β-Thalassemia is a global health issue, caused by mutations in the HBB gene. Among these mutations, HBB -28 （A〉G） mutations is one of the three most common mutations in China and Southeast Asia ...patients with β-thalassemia. Correcting this mutation in human embryos may prevent the disease being passed onto future generations and cure anemia. Here we report the first study using base editor （BE） system to correct disease mutant in human embryos. Firstly, we produced a 293T cell line with an exogenous HBB -28 （A〉G） mutant fragment for gRNAs and targeting efficiency evaluation. Then we collected primary skin fibroblast cells from a β-thalassemia patient with HBB -28 （A〉G） homozygous mutation. Data showed that base editor could precisely correct HBB -28 （A〉G） mutation in the patient＇s primary cells. To model homozygous mutation disease embryos, we consb＇ucted nuclear transfer embryos by fusing the lymphocyte or skin fibroblast cells with enucleated in vitro matured （IVM） oocytes.Notably, the gene correction efficiency was over 23.0% in these embryos by base editor. Although these embryos were still mosaic, the percentage of repaired blastomeres was over 20.0%. In addition, we found that base editor variants, with narrowed deamination window, could promote G-to-A conversion at HBB -28 site precisely in human embryos. Collectively, this study demonstrated the feasibility of curing genetic disease in human somatic cells and embryos by base editor system.
Genome editing tools such as the clustered regularly interspaced short palindromic repeat （CRISPR）-associated system （Cas） have been widely used to modify genes in model systems including animal ...zygotes and human cells, and hold tremendous promise for both basic research and clinical applications. To date, a serious knowledge gap remains in our understanding of DNA repair mechanisms in human early embryos, and in the efficiency and potential off-target effects of using technologies such as CRISPR/Cas9 in human pre-implantation embryos. In this report, we used tripronuclear （3PN） zygotes to further investigate CRISPR/Cas9-mediated gene editing in human cells. We found that CRISPR/Cas9 could effectively cleave the endogenous β-globin gene （HBB）. However, the efficiency of homologous recombination directed repair （HDR） of HBB was low and the edited embryos were mosaic. Off-target cleavage was also apparent in these 3PN zygotes as revealed by the T7E1 assay and whole-exome sequencing. Furthermore, the endogenous delta-globin gene （HBD）, which is homologous to HBB, competed with exogenous donor oligos to act as the repair template, leading to untoward mutations. Our data also indicated that repair of the HBB locus in these embryos occurred preferentially through the non-crossover HDR pathway. Taken together, our work highlights the pressing need to further improve the fidelity and specificity of the CRISPR/Cas9 platform, a prerequisite for any clinical applications of CRSIPR/Cas9-mediated editing.
Malignant tumors continue to remain a main global public health issue. In the past 40 years, due to strides made in multi-disciplinary comprehensive treatment schemes for patients suffering from ...malignant tumors, especially chemotherapy schemes, the survival rate has been greatly improved in such patients. This group can be expected to maintain their fertility or have restored endocrine function following successful malignant tumor treatment. Therefore, focusing on the ovarian damage caused by chemotherapy in women of childbearing age is vital in order to protect their fertility and improve their quality of life.
This study attempted to evaluate whether VX-765 possesses an ovarian protective effect in ovarian injury induced by chemotherapy in the mice model.
Female C57BL/6J mice were administered with VX-765 gavage once a day for 21 consecutive days. Use of cyclophosphamide (Cy) began one week after the last gavage administration of VX- 765. Detailed classification of follicles at various levels was then quantified in each group. Immunohistochemistry and Western blot analysis were then used in order to analyze the expression of key proteins (FOXO3a, mTOR, RPS6 and AKT) as well as their phosphorylation of the PI3K / PTEN / AKT pathways in the ovary. The concentrations of AMH were measured by ELISA.
The follicles at all levels of Cy treated mice were less than those of the normal group (P < 0.05). Meanwhile, mice treated with VX-765 prior to receiving Cy treatment had more primordial follicles (PMF) than mice treated with Cy alone (P < 0.05). In early growing follicles (EGF) and antral follicles (AF), no difference was observed among the experimental groups (P > 0.05), however, they were lower than those in the normal group (P < 0.05). In mice treated with continuous Cy, the total follicle number (TF) of mice combined with VX-765 (C-Cy-Vx765) was higher than that of mice without VX-765, and the TF of the two groups was lower than that of the normal group (P < 0.05). The value of PMF/TF in C-Cy-Vx765 group was significantly higher than that in the other three groups, while that of EGF/TF was significantly lower (P < 0.05). Immunohistochemical results showed that the phosphorylated forms of the main proteins of the PI3K / PTEN / AKT pathway were found to be more positive in Cy treated mice. The Western blot analysis showed that when Cy and VX-765 were cotreated, the increased levels of these phosphorylated proteins decreased compared with those treated with Cy alone. The AMH level of infancy Cy and VX-765 co-treated mice was higher than that of infancy normal mice (P < 0.05). After the mice grew to sexual maturity, the AMH level of Cy and VX- 765 co-treated mice was still higher than that of Cy treated mice (P < 0.05), and there was no significant difference with normal mice (P > 0.05).
VX-765 can maintain the level of AMH and inhibit the recruitment of PMF, thus protecting mice from Cy induced gonadotropic toxicity. Accordingly, VX-765 may play a protective role in mice with ovarian injury caused by chemotherapy.
Polycystic ovary syndrome (PCOS) is a prevalent endocrine disorder associated with infertility and pregnancy complications. The pathogenesis of PCOS and its impact on reproductive function may be ...influenced by the source of androgens, including testosterone, free androgen, dehydroepiandrosterone sulfate (DHEAS). However, the differential effects of these androgen on pregnancy and neonatal outcomes and the cut-off value of East Asian population with PCOS remain unclear.
A retrospective cohort study was conducted at the Reproductive Medicine Center of the First Affiliated Hospital of Sun Yat-sen University from January 2015 to November 2022, involving 636 cycles of in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI). Subgroup analyses were performed using cut-off values of 6.4 for free androgen index (FAI), 9.5 µmol/L for DHEAS. Pregnancy and neonatal outcomes were compared between groups. Restricted cubic spline (RCS) was used to identify significant cut-off values affecting pregnancy.
Higher FAI levels (> 6.4) were associated with decrease in clinical pregnancy rate (PR) (50.61% vs. 41.66%, p = 0.024), live birth rate (LBR) (42.42% vs. 32.35%, p = 0.011). When DHEAS levels exceeded 9.5 µmol/L, there was a significant decrease in clinical PR (51.27% vs. 42.73%, P = 0.039), LBR (42.73% vs. 32.73%, P = 0.012). Negative correlations were also observed between DHEAS levels and cumulative pregnancy rate (70.57% vs 56.62% p = 0.002) and cumulative live birth rate (CLBR) (59.35% vs 43.37%, p = 0.0007). Both FAI and DHEAS elevated is associated with the lowest clinical pregnancy rate (37.84%). Conversely, when solely FAI is elevated, the pregnancy rate increases to 52.38%, while an elevation in DHEAS alone is associated with a pregnancy rate of, both of which are lower than when neither FAI nor DHEAS are elevated (60.68%). The live birth rates exhibit a similar trend (30.00% vs 40.00% vs 41.83% vs 44.48%). RCS revealed a significant decrease in CPR and CLBR when DHEA levels exceeded 7.69 umol/L, while the cut-off value of FAI was 6.36 for CPR and CLBR.
In conclusion, PCOS patients with biochemical hyperandrogenism show unsatisfactory clinical PR and CLBR when undergoing assisted reproductive technology (ART). This may be attributed to the influence of both adrenal-derived DHEAS and ovarian-derived FAI on the unfavorable pregnancy outcomes.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
To evaluate whether the incidence of hypertensive disorders of pregnancy (HDP) in pregnant women was related to endometriosis (EM), ovulation and embryo vitrification technology.
A retrospective ...cohort study was conducted on the clinical data of 3674 women who were treated with IVF / ICSI in the Reproductive Medicine Center of the First Affiliated Hospital of Sun Yat-sen University and maintained clinical pregnancy for more than 20 weeks. All pregnancies were followed up until the end of pregnancy. The follow-up consisted of recording the course of pregnancy, pregnancy complications, and basic situation of newborns.
Compared with NC-FET without EM, HRT-FET without EM was found to have a higher incidence of HDP during pregnancy (2.7% V.S. 6.1%, P<0.001); however, no significant difference was found in the incidence of HDP between NC-FET and HRT-FET combined with EM (4.0% V.S. 5.7%, P>0.05). In total frozen-thawed embryo transfer (total-FET), the incidence of HDP in the HRT cycle without ovulation (HRT-FET) was observed to be higher than that in the NC cycle with ovulation (NC-FET) (2.8% V.S. 6.1%, P<0.001). In patients with EM, no significant difference was found in the incidence of HDP between fresh ET and NC-FET (1.2% V.S. 4.0%, P>0.05).
EM does not seem to have an effect on the occurrence of HDP in assisted reproductive technology. During the FET cycle, the formation of the corpus luteum may play a protective role in the occurrence and development of HDP. Potential damage to the embryo caused by cryopreservation seems to have no effect on the occurrence of HDP.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The goal of the assisted reproductive treatment is to transfer one euploid blastocyst and to help infertile women giving birth one healthy neonate. Some algorithms have been used to assess the ploidy ...status of embryos derived from couples with normal chromosome, who subjected to preimplantation genetic testing for aneuploidy (PGT-A) treatment. However, it is currently unknown whether artificial intelligence model can be used to assess the euploidy status of blastocyst derived from populations with chromosomal rearrangement.
From February 2020 to May 2021, we collected the whole raw time-lapse videos at multiple focal planes from in vitro cultured embryos, the clinical information of couples, and the comprehensive chromosome screening results of those blastocysts that had received PGT treatment. Initially, we developed a novel deep learning model called the Attentive Multi-Focus Selection Network (AMSNet) to analyze time-lapse videos in real time and predict blastocyst formation. Building upon AMSNet, we integrated additional clinically predictive variables and created a second deep learning model, the Attentive Multi-Focus Video and Clinical Information Fusion Network (AMCFNet), to assess the euploidy status of embryos. The efficacy of the AMCFNet was further tested in embryos with parental chromosomal rearrangements. The receiver operating characteristic curve (ROC) was used to evaluate the superiority of the model.
A total of 4112 embryos with complete time-lapse videos were enrolled for the blastocyst formation prediction task, and 1422 qualified blastocysts received PGT-A ( n = 589) or PGT for chromosomal structural rearrangement (PGT-SR, n = 833) were enrolled for the euploidy assessment task in this study. The AMSNet model using seven focal raw time-lapse videos has the best real-time accuracy. The real-time accuracy for AMSNet to predict blastocyst formation reached above 70% on the day 2 of embryo culture, and then increased to 80% on the day 4 of embryo culture. Combing with 4 clinical features of couples, the AUC of AMCFNet with 7 focal points increased to 0.729 in blastocysts derived from couples with chromosomal rearrangement.
Integrating seven focal raw time-lapse images of embryos and parental clinical information, AMCFNet model have the capability of assessing euploidy status in blastocysts derived from couples with chromosomal rearrangement.
F-box proteins play essential roles in various cellular processes of spermatogenesis by means of ubiquitylation and subsequent target protein degradation. They are the substrate-recognition subunits ...of SKP1–cullin 1–F-box protein (SCF) E3 ligase complexes. Dysregulation of F‑box protein‑mediated proteolysis could lead to male infertility in humans and mice. The emerging studies revealed the physiological function, pathological evidence, and biochemical substrates of F-box proteins in the development of male germ cells, which urging us to review the current understanding of how F‑box proteins contribute to spermatogenesis. More functional and mechanistic study will be helpful to define the roles of F-box protein in spermatogenesis, which will pave the way for the logical design of F-box protein-targeted diagnosis and therapies for male infertility, as the spermatogenic role of many F-box proteins remains elusive.
Abstract
m
5
C is one of the longest-known RNA modifications, however, its developmental dynamics, functions, and evolution in mRNAs remain largely unknown. Here, we generate quantitative mRNA m
5
C ...maps at different stages of development in 6 vertebrate and invertebrate species and find convergent and unexpected massive methylation of maternal mRNAs mediated by NSUN2 and NSUN6. Using
Drosophila
as a model, we reveal that embryos lacking maternal mRNA m
5
C undergo cell cycle delays and fail to timely initiate maternal-to-zygotic transition, implying the functional importance of maternal mRNA m
5
C. From invertebrates to the lineage leading to humans, two waves of m
5
C regulatory innovations are observed: higher animals gain cis-directed NSUN2-mediated m
5
C sites at the 5' end of the mRNAs, accompanied by the emergence of more structured 5'UTR regions; humans gain thousands of trans-directed NSUN6-mediated m
5
C sites enriched in genes regulating the mitotic cell cycle. Collectively, our studies highlight the existence and regulatory innovations of a mechanism of early embryonic development and provide key resources for elucidating the role of mRNA m
5
C in biology and disease.
To investigate whether aneuploidy screening in preimplantation genetic testing (PGT) for monogenic diseases improves the ongoing pregnancy/live birth rate of single frozen/thawed embryo transfer ...(FET) cycles in young women.
Retrospective cohort study.
Single university-based fertility center.
From January 2016 to December 2017, 569 FET cycles were selected for analysis. The aneuploidy screening (AS) group included 131 FET cycles from 105 oocyte retrieval cycles in 98 patients who underwent PGT for monogenic diseases with aneuploidy screening, and the non-AS group included 438 FET cycles from 280 oocyte retrieval cycles in 266 patients who underwent PGT for monogenic diseases without aneuploidy screening.
The patient population was all under the age of 35 years and underwent PGT for monogenic diseases with and without AS.
Ongoing pregnancy/live birth rate, live birth rate, implantation rate, and miscarriage rate.
Aneuploidy screening significantly improved the ongoing pregnancy/live birth rate (61.22% vs. 43.98%), implantation rate (64.29% vs. 50.38%), and live birth rate (53.06% vs. 36.09%) of young women carrying monogenic diseases in the first FET cycles. When adjusted for the parity, number of previous miscarriages, and percentage of infertility, the likelihood of implantation was 1.874 times higher (95% confidence interval 1.126–3.119), and an ongoing pregnancy/live birth was 2.139 times more likely (95% confidence interval 1.295–3.534). In addition, the miscarriage rate was significantly decreased (3.17% vs. 11.94%). In the cumulative pregnancy outcomes, the cumulative ongoing pregnancy/live birth rate both per transfer and per patient were significantly higher in the AS group (62.24% vs. 50.38% and 79.59% vs. 68.80%), but no difference existed after adjusting for the parity, number of previous miscarriage, and percentage of infertility. Nevertheless, aneuploidy screening reduced the time interval from the first ET to the achievement a pregnancy.
Aneuploidy screening in PGT significantly improved the ongoing pregnancy/live birth rate of young women carrying monogenic diseases in the first FET cycles.
Papel del cribado de aneuploidías en test genético preimplantacional para enfermedades monogénicas en mujeres jóvenes
Investigar si el cribado de aneuploidías en el test genético preimplantacional (PGT) para enfermedades monogénicas mejora la tasa de embarazo evolutivo/nacido vivo de ciclos de transferencia de embrión único congelado (FET) en mujeres jóvenes.
Estudio retrospectivo de cohortes.
Centro único de fertilidad universitario.
Desde Enero de 2016 a Diciembre de 2017, 569 ciclos de FET fueron seleccionados para el análisis. El grupo de cribado de aneuploidías (AS) incluyó 131 ciclos de FET de 105 ciclos de recuperación ovocitaria en 98 pacientes que realizaron PGT para enfermedades monogénicas con cribado de aneuploidías, y el grupo de no-AS incluyó 438 ciclos de FET de 280 ciclos de recuperación ovocitaria en 266 pacientes que realizaron PGT para enfermedades monogénicas sin cribado de aneuploidías.
La población de pacientes estuvo toda por debajo de los 35 años y realizó PGT para enfermedades monogénicas con y sin AS.
Tasa de embarazo evolutivo/nacido vivo, tasa de nacido vivo, tasa de implantación, y tasa de aborto.
El cribado de aneuploidías mejoró significativamente la tasa de embarazo evolutivo/nacido vivo (61,22% vs. 43,98%), tasa de implantación (64,29% vs. 50,38%), y tasa de nacido vivo (53,06% vs. 36,09%) de mujeres jóvenes portadoras de enfermedades monogénicas en el primer ciclo de FET. Cuando se ajustó por la paridad, número de abortos previos, y porcentaje de infertilidad, la probabilidad de implantación fue 1,874 veces más alta (intervalo de confianza 95% 1,126-3,119), y embarazo evolutivo/nacido vivo fue 2,139 veces más probable (intervalo de confianza 95% 1,295-3,534). Además, la tasa de aborto fue disminuida significativamente (3,17% vs. 11,94%). En los resultados gestacionales acumulativos, la tasa acumulativa de embarazo evolutivo/nacido vivo tanto por transferencia y por paciente fueron significativamente más altas en el grupo AS (62,24% vs. 50,38% y 79,59% vs. 68,80%), pero no existió diferencia después de ajustar según paridad, número de abortos previos, y porcentaje de infertilidad. Sin embargo, el cribado de aneuploidías redujo el intervalo de tiempo desde el primer ET hasta lograr un embarazo.
Cribado de aneuploidías en PGT mejoró significativamente la tasa de embarazo evolutivo/nacido vivo de mujeres jóvenes portadoras de enfermedades monogénicas en el primer ciclo de FET.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Is embryo vitrification associated with a higher risk of adverse perinatal outcomes than slow-freezing?
Embryo vitrification was not associated with increased risks of adverse perinatal outcomes of ...pre-term birth (PTB), low birthweight (LBW), small for gestational age (SGA), large for gestational age (LGA) and macrosomia, as compared to slow-freezing.
Vitrification is becoming a widely adopted technology for embryo cryopreservation with higher embryo survival rate and live birth rate than the slow-freezing technique. However, limited data are currently available on risks of adverse perinatal outcomes following vitrification as compared to that of slow-freezing. The impact of vitrification on perinatal outcomes remains further to be elucidated.
Six large reproductive medical centers in Guangdong province, Southeast of China, took part in this multicenter retrospective cohort study. Cohorts of 3199 live born singletons after Day 3 frozen-thawed embryo transfer (FET) cycles with either vitrification or slow-freezing between January 2011 and December 2015 were included in the study. Each patient only contributed one cycle per cohort and vanishing twins were excluded. Propensity score (PS) matching was used to control for potential confounding factors.
All live-born singletons following either a vitrified or a slow-frozen cleavage FET cycle during the period from 2011 to 2015 were analyzed. Perinatal outcomes of PTB, LBW, macrosomia, SGA and LGA were compared. The vitrified and slow-frozen cohorts were matched by propensity scores with a 1:1 ratio accounting for potential confounding factors associated with perinatal outcomes. These variables included baseline demographics (maternal age, BMI, education level, parity, type of infertility and cause of infertility), as well as IVF characteristics (insemination method, endometrial preparation protocol and embryo cryopreservation duration).
A total of 2858 cases from vitrified embryo transfer (ET) and 341 babies from the slow-freezing group were included. After PS matching, 297 pairs of newborns were generated for comparison. The median gestational age was 39 weeks for both cohorts and the birthweights were comparable (3187.7 ± 502.1 g in the vitrified group vs. 3224.6 ± 483.6 in the slow-freezing group, P>0.05). There were no significant differences between the two groups on the incidence of PTB (5.4% vs. 7.7%), LBW (6.7% vs. 5.7%), macrosomia (5.7% vs. 6.1%), SGA (12.5% vs. 8.4%) and LGA (6.4% vs. 8.1%). Parallel logistic regression analysis indicated that vitrification was non-inferior to slow-freezing method in terms of the occurrence of PTB (OR, 0.68 95% CI, 0.35, 1.31), LBW (OR, 1.190.61-2.32), macrosomia (OR, 0.94 0.48-1.86), SGA (1.550.91-2.64) and LGA (0.780.42-1.45), P>0.05. Sex-stratified PS matching models with multivariable regression analysis further confirmed that vitrification did not increase the risks of above-mentioned adverse perinatal outcomes in either the male or female infant cohort.
Although the analysis was adjusted for a number of important confounders, the hospital dataset did not contain other potential confounders such as the medical history and obstetrics outcomes of women during pregnancy to allow adjustment. In addition, the current findings are only applicable to cleavage stage FET, but not pronuclei stage or blastocyst stage ET.
Vitrified ET, in comparison with slow-frozen ET, was not associated with increased risks of adverse neonatal outcomes. With its superiority on live birth rates and non-inferiority on safety perinatal outcomes, transition from slow-freezing to the use of vitrification for embryo cryopreservation is reassuring. Nonetheless, future research is needed for the long-term effects of vitrification method on offspring's health outcomes.
The study was funded by the National Key Research and Development Program (2016YFC100205), Guangzhou Science and Technology Project (201804020087), Guangdong Province Science and Technology Project (2016A020218008) and Guangdong Provincial Key Laboratory of Reproductive Medicine (2012A061400003). The authors have no conflicts of interest to declare.
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