethodsCouples with one partner carrying translocation or inversion underwent preimplantation genetic testing for chromosomal structural rearrangement (PGT-SR) cycles, including 215 PGT-SR cycles performed in subsequent 164 frozen-thawed embryo transfer cycles and 61 prenatal diagnoses of fetuses and 59 normal live birth babies. A total of 899 samples were processed by whole-genome amplification followed by next-generation sequencing (NGS). Karyotype and chromosome microarray analyses were used to confirm the PGT results from the amniotic fluid samples.ResultsA total of 843 blastocysts from 124 REC, 21 INV, and 35 ROB carriers were diagnosed by PGT-SR. The percentage of unbalanced blastocysts was significantly higher in REC than in INV and ROB carriers (64.31% vs. 28.05% vs. 37.02%). Stratification analysis of female carrier age and gonadotropin doses showed no significant increase in unbalanced chromosomal abnormalities in the three groups. Also, the different breakpoints in chromosomal arms did not affect the rate of unbalanced chromosomes in the embryos. Logistic regression indicated blastocyst quality as a statistically significant risk factor associated with unbalanced chromosomal abnormalities from translocation carriers (P < 0.001). The source of abnormalities in the three groups showed significant differences such that the abnormalities in REC mostly originated from parental translocation but the abnormalities in INV were mainly de novo variations. 164 blastocysts were transferred, and there were no significant differences in the clinical pregnancy rate and miscarriage rate. A total of 59 healthy babies were born, and there were no significant differences in the gender ratio and birth height, except the birth weight of boys between INV and ROB groups (P = 0.02). The results of amniocentesis revealed that more fetuses have normal chromosomal karyotypes than balanced carriers, particularly in the REC group.ConclusionsReciprocal translocation carriers have more risk of unbalanced rearrangement, but embryonic chromosome abnormalities of inversion carriers come mainly from de novo variations. This is the first study specifically comparing three different PGT-SRs using the NGS method and evaluating their reproductive outcomes. Our findings will provide the reciprocal translocation, inversion, and Robertsonian translocation carrier couples with more accurate genetic counseling on the reproductive risk of chromosomal imbalance.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10815-020-02053-5.     

Balanced chromosomal translocations in men: relationships among semen parameters, chromatin integrity, sperm meiotic segregation and aneuploidy

Purpose

To analyse relationships between semen parameters, sperm chromatin integrity and frequencies of chromosomally unbalanced, disomic and diploid sperm in 13 Robertsonian and 37 reciprocal translocation carriers and to compare the results with data from 10 control donors.

Methods

Conventional semen analysis, Sperm Chromatin Structure Assay and FISH with probes for chromosomes involved in the individual translocations and for chromosomes X, Y, 7, 8, 13, 18 and 21.

Results

Normal semen parameters were found in 30.8 % of Robertsonian and 59.5 % of reciprocal translocation carriers. The rates of unbalanced sperm were 12.0 % in Robertsonian and 55.1 % in reciprocal translocation carriers with no difference between normospermic patients and those showing altered semen parameters. Significantly increased frequencies of spermatozoa showing defects in chromatin integrity and condensation, aneuploidy for chromosomes not involved in a translocation and diploidy were detected in translocation carriers with abnormal semen parameters. Normospermic reciprocal translocation carriers showed an increase in chromosome 13 disomy compared to the control group. There was no relationship between gametic and somatic aneuploidy in 12 translocation carriers studied by FISH on sperm and lymphocytes. The frequency of motile sperm was negatively correlated with the frequency of sperm showing disomy, diploidy and defective chromatin condensation.

Conclusions

Abnormal semen parameters can serve as indicators of an additional risk of forming spermatozoa with defective chromatin and aneuploidy in translocation carriers.     

胚胎染色体非整倍体筛查用于平衡易位携带者植入前的遗传学诊断

目的 交信号和1个Y染色体杂交信号者,则诊断为整倍体胚胎;异常杂交信号的胚胎则诊断为非整倍体胚胎.结果 (1)11个平衡易位的PGD周期中,选出杂交信号完整的130个细胞核进行分析,FISH共分析了937个荧光杂交信号,其中整倍体细胞核38个,共有304个杂交信号;其余92个为非整倍体细胞核.(2)在92个非整倍体细胞核中,Ⅰ、Ⅱ及Ⅲ级胚胎的比例分别为20个(22%)、36个(39%)及36个(39%);38个整倍体细胞核中,Ⅰ、Ⅱ及Ⅲ级胚胎的比例分别为13个(34%)、17个(45%)及8个(21%),两者的Ⅰ、Ⅱ及Ⅲ级胚胎数分别比较,差异均无统计学意义(P＞0.05).虽然染色体整倍体率在不同级别胚胎中的分布比较,差异均无统计学意义(P＞0.05),但优质胚胎(Ⅰ级+Ⅱ级)中非整倍体率仍为60%(56/92).(3)平衡胚胎来源的卵裂球细胞核整倍体率(71.4%,30/42)明显高于非平衡胚胎来源的卵裂球细胞核整倍体率(9.1%,8/88),两者比较,差异有统计学意义(P＜0.05).平衡胚胎来源的卵裂球细胞核非整倍体率(包括三体、单体、复杂非整倍体、单倍体、多倍体)明显低于非平衡胚胎来源的卵裂球细胞核非整倍体率(P＜0.05).结论 平衡易位携带者的胚胎中有较高的非整倍体率,因此,胚胎非整倍体筛查在平衡易位携带者的PGD中有重要价值和临床意义.

Abstract：

Objective To determine the importance of aneuploidy screening in preimplantation genetic diagnosis for the couples of chromosome translocation carriers. Methods To perform 11 prenatal genetic disgnosis (PGD) cycles for 7 couples of chromosome translocation carriers from January 2006 to March 2009 in the Reproductive Medical Center, First Affiliated Hospital of Zhengzhou University. To re-analyze the well-fixed, non-multinuclear and non-debris nuclei using the probes of LSI 13, 18, 21,CEPX, CEPY to detect the aneuploidy rate which come from the PGD cycles of the couples of chromosome translocation carriers. The euploid embryo was defined as two fluorescence in situ hybridization (FISH)signals of LSI 13, 18, 21 respectively and two signals of CEPX, or one signal of CEPX and one signal of CEPY. The other abnormal signals were defined as aneuploid embryo. Results (1) A tolal of 130 nuclei from 11 PGD cycles got the integrated re-FISH signals. Nine hundred and thirty-seven FISH signals were analysized, including 304 signals from 38 euploid nuclei and the others from 92 aneuploid nuclei. (2) The number of the aneuploid nuclei from grade Ⅰ , Ⅱ and Ⅲ embryo was 20 (22%), 36(39%), and 36(39%). The number of the euploid nuclei from grade Ⅰ , Ⅱ and Ⅲ embryo was 13(34%), 17(45%),and 8(21%). There was no significant difference of aneupioidy rate between the embryos form different grades (P＞0.05). However, the rate of aneuploid nucleus from good quality embryos (grade Ⅰ + grade Ⅱ) was 60% (59/92). (3) The euploidy rate was 71.4% (30/42) from balanced embryos, while 9.1%(8/88)from unbalanced embryos. There was significant difference between them (x2=53.4, P＜0.05).The rate of aneuploidy from balanced embryos was lower than those from unbalanced embryos (P＜0.05).Conclusions Since higher rate of aneuploidy was detected in embryos of the couples of chromosome translocation carriers. It is advisable to recommend the FISH re-analysis for aneuploidy screening to preimplantation genetic diagnosis for the couples of chromosome translocation carriers.     

The role for preimplantation genetic diagnosis in balanced translocation carriers

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

Analysis of meiotic segregation patterns and interchromosomal effects in sperm from six males with Robertsonian translocations

Purpose To provide more genetic information about meiotic segregation behavior and the possibility of interchromosomal effects (ICE) in spermatozoa from carriers of Robertsonian (Rob) translocations. Materials and methods Meiotic segregation behavior in spermatozoa from six carriers of Rob translocations, four t(13;14), one t(14;22) and one t(13;21), was investigated by dual fluorescence in-situ hybridization (FISH). Aneuploidy for chromosomes 18, X and Y was studied by triple FISH. Results The rate of normal/balanced spermatozoa resulting from alternate segregation ranged from 78.14 to 86.88%. The frequency of unbalanced spermatozoa resulting from adjacent segregation varied between 11.70 and 19.53%. The higher frequencies of aneuploidy for sex chromosome were observed in three Rob translocation carriers. In addition, the increased rates of diploid were found in two t(13;14) carriers. Conclusions Alternate segregation is dominant in the different types of Rob translocations. Some carriers may be at an increased risk for ICE. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users. An increased aneuploidy for sex chromosome observed in three Robertsonian translocation carriers suggests that an interchromosomal effect is likely in some carriers.     

Trophectoderm biopsy protocols may impact the rate of mosaic blastocysts in cycles with pre-implantation genetic testing for aneuploidy

PurposeThis study aimed to analyze the impact of different biopsy protocols on the rate of mosaic blastocysts.MethodsThis is a retrospective cohort study which included 115 cycles with pre-implantation genetic testing for aneuploidy (PGT-A). Two groups were allocated based on the biopsy protocols: method 1 group, the zona pellucida (ZP) was drilled on day 3 embryos followed by trophectoderm (TE) biopsy; and method 2 group, the ZP was opened on day 5 or 6 blastocysts followed by TE biopsy. All biopsy samples were assessed using next-generation sequencing (NGS) at a single reference laboratory. The euploid, aneuploid, and mosaic blastocyst rates and clinical outcomes were compared.ResultsThe mosaicism rate in the method 1 group was 19.58%, significantly higher than the method 2 group (8.12%; P < 0.05). No statistically significant difference was observed in euploid, aneuploid blastocyst rates, and clinical pregnancy rates between the two groups. Logistic regression analysis indicated that the biopsy protocols were independently associated with the mosaicism rates among all the variables.ConclusionsThe present study showed that different biopsy protocols may have an impact on the mosaic blastocyst rate. ZP opening on day 3 combined with TE biopsy might increase the incidence of mosaic blastocysts.     

Preimplantation genetic diagnosis by fluorescence in situ hybridization of reciprocal and Robertsonian translocations

ObjectiveThe presence of reciprocal and Robertsonian chromosomal rearrangement is often related to recurrent miscarriage. Using preimplantation genetic diagnosis, the abortion rate can be decreased. Cases treated at our center were reviewed.Materials and methodsA retrospective analysis for either Robertsonian or reciprocal translocations was performed on all completed cycles of preimplantation genetic diagnosis at our center since the first reported case in 2004 until the end of 2010. Day 3 embryo biopsies were carried out, and the biopsied cell was checked by fluorescent in situ hybridization using relevant informative probes. Embryos with a normal or balanced translocation karyotype were transferred on Day 4.ResultsThirty-eight preimplantation genetic diagnosis cycles involving 17 couples were completed. A total of 450 (82.6%) of the total oocytes were MII oocytes, and 158 (60.0%) of the two-pronuclei embryos were biopsied. In 41.4% of the fluorescent in situ hybridization analyses, the results were either normal or balanced. Embryos were transferred back after 21 cycles. Three babies were born from Robertsonian translocation carriers and another two from reciprocal translocation carriers. The miscarriage rate was 0%. Among the reciprocal translocation group, the live delivery rate was 8.3% per ovum pick-up cycle and 18.2% per embryo transfer cycle. Among the Robertsonian translocation group, the live delivery rate was 14.3% per ovum pick-up cycle and 20.0% per embryo transfer cycle.ConclusionThere is a trend whereby the outcome for Robertsonian translocation group carriers is better than that for reciprocal translocation group carriers. Aneuploidy screening may possibly be added in order to improve the outcome, especially for individuals with an advanced maternal age. The emergence of an array-based technology should help improve this type of analysis.     

Inheritance of imbalances in recurrent chromosomal translocation t(11;22): clarification by PGT-SR and sperm-FISH analysis

Research questionTo analyse why unbalanced viable offspring are derived mainly from the 3:1 segregation mode in t(11;22)(q23;q11.2) reciprocal translocation.DesignRetrospective analysis of 24 pre-implantation genetic testing for chromosomal structural re-arrangements (PGT-SR) cycles was performed on seven male and five female carriers of t(11;22) translocation. Sperm analysis was performed on each male carrier. These patients were directed to the study centre after several years of miscarriages and/or abortions, primary infertility for male carriers or birth of an affected child.ResultsTwenty-four PGT-SR cycles were performed to exclude imbalances in both male and female carriers. The unbalanced embryos derived from the adjacent-1 segregation mode were the most represented in both male and female carriers (68.4% and 50%, respectively). These results were positively related with meiotic segregation analysis of reciprocal translocation in spermatozoa. A thorough analysis of the unbalanced embryo karyotypes determined that the expected viable +der22 karyotype resulting from 3:1 malsegregation was less represented at 5.3%.ConclusionsThese findings highlight the divergence that may exist between meiotic segregation and post-zygotic selection. Post-zygotic selection would be responsible for the elimination of unbalanced embryos derived from the adjacent-1 segregation mode. The combined action of several factors occurs at the beginning of post-zygotic selection. Genetic counselling must consider the risk of a birth related to the adjacent-1 segregation mode, irrespective of the sex of the translocation carrier. These results will allow deeper understanding of the PGT results of t(11;22) carriers, which often include a high number of aneuploid embryos.     

Chromosomal segregation in sperm of Robertsonian translocation carriers

Purpose

To study meiotic segregation patterns of Robertsonian translocations in sperm of male carriers and to assess the frequencies of unbalanced sperm formation.

Methods

FISH with combination of probes to detect all the variants of meiotic segregation was performed on decondensed sperm nuclei of 5 carriers of der(13;14), 3 carriers of der(14;21) and one carrier of a rare der(13;21) translocation.

Results

The frequency of sperm with alternate segregation and normal/balanced chromosomal complement ranged from 68 % to 94.4 % (mean 79.2 ± 8.4). Adjacent segregation was detected in 17.9 ± 7.3 % of sperm (from 5.6 % to 29 %). No significant differences in frequencies of gametes with nullisomies and disomies of chromosomes involved in translocations were observed. The mean frequency of 3:0 segregation products was 2.5 ± 1.4 %.

Conclusions

All analyzed patients showed homogenous segregation pattern with clear predominance of alternate segregation resulting in normal/balanced sperm production. Still, from 5.8–32 % (mean 20.4 ± 8.3 %) of sperm was unbalanced, which is the evidence of the increased risk of unbalanced offspring in carriers of Robertsonian translocations. Our results highlight the importance of genetic counseling of Robertsonian translocation carriers prior to ICSI or IVF.     

植入前遗传学诊断100个周期临床分析

目的 探讨染色体易位对早期胚胎发育的影响,以及植入前遗传学诊断(PGD)技术的诊断效率和可行性.方法 回顾性分析PGD中23个罗伯逊(罗氏)易位周期、19个平衡易位周期(染色体易位组),以及58个α地中海贫血周期(地贫组)共100个周期中的胚胎发育情况、PGD的诊断效率以及临床结局.结果 染色体易位组中有354个胚胎进行PGD,321(90.7%)个胚胎有荧光原位杂交(FISH)结果,其中罗氏易位者中正常和(或)平衡易位胚胎占38.3%(64/167),显著高于平衡易位者的20.8%(32/154).地贫组有537个胚胎进行PGD,单个卵裂球的扩增效率为82.5%(443/537),诊断出正常纯合子140个、杂合子112个、异常纯合子155个、另36个诊断结果不明确,总体诊断效率为75.8%(407/537).染色体易位组中,取卵后第3天卵裂球数≥7的胚胎中,正常和(或)平衡易位发生率(34.4%,77/224)显著高于卵裂球数＜7的胚胎(19.6%,19/97),在取卵后第4天,正常和(或)平衡易位胚胎的细胞融合率为59.4%(57/96),显著高于染色体不平衡胚胎的34.2%(77/225).染色体易位组共在37个周期移植了75个胚胎,获得10例临床妊娠,临床妊娠率27.0%(10/37).地贫组共在58个周期移植了170个胚胎,获得25例临床妊娠,临床妊娠率为43.1%(25/58).结论 PGD技术可有效为染色体易位和地中海贫血基因携带者提供优生选择.染色体易位可能对着床前胚胎的发育有一定的影响.

Abstract：

Objective To investigate influence of chromosomal translocations on early embryo development and to evaluate the efficacy and feasibility of preimplantation genetic diagnosis (PGD)techniques through clinical analysis on PGD cycles. Methods Embryo development, efficacy of PGD and clinical outcome of 100 cycles were studied retrospectively, including 23 cycles with Robertsonian translocations, 19 cycles with reciprocal translocations, and 58 cycles for α-Thalassaemia. Results Among 354 embryos biopsied by PGD for translocations, 321 (90. 7% ) presented fluorescence in situ hybridization (FISH) results. The rate of normal/balanced embryos in the Robertsonian translocation was 38. 3% (64/167),which was significantly higher than 20. 8% (32/154) in the reciprocal translocation group. Amplification was achieved in 443 blastomeres from 537 embryos in Thalassaemia group, which given to an amplification efficiency rate of 82. 5% ( 443/537 ). Totally, 140 normal homozygous, 112 heterozygotes and 155 affected homozygous embryos were identified, while 36 embryos had uncertain result. The successful diagnostic rate was 75.8% (407/537). After 3 days in the translocation groups, the rate of normal and/or balanced translocations in biopsed embryos with ≥7 cells was 34. 4% (77/224), which was significantly higher than 19. 6% ( 19/97 ) of biopsed embryos with ＜ 7 cells. After 4 days, the compaction rate in normal/balanced embryos was 59.4% ( 57/96 ), which was significantly higher than 34. 2% ( 77/225 ) in imbalanced embryos significantly. Seventy-five embryos transferred in 37 cycles with translocations group led to clinical pregnancy rate of 27.0% (10/37), and 170 embryos transferred in 58 cycles with Thalassaemia got a clinical pregnancy rate of 43. 1% ( 25/58 ) . Conclusions PGD can provide management efficiently for both chromosome translocations and Thalassaemia. Translocations might have slightly negative impact on embryo development before implantation.     

Morphokinetic parameters in chromosomal translocation carriers undergoing preimplantation genetic testing

Research question

Can embryo morphokinetic parameters help identify unbalanced embryos in translocation carriers?

Clinical outcome of treatment cycles using preimplantation genetic diagnosis for structural chromosomal abnormalities

OBJECTIVES: To explore oocyte recovery, embryo quality, the number of transferable embryos and pregnancy rate after preimplantation genetic diagnosis (PGD) in patients with structural chromosomal aberrations. METHODS: PGD was performed in seven couples with Robertsonian translocations (Rob), eight couples with reciprocal translocations (Rec), two couples with inversions and one couple with a deletion. A total of 43 treatment cycles were carried out. RESULTS: A total of 14.2 oocytes per cycle were retrieved. Fertilisation and cleavage rates were 63% and 58%, respectively. Of the biopsied embryos 20% were transferable. Comparison of the Rob and Rec group revealed no significant differences in number of oocytes, fertilisation or cleavage rates. The number of transferable embryos after biopsy was significantly higher in the Rob group than in the Rec group. When embryo transfer (ET) was performed the pregnancy rate did not differ between the Rob and the Rec groups. Twenty-eight embryo transfers (one or two embryos) were carried out leading to eight clinical pregnancies (29% per ET): two twins, four singletons, one miscarriage and one ectopic pregnancy. All the children are carriers of balanced chromosomal aberrations. CONCLUSION: An acceptable pregnancy rate can be achieved among couples with structural chromosomal abnormalities.     

The impact of patient,embryo, and translocation characteristics on the ploidy status of young couples undergoing preimplantation genetic testing for structural rearrangements (PGT-SR) by next generation sequencing (NGS)

PurposeTo evaluate the factors that affect the incidence of euploid balanced embryos and interchromosomal effect (ICE) in carriers of different structural rearrangements.MethodsThis retrospective study includes 95 couples with reciprocal translocations (RecT) and 36 couples with Robertsonian translocations (RobT) undergoing Preimplantation Genetic Testing for Structural Rearrangements (PGT-SR) between March 2016 and July 2019. Next-generation sequencing (NGS) was the technique used coupled with trophectoderm (TE) biopsy. Only cases with females under 38 years were included. A total of 532 blastocysts were evaluated.ResultsThe euploidy rate was similar in RobT when compared with RecT carriers [57/156 (36.5%) vs. 112/376 (29.8%), p = 0.127]. The pure ICE rate was significantly higher in RobT carriers [48/156 (30.8%) vs. 53/376 (14.1%), p < 0.001] than it was in RecT carriers. Female age was the independent factor for the probability of obtaining a euploid embryo in RecT and RobT carriers, and increasing female age decreases the probability of obtaining a euploid embryo. In RecT carriers, no significant differences were observed in euploidy rates, pure ICE, or combined ICE according to the length of the translocated fragment and the chromosome group. However, total ICE was significantly lower when there was a breakpoint in the short chromosome arm together with a breakpoint in the long arm [(44/158 (27.8%) for pq or qp, 51/155 (32.9%) for pp and 30/63 (47.6%) for qq; p = 0.02].ConclusionThe incidence of euploid/balanced blastocysts was similar in both types of translocations. However, there was a significant increase in pure ICE in RobT compared to RecT carriers. In RecT carriers, the presence of the breakpoints in the long arm of the chromosomes involved in the rearrangement resulted in a higher total ICE.     

Quantitative analysis of seminiferous epithelium in subfertile carriers of chromosomal translocations.

In order to study the effect of structural chromosomal aberrations on spermatogenesis, quantitative analysis of the germinal epithelium was performed on testicular specimens from 12 carriers of balanced chromosomal translocations diagnosed at male infertility clinics: a Y-autosome translocation, eight reciprocal translocations, and three D/D Robertsonian translocations. Spermatogenic arrest at the spermatocyte stage was revealed in the Y-autosome translocation and four of the reciprocal translocations. The number of spermatids was selectively reduced in three carriers, while normal spermatogenesis was found in three. Thus, the majority of carriers with impaired semen quality showed a complete or partial reduction in the number of spermatids, suggesting that the effect of chromosomal aberrations is to arrest the spermatogenic process at the spermatocyte stage.     

Could PGT-A pick up true abnormalities that have clinical relevance? Retrospective analysis of 1043 embryos

ObjectiveTo determine whether preimplantation genetic testing for aneuploidy (PGT-A) could pick up true abnormalities that have clinical relevance.Materials and methodsThis was a retrospective cohort study of patients who underwent in vitro fertilization with PGT-A from 2015 to 2017. We evaluated the associations of aneuploidy and mosaicism with maternal age, the chromosome abnormalities present in individual chromosomes, and the effect of embryo sex on the proportion of each type of error in the four chromosomes most frequently affected.Result(s)A total of 1043 embryos from 255 patients (mean maternal age = 39 ± 4 years) were included in the initial analysis. Of these, 36% (377/1043) were euploid, 47% (487/1043) were aneuploid, 13% (140/1043) contained mosaicism, and 4% (39/1043) gave no result. We excluded the 39 embryos with no result; thus, 1004 embryos were included in the analysis. Increased aneuploidy was associated with increased maternal age, but the rate of embryo mosaicism was not. A combined analysis of aneuploidy with noncomplex abnormalities and mosaicism showed that chromosomes 22, 21, 16, and 15 were the most frequently involved. Chromosome 22 showed the highest proportion of mosaicism and chromosome 15 showed the highest proportion of aneuploidy. When we included embryo sex in the analysis, embryo sex was associated with these chromosome errors in the most susceptible chromosome, 22.Conclusion(s)PGT-A showed that chromosomes 22, 21, 16, and 15 were the most frequently involved among common chromosome abnormalities, comparable with those of published data analyzed from spontaneous abortion. This result suggested that PGT-A could pick up abnormalities that have clinical relevance to spontaneous abortion. Moreover, we identified a role of embryo sex in these chromosomal errors on chromosome 22.     

Predictability of preimplantation genetic diagnosis of aneuploidy and translocations on prospective attempts

The aim of this study was to determine if the outcomes of aneuploidy and translocation testing by preimplantation genetic diagnosis (PGD) at the 8-cell stage have a predictive value for new genetic diagnosis cycles. In total, 83 cycles (39 patients) undergoing PGD of translocations and 378 cycles (176 patients) of aneuploidy were included. Predictability, defined as having similar rate (+/-20%) of euploid embryos in the first and successive cycles, was found in 66% of patients undergoing aneuploidy testing. Predictability was found significantly more often in patients undergoing PGD of translocations (90%, P = 0.006). In addition, patients with 0, <30 or > or =30% euploid embryos in the first cycle were compared and groups 0 and <30% had significantly fewer euploid embryos in the second cycle (22-26%) than those of the group with > or =30% (37%) (P < 0.05). Patients who did not become pregnant after the first attempt were stimulated more aggressively than those becoming pregnant, producing significantly more embryos in the second than in the first cycle (P < 0.001). Therefore, correlation between euploidy rate and pregnancy rate could not be assessed objectively between cycles. In conclusion, the PGD results of a first cycle can predict the results of the second cycle, but this is likely to be of more value when the condition investigated is translocation rather than aneuploidy. The chance of pregnancy is usually related to the number of euploid embryos.     

Prenatal search for UPD 14 and UPD 15 in 83 cases of familial and de novo heterologous Robertsonian translocations

OBJECTIVES: The presence in the conceptus of a Robertsonian translocation predisposes to UPD formation, mainly by post-zygotic events of chromosome abnormality rescue. This is due to the increased risk of generating aneuploid zygotes because the rearranged chromosome and the respective homologues are prone to non-disjunction errors. Given this, carriers and karyotypically normal individuals conceived from a parent with a Robertsonian translocation are at risk for UPD. Abnormal phenotypes due to an imprinting effect have been found to be associated with UPD 14 and 15.The aim of the study was to refine, at the time of prenatal diagnosis, the risk for UPD 14 and 15 in a population with Robertsonian translocations involving these chromosomes. METHODS: Sixty-five cases of familial and de novo heterologous Robertsonian translocations involving chromosomes 14 and 15 and 18 fetuses with a normal karyotype, but conceived by a Robertsonian translocation carrier were prenatally studied to investigate the presence of UPD for chromosomes 14 and 15. RESULTS: Of the 65 Robertsonian translocation carriers, one fetus with a de novo der(14;21) showed maternal UPD 14. None of the 18 fetuses with a normal karyotype had UPD. CONCLUSION: Our data, combined with other previous prenatal investigations provide a general risk estimate for UPD 14 and 15 of 0.6%. Nevertheless, combining our data and those previously reported, all three fetuses with UPD had a de novo Robertsonian translocation, thus suggesting a risk of UPD formation of about 3% for this specific group of translocation carriers.