Preimplantation Genetic Testing Research Articles

Defining ethical criteria to guide the expanded use of Noninvasive Prenatal Screening (NIPS): Lessons about severity from preimplantation genetic testing.

We hypothesized that ethical criteria that guide the use of preimplantation genetic testing (PGT) could be used to inform policies about expanded use of non-invasive prenatal screening (NIPS). We used a systematic review of reasons approach to assess ethical criteria used to justify using (or not using) PGT for genetic conditions. Out of 1135 identified documents, we retained and analyzed 216 relevant documents. Results show a clear distinction in acceptability of PGT for medical vs. non-medical conditions. Criteria to decide on use of PGT for medical conditions are largely based on their severity, but there is no clear definition of "severity". Instead, characteristics of the condition that relate to severity are used as sub-criteria to assess severity. We found that characteristics that are used as sub-criteria for assessing severity include monogenic etiology, high penetrance, absence of treatment, early age of onset, shortened lifespan, and reduced quality of life. Consensus about the use of PGT is highest for conditions that meet most of these criteria. There is no consensus around the acceptability of using PGT to detect non-medical conditions. We propose that the same severity criteria could be used by policymakers to assess the acceptability of using other genetic tests in screening and practice, including for the use of NIPS for additional conditions as indications broaden.

Outcomes for Female Physicians Compared With Nonphysicians After Assisted Reproductive Technology.

To evaluate outcomes of female physicians after assisted reproductive technology (ART). We conducted a retrospective cohort study using data from 248 physician patients and 3,470 nonphysician patients who underwent a total of 10,095 fresh or frozen ART cycles at a single academic center in an insurance-mandated state between January 2015 and March 2022. The primary outcome was live-birth rate. The secondary outcomes were implantation rate, early pregnancy loss rate, and time to pregnancy. Models were adjusted for confounders where appropriate. Both groups were similar in age (mean physician age 36.29 years; mean nonphysician age 35.96 years, P=.35). Physicians had lower body mass index (BMI) (mean physician BMI 23.51, mean nonphysician BMI 26.37, P<.01), and a higher proportion were diagnosed with unexplained infertility (physician 33.9%, nonphysician 25.9%, P<.01) and used preimplantation genetic testing for aneuploidy (physician 21.5%, nonphysician 12.7%). Physicians and nonphysicians had similar live-birth rates (physician 39.3%, nonphysician 38.2%; adjusted relative risk [aRR] 1.01 95% CI, 0.91-1.13), implantation rates (physician 34.7%, nonphysician 33.7%; relative risk 1.03 95% CI, 0.94-1.14), and early pregnancy loss rates (physician 21.9%, nonphysician 19.8%; aRR 1.18 95% CI, 0.99-1.41) per transfer. Physicians had a shorter time from initial ART cycle to pregnancy (physician 21.82 weeks, nonphysician 25.16 weeks; aRR 0.86, 95% CI, 0.83-0.89). There was no difference between female physicians and nonphysicians in assisted reproduction cycle outcomes. Physicians become pregnant slightly faster than nonphysicians.

Two-year cumulative live-birth rates and maximum number of transfer cycles in women aged ≥ 40 years

This retrospective study aimed to clarify the cumulative live-birth rates (CLBRs) and cost per live-birth (LB) to evaluate the validity of frozen–thawed embryo transfer without preimplantation genetic testing for aneuploidy (PGT-A) in women aged ≥ 40 years. The study included 1,011 patients aged ≥ 40 years who underwent their first oocyte retrieval at our hospital between January 2010 and September 2017. They were followed up for up to two years or until either treatment discontinuation or a pregnancy that resulted in a live birth. The 2-year CLBRs were 55.6%, 39.0%, 31.3%, 19.1%, 10.6%, 4.4%, and 0% for patients aged 40, 41, 42, 43, 44, 45, and > 46 years, respectively. In approximately 80% of LB cases, patients aged 40–42 years and 43–44 years became pregnant by the fourth and second transfers, respectively. Costs per LB were $30,207, $49,034, $66,345, $102,759, and $195,862 for patients aged 40, 41, 42, 43, and 44, respectively. Cost per LB for each number of transfers reached $300,000 and $ 450,000 for the third transfer at 42 and 43 years of age, respectively. For cost-effectiveness, up to two ET cycles are recommended for patients aged 42–43, and none for patients aged ≥ 44 years.

Comparative Analysis of Fluorescence In Situ Hybridization and Next-Generation Sequencing in Sperm Evaluation: Implications for Preimplantation Genetic Testing and Male Infertility

Pre-implantation genetic testing (PGT) is a crucial process for selecting embryos created through assisted reproductive technology (ART). Couples with chromosomal rearrangements, infertility, recurrent miscarriages, advanced maternal age, known single-gene disorders, a family history of genetic conditions, previously affected pregnancies, poor embryo quality, or congenital anomalies may be candidates for PGT. Preimplantation genetic testing for aneuploidies (PGT-A) enables the selection and transfer of euploid embryos, significantly enhancing implantation rates in assisted reproduction. Fluorescence in situ hybridization (FISH) is the preferred method for analyzing biopsied cells to identify these abnormalities. While FISH is a well-established method for identifying sperm aneuploidy, NGS offers a more comprehensive assessment of genetic material, potentially enhancing our understanding of male infertility. Chromosomal abnormalities, arising during meiosis, can lead to aneuploid sperm, which may hinder embryo implantation and increase miscarriage rates. This review provides a comparative analysis of fluorescence in situ hybridization (FISH) and next-generation sequencing (NGS) in sperm evaluations, focusing on their implications for preimplantation genetic testing. This analysis explores the strengths and limitations of FISH and NGS, aiming to elucidate their roles in improving ART outcomes and reducing the risk of genetic disorders in offspring. Ultimately, the findings will inform best practices in sperm evaluations and preimplantation genetic testing strategies.

A method for determining potential parental contamination: linkage disequilibrium-based log-likelihood ratio analysis for IVF-PGT

BackgroundAt present, embryologists are attempting to use conventional in vitro fertilization (cIVF) as an alternative to intracytoplasmic sperm injection (ICSI) for preimplantation genetic testing (PGT). However, the potential parental contamination origin of sperm cells and cumulus cells is considered the main limiting factor in the inability of cIVF embryos to undergo PGT.MethodsIn this study, we established an IVF-PGTA assay for parental contamination tests with a contamination prediction model based on allele frequencies and linkage disequilibrium (LD) to compute the log-likelihood ratio (LLR) under competing ploidy hypotheses, and then verified its sensitivity and accuracy. Finally, comparisons of the effectiveness of SNP-based analysis and LLR-based IVF-PGTA among 40 cIVF embryos was performed, based on both statistical analysis of the parental contamination rate and chromosomal ploidy concordance rate between TE biopsy and ICM isolations.ResultsWith IVF-PGTA assay, biopsies with 10% maternal contamination could be detected accurately, and contamination caused by sperm cells could be eliminated completely. Utilizing LLR-based or single Nucleotide Polymorphism (SNP) -based analyses, our comprehensive examination of 40 clinically discarded fresh cIVF embryos revealed an absence of paternal contamination. Strikingly, the LLR-based analysis uniquely revealed a mere instance of 24% maternal contamination within the trophectoderm cell (TE) biopsy of 5* embryo. Furthermore, it was solely through this analysis that embryo (9-F) was identified as a triploid of paternal origin.ConclusionsIn this study, we developed a new bioinformatics analysis method for identifying parental contamination during IVF-PGT, especially for couples with nonmale factor infertility.

Preimplantation Genetic Testing for Aneuploidy (PGT-A) in In-Vitro Fertilisation (IVF) Treatment: Study Protocol for Pilot Phase of a Randomised Controlled Trial.

Introduction: Poor outcomes following IVF treatments are speculated to be due to the transfer of aneuploid embryos that cannot be identified based on morphological evaluation alone. This leads to patients requiring numerous embryo transfers and, consequently, a prolonged time interval before live birth. Embryo selection following preimplantation genetic testing for aneuploidy (PGT-A) with next-generation sequencing (NGS) has been suggested as an intervention to shorten time to pregnancy in women undergoing in vitro fertilisation (IVF). Past studies assessing the clinical efficacy of PGT-A in improving clinical outcomes have been conflicting and the associated clinical pregnancy rates and live birth rates following the transfer of a mosaic embryos have yet to be determined. None of the existing studies solely included women of advanced reproductive age (ARA). The pilot study and proposed RCT will determine if, compared to morphological evaluation alone, the use of PGT-A through NGS is a more clinically effective, safer, and more cost-effective way to provide IVF treatment in women of advanced reproductive age. Method and Analysis: The proposed pilot study will aim to randomise 100 patients within a single-centre study to evaluate recruitment, randomisation, and adherence to study protocol and allocated trail arms by participating patients. The results of the pilot study will enable us to determine the sample size for a larger study to establish the effectiveness of PGT-A in ARA women. Ethics and Dissemination: The study (Integrated Research Application System Number 236067) received approval from the Health Research Authority and Health and Care Research Wales (HCRW) and the East Midlands-Leicester South Research Ethics Committee (20/EM/0290). The results will be made available to patients, the funders, the Reproductive Medicine societies, and other researchers. Trial registration: ClinicalTrials.gov Identifier: NCT05009745, n.

Multi-omics PGT: re-evaluation of euploid blastocysts for implantation potential based on RNA sequencing.

In addition to chromosomal euploidy, can the transcriptome of blastocysts be used as a novel predictor of embryo implantation potential? This retrospective analysis showed that based on differentially expressed genes (DEGs) between euploid blastocysts which resulted and did not result in a clinical pregnancy, machine learning models could help improve implantation rates by blastocyst optimization. Embryo implantation is a multifaceted process, with implantation loss and pregnancy failure related not only to blastocyst euploidy but also to the intricate dialog between blastocyst and endometrium. Although in vitro studies have revealed the characteristics of trophectoderm (TE) differentiation in implanted blastocysts and the function of TE placentation at the implantation site, the precise molecular mechanisms of embryo implantation and their clinical application remain to be fully elucidated. This study involved 102 patients who underwent 111 cycles for preimplantation genetic testing for aneuploidies (PGT-A) between March 2022 and July 2023. The study included 412 blastocysts biopsied at Day 5 [D5] or Day 6 [D6] for patients who underwent PGT-A. The biopsy lysates were split and subjected to DNA and RNA sequencing (DNA- and RNA-seq). One part was used for PGT-A to detect DNA copy number variations, whereas the other part was assessed simultaneously by RNA-seq to determine the transcriptome characteristics. To validate the reliability and accuracy of RNA-seq obtained from this strategy, we initially analyzed the transcriptome of blastocysts with chromosomal aneuploidies. Subsequently, we compared the transcriptomic features of blastocysts with different rates of formation (D5 vs D6) and investigated the network of interactions between key blastulation genes and the receptive endometrium. Then to evaluate the implantation potential of euploid blastocysts, we identified DEGs between euploid blastocysts that resulted in clinical pregnancy (defined as the presence of a gestational sac detected by ultrasound after 5 weeks) and those that did not. These DEGs were then employed to construct a predictive model for optimizing blastocyst selection. The successful detection rate of PGT-A was remarkably high at 99.8%. The RNA data may infer aneuploidy for both trisomy and monosomy. Between the euploid blastocysts that formed on D5 and D6, 187 DEGs were predominantly involved in cell differentiation for embryonic placenta development, the PPAR signaling pathway, and the Notch signaling pathway. These D5/D6 DEGs also exhibited a functional dialog with the receptive phase endometrium-specific genes through protein-protein interaction networks, indicating that the embryo undergoes further differentiation for post-implantation development. Furthermore, a modeling strategy using 280 DEGs between blastocysts leading to successful clinical pregnancies or failing to produce clinical pregnancies was implemented to refine the euploid embryo optimization, achieving areas under the curves of 0.88, 0.71, and 0.84 for the random forest (RF), support vector machine, and linear discriminant analysis models, respectively. Finally, a retrospective analysis of 83 transferred euploid blastocysts using the RF model identified three types of euploid embryos with a decreasing trend in implantation potential. Notably, the implantation rate of the good group was significantly higher than that of the moderate group (88.6% vs 50.0% P = 0.001) and that of the moderate group was higher than that of the poor group (50.0% vs 20.8%, P = 0.035). The sample size was insufficient; thus, a prospective study is needed to verify the clinical effectiveness of the above model. Because we did not analyze blastocysts that led only to biochemical pregnancies but failed clinical pregnancies separately, our classification system still must be modified to screen these embryos. Transcriptomic analysis of blastocysts offers a novel approach for predicting embryo implantation potential, which can be utilized to optimize clinical embryo selection. The ranking system may be effective in reducing the times and costs involved in achieving a clinical pregnancy. This study was funded by the "Pioneer" and "Leading Goose" R&D Program of Zhejiang (No. 2023C03034), the National Natural Science Foundation of China (82101709), and the National Key Research and Development Program for Young Scientists of China (No. 2022YFC2702300). The authors state no competing interests. N/A.

The kinetics of nucleolar precursor bodies clustering at the pronuclei interface: Positive correlations with the morphokinetic characteristics of cleaving embryos and euploidy in preimplantation genetic testing programs

Objective: This study investigated potential relationships between the kinetics of nucleolar precursor bodies (NPBs) in the pronucleus and developmental morphokinetics and euploidy in human preimplantation genetic testing for aneuploidy (PGT-A) cycles.Methods: The morphokinetic analysis of 200 blastocysts obtained from 53 PGT-A cycles was performed retrospectively in a time-lapse incubator. At the time of pronuclear breakdown (PNBD), we categorized the blastocysts into two groups based on the kinetic degree of clustering NPBs at the interface of the two pronuclei: clustered NPBs (CL) and non-clustered NPBs (NCL). We then compared morphokinetic parameters, abnormal behavioral events, and the rate of aneuploidy between the two groups.Results: Pronuclear fading and the first cleavage occurred earlier in the NCL group than in the CL group. However, the initiation of blastocyst formation and blastocyst expansion was delayed in the NCL group relative to the CL group. No differences were found in the rate of abnormal cleavage events, such as multinucleation at the 2-cell stage, direct cleavage from one to three cells, and from two to five cells between the CL and NCL groups. However, the fragmentation rate at the 8-cell stage was higher in the NCL group than in the CL group (10.3% vs. 1.9%, p&lt;0.05). Additionally, the euploid rate in the CL group was significantly higher than in the NCL group (37.9% vs. 12.4%, p&lt;0.05).Conclusion: These results demonstrate the effectiveness of combining NPB clustering at PNBD with morphokinetics as a parameter for selecting embryos with higher developmental potential in in vitro fertilization.

Comparison of live birth rates following the transfer of day-6 blastocysts on the 6th versus 7th day of progesterone exposure in hormone replacement treatment–frozen embryo transfer cycles

Objective: The duration of progesterone exposure is critical for establishing and maintaining a pregnancy in hormone replacement treatment (HRT)–frozen embryo transfer (FET) cycles. This study compared the live birth rates (LBRs) of patients undergoing day-6 warmed blastocyst transfer on either the 6th or 7th day of progesterone administration in HRT-FET cycles.Methods: A retrospective cohort study was conducted using data from the central registry of Indira IVF Hospital Private Limited. In total, 2,633 patients who underwent day-6 blastocyst transfer cycles with HRT-FET were identified. The cycles were categorized based on the timing of the day-6 blastocysts transfer: P+6 vs. P+7 (initiation date: P+1). Propensity scores were calculated. The primary outcome measure was the LBR.Results: Following propensity score matching, a total of 1,401 patients were included in the final analysis (P+6: n=1,212; P+7: n=189). The number of previous attempts was significantly higher in the P+6 group, whereas the rate of preimplantation genetic testing for aneuploidy was significantly higher in the P+7 group. The LBRs were 59.2% and 54.5% in the P+6 and P+7 groups, respectively (p=0.21). Multivariate regression analysis revealed that the number of previous in vitro fertilization attempts (odds ratio [OR], 3.246; 95% confidence interval [CI], 2.429 to 4.337; p&lt;0.001), the number of blastocysts transferred (OR, 2.011; 95% CI, 1.461 to 2.768; p&lt;0.01), and endometrial thickness on the day of progesterone initiation (OR, 1.143; 95% CI, 1.022 to 1.28; p=0.02) were independent predictors of live birth.Conclusions: The LBRs were comparable in patients who underwent day-6 warmed blastocyst transfer on day 6 or 7 post-progesterone initiation in HRT-FET cycles.

Healthy live births achieved from embryos diagnosed as non-mosaic segmental aneuploid.

To investigate pregnancy outcomes resulting from transfer of embryos with non-mosaic (NM) segmental aneuploid (SA) results following preimplantation genetic testing for aneuploidy (PGT-A). All patients who underwent frozen embryo transfer (FET) of at least one embryo with a NM-SA between March 2021 and April 2024 were retrospectively reviewed. Primary outcomes included live birth rate (LBR) and results of prenatal diagnosis. Embryos with NM-SA results were also compared to those with NM whole chromosome aneuploid (WCA) and mosaic SA results. Out of 25 NM-SA embryos transferred, the LBR was 24%. Prenatal diagnosis by amniocentesis and/or chorionic villus sampling was performed in 3/6 pregnancies, and results were normal. Embryos with duplications produced more live births compared to those with deletions. NM-SA embryos had a significantly higher ongoing pregnancy (OP)/LBR compared to embryos with NM-WCA results and a significantly lower OP/LBR compared to embryos with mosaic SA results; however, when compared to embryos with high-level SA mosaicism > 40%, the OP/LBR was not significantly different. Embryos with NM-SAs can result in euploid live births, albeit at reduced rates compared to those with mosaic SAs. These data can be used to aid in patient counseling about PGT-A results and embryo transfer decisions.

Effects of Carrier’s sex on the outcome of embryos and pregnancies in 412 couples undergoing preimplantation genetic testing for structural rearrangements

Study designTo ascertain whether the carrier’s sex affects the outcome of embryos and pregnancies in couples undergoing preimplantation genetic testing for structural rearrangements (PGT-SR). MethodsThis retrospective study comprised 412 couples with reciprocal translocations (RecT), Robertsonian translocations (RobT), or inversions (INV) between January 2017 and October 2022. We applied next-generation sequencing (NGS) on 2588 embryos after trophectoderm (TE) biopsy. ResultsGenetically transferable blastocyst rate was higher in the male carrier group (34.0 % vs 31.7 %, P = 0.013) relative to the female carrier group whereas other embryo and pregnancy outcomes remained similar. Further analysis revealed that this result was primarily due to the alteration of segregation patterns in the RobT subgroup, in which the proportion of alternate segregation was higher (84.3 % vs 66.4 %, P < 0.001) in male carriers compared with female carriers. In the RecT subgroup, the genetically transferable blastocyst rate between male and female carriers was similar although the segregation models also changed, such that the frequency of the adjacent-1 segregation pattern was higher in male carriers than in female carriers (42.5 % vs 34.7 %, P = 0.002). In addition, interchromosomal effect (ICE) did not differ between male and female carriers although ICE was lower in male carriers of the RobT subgroup (pure ICE: 35.50 % vs 44.30 %, P = 0.14; total ICE: 35.50 % vs 40.30 %, P = 0.32) and higher in male carriers of the INV subgroup (pure ICE: 42.3 % vs 37.20 %, P = 0.33; total ICE: 40.90 % vs 36.00 %, P = 0.36). ConclusionsThe carrier’s sex was closely associated with the genetically transferable embryo rate in couples undergoing PGT-SR, principally resulted from the change in segregation pattern in the RobT subgroup but not in the RecT and INV subgroups.

Incidence of haploidy and triploidy in trophectoderm biopsies of blastocysts derived from normally and abnormally fertilized oocytes.

We aimed to identify the correlation between morphological pronuclear (PN) status and the genetically determined ploidy configuration in preimplantation embryos. A retrospective observational study was conducted on 1982 embryos displaying normal fertilization and 380 embryos showing an atypical PN pattern, tested for aneuploidies and ploidy status via preimplantation genetic testing (PGT) between May 2019 and May 2024. Ploidy prediction was performed using a validated targeted-NGS approach and a proprietary bioinformatic pipeline analyzing SNPs B-allele frequency information. Ploidy results were obtained in relation to the morphological PN pattern and further stratified by mode of PN observation, maternal age, and embryo quality parameters. Abnormal ploidy results in 2PN-derived embryos were 1% (n = 20/1982): 0.8% showed triploidy and 0.2% haploidy. Ploidy results in relation to PN number in atypical fertilization were as follows: 0PN (n = 150/380) associated with 87.3% of diploidy, 8.7% of haploidy, and 4.0% of triploidy; 1PN-derived blastocysts (n = 73/153) were haploid in 47.7% of cases, 6.5% were triploid, and 45.7% diploid; 2.1PN (n = 23/280) and 3PN patterns (n = 54/280) predicted a triploid result in 34.8% and 74.1% of cases, respectively. PN observation with time-lapse increased ploidy status predictivity from 28.3% to 80.4% (p < 0.01) and reduced expected diploid rates to 19.6% (p < 0.01). Diploidy rate was higher for maternal age ≤ 35years and for morphologically high-grade embryos. Morphological PN check can be improved by incorporating ploidy analysis within the conventional PGT workflow. Euploid 2PN-derived embryos can be further selected removing haploids and triploids, and some atypical PN pattern can be better classified.

Impacts of double biopsy and double vitrification on the clinical outcomes following euploid blastocyst transfer: a systematic review and meta-analysis.

Compared to the 'single biopsy + single vitrification' approach, do 'double biopsy + double vitrification' or 'single biopsy + double vitrification' arrangements compromise subsequent clinical outcomes following euploidy blastocyst transfer? Both 'double biopsy + double vitrification' and 'single biopsy + double vitrification' led to reduced live birth/ongoing pregnancy rates and clinical pregnancy rates. It is not uncommon to receive inconclusive results following blastocyst biopsy and preimplantation genetic testing for aneuploidy (PGT-A). Often these blastocysts are warmed for re-test after a second biopsy, experiencing 'double biopsy + double vitrification'. Furthermore, to achieve better workflow, IVF laboratories may choose to routinely vitrify all blastocysts and schedule biopsy at a preferred timing, involving 'single biopsy + double vitrification'. However, in the current literature, there is a lack of systematic evaluation of both arrangements regarding their potential clinical risks in reference to the most common 'single biopsy + single vitrification' approach. A systematic review and meta-analysis were performed, with the protocol registered in PROSPERO (CRD42023469143). A search in PUBMED, EMBASE, and the Cochrane Library for relevant studies was carried out on 30 August 2023, using the keywords 'biopsy' and 'vitrification' and associated variations respectively. Only studies involving frozen transfers of PGT-A tested euploid blastocysts were included, with those involving PGT-M or PGT-SR excluded. Study groups included blastocysts having undergone 'double biopsy + double vitrification' or 'single biopsy + double vitrification', with a 'single biopsy + single vitrification' group used as control. The primary outcome was clinical pregnancy, while secondary outcomes included live birth/ongoing pregnancy, miscarriage, and post-warming survival rates. Random effects meta-analysis was performed with risk ratios (RR) and 95% CIs were used to present outcome comparisons. A total of 607 records were identified through the initial search and nine studies (six full articles and three abstracts) were eventually included. Compared to 'single biopsy + single vitrification', 'double biopsy + double vitrification' was associated with reduced clinical pregnancy rates (six studies, n = 18754; RR = 0.80, 95% CI = 0.71-0.89; I2 = 0%) and live birth/ongoing pregnancy rates (seven studies, n = 20964; RR = 0.72, 95% CI = 0.63-0.82; I2 = 0%). However, no significant changes were seen in miscarriage rates (seven studies, n = 22332; RR = 1.40, 95% CI = 0.92-2.11; I2 = 53%) and post-warming survival rates (three studies, n = 13562; RR = 1.00, 95% CI = 0.99-1.01; I2 = 0%) following 'double biopsy + double vitrification'. Furthermore, 'single biopsy + double vitrification' was also linked with decreased clinical pregnancy rates (six studies, n = 13284; RR = 0.84, 95% CI = 0.76-0.92; I2 = 39%) and live birth/ongoing pregnancy rates (seven studies, n = 16800; RR = 0.79, 95% CI = 0.69-0.91; I2 = 70%), and increased miscarriage rates (five studies, n = 15781; RR = 1.48, 95% CI = 1.31-1.67; I2 = 0%), but post-warming survival rates were not affected (three studies, n = 12452; RR = 0.99, 95% CI = 0.97-1.01; I2 = 71%) by 'single biopsy + double vitrification'. All studies included in this meta-analysis were retrospective with varying levels of heterogeneity for different outcomes. Not all studies had accounted for potential confounding factors. Only one study reported neonatal outcomes. Our data indicated adverse impacts of 'double biopsy + double vitrification' and 'single biopsy + double vitrification' on clinical outcomes following euploid blastocyst transfers. Patients should be carefully consulted about the risks when offered such approaches. The biopsy process should be carried out as carefully and competently as possible to minimize an inconclusive diagnosis. R.W. is supported by a National Health and Medical Research Council Emerging Leadership Investigator Grant (2009767). There is no other external funding to report. All authors report no conflict of interest. CRD42023469143.

Current research status and clinical applications of noninvasive preimplantation genetic testing: A review.

Noninvasive preimplantation genetic testing (ni-PGT) is conducted by obtaining genetic information from embryos through the analysis of free DNA released by embryos in spent embryo culture medium or blastocoel fluid. Compared to conventional preimplantation genetic testing relying on trophectoderm biopsy, ni-PGT is characterized by its noninvasiveness. It has demonstrated early advancements in the detection of embryonic chromosomal aneuploidies and the diagnosis of monogenic diseases, showcasing considerable potential for clinical application. However, there are substantial controversies in the literature concerning the reliability of ni-PGT, the source of cell-free DNA, and maternal contamination. This paper elaborates on the principles, research advancements, effectiveness, and limitations of ni-PGT to provide a basis for clinical applications.

Effects of ovarian stimulation on embryo euploidy: an analysis of 12874 oocytes and 3106 blastocysts in cycles with preimplantation genetic testing for monogenic disorders.

Does ovarian stimulation and the ovarian response affect embryo euploidy? Ovarian stimulation and the ovarian response in women undergoing preimplantation genetic testing for monogenic disorders (PGT-M) cycles did not affect the rates of blastocyst euploidy. Whether or not ovarian stimulation in IVF-embryo transfer has potential effects on embryo euploidy is controversial among studies for several reasons: (i) heterogeneity of the study populations, (ii) biopsies being performed at different stages of embryo development and (iii) evolution of the platforms utilized for ploidy assessment. Patients who undergo PGT-M cycles typically have no additional risks of aneuploidy, providing an ideal study population for exploring this issue. A retrospective cohort study including embryos undergoing PGT-M was conducted at a single academically affiliated fertility clinic between June 2014 and July 2021. A total of 617 women with 867 PGT-M cycles involving 12874 retrieved oocytes and 3106 trophectoderm biopsies of blastocysts were included. The primary outcome of the study was median euploidy rate, which was calculated by dividing the number of euploid blastocysts by the total number of biopsied blastocysts for each cycle. Secondary outcomes included the median normal fertilization rate (two-pronuclear (2PN) embryos/metaphase II oocytes) and median blastulation rate (blastocyst numbers/2PN embryos). Comparable euploidy rates and fertilization rates were observed across all age groups, regardless of variations in ovarian stimulation protocols, gonadotropin dosages (both the starting and total dosages), stimulation durations, the inclusion of human menopausal gonadotrophin supplementation, or the number of oocytes retrieved (all P > 0.05). Blastulation rates declined with increasing starting doses of gonadotropins in women aged 31-34 years old (P = 0.005) but increased with increasing gonadotrophin starting doses in women aged 35-37 years old (P = 0.017). In women aged 31-34, 35-37, and 38-40 years old, blastulation rates were significantly reduced with increases in the number of oocytes retrieved (P = 0.001, <0.001, and 0.012, respectively). Limitations include the study's retrospective nature and the relatively small number of patients of advanced age, especially patients older than 40 years old, leading to quite low statistical power. Second, as we considered euploidy rates as outcome measures, we did not analyze the effects of ovarian stimulation on uniform aneuploidy and mosaicism, respectively. Finally, we did not consider the effects of paternal characteristics on embryo euploidy status due to the fact that blastocyst aneuploidy primarily originates from maternal meiosis. However, sperm factors might have an effect on embryo development and the blastulation rate, and therefore also the number of blastocysts analyzed. The exclusion of patients with severe teratozoospermia and the fact that only ICSI was used as the insemination technique for women undergoing PGT-M contributed to minimize the effect of paternal factors. Ovarian stimulation and response to stimulation did not affect blastocyst euploidy rates in women undergoing PGT-M cycles. However, in women aged 31-40 years old, there was a significant decline in blastulation rates as the number of retrieved oocytes increased. This study was supported by the National Natural Science Foundation of China (Grant No. 81701407, 82301826); the National Key Research and Development Program of China (2022YFC2702901, 2022YFC2703004); China Postdoctoral Science Foundation (2022M710261), and China Postdoctoral Innovation Talent Support Program (BX20220020). There is no conflict of interest. N/A.

Cytoplasmic strings in human blastocysts: hypotheses of their role and implications for embryo selection.

What are the implications of the presence cytoplasmic strings (Cyt-S) and their quantity and dynamics for the pre-implantation development of human blastocysts? Cyt-S are common in human embryos and are associated with faster blastocyst development, larger expansion, and better morphological quality. Cyt-S are dynamic cellular projections connecting inner cell mass and trophectoderm (TE) cells, that can be observed during blastocyst expansion. Their prevalence in human embryos has been estimated to be between 44% and 93%. Data relevant to their clinical implications and role in development are lacking, limited, or controversial. Retrospective study conducted at a single IVF center between May 2013 and November 2014 and involving 124 pre-implantation genetic testing for aneuploidy cycles in a time-lapse incubator with ≥1 blastocyst biopsied and vitrified (N = 370 embryos assessed). These cycles resulted in 87 vitrified-warmed single-euploid blastocyst transfers. ICSI, continuous blastocyst culture (Days 5-7), TE biopsy of fully expanded blastocysts without Day 3 zona pellucida drilling, qPCR to assess uniform full-chromosome aneuploidies, and vitrification were all performed. Only vitrified-warmed euploid single-embryo-transfers were conducted. Blastocyst morphological quality was defined according to Gardner's criteria. The AI-based software CHLOE™ (Fairtility) automatically registered timings from time of starting blastulation (tSB) to biopsy (t-biopsy, i.e. blastocyst full-expansion) as hours-post-insemination (hpi), embryo area (including zona pellucida in µm2), and spontaneous blastocyst collapses. One senior embryologist manually annotated Cyt-S presence, quantity, timings, and type (thick cell-to-cell connections and/or threads). All significant associations were confirmed through regression analyses. All couples', cycles', and embryos' main features were also tested for associations with Cyt-S presence, quantity, and dynamics. About 94.3% of the patients (N = 117/124) had ≥1 embryo with Cyt-S. Out of a total of 370 blastocysts, 55 degenerated between blastulation and full-expansion (N = 55/370, 14.9%). The degeneration rate among embryos with ≥1 Cyt-S was 10.8% (N = 33/304), significantly lower than that of embryos without Cyt-S (33.3%, N = 22/66, P < 0.01). Of the remaining 315 viable blastocysts analyzed, 86% (N = 271/315; P < 0.01) had ≥1 Cyt-S, on average 3.5 ± 2.1 per embryo ranging 1-13. The first Cyt-S per viable embryo appeared at 115.3 ± 12.5 hpi (85.7-157.7), corresponding to 10.5 ± 5.8 h (0.5-31) after tSB. Overall, we analyzed 937 Cyt-S showing a mean duration of 3.8 ± 2.7 h (0.3-20.9). Cyt-S were mostly threads (N = 508/937, 54.2%) or thick cell-to-cell connections becoming threads (N = 382/937, 40.8%) than thick bridges (N = 47/937, 5.0%). The presence and quantity of Cyt-S were significantly associated with developmentally faster (on average 6-12 h faster) and more expanded (on average 2700µm2-larger blastocyst's area at t-biopsy) embryos. Also, the presence and duration of Cyt-S were associated with better morphology. Lastly, while euploidy rates were comparable between blastocysts with and without Cyt-S, all euploid blastocysts transferred from the latter group failed to implant (N = 10). Cyt-S presence and dynamics were assessed manually on seven focal planes from video frames recorded every 15 min. The patients included were mostly of advanced maternal age. Only associations could be reported, but no causations/consequences. Lastly, larger datasets are required to better assess Cyt-S associations with clinical outcomes. Cyt-S are common during human blastocyst expansion, suggesting their physiological implication in this process. Their presence, quantity and dynamics mirror embryo viability, and morphological quality, yet their role is still unknown. Future basic science studies are encouraged to finally describe Cyt-S molecular nature and biophysical properties, and Artificial Intelligence tools should aid these studies by incorporating Cyt-S assessment. None. N/A.

Feasibility of preimplantation genetic testing for aneuploidy on frozen-thawed embryos following conventional IVF insemination.

Intracytoplasmic sperm injection (ICSI) is commonly employed in preimplantation genetic testing (PGT) to minimize the risk of foreign sperm DNA contamination. Cryopreserved embryos from patients with recurrent miscarriage or repeated implantation failure, who have undergone conventional in vitro fertilization (IVF), can be thawed and biopsied for PGT. Therefore, we aimed to assess the accuracy and effectiveness of preimplantation genetic testing for aneuploidy (PGT-A) on frozen embryos using conventional IVF (c-IVF) insemination methods. From January 2021 to November 2023, our center conducted 107 thawed cryopreserved embryo biopsy cycles to screen for PGT-A. Among them, 58 cycles used c-IVF insemination, and 49 used ICSI insemination. Basic patient clinical information, laboratory data, PGT test results, and clinical outcome data were collected. To minimize the confounding effects of patient characteristics and embryo quality on PGT-A outcomes, clinical outcomes, and contamination assessment, these variables were included in the analysis. We then evaluated the blastocyst euploidy rate, clinical outcomes, and accuracy of PGT-A results between the two groups and analyzed potential contamination in the c-IVF insemination group. A total of 320 blastocysts underwent PGT-A testing, with 179 blastocysts from c-IVF insemination and 141 from ICSI insemination. Considering participants' baseline characteristics and embryological outcomes, no significant differences were found between the two groups regarding infertility type, average age, body mass index, percentage of PGT-A indications, or quality of embryonic development. Regarding PGT-A results, all 320 biopsy samples were successfully analyzed, showing no statistical variance in chromosomal euploidy, abnormality, or mosaicism rates between the two insemination methods. No parental contamination was detected in the c-IVF insemination group. When assessing clinical outcomes, parameters such as biochemical pregnancy, clinical pregnancy, and miscarriage rates did not exhibit significant discrepancies between the two groups, and no misdiagnoses were reported during the study period. Embryo transfer and PGT-A results are not affected by potential parental contamination in frozen-thawed embryos conceived via c-IVF. PGT-A guided embryo transfer in thawed embryos conceived by c-IVF is a viable and clinically effective approach.

TEMPORARY REMOVAL: Is it time for New Guidelines for Recurrent Pregnancy Loss integrating Genetic Testing of Products of Conception and Preimplantation Genetic Testing?

TEMPORARY REMOVAL: Is it time for New Guidelines for Recurrent Pregnancy Loss integrating Genetic Testing of Products of Conception and Preimplantation Genetic Testing?

Agreement with “The ethical guidelines on the provision and practice of advanced reproductive technology and intrauterine insemination 2023” by the Philippine Society of Reproductive Medicine using online Delphi technique

The ethical guidelines of the National Society of Certified Fertility Specialists, the Philippine Society for Reproductive Medicine, were reviewed 5 years after its publication to determine if there was a change in the views of the current members. The 2016 guideline document was converted into 75 Delphi statements, and the level of agreement with each statement was determined after 3 rounds. One hundred twenty-six members of the national society were invited to participate. Voting was conducted using an online Google form or using a self-administered printed questionnaire. The level of agreement with each statement was measured using a 5-point Likert scale. A panel discussion of speakers in theology, law, and medical ethics, as well as stakeholders represented by an infertility patient and from the LGBTQ community, was held before the last Delphi round. The response rate for the first Delphi round was 94%, and the second and third rounds were at 98%. A consensus was achieved in 66 of the 75 Delphi statements. Statements on preimplantation genetic testing, posthumous reproduction, cryopreservation, and third-party reproduction failed to achieve consensus.

The influence of trophectoderm biopsy for preimplantation genetic testing in the serum level of first trimester biomarkers: PAPP-a, βhCG and PIGF

Research questionDoes trophectoderm biopsy for preimplantation genetic testing for aneuploidies (PGT-A) affect maternal serum first trimester pregnancy biomarkers (PAPP- A, free β-hCG, PlGF)? DesignRetrospective cohort study, including all singleton pregnancies (n=9794) following either spontaneous conceived (SC) (n=8005) in vitro fertilization and fresh embryo transfers (IVF) (n=478) or frozen embryo transfer of non PGT-A (FET)(n=963) or PGT-A tested embryos (FET+PGT-A) (n=348). In all women with a viable pregnancy at 8-13.6 weeks of pregnancy, we measured serum levels of free β-hCG and PAPP-A . PlGF was measured in 3784 patients. These biomarkers were converted to a multiple of the expected normal median (MoM) for a pregnancy of the same gestational day. We calculated the medians for the multiple of the median and compared them. ResultsFree β-hCG did not differ according to the mode of conception. PAPP-A concentrations were significantly lower in in vitro fertilization and fresh embryo transfers (-0,1 Log10 MoM raw PAPP-A) when comparing to FET+PGT-A (-0,04 Log 10 MoM raw PAPP-A) and SC (-0,0187 Log 10 MoM raw PAPP-A) (p<0,05). PlGF levels were significantly lower in the FET+PGTA group (p<0.05). The difference in means adjusted by CRL was 4.6 pg/ml 95%CI [2.7-6.6] for SC, 3.5 pg/ml 95%CI [0.34-6.6] for IVF and 2.2 pg/ml 95%CI [0.06-4.4] for FET. ConclusionThis study demonstrates that trophectoderm biopsy for PGT-A has a significant impact on first trimester maternal serum PAPP-A and PIGF. This needs to be further validated since it may mislead the estimation of the first trimester risk of aneuploidies and preeclampsia.