Several reports have presented that balanced chromosomal rearrangements (BCRs) carriers with normal phenotypes may be carriers of complex rearrangements. However, the incidence and PGT clinical outcomes of cryptic complex chromosome rearrangements (CCCRs) in individuals with BCRs is remain unknown. We recruited a cohort of 1,264 individuals with BCR carriers from 2016 to 2021 at the Reproductive and Genetic Hospital of CITIC Xiangya. Peripheral blood was collected for karyotyping and genomic DNA extraction and the PGT-SR clinical outcomes of CCCRs carriers were analyzed and compared with those of BCR carriers. Our findings revealed that 3.6% (45/1,264) of BCR carriers had CCCRs, involving 3-25 breakpoints on 1-3 chromosomes. Furthermore, when mate-pair sequencing was employed, 63.3% (19/30) of CCCR carriers were found to have chromosome rearrangements that were different from those identified by the MicroSeq technique. And the transferable embryo rate of CCCR carriers with 3 chromosomes was significantly lower than that of CCCR carriers with only 1-2 chromosomes. In this research, we revealed that some of the BCR carriers were actually CCCR carriers, and the prognosis of PGT in CCCR carriers with one or two chromosomes is better than that of CCCR carriers with three chromosomes.

OBJECTIVE: The preimplantation genetic testing for aneuploidy (PGT-A) platform is not currently available for small copy-number variants (CNVs), especially those < 1 Mb. Through strategies used in PGT for monogenic disease (PGT-M), this study intended to perform PGT for families with small pathogenic CNVs.
METHODS: Couples who carried small pathogenic CNVs and underwent PGT at the Reproductive and Genetic Hospital of CITIC-Xiangya (Hunan, China) between November 2019 and April 2023 were included in this study. Haplotype analysis was performed through two platforms (targeted sequencing and whole-genome arrays) to identify the unaffected embryos, which were subjected to transplantation. Prenatal diagnosis using amniotic fluid was performed during 18-20 weeks of pregnancy.
RESULTS: PGT was successfully performed for 20 small CNVs (15 microdeletions and 5 microduplications) in 20 families. These CNVs distributed on chromosomes 1, 2, 6, 7, 13, 15, 16, and X with sizes ranging from 57 to 2120 kb. Three haplotyping-based PGT-M strategies were applied. A total of 89 embryos were identified in 25 PGT cycles for the 20 families. The diagnostic yield was 98.9% (88/89). Nineteen transfers were performed for 17 women, resulting in a 78.9% (15/19) clinical pregnancy rate after each transplantation. Of the nine women who had healthy babies, eight accepted prenatal diagnosis and the results showed no related pathogenic CNVs.
CONCLUSIONS: Our results show that the extended haplotyping-based PGT-M strategy application for small pathogenic CNVs compensated for the insufficient resolution of PGT-A. These three PGT-M strategies could be applied to couples with small pathogenic CNVs.

This retrospective cohort study aimed to assess the effect of chromosomal reciprocal translocation on meiotic segregation products of non-translocation chromosomes. A total of 744 reciprocal translocation carriers and 875 non-carriers were included in this study. A total of 6,832 blastocysts were biopsied and tested by next-generation sequencing. Blastocysts from the carrier group were classified into five subgroups according to the theoretical segregation pattern of quadrivalent structure. For carrier patients, normal meiotic segregation products of the non-translocation chromosome were classified after excluding the segregation modes of the quadrivalent structure. The proportion of normal non-translocation chromosome meiotic segregation products was similar between the carrier and noncarrier groups (p = 0.69). The generalized Estimation Equation revealed that there was no correlation between reciprocal translocation and meiotic segregation products of non-translocation chromosomes. Moreover, subgroup analyses showed that the segregation modes of quadrivalent structure (p = 0.00) and carrier\'s gender (p = 0.00) may affect the meiotic segregation products of non-translocation chromosomes. In conclusion, reciprocal translocation does not directly reduce the proportion of normal segregation products of non-translocation chromosomes. The difference among subgroups of different quadrivalent segregation patterns implied that interchromosomal effect may exist but the high incidence of chromosomal abnormalities for reciprocal translocation carriers should not be attributed to interchromosomal effect.

Hereditary spherocytosis (HS), the most common inherited hemolytic anemia disorder, is characterized by osmotically fragile microspherocytic red cells with a reduced surface area on the peripheral blood smear. Pathogenic variants in five erythrocyte membrane structure-related genes ANK1 (Spherocytosis, type 1; MIM#182900), SPTB (Spherocytosis, type 2; MIM#616649), SPTA1 (Spherocytosis, type 3; MIM#270970), SLC4A1 (Spherocytosis, type 4; MIM#612653) and EPB42 (Spherocytosis, type 5; MIM#612690) have been confirmed to be related to HS. There have been many studies on the pathogenic variants and mechanisms of HS, however, studies on how to manage the transmission of HS to the next-generation have not been reported. In this study, we recruited a patient with HS. Targeted next-generation sequencing with a panel of 208 genes related to blood system diseases detected a novel heterozygous variant in the SPTB: c.300+2dup in the proband. Sanger sequencing of variant alleles and haplotype linkage analysis of single nucleotide polymorphism (SNP) based on next-generation sequencing were performed simultaneously. Five embryos were identified with one heterozygous and four not carrying the SPTB variant. Single-cell amplification and whole genome sequencing showed that three embryos had varying degrees of trisomy mosaicism. One of two normal embryos was transferred to the proband. Ultimately, a healthy boy was born, confirmed by noninvasive prenatal testing for monogenic conditions (NIPT-M) to be disease-free. This confirmed our successful application of PGT in preventing transmission of the pathogenic variant allele in the HS family.

UNASSIGNED: This study aimed to determine whether controlled ovarian hyperstimulation (COH) parameters influence the incidence of de novo chromosomal abnormalities (> 4 Mb) in blastocysts and, thus, clinical pregnancy outcomes in preimplantation genetic testing (PGT).
UNASSIGNED: Couples who underwent preimplantation genetic testing for structural chromosome rearrangements (PGT-SR) and monogenic disorders (PGT-M) were included in this study. The relationships of maternal age, paternal age, stimulation protocol, exogenous gonadotropin dosage, duration of stimulation, number of oocytes retrieved and estradiol (E2) levels on human chorionic gonadotropin (hCG) trigger day with the incidence of de novo chromosomal abnormalities were assessed. Blastocysts were biopsied, and nuclear DNA was sequenced using next-generation sequencing (NGS). Clinical pregnancy outcomes after single euploid blastocyst transfers under different COH parameters were assessed.
UNASSIGNED: A total of 1,710 and 190 blastocysts were biopsied for PGT-SR and PGT-M, respectively. The rate of de novo chromosomal abnormalities was found to increase with maternal age (p< 0.001) and paternal age (p = 0.019) in the PGT-SR group. No significant differences in the incidence of de novo chromosomal abnormalities were seen for different maternal or paternal age groups between the PGT-SR and PGT-M groups (p > 0.05). Stratification analysis by gonadotropin dosage, stimulation protocol, duration of stimulation, number of retrieved oocytes and E2 levels on hCG trigger day revealed that de novo chromosomal abnormalities and clinical pregnancy outcomes were not correlated with COH parameters after adjusting for various confounding factors.
UNASSIGNED: The rate of de novo chromosomal abnormalities was found to increase with maternal or paternal age. COH parameters were found to not influence the incidence of de novo chromosomal abnormalities or clinical pregnancy outcomes.

This study aimed to analyze the clinical outcomes of blastocyst which undergo the preimplantation genetic testing (PGT) transplantation from frozen-thawed D5 and D6. In addition, the effect of blastocyst grade on clinical and neonatal outcomes was also investigated in this study.
The pregnancy and miscarriage rates of 1130 cycles of frozen embryo transfer, including 784 D5 frozen embryos and 346 D6 frozen embryos in the Reproductive Hospital of Shandong University from January to December 2020 were analyzed. Gardner blastocyst scoring was used for blastocyst evaluation.
The pregnancy rate of D5 blastocyst was significantly higher, whereas the miscarriage rate of D5 blastocyst was lower, than that of D6 blastocyst tissue biopsy. No significant difference was observed in birth weight and low birth weight of D5 blastocyst and D6 blastocyst, preterm birth, gestational age, and neonatal sex. Frozen-thawed D5 blastocysts have higher pregnancy success rates and lower miscarriage rates compared to D6 blastocysts.
Therefore, both blastocyst grade and embryo biopsy date must be considered when transferring frozen embryos.

OBJECTIVE: To compare aneuploidy rates in early aborted tissues or blastocysts between in vitro fertilization (IVF) cycles after the gonadotropin-releasing hormone (GnRH) antagonist (GnRH-ant) protocol or the GnRH agonist (GnRH-a) long protocol.
METHODS: This was a retrospective cohort study from a university-affiliated fertility center. In total, 550 early miscarriage patients who conceived through IVF/intracytoplasmic sperm injection (ICSI) after receiving the GnRH-ant or GnRH-a long protocol were analyzed to compare aneuploidy rates in early aborted tissues. To compare aneuploidy rates in blastocysts, 404 preimplantation genetic testing for aneuploidy (PGT-A) cycles with the GnRH-ant protocol or GnRH-a long protocol were also analyzed.
RESULTS: For early miscarriage patients who conceived through IVF/ICSI, compared to the GnRH-a long protocol group, the GnRH-ant protocol group had a significantly higher rate of aneuploidy in early aborted tissues (48.51% vs. 64.19%). Regarding PGT-A cycles, the rate of blastocyst aneuploidy was significantly higher in the GnRH-ant protocol group than the GnRH-a long protocol group (39.69% vs. 52.27%). After stratification and multiple linear regression, the GnRH-ant regimen remained significantly associated with an increased risk of aneuploidy in early aborted tissues and blastocysts [OR (95% CI) 1.81 (1.21, 2.71), OR (95% CI) 1.65 (1.13, 2.42)]. Furthermore, the blastocyst aneuploidy rate in the GnRH-ant protocol group was significantly higher but only in young and normal ovarian responders [OR (95% CI) 5.07 (1.99, 12.92)].
CONCLUSIONS: Compared to the GnRH-a long protocol, the GnRH-ant protocol is associated with a higher aneuploidy rate in early aborted tissues and blastocysts. These results should be confirmed in a multicenter, randomized controlled trial.

OBJECTIVE: To investigate use of the third-generation sequencing (TGS) Oxford Nanopore system as a new approach for preimplantation genetic testing (PGT).
METHODS: Embryos with known structural variations underwent multiple displacement amplification to create fragments of DNA (average ~ 5 kb) suitable for sequencing on a nanopore.
RESULTS: High-depth sequencing identified the deletion interval for the relatively large HBA1/2--SEA alpha thalassemia deletion. In addition, STRs were able to be identified in the primary sequence data for potential use in conventional PGT-M linkage confirmation. Sequencing of amplified embryo DNA carrying a translocation enabled balanced embryos to be identified and gave the precise identification of translocation breakpoints, offering the opportunity to differentiate carriers from non-carrier embryos. Low-pass sequencing gave reproducible profiles suitable for simple identification of whole-chromosome and segmental aneuploidies.
CONCLUSIONS: TGS on the Oxford Nanopore is a possible alternative and versatile approach to PGT with potential for performing economical workups where the long read sequencing information can be used for assisting in a traditional PGT workup to design an accurate and reliable test. Additionally, application of TGS has the possibility of providing combined PGT-A/SR or in selected stand-alone PGT-M cases involving pathogenic deletions. Both of these applications offer the opportunity for simultaneous aneuploidy detection to select either balanced embryos for transfer or additional carrier identification. The low cost of the instrument offers new laboratories economical entry into onsite PGT.

Studies have shown that early-follicular phase long-acting gonadotropin-releasing hormone (GnRH) agonist long protocol (EFLL), a popular controlled ovarian hyperstimulation protocol widely used in China, leads to higher rates of implantation and clinical pregnancy, as well as lower rates of spontaneous abortion and ectopic pregnancy in patients undergoing in vitro fertilization treatment. However, the impact of EFLL on euploid embryos and its underlying mechanisms remain unclear. To address these gaps of knowledge, we conducted a retrospective comparative study of 310 preimplantation genetic testing (PGT) cycles with a total of 1,541 embryos using the EFLL protocol or midluteal short-acting GnRH agonist long protocol (MLSL). Patients were matched by PGT subtype [aneuploidies (PGT-A) vs. PGT for chromosomal structural rearrangements (PGT-SR)], age (±2 years), and body mass index (±1 kg/m2). For PGT-A, there was no significant difference in the number of euploid embryos (1.80 ± 1.47 for EFLL vs. 1.84 ± 2.03 for MLSL, p > 0.05) or the rate of euploidy (44.6 vs. 36.9%, p > 0.05). For PGT-SR, the number of euploid embryos in the EFLL group was significantly higher than that in the MLSL group (1.76 ± 1.54 vs. 1.21 ± 1.24, p < 0.05). A higher euploidy rate was also observed with the EFLL protocol compared with that obtained in MLSL (31.9 vs. 25.7%), although the difference was not statistically significant (p > 0.05). Compared with the MLSL protocol, more euploid embryos were achieved when using the EFLL protocol in PGT-SR, demonstrating the value in PGT-SR. To the best of our knowledge, this study is the first one to compare embryonic outcomes between EFLL and MLSL, providing key insights into the clinical application of EFLL in PGT cycles. In the light of the limited sample size of our study, we recommend that these questions be explored using a larger prospective study.

The operations involved in preimplantation genetic testing (PGT) occur during the key stages of gametogenesis and early embryonic development, and the health of progeny following PGT (PGT-born) is worthy of attention. In order to fully assess the potential risk of abnormal glucose metabolism in adult PGT-born offspring and to evaluate possible mechanisms, we compared a mouse model of PGT (in vitro cultured embryos with biopsy, hereafter \"PTG-born mice\"), an in vitro embryo manipulation mouse model (in vitro cultured embryos without biopsy), and normal mice. PGT-born mice displayed increased fasting glucose, and decreased glycogen synthesis and glucose oxidative utilization in the liver. Moreover, PGT-born mice also displayed reduced expression of insulin receptor, AKT, and insulin-stimulated Akt phosphorylation (pAkt) in the liver. These results suggest a potential risk of insulin resistance in adult PGT-born mice. By analyzing the DNA methylation profiles of 7.5 days postconception (dpc) embryos, we identified differentially methylated genes associated with liver development between PGT-born and control groups; some of these genes are associated with glucose homeostasis and insulin response. These results suggest that abnormal methylation in embryos that develop after PGT may be a potential mechanism occurring during embryonic development that can influence the risk of liver-derived insulin resistance in adulthood.