Preimplantation Genetic Diagnosis Research Articles

P-180 Trophectoderm biopsy technique and its effect on life birth rates: a prospective study

Abstract Study question Do life birth rates of euploid embryos vary based on factors such as laser intensity, number of laser shots, and the biopsy technique employed? Summary answer This study found no correlation between biopsy technique parameters and life birth rates of euploid embryos, underscoring the robustness of current trophectoderm biopsy methods. What is known already Embryo biopsy for Preimplantation Genetic Testing (PGT) must achieve two primary objectives: ensuring that the biopsied material accurately reflects the genetic makeup of the embryo and maintaining the viability of the embryo without compromise. The strategy that most closely aligns with accomplishing these dual goals is trophectoderm (TE) biopsy. However, it’s important to note that there is currently no standardized TE biopsy procedure, leading to the coexistence several techniques. Given the potential impact of TE biopsy on trophectoderm quality and integrity, and consequently on implantation, there is a critical need for studies to assess the effects of different biopsy techniques. Study design, size, duration This prospective observational study spanned from 2019 to 2021 and involved 298 Preimplantation Genetic Testing for Aneuploidy (PGT-A) treatments, incorporating a total of 1187 trophectoderm (TE) biopsies. Embriologyst were trained in both Pulling and Flicking techniques and choose between the two techniques according to their criteria. Pulling involved laser pulses weakening junctions, facilitating TE detachment with moderate tension. Flicking substituted tension with a cutting motion between holding and biopsy pipettes. Participants/materials, setting, methods Biopsy technique (pulling/flicking), number of laser shots (1-10), laser intensity (200µs-600µs), and the embriologyst responsible for the biopsy were annotated for every biopsied embryo. PGT indication, stimulation protocol, patient age (39.32±4.22), embryo quality, and sperm diagnosis were annotated and used as possible confounding factors in a logistic regression model to understand the impact of each independent variable on the log-odds of the outcome. Main results and the role of chance The 1187 trophectoderm biopsies resulted in a total of 410 (34.5%) euploid embryos. Among these, 237 euploid embryos underwent individual transfers, with 153 subjected to Pulling technique and 84 to Flicking technique leading to a total of 120 healthy babies (50.6% life birth rate). Logistic regression, considering potential confounding factors failed to identify any variable significantly influencing the likelihood of life births (p < 0.05). Both Pulling and Flicking techniques demonstrated comparable success rates (52.9% vs 46.4%), with no discernible impact from variations in laser shots (1-3=65.0%; 4-6=45.6%; >6=66.7%) or intensity (200µs=75.0%; 300µs=45.0%; 400µs=50.6%; 500µs=50.94%; 600µs=50.0%). Furthermore, when evaluating the embryologist conducting the biopsy, no significant differences were identified among operators (p < 0.05). This pattern persists for miscarriage rates, indicating no influence from biopsy technique, laser shots, or laser intensity (p < 0.05). Thus, our findings underscore that variations in trophectoderm biopsy variables, when performed by an experienced embryologist, do not adversely impact trophectoderm quality and do not compromise success rates. Limitations, reasons for caution This is a single-center, observational study, conducted from 2019 to 2021, and this may limit generalizability. The sample size variation between biopsy techniques and potential unmeasured confounders pose challenges. The assumption of technique homogeneity, and reliance on binary outcomes underscore caution in interpreting findings. Wider implications of the findings The lack of correlation between biopsy parameters and life birth rates in our study underscores the effectiveness and reliability of current biopsy techniques. This discovery offers reassurance regarding the proficiency of trophectoderm biopsy methods for achieving success in IVF treatments. Trial registration number Not Applicable

P-530 Precise quantification of DNA in spent culture media sheds light on associations between DNA quantity, embryonic ploidy status and morphological quality

Abstract Study question Is it possible to accurately measure cell-free DNA (cfDNA) quantity in embryo spent culture media, and are amounts correlated with embryo morphological and/or chromosomal status? Summary answer A sensitive DNA quantification method was created, demonstrating that cfDNA quantity is not associated with embryo ploidy, but increases when embryos are of poor morphology. What is known already Preimplantation genetic testing involves embryo biopsy, necessitating specialist equipment and highly trained staff, increasing costs. Additionally, there have been concerns over potential embryo damage to the embryo resulting from biopsy. The discovery of cfDNA in medium in which embryos were cultured, led to suggestions that non-invasive PGT (niPGT) might be possible. However, the amount of cfDNA in media samples, and potential correlations with other aspects of embryo biology, require clarification. We aimed to provide the first accurate measurement of the amount of DNA in spent culture medium, providing insights into whether niPGT is likely to provide acceptable accuracy rates. Study design, size, duration This prospective analysis involved media samples from 151 fresh embryos, which were produced at a single IVF clinic over the course of one year. Embryos were cultured individually, with a medium change on day-3 of development. Trophectoderm biopsy was undertaken on day-5/-6, followed by PGT-A. Embryo morphology was assessed according to ASEBIR criteria (ranked A-C). Participants/materials, setting, methods The culture and biopsy protocols used were standard for the clinic. Media was collected prior to biopsy and underwent a novel quantitative whole genome amplification method, allowing precise measurement of DNA with respect to a standard curve generated using samples containing known amounts of DNA. PGT-A involved the use of a highly validated method, based upon next generation sequencing. Correlation between DNA quantity and embryo morphological quality was also studied. Data were analyzed using ANOVA. Main results and the role of chance There was no difference in the quantity of DNA in media samples associated with euploid and aneuploid embryos (mean DNA quantity and standard deviations were 1.72 ± 3.23 pg and 1.54 ± 3.66 pg for aneuploid and euploid embryos, respectively; p = 0.75). Overall, 91% of media samples contained less than the equivalent of one cell (<5pg of DNA) and almost two-thirds of samples (65%) contained less than 1pg of DNA. Given such low quantities of embryonic DNA, achieving accurate genetic testing is likely to be challenging. Drops that held embryos until day-6 had significantly higher levels of DNA than those associated with culture to day-5 (P < 0.0001), suggesting cfDNA accumulates with time. Thus, longer culture periods are likely to yield more DNA for analysis. Comparing the amount of cfDNA with morphological status revealed a significant association. Embryos of fair quality (C) tended to have higher quantities of DNA in spent culture medium, whereas top-quality embryos (classified as A or B) typically had less (mean and standard deviation of DNA quantity: 1.23 ± 3.14 pg for class A and B versus 2.60 ± 3.85 pg for class C; p < 0.05). This may reflect increased cell death and DNA release in poorer quality embryos. Limitations, reasons for caution This project is limited by its single-centre nature and relatively small sample size. A large-scale, multicentre study would be necessary to confirm these results. Wider implications of the findings It has been hypothesised that cfDNA quantity in culture medium may be predictive of embryo chromosomal status. We found no evidence to support this. Although DNA quantities were higher after culture to day-6, levels remained low, calling into question the ability of niPGT to deliver high accuracy diagnosis of embryos. Trial registration number not applicable

P-249 Morphokinetic parameters comparison between balanced and unbalanced embryos from patients with structural chromosome abnormalities

Abstract Study question Are there morphokinetic differences between balanced and unbalanced embryos after PGT-SR? Summary answer The total time embryos remain at the morula stage (TiCAV- TM) could be a morphokinetic marker to consider in PGT-SR cycles. What is known already Due to the preimplantation genetic testing for structural rearrangements (PGT-SR) it is possible to identify embryos with an excess or defect of genetic material associated with a parental structural rearrangement. This technique is suitable for patients with a normal phenotype but a balanced chromosomal rearrangement. These patients have a higher risk of spontaneous abortion, as well as higher aneuploidy rates. Within structural chromosomal alterations, translocations are the most common presentation of chromosomal rearrangement. Study design, size, duration Within this multicentre retrospective study, we evaluated 133 embryos, from 39 cycles in which one of the partners had a karyotype with balanced reciprocal or Robertsonian translocations, from January 2021 to September 2023. After PGT-SR was carried out, we obtained 40 balanced embryos and 93 unbalanced embryos. Participants/materials, setting, methods We compared the conventional morphokinetic parameters and the duration of each state between two groups after PGT-SR samples were analysed. All embryos were cultured in Geri® time-lapse incubators, and underwent embryo biopsy on day 5-7. Statistical analysis was conducted using R software (v.4.3.1), including the following confounding variables: origin of karyotype alteration (maternal or paternal), type of translocation (Robertsonian or reciprocal), female and male gamete age, type of oocyte (fresh or frozen) and oocyte origin. Main results and the role of chance We observed that the mean age of the oocyte was significantly higher in the unbalanced embryos compared to the balanced ones (32.85±5.32 years vs.30.45±5.01 years). In terms of the altered karyotype, the paternal origin is more prevalent than the maternal one (60.9% vs. 39.1%). Regarding the type of translocation, reciprocal translocation is more predominant than Robertsonian translocation (83.5% vs. 16.5%) in our study population. When comparing morphokinetic parameters between both groups, we observed that unbalanced embryos tend to reach an expanded blastocyst stage later than balanced embryos (107.62±10.02h vs. 102.48±8.87h, respectively; p = 0.008, corrected p-value=0.186). We observed the same tendency with hatching initiation time (110.64±10.49h vs. 105.91±9.38h; p = 0.013, corrected p-value=0.296). We observed that unbalanced embryos have a slower development than balanced embryos (TiCAV-TFB 10.10±4.46h vs. 8.35±3.79h; p = 0.006, corrected p-value=0.437; TiH-TiCAV 13.03±5.98h vs. 11.79±5.50h; p = 0.017, corrected p-value=0.816). We did observe significant differences in total time at morula stage (TiCAV – TM) when comparing unbalanced and balanced embryos (12.60±6.94 vs. 8.88±5.65; p = 0.003, corrected p-value=0.001). Limitations, reasons for caution A higher number of embryos should be evaluated in order to enhance the statistical power of this study, as there is an important difference in sample size between balanced and unbalanced embryos in the PGT-SR cycles included in this study. Wider implications of the findings Further morphokinetic study of embryos from patients with structural chromosomal abnormalities can open up new lines of research and can help us to predict the final outcome of the cycle, accompanied by a genetic study. Trial registration number Not applicable

P-074 The effect of sperm from male patients who use electronic cigarettes on the euploidy ratio of embryos during in vitro fertilization procedures

Abstract Study question What is the effect of electronic cigarettes as a substitute for regular cigarettes on sperm and subsequent embryo development in IVF patients? Summary answer E-cigarettes are just as harmful to male fertility as regular cigarettes, reduce the euploidy rate of the embryo, and have negative effects on pregnancy. What is known already Many harmful substances from smoking are known to negatively affect sperm development and function. It causes high elevations in sperm DNA fragmentation (SDF), which are associated with embryo euploidy rates. Recently, the use of e-cigarettes is increasing as an alternative to regular cigarettes because they are less harmful and safer. However, several animal studies have shown that e-cigarettes are just as harmful as regular cigarettes and can have negative effects on sperm and fertility. Preliminary data showed that SDF increased with e-cigarettes compared to regular cigarettes. Therefore, we would like to find out whether e-cigarettes affect embryo euploidy. Study design, size, duration In this study, from January 2022 to December 2023, 1,725 cycles of PGT-A (PGT for aneuploidy) were analyzed among the patients of IVF. The average ages of men and women were 40.0±4.6 and 38.7±4.0. A total of 4,796 embryos were analyzed (2.8 ± 2.0 per case) for euploidy rates. Participants/materials, setting, methods Blastocyst biopsy and PGT-A were performed, and embryo euploidy rates was confirmed. In male, the number of no smoking IVF cycles was 1061 and the number of embryo biopsies was 2.9 ± 2.1. The number of IVF cycles using regular cigarettes is 370, and the number of embryo biopsies is 2.8 ± 2.1. The number of IVF cycles using e-cigarettes was 294, and the number of embryo biopsies was 2.5 ± 1.6. Main results and the role of chance Based on the total female age, the embryo euploidy rate was nonsmoking (27.3%), regular cigarettes (23.6%), and e-cigarettes (18.8%). Among women under 38 years of age, e-cigarettes (29.9%) had a lower euploidy rate than regular cigarettes (37.4%), and nonsmoking (38.2%) had the highest rate. Among women over 38, the euploidy rate dropped significantly compared to those under 38, including nonsmoking (18.9%), but it was also lower for e-cigarettes (11.6%) than regular cigarettes (17.7%). Based on female age under 38 years old, the embryo euploidy rate among male age under 39 years old was nonsmoking (39.0%), regular cigarettes (38.3%), and e-cigarettes (31.7%). And among men over 40, the group with the lowest embryo euploidy rate was those who used e-cigarettes (no smoking (35.0%), regular cigarettes (32.6%), e-cigarettes (27.7%)). Based on the age of women over 38 years old, the euploidy rate among men under 39 years of age is nonsmoking (23.9%), regular cigarettes (21.6%), and e-cigarettes (12.7%). And the euploidy rate among men over 40 years of age is nonsmoking (16.8%), regular cigarettes (16.5%), and e-cigarettes (11.8%). nonsmoking is essential to maintain a high euploidy rate in embryos, and e-cigarettes may have a more adverse effect on pregnancy than regular cigarettes. Limitations, reasons for caution Because there are various types of e-cigarettes, it is expected that the degree of harmfulness will vary, but the number of patients was not sufficient to conduct research on each type of e-cigarette. Therefore, it is believed that the above problems should be solved through additional research. Wider implications of the findings This study is a study that links the harmful effects of cigarettes and the polyploidy of embryos. Electronic cigarettes, used as an alternative to regular cigarettes, may reduce embryo euploid production and cause recurrent pregnancy failure. Therefore, it is believed that electronic cigarettes should also be managed appropriately. Trial registration number not applicable

O-316 Clinical application of PGT-A for recurrent pregnancy loss, what effects live birth after single euploid blastocyst transfer?

Abstract Study question Which factors effect the likelihood of live birth after euploid frozen embryo transfer (FET) in recurrent pregnancy loss (RPL)? Summary answer Female age and number of previous pregnancy loss do not effect the live birth rates after euploid blastocyst FET. What is known already PGT-A is helpful to discard the aneuploid embryos and has been proven to improve pregnancy outcomes and reduce miscarriage rate per embryo transfer. As approximately 50% of clinical miscarriages have chromosomal abnormalities, PGT-A has been suggested as a treatment option for RPL. However it is questionable on the cost effectiveness, effect on prognosis and labor-intensive. In addition, currently PGT-A use is not recommended by any society guidelines in RPL patients. ESHRE guideline on RPL stated ‘All couples with results of an abnormal fetal or parental karyotype may be informed about the possible treatment options available including their advantages and disadvantages.’ Study design, size, duration This retrospective cohort study from January 2018 to January 2023 was conducted at Bahçeci Health Group included patients underwent PGT-A/ICSI cycles. Patients with RPL (2 or more clinical pregnancy loss) had a complete RPL workup recommended by ASRM and reported no etiology for RPL was included in the study. Patients with uterine synechia, hydrosalpinx, endocrinological disorders and thin endometrium (endometrial thickness <6mm) were excluded Participants/materials, setting, methods A total of 366 single euploid blastocyst FETs employing hormone replacement therapy for endometrial preparation were evaluated. Patients were categorized into 2 group regarding female age (female age< 35 years and female age≥35 years. Demographic, embryological and endocrinological parameters were analyzed. Live birth rate (LBR) was the primary outcome. Clinical pregnancy rate (CPR) and miscarriage rate (MR) were the secondary outcome. Main results and the role of chance The mean female age of the study population was 33.9±4.6 years, the number of previous pregnancy loss was 3.2±1.6 and BMI was 23.8 ±7.9 kg/m2. Overall CPR, MR and LBR were 61.7% (226/366), 24.8% (56/226) and 46.5% (170/366) respectively. Comparison of clinical characteristics of the 195 women <35 years and 171 women ≥35 years revealed no significant deference in BMI, number of previous live birth, and number of previous pregnancy loss. CPR, MR and LBR were similar among two groups [ 63.6% (124/195) vs 59.6% (102/171), p = 0.44; 27.4% (34/124) vs 21.6% (22/102), p = 0.31; 46.2% (90/195) vs 46.8% (80/171), p = 0.9; respectively]. Binary logistic regression evaluating the independent factors that effect live birth revealed that female age and number of previous pregnancy loss were not found to be statistically significant factors. The only independent factor for live birth was day of embryo biopsy, transferring the day 6 biopsied embriyo significantly decreased the live birth [p = 0.003, OR: 0.47, 95% CI (0.29-0.77)]. Limitations, reasons for caution The maim limitation of the study is its retrospective nature. Wider implications of the findings PGT-A has been suggested to be a treatment for RPL. However there is insufficient data to call PGT-A as an efficient treatment for RPL. Investigating the factors that effect live birth after PGT-A may be the first step for selecting the most favorable group Trial registration number Not applicable

P-511 Euploidy potential of day 7 embryos and the relevance in Assisted Human Reproduction treatments

Abstract Study question Is it worth waiting embryos with late blastulation (Day 7) for preimplantation genetic analysis? Summary answer Embryo biopsy on Day 7 (D7) benefits cases of ovarian insufficiency and severe male factor with a euploidy rate of 15.1%. What is known already New technologies and improvements in IVF laboratories has allowed the extended cultivation of blastocysts to increase the pool of embryos per IVF cycle, resulting in greater chances, especially in cases of low blastulation rate, blastocysts with low quality or with few cells at Day 5(D5) and Day 6(D6). The variation in the time of the compaction stage depends on many factors, which may take more time, leading some embryos to reach the blastocyst stage only on D7 of development. These embryos have a lower euploidy rate, but there is embryonic competence and these embryos can be eligible for embryo transfer. Study design, size, duration Retrospective study that evaluated the chromosomal status (by Next Generation Screening - NGS) of 33 blastocysts biopsied on day 7 of development, between 2016 and 2023. Subsequently, it was compared the rate of euploidy and mosaicism with 295 blastocysts biopsied at D5 and D6, biopsied from January to October 2023. D7 embryos were originated from IVF cycles of couples with female age between 26 and 44 years, and males age between 29 and 56 years. Participants/materials, setting, methods D7 blastocysts from fresh and own oocytes fertilized by ICSI. The euploidy and mosaicism rates was compared, as well as the morphology of the ICM and trophectoderm (A-C; by Gardner Blastocyst Grade), the day which biopsy was performed and the respective infertility factors, in the groups: Group 1: D7 euploid and Group 2: D7 aneuploid. Subsequently, the rate of euploidy and mosaicism was compared between D7 embryos (Group 3) and D5/D6 embryos (Group 4). Main results and the role of chance There was no difference (p > 0.05) between maternal age for group D7 euploid (38.4 years) vs group D7 aneuploid (37.9 years) and group 3 (38.4 years) vs group 4 (38.2 years). Embryonic morphological evaluation of D7 embryos showed that ICM was classified as B (18.2%) or C (81.8%) and trophectoderm as A (6.1%), B (24.2%) or C (69.7%). Comparisons of ICM and trophectoderm classifications between euploid (group 1) and aneuploid (group 2) in D7 blastocysts showed no difference (p > 0.05). There was a predominance of 5CC embryos in both groups (Group 1: 40.0% vs Group 2: 42.4%), with no difference between them (p > 0.05). In most cases, the biopsy was performed on two days (D5+D7 – 39.3%; D6+D7 – 35.7%), while in 10.7% of cases only on Day 7, when the infertility factor was 100% ovarian insufficiency. In cases of biopsy within 3 days (D5+D6+D7), we observed that 100% of cases were related to severe male factor. When comparing the euploidy rate of D7 embryos – group 3 (15.1%) with that of D5/D6 – group 4 (34.2%), we observed a statistical difference (p < 0.05), while, for in mosaicism rates, D7 embryos represented 6.1% and D5/D6 embryos 9.1%, with no statistical difference (p > 0.05). Limitations, reasons for caution The retrospective nature of our study may lead to bias derived from clinical practices and variability in relation to patient age, embryonic quality and infertility factor. Wider implications of the findings Extended culture and biopsy on D7 benefits cases of ovarian insufficiency and severe male factor by optimizing the IVF cycle. Although the euploidy rate in D7 embryos is lower than D5/D6 embryos, 15% of D7 embryos are euploid, demonstrating that the slower development is not caused exclusively by aneuploidy Trial registration number NA

P-557 The contribution of PGT-A to PGT-M and PGT-SR clinical outcomes – A single centre retrospective analysis

Abstract Study question To determine if the introduction of concurrent testing with PGT-A has improved clinical outcomes for PGT-M/SR patients. Summary answer This study demonstrates that the introduction of concurrent testing may have improved clinical outcomes for this patient demographic irrespective of age. What is known already Preimplantation Genetic Testing (PGT) is utilised to investigate the genetic constitution of embryos. PGT-M (monogenic) and PGT-SR (structural rearrangements) are screening techniques considered in treatment for couples at risk of conceiving offspring with known genetic or structural chromosomal abnormalities respectively. Preimplantation Genetic testing for Aneuploidy (PGT-A) was developed in order to screen embryos for numerical chromosomal abnormalities, a leading cause of failed implantation. Nevertheless, routine use of PGT-A is discouraged due to lack of evidence regarding its efficacy. However, PGT-A is routinely used alongside PGT-M and PGT-SR despite the lack of evidence of its effectiveness in improving patient outcomes. Study design, size, duration The retrospective analysis of 496 PGT-M/SR single embryo transfer patient cycles between December 2013 and December 2021. Participants/materials, setting, methods The retrospective analysis of 496 PGT-M/SR single embryo transfer cycles at CRGH Portland was undertaken (single testing n = 77; concurrent testing n = 419). The clinical outcomes (positive urine pregnancy test, clinical pregnancy, live birth and miscarriage rates) were compared between groups using IBM SPSS Statistics (premium 27). Binary logistic regression was performed for statistical analysis, results with p < 0.05 were determined statistically significant. Main results and the role of chance Overall, concurrent testing was associated with statistically significant improved clinical outcomes compared to the reference group. Statistical significance was seen in positive urine pregnancy test (49% vs 68%, p = 0.012), clinical pregnancy (32.5% vs 63%; p = 0.000013), live birth rate (32.5% vs 59%; p = <0.00016) and miscarriage rates (34.2% vs 13%; p = 0.001). The results imply that the introduction of concurrent testing has improved the probability of obtaining a positive clinical outcome and has reduced the chance of miscarriage in this patient demographic. Further analysis showed concurrent testing improved the chances of obtaining a positive clinical outcome irrespective of the patients age or quality of the embryo. Limitations, reasons for caution This study has produced promising results. However, there are several limitations such as the small sample size, embryo biopsy techniques used, and the different molecular diagnostic techniques and providers utilised. Wider implications of the findings The results imply a justification for the use of PGT-A alongside PGT-M/SR screening techniques to improve embryo selection and clinical outcomes for this cohort of patients. Additionally, they demonstrate the efficacy of PGT-A in a previously underrepresented demographic. However, further research is required to confirm the clinical value of PGT-A. Trial registration number not applicable

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

À propos du diagnostic préimplantatoire des aneuploïdies (DPI-A). Les inconvénients de mots inappropriés et d’une réglementation inadaptée

Finding biological markers to assess the ability of embryos to develop is a major objective of research being carried out to improve the results of in vitro fertilization (IVF). Among these markers, the chromosomal content of embryonic cells has been the subject of numerous studies to identify euploid embryos which will be transferred as a priority into the uterus. In France, a clinical research program funded by the Ministry of Health was undertaken to evaluate the benefit of this strategy when IVF is carried out in women aged 35 to 41. This program was interrupted because its authorization was canceled by a court on the grounds that the search for aneuploidy in the embryo is a preimplantation genetic diagnosis (PGD) and that this type of analysis is not provided for in the governing law. The law says in fact that a PGD can only be carried out if “has been previously and precisely identified, in one of the parents or one of their immediate ascendants in the case of a seriously disabling illness, with late revelation and prematurely endangering the vital prognosis”. But the chromosomal analysis provided for in the research protocol is not a diagnosis, it is a test which identifies the embryos most capable of developing. Like all other techniques with the same goal, this test is not the responsibility of the law but of the good practice guide. Studies seeking to improve the results of IVF for the benefit of couples who use it should not be prevented.

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.

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