Preimplantation Genetic Diagnosis Research Articles

Association Between Anti-Müllerian Hormone and Early Spontaneous Abortion in Assisted Reproduction Treatment: A Case-Control Study Integrated with Biological Evidence.

Early spontaneous abortion (ESA) is a common adverse pregnancy outcome mainly attributed to embryo chromosomal abnormalities. However, as a quantitative marker, whether the anti-Müllerian hormone (AMH) can reflect oocyte quality is still controversial. By integrating biological evidence and adjusting many cofounders, this study aimed to clarify the controversies about the association between AMH and ESA caused by embryo aneuploidy during assisted reproductive technology (ART) treatment. We strictly preselected 988 patients receiving first ART treatment for analyzing clinical data, while 55 of them acquired chorionic villi karyotype results. In addition, 373 biopsied embryos from 126 patients receiving preimplantation genetic diagnosis (PGT) were tracked to compare embryo karyotypes. Univariate and multiple factor regressions were applied to analyze the risk factors leading to ESA. As covariates unadjusted, AMH (odds ratio 0.87, 95% CI 0.82-0.93) was the significant variable contributing to ESA. However, AMH played no significant role in the following regression models after age was adjusted. Also, AMH had no significant association with ESA in most age-adjusted subgroups, except in the male factors engaged subgroup. Additionally, compared to the patients with euploid chorionic villi karyotypes, those with aneuploid karyotypes were older and acquired fewer oocytes, yet their AMH levels were not significantly different. Furthermore, the embryo aneuploidy was independent of AMH while associated with maternal age, retrieved oocyte number, and embryo quality. This study suggested that AMH was unassociated with the ESA caused by embryo aneuploidy in ART therapy. As a critical cofounder, age remains the variable closely related to ESA.

The first clinical validation of whole-genome screening on standard trophectoderm biopsies of preimplantation embryos

The first clinical validation of whole-genome screening on standard trophectoderm biopsies of preimplantation embryos

Comparison of outcomes between intracytoplasmic sperm injection and in vitro fertilization inseminations with preimplantation genetic testing for aneuploidy, analysis of Society for Assisted Reproductive Technology Clinic Outcome Reporting System data

To evaluate whether insemination via intracytoplasmic sperm injection provides any benefit over in vitro fertilization for non-male factor infertility with respect to pre-implantation genetic testing results and pregnancy outcome. Retrospective Cohort Study of the Society for Assisted Reproductive Technology database. Patients undergoing in vitro fertilization or intracytoplasmic sperm injection in non-male factor pre-implantation genetic testing for aneuploidy cycles. In vitro fertilization vs. intracytoplasmic sperm injection MAIN OUTCOME MEASURES: Primary outcomes were percentage of embryos suitable for transfer and live birth rates. Secondary outcomes included subgroup analysis for embryos suitable for transfer on cycles from patients ≥ 35 y/o vs. < 35 y/o, ≤ 6 oocytes retrieved vs. > 6 oocytes retrieved, and unexplained infertility. Additionally, gestational age at delivery and birthweight between in vitro fertilization and intracytoplasmic sperm injection were evaluated. A total of 30,446 non-male factor preimplantation genetic diagnosis for aneuploidy cycles were evaluated, of which 4867 were in vitro fertilization and 25,579 were intracytoplasmic sperm injection. Following exclusion criteria and adjustment for any necessary confounding variables, no significant differences existed in embryos suitable for transfer between in vitro fertilization and intracytoplasmic sperm injection cycles, 41.6% (40.6%, 42.6%) vs. 42.5% (42.0%, 42.9%), respectively or live birth rates, 50.1% (37.8, 62.4%) vs. 50.8% (38.5%, 62.9%), respectively. There were no significant differences in the rates of embryos suitable for transfer and live birth rates between in vitro fertilization and intracytoplasmic sperm injection in non-male factor cycles undergoing pre-implantation genetic testing for aneuploidy.

Preimplantation Genetic Diagnosis of Androgen Resistance Syndrome Caused by Mutation on the AR Gene in Vietnam.

Androgen resistance syndrome or androgen insensitivity syndrome (AIS - Androgen Insensitivity Syndrome, OMIM 300068) is an X-linked recessive genetic syndrome causing disorders of sexual development in males. This disease is caused by mutations in the AR gene located on the X chromosome, which encodes the protein that structures the androgen receptor, with the role of receiving androgens. Mutation of the AR gene causes complete or partial loss of androgen receptor function, thereby androgen not being obtained and exerting its effect on target organs, resulting in abnormalities of the male reproductive system due to this organ system, differentiating towards feminization under the influence of estrogen. Disease prevention can be achieved by using pre-implantation genetic diagnosis, which enables couples carrying the mutation to have healthy offspring. To carry out preimplantation genetic diagnosis of androgen resistance syndrome. Sanger sequencing was used to detect the mutation in the blood samples of the couple, their son, and 01 embryo that were biopsied on the fifth day based on the findings of next-generation sequencing (NGS) of the affected son. We combined Sanger sequencing and linkage analysis using short tandem repeats (STR) to provide diagnostic results. We performed preimplantation genetic diagnosis for AIS on an embryo from a couple who had previously had an affected son. Consequently, one healthy embryo was diagnosed without the variant NM_000044: c.796del (p.Asp266IlefsTer30). We report on a novel variant (NM_000044: c.796del (p.Asp266IlefsTer30)) in the AR gene discovered in Vietnam. The developed protocol was helpful for the preimplantation genetic diagnosis process to help families with the monogenic disease of AIS but wish to have healthy children.

Revisiting the Liberal Case against Liberal Eugenics

In his book The Future of Human Nature, Habermas argues against the moral and legal permissibility of any future practices of genetic human enhancement as well as against current practices such as embryonic research or preimplantation genetic diagnosis. After analyzing the core of Habermas’s argument against positive eugenics, I argue that his attempt to derive a principle of abstention under uncertainty from the principle of counterfactual consent assumes that non-interference is the proper default norm in the absence of consent. Yet, this cannot be a plausible default norm for parental relationships. Moreover, since Habermas agrees that the no-harm principle justifies negative eugenics, once technical possibilities of genetic manipulation become available, non-interference becomes as much in need of normative justification as interference. I conclude that if our normative innocence regarding positive eugenics must be lost for the sake of negative eugenics, then it is an innocence well lost.

Gendering the beginning of life: Taiwanese gay fathers' navigation of preimplantation genetic diagnosis-assisted sex selection in transnational third-party reproduction.

Preimplantation genetic diagnosis (PGD) has been used not only to avoid genetic diseases and increase conception success rates but also to perform non-medical sex selection, particularly in the surging cross-border reproductive care (CBRC). In the context of commercialised biomedicine, assisted reproductive technologies, such as lifestyle sex selection, have been tailored to meet intended parents' preferences. However, there is a lack of analysis on how individuals' reproductive decisions on PGD-assisted sex selection were shaped within the sociocultural norms and CBRC. This article explores Taiwanese gay fathers' navigations on sex selection while seeking third-party reproduction overseas because of local legal constraints. Drawing on in-depth interviews with 53 gay fathers (to-be), I analysed how 'individual preferences' were dynamically shaped by local sociocultural norms and embedded within transnational settings of routinising PGD in chosen repro-destinations. The findings showed that gay fathers mobilised strategic discourses on non-medical sex selection from both the local and the global to negotiate their decisions in coherence with their LGBTQ+ identity and their role as sons carrying familial responsibility to procreate male heirs. This article proposed a nuanced understanding of gay fathers' reproductive practices of 'gendering the beginning of life' through PGD-assisted sex selection.

28 Evaluation of cell-free DNA in spent holding media as a noninvasive technique for pre-implantation genetic diagnosis on in vivo-produced equine embryos

28 Evaluation of cell-free DNA in spent holding media as a noninvasive technique for pre-implantation genetic diagnosis on in vivo-produced equine embryos

#176 : The Relationship Between Blastocyst Morphology and PGD Result

Background and Aims: Blastocyst morphology has been clinically assessed during the selection of blastocyst for transfer and cryopreservation, and the morphological grading scheme has gained wide acceptance. Blastocyst grading according to Gardner et al. (20) early blastocyst, blastocoel less than half of the blastocyst (1AA), blastocyst with blastocoel more than half of the blastocyst (2AA), blastocyst with blastocoel fill the blastocyst (3AA), and Expanded Blastocyst (4AA/4BB/4CC). Morphology blastocyst with good quality determining the success of PGD. Preimplantation genetic diagnosis (PGD) are routine procedures performed in many in vitro fertilization (IVF) clinics. In patients with genetic or chromosomal abnormalities, PGD is an integral part of the IVF program. This study aims to determine whether embryos with normal blastocyst morphology have euploidy result on the PGD test. Method: Observational research study. Results: Dari 22 embryo blastocyst yang di PGD, 59% embryo euploidy 4AA/4BB, 22,7% embryo low level mosaicism 4AA/4BB, 13% embryo aneuploidy 2AA/3AA and 4,5% embryo multiple aneuploidy 3AA. The result suggested that the euploidy rate of blastocyst grade 4AA was the highest. Conclusion: Based on data, embryo morphology with quality 4AA/4BB show PGD result Euploidy was the highest. Euploidy of embryo is one of the important factors affecting embryo implantation and development. Euploidy of blastocyst during PGD treatment was found to be correlated with morphological assessment (Gardner grade)

Modifying Our Genes: Theology, Science and “Playing God”

Modifying Our Genes: Theology, Science and “Playing God”

Common Mistakes While Performing an Embryo Biopsy

Embryo biopsy for Pre-Implantation Genetic Testing (PGT) is an advanced intervention in embryology lab requiring technical preciseness. Improper technique can influence the process of genetic evaluation, genetic report, and the reproductive outcomes. Following are few tips and tricks which help in optimizing the embryo biopsy technique : — It is not recommended to perform embryo biopsy in the same lab where regular embryo cultures are done. Use of UV light and Ethanol for sterilization of consumables might increase the VOCs (volatile organic compounds) and this might affect the embryo development. — Skill of micro-manipulation for an embryologist is very essential for a smooth biopsy technique — Additional care needs to be exercised when inner cell mass is at the site of biopsy or embryo has completely hatched — Negative suction pressures in the biopsy and holding pipettes during biopsy technique have to be well controlled. This avoids damage to the tissues. — Minimum of 6-8 trophectoderm cells need to be biopsied — Post biopsy we need to ensure that there is no remnant tissue on the biopsy pipette. This might bring about cross contamination of DNA. — While tubing care needs to be exercised to avoid excess buffer in the PCR tube. This can increase the chances of “NO DNA” detection. — Post biopsy its preferable to wait for one hour to assess significant damages to the embryo before vitrification — Embryos that have completely hatched need additional care at vitrification. Trophectoderm tissue is very sticky and might get adherent to the culture dish or the cryo-device. — Biopsied tissue post tubing needs to be stored at minus 20 degrees C till it gets shipped for genetic evaluation.

#237 : The Application of Noninvasive Implantation Capability Screening (NICS®) Resulted in Improved Clinical Outcomes in Patients with Recurrent Pregnancy Loss or Repeated Implantation Failure

Background and Aims: Chromosomal abnormalities are frequently observed in early human embryos, particularly in patients experiencing repeated pregnancy loss (RPL) or repeated implantation failure (RIF). Although preimplantation genetic testing for aneuploidy (PGT-A) is a common technique for screening embryo ploidy, it comes with the risks associated with embryo biopsy. To address this issue, we developed the NICS assay, which evaluates embryos using spent culture medium (SCM) and thus reduces the risks associated with embryo biopsy. However, further evaluation is required to determine the clinical applicability of NICS for patients with RPL or RIF. Method: This retrospective cohort study analyzed 303 patients with RPL or RIF between July 2018 and May 2021. These patients underwent aneuploidy screening using the NICS assay and were thus categorized into the NICS group. The non-NICS group consisted of patients who received conventional morphology embryo transfer during the same period. Routine intracytoplasmic sperm injection (ICSI) was performed, and the embryos were placed in droplets. Approximately 30 [Formula: see text] L of blastocyst medium from each embryo was transferred into cell lysis buffer, followed by DNA library preparation using the ChromInst kit from Yikon Genomics. Results: In patients with RPL, the NICS group demonstrated significantly lower rates of miscarriage per frozen embryo transfer (FET) than the non-NICS group, 17.9% vs 42.6%. Furthermore, the NICS group exhibited significantly higher rates of ongoing pregnancies (40.7% vs 25.0%) and live births (38.9% vs 20.6%) compared to the non-NICS group. In the case of patients with RIF, the NICS group showed higher clinical pregnancy rates per FET compared to the non-NICS group. Specifically, the clinical pregnancy rates, 46.9% vs 28.7%. Conclusion: The present study demonstrates that the use of NICS for selecting euploid embryos can effectively reduce the incidence of miscarriage in patients experiencing RPL, as well as improve the clinical pregnancy rate in those experiencing RIF.

Preimplantation Genetic Testing for Single Gene Conditions and Aneuploidy

It is now over 30 years since the first pregnancies and live births following IVF, embryo biopsy and preimplantation genetic testing (PGT) were reported in 1990. Since then, methods for genetic analysis at the single-cell level have evolved alongside the rapid advances in human genetics and sequencing of the human genome. With the development of methods for whole genome amplification, such as multiple displacement amplification (MDA), genome-wide analysis is now possible. This has led to the development of microarray or next generation sequencing (NGS) based methods for genome-wide haplotyping of parental chromosomes in embryo samples and enabled universal linkage-based testing for monogenic diseases combined with molecular cytogenetic analysis of chromosome abnormalities. These comprehensive methods have also recently been exploited to calculate polygenic risk scores for common multifactorial conditions, such as diabetes, cardiovascular disease, and cancer. Although controversial, polygenic risk scoring may have a role for reducing residual risk of breast cancer, for example, in combination with PGT for pathogenic variants. Finally, there is now evidence that cell-free DNA in spent culture medium at the blastocyst stage may originate from nuclear DNA shedding from the trophectoderm. In the future, therefore, it may be possible to use this cell-free DNA for genome-wide analysis and comprehensive PGT to avoid embryo biopsy.

Micromanipulator Set-up - Taking Control of This System

Embryo biopsy is a precise procedure, demanding adept manipulation skills and the finest instruments to ensure high-quality results. One of the most crucial elements to gain mastery over is the micromanipulator system. In this session attendees will delve into the practical facets of setting up and controlling a micromanipulator within the context of a blastocyst biopsy. The session will also cover the important aspects of system set-up, instrument alignment, and objective positioning, aimed to maximize control, precision, and stability during the biopsy procedure. Moreover, the discussion will touch on the common challenges faced during the set-up process, offering practical solutions, and troubleshooting tips to manage these effectively.

#121 : The Easier Blastocyst Biopsy, The More Trophectoderm Cells We Got

Background and Aims: Several studies showed various factors associated with pregnancy outcomes after the euploid embryo transfer, including parental, endometrial, and embryo biopsy. The consensus of the most suitable biopsied cell number is 5-10 cells among IVF labs. Therefore, for embryologists, how to have a steadily manipulated blastocyst to get an appropriate biopsied trophectoderm cell number is crucial. However, the factors related to biopsy manipulation are still being determined. Here, we retrospectively analyze the DNA concentrations from biopsied cells to test the hypothesis TE grading, hatching status, and biopsy day correlated with embryologists’ biopsy handling. Method: A retrospective study enrolled 212 blastocysts from 49 PGT-A cycles (NGS platform) from September 2021 and March 2023 at the Fertility and Reproductive Genetic Center of Linkou Chang Gung Memorial Hospital, Taoyuan, Taiwan. All the TE cells were removed by mechanical cutting by embryologists following the lab standard operating procedure. This study excluded five blastocysts (2.4%, 5/212) for the unsuccessful whole genome amplification (WGA), i.e., DNA concentration was less than 10 ng/[Formula: see text] l. Results: No difference in average DNA concentration was found between embryo ploidy status (Fig. A, P=0.8), embryologist (Fig. A, P=0.65), and TE grading (Gardner TE grading B vs. C, P=0.8). However, combined hatching status and day of the biopsy revealed a significantly higher DNA concentration in the hatching Day 5 to Day 7 groups than in the hatched Day 5 to Day 7 group (Fig. B, P[Formula: see text]0.0001). Conclusion: Our data suggest that the embryo ploidy status and Gardner grading of TE cells did not affect embryologists’ steadily performing biopsies. Interestingly, the easier biopsy, the more TE cells we got. This result implies that the hatching (Gardner expansion grade 5) embryo has a higher potential for damage after trophectoderm biopsy.

Aberrant landscapes of maternal meiotic crossovers contribute to aneuploidies in human embryos.

Meiotic recombination is crucial for human genetic diversity and chromosome segregation accuracy. Understanding its variation across individuals and the processes by which it goes awry are long-standing goals in human genetics. Current approaches for inferring recombination landscapes rely either on population genetic patterns of linkage disequilibrium (LD)-capturing a time-averaged view-or on direct detection of crossovers in gametes or multigeneration pedigrees, which limits data set scale and availability. Here, we introduce an approach for inferring sex-specific recombination landscapes using data from preimplantation genetic testing for aneuploidy (PGT-A). This method relies on low-coverage (<0.05×) whole-genome sequencing of in vitro fertilized (IVF) embryo biopsies. To overcome the data sparsity, our method exploits its inherent relatedness structure, knowledge of haplotypes from external population reference panels, and the frequent occurrence of monosomies in embryos, whereby the remaining chromosome is phased by default. Extensive simulations show our method's high accuracy, even at coverages as low as 0.02×. Applying this method to PGT-A data from 18,967 embryos, we mapped 70,660 recombination events with ∼150 kbp resolution, replicating established sex-specific recombination patterns. We observed a reduced total length of the female genetic map in trisomies compared with disomies, as well as chromosome-specific alterations in crossover distributions. Based on haplotype configurations in pericentromeric regions, our data indicate chromosome-specific propensities for different mechanisms of meiotic error. Our results provide a comprehensive view of the role of aberrant meiotic recombination in the origins of human aneuploidies and offer a versatile tool for mapping crossovers in low-coverage sequencing data from multiple siblings.

The Different Biopsy Techniques

Preimplantation genetic testing needs access to genomic DNA in order to execute a genetic test on a preimplantation embryo. Normally a few cells must be extracted or ‘biopsied’ from the embryo to achieve this. Embryo biopsy evolved from attempts in the 1960s and early 1970s to sex farm animal embryos to now utilizing it to screen and select human embryos based on Ploidy, or avoid monogenic disorders. In October 1989, the first clinical cleavage stage biopsies and single cell PGT to determine the sex of the embryos were conducted. It has now evolved to utilize Trophectoderm cells. The advent of Laser has allowed the biopsy process to shift away from mechanical and chemical hatching and techniques also now involve Laser to separate the cells from the blastocyst. Here is a short summary of the different biopsy stages. Polar Body Biopsy : — Polar bodies are small cells produced during the process of oogenesis (egg development) in females. — Since polar bodies contain genetic material from the egg, they can be biopsied to indirectly infer the genetic status of the egg. — This technique is less common and has limitations, but it can be used in certain situations. Blastomere Biopsy (Day 3 Biopsy) : — Performed on the third day after fertilization when the embryo has around 8 cells. — A single cell (blastomere) is removed from the embryo for genetic testing. — Mosaicism cannot be ruled out thus questioning the efficacy of this technique. Trophectoderm Biopsy (Day 5-7 Biopsy) : — Performed at the blastocyst stage, typically on the fifth or sixth day of embryonic development. — Usually, 5-10 cells from the trophectoderm are removed for genetic testing. — This technique is now gold standard and allows determination of mosaicism. The embryo biopsy revolution’s 30-year saga is still unfolding, and non-invasive genetic testing holds potential to revolutionize the way embryos are tested for genetic disorders.

Preimplantation Genetic Testing within the Public Healthcare System in Slovenia.

Preimplantation genetic testing (PGT) is the earliest form of prenatal diagnosis that has become an established procedure for couples at risk of passing a severe genetic disease to their offspring. At UMC Ljubljana, we conducted a retrospective register-based study to present 15 years of PGT service within the public healthcare system in Slovenia. We collected the data of the PGT cycles from 2004 to 2019 and compared clinical outcomes for chromosomal and monogenic diseases using different embryo biopsy and testing approaches. In addition, we assessed the extent to which PGT has become the preferred option compared to classic prenatal diagnostics. We treated 211 couples, 110 with single gene disorder, 88 with structural chromosome rearrangement and 13 for numerical chromosome aberration. There were 375 PGT cycles with oocyte retrieval, while embryo transfer was possible in 263 cases resulting in 78 deliveries and 84 children. Altogether, the clinical pregnancy rate per embryo transfer was 31% in 2004-2016 (blastomere biopsy) and 43% in 2017-19 (blastocyst biopsy), respectively. We assessed that approximately a third of couples would opt for PGT, while the rest preferred natural conception with prenatal diagnosis. Our results show that providing a PGT service within the public healthcare system has become a considerable option in pregnancy planning for couples at risk of transmitting a severe genetic disease to their offspring. In Slovenia, approximately a third of couples would opt for PGT. Although the number of cycles is small, our clinical results are comparable to larger centres.

Biopsy Procedure and Preparation (Including Biopsy Medium and Biopsy Dish)

This lecture is a concise introduction designed to equip participants with essential knowledge and practical skills in performing embryo biopsy for PGT. The workshop emphasizes the significance of procedure and preparation which may impact the blastocyst biopsy and its role in selecting embryos. During the lecture, attendees are introduced to the fundamental steps of the blastocyst biopsy, starting with the optimal time frame for performing the biopsy, followed by techniques for safe and accurate zona drilling without compromising the embryo’s viability, as well as the importance of using a suitable biopsy medium that provides the necessary nutrients and support for the embryo during the biopsy. This lecture also delves into the selection and preparation of the biopsy dish, outlining the key factors to consider and practical tips on maintaining optimal conditions for the embryo within the biopsy dish and minimizing potential stress, to ensure successful outcomes. The instructor will share experiences and provide valuable insights into troubleshooting potential challenges that may arise during the biopsy process. Participants are encouraged to engage in interactive discussions and following hands-on sessions, allowing for a comprehensive understanding of the procedure, and fostering a collaborative learning environment. In conclusion, this lecture offers a valuable platform for embryologists to enhance their knowledge and skills in performing blastocyst biopsies with precision and confidence.

Evaluation of Couples at Risk for Hemoglobin Disorders in Italy: The Results of an 18-Month Retrospective Observational Single-Center Study

Background: Hemoglobinopathies are genetic disorders caused by mutations in the genes encoding globin chains. These conditions are the most common monogenic disorders worldwide, resulting in significant morbidity and mortality among those affected by the most severe forms. Antenatal evaluation for thalassemia and sickle cell disease (SCD) enables couples to know their reproductive risk, thus allowing an informed reproductive choice. However, in certain areas, couples do not often receive the appropriate counseling at the appropriate time. Aim: The aim of this study is to describe the characteristics of couples referred to our tertiary referral center in Milan, Italy, for evaluation of hemoglobinopathy risk and their management. Methods: in our Institution, a diagnostic-therapeutic pathway involving the Hemoglobinopathies unit, the Medical Genetic unit, and the central laboratory has been implemented. Subjects referred to the Institution, and their partner, underwent the following laboratory tests: complete blood count, hemolysis and iron parameters, vitamin pattern, and hemoglobin (Hb) pattern by high-performance liquid chromatography (HPLC). The couples were then evaluated by a hemoglobinopathy expert, and those at risk for Hb disorders were offered a molecular analysis. Couples were then referred to the geneticist to receive the results of the molecular analysis, discuss the genetic risk for the fetus and, in case of high risk, discuss possible reproductive choices (i.e., prenatal diagnosis, pre-implantation diagnosis). We retrospectively collected and analyzed couples' data referred to our center from the 1 st of July 2021 to the 31 st of December 2022. Results: Three hundred and fifty-one couples were evaluated for hemoglobinopathies over 18 months. The median age was 34 years, with a tendency for younger age in female partners; 67% (234/351) of the women were already pregnant at the time of evaluation (median gestational age 18 weeks, range 7-37), while 33% (117/351) were referred for preconception counseling. Individuals from more than 48 different countries were screened, and the most represented countries of origin included: Italy (50%), Nigeria (6%), Bangladesh (4%), and Ivory Coast (4%), underpinning a vastly diverse genomic background. The referral was triggered by either evidence of microcytic anemia (58%) or HPLC anomalies (42%), including Hb variants, HbA2 anomalies, or elevated HbF in at least one partner. 55% of the referrals did not require additional testing. The remaining 45% (158/351) underwent advanced molecular tests to determine the nature of the Hb defect, guided by the results of the first-level panel. Among these, 33 couples (12% of the total) were found to be at risk for transmission of severe clinical forms of β-thalassemia, sickle cell anemia, or compound β-disorder heterozygosity (HbS/β-thal, HbC/HbS). One additional couple (0.4%) was at risk for HbH disease. In addition, 4 couples (1.5%) were found to be at risk for thalassemia intermedia combining a β-thalassemia mutation and α-gene triplication. Of the above, all 34 couples underwent invasive fetal evaluation: 24 women underwent chorionic villous sampling, while 10 received amniocentesis. A diagnosis of thalassemia in the fetus was reported in 6/34 (18%) cases and 3 (9%) of SCD. Interestingly, during the 18 months of observation, among the evaluated adult subjects, we found 4 cases of non-transfusion dependent thalassemia (NTDT) and 1 of HbS/β-thal in otherwise healthy subjects. All 4 cases of NTDT resulted from α-gene triplication paired with a β-thalassemia heterozygous defect. Furthermore, α triplication was found alone in 4 subjects without additional Hb gene defects. Overall, α triplication was detected in 9 out of 154 (5.8%) individuals who underwent HBA gene analysis. Conclusion: The median gestational age at presentation was high, leading to a limited time for couples to receive the appropriate counseling to make an informed reproductive choice. Increasing awareness across all involved specialists remains the mainstay for an effective prevention program to promptly identify at-risk couples and offer counseling at the right time. In addition, the relatively high incidence of α triplication in our population of asymptomatic and hematologically normal subjects and the new diagnosis of 4 NTDT warrants further studies to confirm the prevalence of α triplication in the general population.

Myopathy with Lactic Acidosis and Sideroblastic Anemia Caused By a Novel PUS1mutation

Mutations in the mitochondrial aminoacyl tRNA synthetases (ARSs) are associated with various clinical phenotypes, including myopathy, lactic acidosis, and sideroblastic anemia (MLASA), which is a rare autosomal recessive disorder. Due to oxidative phosphorylation disorder and abnormal iron metabolism in the skeletal muscle and erythroid bone marrow lines, patients show progressive muscle weakness, exercise intolerance, lactic acidosis, siderocyte anemia, and growth retardation, etc. Herein, we report a case of MLASA caused by a novel homozygous missense mutation (c.597G&amp;gt;T p.R199S) in the pseudouridine synthase 1 ( PUS1) gene, causing the loss of protein function. The patient's parents had a consanguineous marriage, with a grandmother in common. All mutations were heterozygous without disease. While the patient was homozygous for the mutation and developed symptoms of anemia, no specific treatment exists for this genetic disease. Deferrization therapy can reduce iron overload, which leads to reduced heme synthesis in such patients but cannot treat the disease. Consanguineous marriage often causes autosomal recessive genetic diseases and prohibiting it can reduce the incidence of these diseases. The present patient's condition was caused by a mutation in nuclear PUS1, which manifested as SA, hyperlactatemia, iron overload, and muscle damage. PUS1 mutations can be missense, nonsense, or frameshift. We identified a novel missense mutation type in PUS1 in this patient, located on chromosome 12, c.597G&amp;gt;T (p.R199S), which is a homozygous mutation. This was confirmed by the PUS1 analysis revealing homozygosity for the novel mutation. Both the parents and grandmothers were heterozygous for the mutation, but none of them had a clinical manifestation. Combining the patient's family history, medical history, laboratory examination, and genetic characteristics of the disease, the mutation was identified as a pathogenic variant, and the clinical manifestations and genetic findings of the patient expanded the genotype-phenotype spectrum of MLASA. The possibility of MLASA should be suspected in cases of progressive muscle weakness, growth retardation, exercise intolerance in childhood, SA, and lactic acidosis before or after puberty. With the recognition of MLASA and the advent of NGS, identifying patients with PUS1 mutations characterizing incomplete MLASA syndrome became easier, thus further expanding the phenotypic and genotypic heterogeneities of these mitochondrial disorders. Targeting NGS may also significantly improve the prenatal and preimplantation genetic diagnosis of MLASA, providing better genetic counseling for parents. Genetic diseases lack effective treatment measures, and the curative effect is unsatisfactory. Therefore, prevention is more important. Prevention strategies include avoiding consanguineous marriage, genetic counseling, genetic testing for carriers, and a prenatal diagnosis.