Preimplantation Genetic Diagnosis Research Articles

P-528 Establishing sequencing coverage depth for preimplantation genetic testing for monogenic disorders (PGT-M) using a specific targeted next generation sequencing (tNGS)

Abstract Study question What is the sufficient sequencing coverage read depth (RD) for direct variant analysis for PGT-M using a specific tNGS assay? Summary answer Downsampling modelling experiments identified a minimum sequencing depth of 50X that is recommended for PGT-M with this specific tNGS assay. What is known already Properly developed and validated multiplex PCR amplification and NGS-based analysis reliably produces PGT-M diagnoses with low allele and amplification failure at RD of hundreds of reads when analyzing a 3-10 cell trophectoderm (TE) biopsy. However, the RD can be affected by amplification efficiency in a specific genomic region and therefore affect the reproducibility of variant detection: the higher the number of aligned reads, the higher the confidence of base calling at a particular position. Downsampling is a powerful bioinformatics tool for modelling comparisons between samples or between replicates, identifying analytical sequencing depth limits for accurate clinical diagnosis. Study design, size, duration This is a retrospective comparative diagnostic test accuracy (DTA) and verification study. Twenty-three embryos from 8 patients that had undergone PGT-M with clinically established Taqman qPCR genotyping were used as positive controls. The primary outcome was to establish the lowest RD point of down sampling where allelic frequency is reliably detected at heterozygosity between 20-80% in known positive controls. A further 30 PGT-M clinical cases were used to verify analytical performances under clinical conditions. Participants/materials, setting, methods Embryo biopsies were pre-amplified with a multiplex primer pool for PGT-M and PGT-A, using a two-step PCR strategy. Sequencing was performed on Illumina NextSeq550. Reads were aligned to a human reference genome (GRCh37/hg19) and processed with an in house bioinformatic pipeline. The RD and alternative allele frequencies (AAF) for PGT-M was calculated. The reads for the heterozygous variants were downsampled to 10X, 20X, 30X, 50X, 100X, 200X, 300X, 500X, and 1000X to establish the cutoff. Main results and the role of chance One or two TE biopsies (N = 44) of 23 embryos that were 100% concordant with previous PGT-M results were tested for 1 to 3 of the 12 variants (MLH1 c.199G>A, RB1 c.1589A>G, SLC16A2 c.604G>A, GNPTAB c.1123C>T, GNPTAB c.3314A>G, WFS1 c.1391C>G, USH2A c.10073G>A, USH2A del exons 5-11, F5 c.1601G>A, GAA c.693-1G>C, GAA c.875A>G, and HEXA c.1274_1277dupTATC). PGT-A analysis of multiple biopsies from the same embryo demonstrated consistent karyotypes (N = 44). The 19 heterozygous variants from 17 biopsies exhibited average RD of 763X (238X -1980X) with average AAF of 50% (44%- 56%). The reads for the heterozygous variants were downsampled to different levels (100,000 replicates). The distribution of the AAF were compared among the different depth levels. At a RD of 50X, the AAF displayed an interquartile range (IQR) of 8% (46%-54%) between the maximum and minimum values from 20% to 78%. Compared to 30X (IQR: 43%-57%, with minimum 13% and maximum 90%), and 100X (IQR: 47%-54%, with minimum 30% and maximum 71%), the RD at 50X generated reliable heterozygous calls with minimum depth requirement. 245 biopsies from 30 clinic cases were analyzed by tNGS-based PGT-M. 90 samples with heterozygous variant calls showed average RD of 979X (57X-5803X) with average AAF of 47% (29%-585). Limitations, reasons for caution The 50X cutoff is based on the heterozygous variants with theoretical 50% AFF and is specific for this PGT-A assay. This evaluation should be corroborated for complicated variants with homologous genes, which might require different thresholds. The aneuploidy at specific chromosome can cause the deviation of AFF from 50%. Wider implications of the findings This specific tNGS-based PGT-M can provide direct variant analysis at a 50X read depth with a high level of consistency compared to established methodologies while allowing for simultaneous aneuploidy screening using the same biopsy and procedural optimization. Trial registration number not applicable

P-545 Early pregnancy outcomes associated with age and AMH following Next-Generation Sequencing(NGS)-based Preimplantation Genetic Testing for Aneuploidy(PGT-A) in single euploid embryo transfers for advanced age patients

Abstract Study question Does age and AMH impact early pregnancy outcomes in euploid single embryo transfers using NGS-based PGT-A for individuals aged 35 and above? Summary answer In this NGS-based PGT-A study, no significant differences in early pregnancy rates were observed based on age or AMH among individuals aged 35 and above. What is known already A recent meta-analysis suggest a decline in pregnancy rates with advancing age in the context of euploid embryo transfer following PGT-A. However, it’s crucial to acknowledge variations in PGT-A techniques across studies. While individuals with low AMH levels may encounter challenges in obtaining euploid embryos, the precise impact of AMH on pregnancy rates post-embryo acquisition remains unclear. Study design, size, duration A retrospective cohort study was conducted on 428 cycles of single euploid embryo transfers in patients aged 35 and above, performed at the single infertility center from January to December 2022. Participants/materials, setting, methods The study examined frozen-thawed embryo transfer cycles in patients aged 35 and above, analyzing early pregnancy outcomes in single euploid embryo transfers. Participants were grouped by age(35-38, 38-42, 42 and above) and AMH levels(<1, 1-3, >3 ng/ml). NGS-based PGT-A conducted embryo biopsy. The analysis aimed to detect differences in initial pregnancy rates among age and AMH subgroups. The differences between groups were compared using Pearson’s chi-squared test. Main results and the role of chance The average age at transfer was 39.05 ± 2.53 years, with a mean AMH of 2.60 ± 1.86(ng/ml). The mean number of prior frozen embryo transfer (FET) cycles was 1.9 ± 2.04, with 119 patients (27.8%) having undergone three or more transfer cycles. Transfer cycles comprised 363 (84.8%) artificial cycles and 65 (15.2%) ovulatory cycles, with an average endometrial thickness of 8.95 ± 1.73 mm at the time of transfer. The β-hCG positive rate was 57.7% (247/428), and the clinical pregnancy rate was 53.5% (229/428). When analyzing positive b-hCG results by age, rates were 59.9% (82/137) for ages 35-38, 55.5% (121/218) for ages 38-42, and 60.3% (44/73) for ages 42 and above (p = 0.641). In terms of AMH, β-hCG positive rates were 52.1% (37/71) for <1, 59.1% (120/203) for 1-3, and 58.0% (83/143) for ≥3, with no statistically significant differences (p = 0.584). Clinical pregnancy rates were 54.0% for ages 35-38, 51.8% for ages 38-42, and 57.5% for ages 42 and above. For AMH, rates were 50.7% for <1, 53.2% for 1-3, and 54.5% for ≥3, with no statistically significant differences (p = 0.869). Limitations, reasons for caution Being a retrospective study analyzing patients undergoing embryo transfer, our study overlooks variables related to oocyte retrieval. Given the challenges of obtaining euploid embryos in advanced age, further investigation is warranted to explore the probability and duration of embryo acquisition in this population. Wider implications of the findings In this study, utilizing NGS-based PGT-A after generating euploid embryos, no significant differences in early pregnancy outcomes were observed for women aged 35 and older, considering age and AMH levels. The study suggests further research is needed to explore if NGS-based PGT-A can address limitations of traditional chromosomal testing methods. Trial registration number not applicable

P-245 Evaluating the impact of embryos undergoing multiple freeze-thaw cycles in preimplantation genetic testing for aneuploidy (PGT-A) on treatment outcomes in IVF

Abstract Study question Do cryopreserved embryos subjected to thaw, biopsy, re-freeze (TBR) exhibit inferior pregnancy outcomes compared to embryos undergoing standard PGT-A with a single vitrification-warming cycle? Summary answer Transfer of twice-thawed euploid embryos demonstrated comparable clinical pregnancy outcomes, yet elevated miscarriage rates following a repeated vitrification-warming cycle warrants further scientific inquiry. What is known already There is often hesitancy to test cryopreserved embryos that were not initially PGT-A tested due to concerns about embryo survival during the additional freeze-thaw cycle those embryos will endure. However, the TBR technique has shown efficacy, particularly for IVF patients with recurrent failed attempts using fresh, untested embryos. Previous studies on TBR consistently report comparable embryo survival rates to fresh cycles. Nevertheless, there is no consensus on whether transferring euploid TBR embryos adversely affects pregnancy outcomes compared to euploid fresh biopsy embryos, potentially due to the relatively low number of transferred TBR embryos in such studies. Study design, size, duration This study retrospectively analysed euploid freeze-thaw single embryo transfer (SET) data from embryos that underwent PGT-A at a large fertility clinic in London, between January 2019 and June 2022. Pregnancy outcomes were compared between freshly biopsied embryos (control group, n = 349) and cryopreserved embryos that underwent thaw and biopsy before freeze-thaw SET (twice-thawed group, n = 35). Embryos previously biopsied with PGT-A, subsequently cryopreserved, and then subjected to thaw and re-biopsy were classified in the twice-thawed group. Participants/materials, setting, methods Blastocysts cultured from fresh embryos (control group) or derived from frozen-thawed blastocysts (twice-thawed group) underwent PGT-A using standard protocols before freeze-thaw SET. The study assessed biopsy rates, post first thaw and survival rates post second thaw in the twice-thawed group, comparing to the control group. The clinical outcomes evaluated were Implantation rate (IR), Live Birth Rate (LBR), and Miscarriage Rate (MR). Statistical analysis included one-way ANOVA for continuous and Pearson’s chi-squared test for categorical variables Main results and the role of chance Blastocyst characteristics, including morphological quality (characterised into ‘GOOD’, ‘FAIR’ and ‘POOR’) and day of biopsy (5 or 6), did not significantly differ between blastocyst transfers of the twice-thawed and control group. Furthermore, maternal factors including age, number of previous PGT-A attempts and endometrial thickness did not significantly differ between groups. However, compared with the control group, the number of eggs collected was significantly higher in the twice-thawed group (15.8±8.20 vs. 25.8±18.7, p = <0.001). The biopsy rate of previously cryopreserved embryos that underwent TBR was 79%. However, there was no significant difference between the survival rate after the second thaw and before SET between the twice-thawed and control groups. There was no difference in IRs (71.1% vs. 74.3%, p = 0.687) and LBRs (56.4% vs. 40.0%, p = 0.062) between groups. Although, compared to the control, MRs were significantly higher in the twice-thawed group (20.6% vs. 46.2% p = 0.003). Limitations, reasons for caution The twice-thawed group includes patients with diminished ovarian reserve, previously undergoing single freeze-thaw SET resulting in recurrent miscarriages. Thus, further research is essential to clarify whether elevated miscarriage rates result from repeated freeze-thaw cycles. Additionally, as single freeze-thaw SET is preferred, small sample size in the twice-thawed group limits conclusions. Wider implications of the findings This study suggests employing TBR on previously frozen blastocysts yields non-inferior IRs and LBRs, with an additional freeze-thaw cycle not adversely affecting embryo survival before SET. Therefore, such techniques should be considered in previously frozen IVF embryos to reduce wastage, mitigate unforeseen fertility complications, and concurrently achieve favourable pregnancy outcomes. Trial registration number Not applicable

P-535 Effect of advanced female age (AFA) on the ratio of patients receiving mosaic embryo transfers

Abstract Study question How does female age affect ratio of patients with at least one euploid embryo (≥1EE) and at least one mosaic embryo without any euploid ones(≥1ME)? Summary answer Percentage of patients receiving mosaic embryo transfers increases with female age. What is known already Introduction of Next Generation Sequencing (NGS) to the area of Preimplantation Genetic Testing for Aneuploidy (PGT-A) has enabled detection of mosaicism in human embryos. Studies reporting the birth of healthy babies after the transfer of embryos with a chromosomal mosaic result on PGT-A have been published (Greco et al., 2015; Kahraman et al., 2020; Viotti et al., 2021 and 2023). Data suggested lower implantation rates and higher miscarriage rates when mosaics were compared with euploid embryo transfers. The percentage of mosaic embryos in overall tested embryos decreases with increased maternal age, similar to euploid embryos’ decline (Sanders et al., 2023). Study design, size, duration This retrospective study includes 7287 cycles with PGT-A performed between January 2017 and December 2023. Number of biopsied blastocysts was 21,277 and all were tested with NGS. All PGT-A testing was conducted on Ion Torrent S5 (Thermo Fisher Scientific) according to the manufacturer guidelines. The mosaic embryo reporting thresholds were determined as 20 to 80%. Participants/materials, setting, methods Euploidy ratio (ER) was defined as (euploid embryos (n)/ all tested embryos(n)). Mosaicism ratio (MR) was defined as (mosaic embryos (n)/all tested embryos(n)). Alternative mosaicism ratio (AMR) was defined as the number of mosaic embryos divided by the sum of euploid embryos and mosaic embryos. In other words, AMR can be defined as the number of mosaic embryos divided by non-aneuploid embryos. Female ages were divided into five categories: FAG1:<35, FAG2:35-37, FAG3:38-40, FAG4:41-42, FAG5:>42. Main results and the role of chance The number of cycles, total number and average biopsied embryos with standard deviations were highest in FA1 and declined with AFA: FAG1:2537, 9298, 3.7±2.2; FAG2:1348, 4140, 3.1±1.7; FAG3:1701, 4298, 2.5±1.6; FAG4: 958,2116, 2.2±1.5; FAG5:738, 2407, 1.9±1.2. ER and MR were observed to decrease with AFA: FAG1:%48.4, %15.2; FAG2:%39.0, %12.7; FAG3:%26.5, %10.5; FAG4:%14.9, %6.0; FAG5:%5.0, %2.6. This decrease was statistically significant (p < 0.0001, Chi-Square Test). However, when AMR was calculated among age groups, AMR was increasing with AFA: FAG1:%23.9; FAG2:%24.6; FAG3:%28.5;FAG4:%28.7; FAG5:%34.5 and this was also statistically significant (p = 0.0002, Chi-Square Test). Correlation of MR and AMR with female age groups were tested with Pearson correlation coefficient and MR was found to be negatively correlated with AFA whereas AMR was positively correlated (r=-0.19, p < 0.0001; r = 0.07, p < 0.0001, respectively). The ratio of patients with ≥1EE and ≥1ME was found to be decreasing with AFA when compared to overall tested cycles: FAG1: %83%, %7; FAG2: %69, %9; FAG3: %47, %11; FAG4: %28, %9; FAG5: %9, %4. However, the ratio of ≥ 1ME in cycles with transferrable embryos significantly increased with AFA consisting of more than one fourth of embryo transfer cycles above 38 years of age: FAG1: %7; FAG2: %12; FAG3: %19; FAG4: %24; FAG5: 31%. Limitations, reasons for caution These are the results of a single ART and Reproductive Genetics Center with its strictly defined standard operating procedures for PGT-A testing and ovarian stimulation protocols. Although not expected, different PGT-A testing methodologies or stimulation protocols may influence the above-mentioned outcomes. Wider implications of the findings The alternative mosaicism ratio defined here is generally overlooked but impacts the treatment strategies of ART patients, specifically for the advanced maternal age groups. Trial registration number not applicable

In Vitro Fertilization and Assisted Reproduction – Basics, Advances and Current Scenario

These changing scenarios have expanded indication for In vitro fertilization (IVF) where the oocyte is fertilized outside the body. Indications of fertility preservation in cancer patients, embryo biopsy to rule out genetic disease, and viral infections like HIV and Hepatitis to decrease transmission are some of the new indications. Special interventions like blastocyst culture, laser assisted hatching, testicular sperm retrieval by Microtese, ovarian tissue freezing for fertility preservation in young girls and rejuvenative medicines with stem cells have come into practice recently. IVF is an acceptable procedure with a high success rate. It is now more easily accessible with wider indication as with time its advantages have been given credit.

Advanced strategies for single embryo selection in assisted human reproduction: A review of clinical practice and research methods

Among the primary objectives of contemporary assisted reproductive technology research are achieving the births of healthy singletons and improving overall fertility outcomes. Substantial advances have been made in refining the selection of single embryos for transfer, with the aim of maximizing the likelihood of successful implantation. The principal criterion for this selection is embryo morphology. Morphological evaluation systems are based on traditional parameters, including cell count and fragmentation, pronuclear morphology, cleavage rate, blastocyst formation, and various sequential embryonic assessments. To reduce the incidence of multiple pregnancies and to identify the single embryo with the highest potential for growth, invasive techniques such as preimplantation genetic screening are employed in in vitro fertilization clinics. However, new approaches have been suggested for clinical application that do not harm the embryo and that provide consistent, accurate results. Noninvasive technologies, such as time-lapse imaging and omics, leverage morphokinetic parameters and the byproducts of embryo metabolism, respectively, to identify noninvasive prognostic markers for competent single embryo selection. While these technologies have garnered considerable interest in the research community, they are not incorporated into routine clinical practice and still have substantial room for improvement. Currently, the most promising strategies involve integrating multiple methodologies, which together are anticipated to increase the likelihood of successful pregnancy.

Preimplantation Genetic Testing for Aneuploidy in In Vitro Fertilization Using Comprehensive Chromosome Screening: A Systematic Review and Meta-Analysis.

The utility of pre-implantation genetic testing (PGT-A) is controversial, with older meta-analyses demonstrating improved pregnancy outcomes, while newer trials have not shown benefit. Therefore, we performed a meta-analysis which aimed to evaluate the benefits of PGT-A using comprehensive chromosome screening (CCS) and its effects on in vitro fertilization (IVF) outcomes among randomized controlled trials (RCTs). We conducted a systematic search to identify RCTs comparing women undergoing PGT-A with CSS with women not undergoing PGT-A, from inception to December 2020. Random effects meta-analysis was utilized to calculate average odds ratios (OR) for clinical pregnancy rate (CPR), ongoing pregnancy rate (OPR), and miscarriage rate (MR). The heterogeneity of exposure was assessed using Forest plots and I2 statistics. Publication bias was evaluated using Egger's test. Among 1251 citations, seven RCTs met the inclusion criteria. Biopsies of embryos were carried out at various developmental stages, including polar body, day 3, and day 5-6 of culture. Data was analyzed as all studies and blastocyst only. Meta-analysis failed to show improvement in OPRs using PGT-A in the all ages, <35 years old and ≥35 years old age groups. There was also no significant difference in CPRs in any group. The MR decreased with the use of PGT-A (among all biopsy types and among blastocyst biopsies) in the all-ages group, but not when stratifying according to patient age <35 and ≥35 years old. More data regarding the risks and advantages of PGT-A are needed to make a final decision on the value of this intervention in clinical practice. The exact magnitude of the benefit of PGT-A selection cannot be correctly determined until multiple standardized protocol IVF PGT-A trials are conducted.

Homozygous TNNI3 frameshift variant in a consanguineous family with lethal infantile dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is characterized by dilatation of the left ventricle, systolic dysfunction, and normal or reduced thickness of the left ventricular wall. It is a leading cause of heart failure and cardiac death at a young age. Cases with neonatal onset DCM were correlated with severe clinical presentation and poor prognosis. A monogenic molecular etiology accounts for nearly half of cases. Here, we report a family with three deceased offspring at the age of 1 year old. The autopsy of the first deceased infant revealed a DCM. The second infant presented a DCM phenotype with a severely reduced Left Ventricular Ejection Fraction (LVEF) of 10%. Similarly, the third infant showed a severe DCM phenotype with LVEF of 30% as well, in addition to eccentric mitral insufficiency. Exome sequencing was performed for the trio (the second deceased infant and her parents). Data analysis following the autosomal dominant and recessive patterns of inheritance was carried out along with a mitochondrial pathways-based analysis. We identified a homozygous frameshift variant in the TNNI3 gene (c.204delG; p.(Arg69AlafsTer8)). This variant has been recently reported in the ClinVar database in association with cardiac phenotypes as pathogenic or likely pathogenic and classified as pathogenic according to ACMG. Genetic counseling was provided for the family and a prenatal diagnosis of choronic villus was proposed in the absence of pre-implantation genetic diagnosis possibilities. Our study expands the case series of early-onset DCM patients with a protein-truncating variant in the TNNI3 gene by reporting three affected infant siblings.

Metabolic Profiles of Offspring Born From Biopsied Embryos from Toddlerhood to Preschool Age.

Embryo biopsy, which is necessary for preimplantation genetic testing (PGT), has not been fully investigated regarding its potential influences and safety. Previous studies of children born from biopsied embryos (PGT children) have primarily centered around their growth and neuropsychological development, while there remains limited knowledge concerning their endocrine and metabolic parameters. This study aims to examine the effect of trophectoderm (TE) biopsy on metabolic outcomes for PGT children. A total of 1267 children from the Center for Reproductive Medicine, Shandong University, who were conceived through in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) with and without PGT, were analyzed in this study. Three sets of measurements pertaining to growth and metabolism were taken at each predetermined follow-up time point. The linear regression models within a generalized estimating equation were employed to examine the associations between the PGT and each outcome measure and the approach of false discovery rate was used to correct for multiple comparisons. After controlling for confounding factors and correcting for multiple comparisons, no statistically significant difference was identified in any of the measured variables between the PGT children and children conceived by IVF alone (IVF children) and children conceived through IVF using ICSI (ICSI children). The same is true also for age- or sex-based subgroup analyses. Between the ages of 1 and 5 years, there are no clinically adverse metabolic outcomes observed in PGT children, and their metabolic profiles are essentially identical to those of IVF children and ICSI children.

Sex Reassignment Dilemma in 4 Yemeni Siblings with Five Alpha-Reductase Type 2 Deficiency

5α-Reductase two deficiency (5αR2D) is an autosomal recessive 46, XY disorder impacting the HeRD5A2 gene, causing ambiguous genitalia. Four Yemeni siblings with this condition sought guidance from the Sudanese Intersex Working Group (SIWG) in adulthood. A multidisciplinary evaluation was conducted, encompassing physical, mental, hormonal, and imaging examinations, although genetic mutation analysis of the srd5a2 gene was precluded due to limited facilities. Participants were initially assigned as females by an untrained birth attendant; the siblings later manifested virilisation before puberty, prompting a reassignment to the male sex. Despite presenting with 46 XY DSD due to 5αR2D, their sexual identity conflict led them to seek counsel from the SIWG. Comprehensive assessments confirmed the 5αR2D diagnosis. Extensive counselling with the family revealed their resolute decision to maintain the male sex despite concerns highlighted by the SIWG regarding future sexual function and fertility. This case underscores the significant challenges stemming from inadequate knowledge and interventions, emphasising the critical need for early diagnosis, proper genetic counselling, and expert management. Timely referrals to specialised facilities and consideration of options such as in vitro fertilisation (IVF) and preimplantation genetic diagnosis (PGD) for subsequent children are imperative. This report advocates for proactive measures in similar cases, emphasising the importance of tertiary care facilities for accurate diagnosis, informed decision-making, and optimal management of 5αR2D-related dilemmas.

Higher Concentrations of Essential Trace Elements in Women Undergoing IVF May Be Associated with Poor Reproductive Outcomes Following Single Euploid Embryo Transfer.

Essential trace elements are micronutrients whose deficiency has been associated with altered fertility and/or adverse pregnancy outcomes, while surplus may be toxic. The concentrations of eight essential trace elements were measured using inductively coupled mass spectrometry (ICP-MS) and assessed with respect to clinical in vitro fertilization (IVF) outcomes in a population of 51 women undergoing IVF with intracytoplasmic sperm injection (ICSI), pre-implantation genetic screening for aneuploidy (PGT-A), and single frozen euploid embryo transfer (SET/FET). Specifically, copper (Cu), zinc (Zn), molybdenum, selenium, lithium, iron, chromium, and manganese were quantified in follicular fluid and whole blood collected the day of vaginal oocyte retrieval (VOR) and in urine collected the day of VOR and embryo transfer. We found that the whole blood Cu/Zn ratio was significantly associated with superior responses to ovarian stimulation. Conversely, the whole blood zinc and selenium concentrations were significantly associated with poor ovarian response outcomes. Higher levels of whole blood zinc and selenium, urinary selenium, lithium, and iron had significant negative associations with embryologic outcomes following IVF. Regarding clinical IVF outcomes, higher urinary molybdenum concentrations the day of VOR were associated with significantly lower odds of implantation and live birth, while higher urinary Cu/Mo ratios on the day of VOR were associated with significantly higher odds of implantation, clinical pregnancy, and live birth. Our results suggest that essential trace element levels may directly influence the IVF outcomes of Spanish patients, with selenium and molybdenum exerting negative effects and copper-related ratios exerting positive effects. Additional studies are warranted to confirm these relationships in other human populations.

Comparing the advantages, disadvantages and diagnostic power of different non-invasive pre-implantation genetic testing: A literature review.

To improve embryo transfer success and increase the chances of live birth in assisted reproductive methods, there is a growing demand for the use of pre-implantation genetic testing (PGT). However, the invasive approaches used in PGT have led to in vitrofertilization failure and abortions, increasing anxiety levels for parents. To address this, non-invasive PGT methods have been introduced, such as the detection of DNA in blastocoel fluid of blastocysts and spent culture media (SCM). These methods have proven to be minimally invasive and effective in detecting aneuploidy in the chromosomes of human embryos. This review aims to explore the different approaches to pre-implantation diagnosis, including invasive and non-invasive methods, with a particular focus on non-invasive PGT (niPGT). The search strategy involved gathering data from scientific databases such as PubMed, Google Scholar, and Science Direct using relevant keywords. The search was conducted until January 2023. In total, 22 studies have successfully reported the detection and amplification of cell-free DNA in the embryonic SCM. It is important to note that niPGT has some limitations, which include differences in indicators such as cell-free DNA amplification rate, concordance, level of maternal DNA contamination, sensitivity, and specificity between SCM samples and biopsied cells. Therefore, more extensive and detailed research is needed to fully understand niPGT's potential for clinical applications.

Impact of double trophectoderm biopsy on reproductive outcomes following single euploid blastocyst transfer

ObjectivesTo study the effect of double trophectoderm biopsy on clinical outcomes following single euploid blastocyst transfer. Study designRetrospective cohort study of 2046 single euploid frozen-thawed blastocyst transfers from January 2015 to June 2022 in a single centre.All patients undergoing a frozen-thawed embryo transfer (FTET) cycle with euploid blastocysts, biopsied for any indication, were included. The outcomes were compared for blastocysts which were biopsied and vitrified once (Group 1, n = 1684), biopsied once but vitrified twice (Group 2, n = 312) and biopsied and vitrified twice (Group 3n = 50). We adjusted for confounders and performed subgroup analysis for PGT-A, PGT-M and PGT-SR cycles. The primary outcome was live birth rate. Secondary outcomes included pregnancy, clinical pregnancy, birthweight and sex ratio. ResultsAfter adjusting for confounders (previous failed euploid implantations, embryo quality and day of biopsy), embryos which were biopsied twice had lower OR for clinical pregnancy (0.48, CI 0.26–0.88, p = 0.019) and for live birth (0.50 CI 0.27–0.92, p = 0.025) compared to controls. Embryos which were biopsied once but vitrified twice had no different ORs for all reproductive outcomes compared to controls. No significant difference was observed for neonatal birthweight or sex ratio amongst the three groups. This is a retrospective single centre study with inherent bias and results may not be transferable to all settings. ConclusionThis study is the largest to date assessing the outcomes of FTET cycles following double trophectoderm biopsy. The results are in keeping with the existing literature and can be incorporated into patient counselling. Whilst double biopsy seems to adversely impact LBR, it is only one of the many factors that can affect success rates. The subfertility background and embryo characteristics should not be overlooked. This study provides reassuring evidence since double biopsied embryos still result in live births with no difference in sex ratio or birthweight. However, long term follow up of the off-springs is lacking and should be reported in future studies.

Preimplantation diagnosis and embryo selection in a patient with severe hereditary coproporphyria

Preimplantation diagnosis and embryo selection in a patient with severe hereditary coproporphyria

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.

Ovarian reserve in patients with FMR1 gene premutation and the role of fertility preservation

IntroductionWomen with premutation (PM) of the FMR1 gene may suffer from reduced ovarian reserve or even premature ovarian insufficiency (POI). We studied hormonal and ultrasound ovarian reserve, fertility and fertility preservation outcomes in these patients. Patients and methodRetrospective cohort study of 63 female FMR1 premutation carriers. ResultsSixty-three female patients bearing an FMR1 premutation were included. Median age was 30 years [26.5–35]. Median number of CGG triplets was 83 [77.2–92]. Before diagnosis of PM, 19 women (30%) had had in all 35 pregnancies, resulting in 20 births, including 7 affected children. After diagnosis of PM, 17 women (26.1%) had in all 23 pregnancies, at a median age of 34.5 years [32.2–36.0]: 2 after pre-implantation genetic diagnosis, 3 after oocyte donation, 18 spontaneously, and 5 ending in medical termination for fragile X syndrome. Thirty-three patients (52.4%) had POI diagnosis (median age, 30 years [27–34]) with median FSH level 84 IU/L [50.5–110] and median AMH level 0.08ng/mL [0.01–0.19]. After POI diagnosis, 8 women had in all 9 pregnancies: 3 following oocyte donation, and 6 spontaneous in 5 women (15.1%). Eight of the 9 pregnancies resulted in a live birth (including 2 affected children) and 1 in medical termination for trisomy 13. The median age of the 30 patients without POI was 31 years [25.2–35.0].Thirteen women (20.6%) underwent fertility preservation, at a median age of 29 years [24–33]: FSH 7.7 IU/L [6.8–9.9], AMH 1.1ng/mL [0.95–2.1], antral follicle count 9.5 [7.7–14.7]. A median 15 oocytes [10–26] were cryopreserved in a median 2 cycles [1–3]. At the time of writing, no oocytes had yet been thawed for in-vitro fertilization. ConclusionsThis study shows the importance of early fertility preservation after diagnosis of FMR1 premutation in women, due to early deterioration of ovarian reserve. Genetic counseling is essential in these patients, as spontaneous pregnancies are not uncommon, even in cases of impaired ovarian reserve, and can lead to birth of affected children.

Repeated pregnancy losses with multiple aneuploidies and major genomic imbalance: A case report

Rationale: If one of the partners is having balanced autosomal translocation, it is usually observed that the offspring inherit either normal chromosomes, balanced translocation identical to one of the parent or unbalanced chromosomal rearrangements of the same parental chromosome having translocation. Concern: A unique case presented with history of 8 miscarriages for genetic counseling. The last abortus material evaluation showed monosomy of chromosome X (Turner syndrome) in all the analyzed cells. There was a history of infertility and also repeated second trimester abortions on the paternal side. On the maternal side, there was a history of intellectual disability. Diagnose: History of repeated abnormal pregnancy outcomes. Wife’s karyotype is normal; however, husband shows translocation between chromosome 4 and 22. Intervention: Peripheral blood sample around 3 mL was collected for karyotype. Embryo biopsy was done and DNA was extracted and processed for whole exome sequencing. Outcomes: Wife’s karyotype is normal and husband has translocation between chromosome 4 and 22. Surprisingly, the entire pregnancy outcome including embryo screening has different, complete or partial aneuploidies of chromosomes other than chromosome 4 and 22. Main lesson: Though the translocation in one of the parent is balanced, we have to think beyond traditional ways for evaluating a couple with repeated pregnancy loss as we cannot predict the errors at cell division. Option of in vitro fertilization and preimplantation genetic diagnosis in couples having balanced translocations should be discussed so that early intervention can prevent the agony of pregnancy loss.

Supporting couples with polycystic kidney disease in a medically assisted reproduction process

Preimplantation diagnosis process for couples with polycystic kidney disease involves several steps: genetic and gynecological counseling, in vitro fertilization (IVF/ICSI), embryo biopsy, genetic analysis, selection of healthy embryos and implantation. The importance of preimplantation diagnosis for couples affected by polycystic kidney disease (PKD) lies in several key factors: Prevention of Inherited Disease: PKD is a genetic disorder with a high likelihood of being passed from parent to offspring. Preimplantation diagnosis offers couples the opportunity to identify and avoid passing on the disease to their children. Family Planning Options: By undergoing preimplantation diagnosis, couples can make better decisions about their family planning options. They can select embryos that do not carry the PKD gene mutation and reduce the risk of having a child affected by the disease. Emotional Burden Reduction: PKD can lead to significant emotional burdens for affected individuals and their families. Preimplantation diagnosis provides a proactive approach to avoiding these burdens by preventing the transmission of the disease to next generations. Enhanced Reproductive Choices: With preimplantation diagnosis, couples have greater control over their reproductive choices. They can pursue assisted reproductive techniques such as in vitro fertilization (IVF) while simultaneously minimizing the risk of passing on PKD to their offspring. Improved Pregnancy Outcomes: By selecting embryos that are free of the PKD gene mutation, couples increase the likelihood of having a healthy pregnancy and a child free of the disease. It can lead to improved pregnancy outcomes and the birth of healthy children. Overall, preimplantation diagnosis offers hope and empowerment to couples affected by PKD by providing them with the means to build a family while minimizing the risk of passing the disease to next generations.

The impact of a second embryo biopsy for preimplantation genetic testing for monogenic diseases (PGT-M) with inconclusive results on pregnancy potential: results from a matched case-control study.

To evaluate whether a second biopsy, following a first diagnostic failure on blastocysts tested for preimplantation genetic testing for monogenic diseases (PGT-M), allows to obtain genetic diagnosis and to what extent this procedure can influence clinical pregnancy and live birth rates compared to the PGT-M process with a successful genetic diagnosis from the first biopsy. Embryos from women who underwent PGT-M in an infertility centre and who had been transferred after two biopsies for genetic analysis (n = 27) were matched in a 1:1 ratio accordingly to women's age (± 1year) and fertility status (fertilevsinfertile), as well as with the study period, with embryos who were transferred after receiving a conclusive PGT result straight after the first biopsy (n = 27). The main evaluated outcome was clinical pregnancy rate following embryo transfers in which healthy embryos were transferred after only one biopsy and those in which an embryo was transferred after being re-biopsied. Live birth rate was the secondary outcome. Clinical pregnancy rate was 52% (95% CI: 34-69) following the transfer of a single-biopsy blastocyst and 30% (95% CI: 16-48) following the transfer of a re-biopsied blastocyst. The likelihood to have a healthy baby was 33% (95% CI: 19-52) following the transfer of a blastocyst biopsied once and 22% (95% CI: 11-41) following the transfer of a re-biopsied blastocyst. The re-biopsy intervention seems to considerably reduce the pregnancy potential of a blastocyst. However, a greater sample size is necessary to clarify this issue definitively.

Оцінка впливу гінекологічної патології на шанси живонародження серед жінок із повторними невдачами імплантації

Aim - to study the prognostic impact of the presence of gynecological pathology on live birth in women with multiple negative implantation attempts in the context of the proposed methods of examination and treatment, such as preimplantation genetic diagnosis of embryos and individualization of the endometrial preparation protocol, taking into account the results of the assessment of the implantation window. Materials and methods. The prospective cohort study included 93 women with infertility treated by in vitro fertilization (IVF). Patients had repeated failed implantation attempts and were divided into three groups: the Group 1 - patients who were treated with genetically untested embryos according to a standard fixed stimulation protocol, the Group 2 - patients who were treated with euploid embryos after preimplantation genetic screening according to a standard fixed protocol; the Group 3 - patients treated with euploid embryos after preimplantation genetic screening and determination of the implantation window with subsequent modification of the stimulation protocol, according to the results of the endometrial examination. The implantation window was determined by three-time aspiration biopsy of the endometrium during the luteal phase of the menstrual cycle. The samples were analyzed by scanning electron microscopy. Based on the results, the endometrial preparation protocol was individualized for the next IVF attempt. Preimplantation diagnostics of embryos was performed using the next generation method. Statistical analysis was performed using IBM SPSS V25.0 for Windows software. Results. According to the results obtained, the presence of genital pathology reduced the chance of live birth by 30% in patients with repeated failed implantation attempts. Pathologies such as endometriosis, including endometrioid ovarian cysts, benign breast disease and Asherman syndrome have a negative effect on the chance of live birth among patients who have undergone infertility treatment with IVF. Conclusions. The presence of gynecologic pathology reduces the chances of live birth in patients with repeated failed implantation attempts undergoing IVF treatment. The study was conducted in accordance with the principles of the Helsinki Declaration. The study protocol was approved by the Local Ethics Committee for all participants. Informed consent was obtained from the patients. No conflict of interests was declared by the authors.