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To explore the relationship between serum 25(OH)D level and pregnancy outcomes (clinical pregnancy rate [CPR] and live birth rate [LBR]) in Chinese women receiving in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI)-embryo transfer (ET) (IVF/ICSI-ET).A total of 612 patients included in the study were divided into four cohorts according to serum 25(OH)D with the threshold of 20 ng/ml, 25 ng/ml, 30 ng/ml, and retrospectively analyzed.None of the baseline characteristics of participants was significantly different in the four cohorts except gravid status. The trend of 25(OH)D concentration was positively correlated with CPR and LBR. The younger (age: p 0.001 both in CPR and LBR) women with primary infertility (infertility type: p = 0.004 in LBR) were more likely to get a better pregnancy outcome under the same 25(OH)D concentration stages. As shown on heatmap plots, CPR, and LBR were significantly increased for 25(OH)D concentrations above 30.00 ng/ml and women younger than 30 years old. The adjusted binary logistic regression and restricted cubic spline (RCS) showed that there existed a nonlinear positive correlation between 25(OH)D concentration and pregnancy outcome (CPR and LBR) (PIn Chinese women receiving IVF/ICSI-ET, the serum level of 25(OH)D demonstrated a nonlinear positive correlation with pregnancy outcomes (CPR and LBR), with stronger correlations above 25 ng/ml and worse yields below 30 ng/ml. However, it could not yet be considered different in distinct ages.

Adenine base editors (ABEs) are novel genome-editing tools, and their activity has been greatly enhanced by eight additional mutations, thus named ABE8e. However, elevated catalytic activity was concomitant with frequent generation of bystander mutations. This bystander effect precludes its safe applications required in human gene therapy. To develop next-generation ABEs that are both catalytically efficient and positionally precise, we performed combinatorial engineering of NG-ABE8e. We identify a novel variant (NG-ABE9e), which harbors nine mutations. NG-ABE9e exhibits robust and precise base-editing activity in human cells, with more than 7-fold bystander editing reduction at some sites, compared with NG-ABE8e. To demonstrate its practical utility, we used NG-ABE9e to correct the frequent T17M mutation in Rhodopsin for autosomal dominant retinitis pigmentosa. It reduces bystander editing by ∼4-fold while maintaining comparable efficiency. NG-ABE9e possesses substantially higher activity than NG-ABEmax and significantly lower bystander editing than NG-ABE8e in rice. Therefore, this study provides a versatile and improved adenine base editor for genome editing.

To investigate whether high estrogen (E2) levels caused by controlled ovarian hyperstimulation affect the birth defect rate in singleton assisted reproductive technology (ART) birth after conceived by fresh embryo transfer and frozen embryo transfer (FET).This was a retrospective cohort study. A total of 581 women with singletons, as well as those who have become pregnant and have had an unwanted abortion under high E2 levels on trigger day were divided into three groups. Group A received FET and the E2 levels on trigger day were higher than 5000 pg/ml. Group B received fresh embryo transfer and the E2 levels were between 3000 and 5000 pg/ml. Group C received FET and the E2 levels were between 3000 and 5000 pg/ml.There were no significant differences in birth weight, delivery mode, preterm birth rate, and fetal sex between the three groups (Fresh embryo transfer under supraphysiological level of estrogen exposure may increase the birth defect rate of ART singletons. Even after prenatal screening and diagnosis, a part of birth defect could not be detected during pregnancy. When the estrogen levels on trigger day were no lower than 3000 pg/ml, FET should be advocated to reduce the occurrence of such risk.

CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated endonuclease Cas9) nucleases have been widely applied for genome engineering. Staphylococcus aureus Cas9 (SaCas9) is compact, which can be packaged in AAV (adeno-associated virus) vector for in vivo gene editing. While, wild-type SaCas9 can induce unwanted off-target mutations and substantially limits the applications. So far, there are two reported SaCas9 variants with high-fidelity, including efSaCas9 from our previous study and SaCas9-HF. However, it remains unknown which one possessing the better fidelity and higher activity. Here, we performed a parallel comparison of efSaCas9 and SaCas9-HF in human cells through fluorescent reporter system and target deep sequencing, respectively. The results demonstrated that efSaCas9 possesses higher cleavage activity and fidelity than SaCas9-HF at the most endogenous sites in human cells. Collectively, our study provides insights for the rational selection of suitable SaCas9 for human genome editing.

Background Ovarian hyperstimulation syndrome (OHSS) is a severe disease that can lead to serious complication. Letrozole has been applied during controlled ovarian hyperstimulation (COH) to reduce the rate of OHSS in women undergoing long-term Gonadotropin-releasing Hormone Analog (GnRHa) treatment for assisted fertility. Prednisone can prevent vasodilatation and increased vascular permeability, which is common during OHSS. However, few studies have evaluated the combined effect of letrozole and prednisone in preventing severe OHSS and is the aim of our retrospective study of patients receiving GnRHa treatment. Methods A total of 296 women who accepted autologous in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) treatments were included in this retrospective study. There were three groups: 146 women had letrozole, including letrozole alone (LE group, n=60) and letrozole with prednisone (LE + Pre group, n=86), and 150 women had no treatment (C group). Severe OHSS was diagnosed according to clinical evidence of hydrothorax, severe dyspnea, oliguria/anuria, and intractable nausea/vomiting. Results The addition of prednisone to letrozole successfully reduced the occurrence rate of severe OHSS than those women administered letrozole alone (55.0% vs. 70.6%, P=0.022). However, the ongoing pregnancy rate was lower in the LE + Pre group than that in the LE-alone group (64.3% vs. 87.0%, P=0.025). Surprisingly, progesterone level on the trigger day (>0.895 ng/mL) is a strong predictor for pregnancy failure with a specificity of 68.3% and sensitivity of 65.7% in the LE-alone group. Conclusions Treatment with a combination of letrozole and prednisone may lower the rate of severe OHSS in women with prolonged gonadotropin-releasing hormone agonist protocol during assisted fertility treatment. When the progesterone level on trigger day is over 0.895 ng/mL, letrozole treatment may negatively affect clinical pregnancy.

Cytosine base editor (CBE) enables targeted C-to-T conversions at single base-pair resolution and thus has potential therapeutic applications in humans. However, the low efficiency of the system limits practical use of this approach. We reported a high-throughput human cells-based reporter system that can be harnessed for quickly measuring editing activity of CBE. Screening of 1813 small-molecule compounds resulted in the identification of Ricolinostat (an HDAC6 inhibitor) that can enhance the efficiency of BE3 in human cells (2.45- to 9.21-fold improvement). Nexturastat A, another HDAC6 inhibitor, could also increase BE3-mediated gene editing by 2.18- to 9.95-fold. Ricolinostat and Nexturastat A also boost base editing activity of the other CBE variants (BE4max, YE1-BE4max, evoAPOBEC1-BE4max and SpRY-CBE4max, up to 8.32-fold). Meanwhile, combined application of BE3 and Ricolinostat led to >3-fold higher efficiency of correcting a pathogenic mutation in ABCA4 gene related to Stargardt disease in human cells. Moreover, we demonstrated that our strategy could be applied for efficient generation of mouse models through direct zygote injection and base editing in primary human T cells. Our study provides a new strategy to improve the activity and specificity of CBE in human cells. Ricolinostat and Nexturastat A augment the effectiveness and applicability of CBE.

Research questionTo explore the relationship between serum 25(OH)D level and Clinical Pregnancy Rate (CPR) as well as Live Birth Rate (LBR) in Chinese women receiving IVF/ICSI-ET.DesignThe data of 612 patients who underwent IVF/ICSI-ET in the Reproduction Center of the Second Affiliated Hospital of Wenzhou Medical University were retrospectively analyzed. ResultsNone of the baseline characteristics of participants were significantly different between the two cohorts. The trend of 25(OH)D concentration was positively correlated with CPR and LBR. Moreover, our results indicated that the younger (Age: pp=0.004 in LBR) were more likely to get a better pregnancy outcome under the same 25(OH)D concentration stages. As shown on heatmap plots, CPR and LBR were significantly increased for 25(OH)D concentrations above 30.00ng/ml and women younger than 30 years old. The adjusted binary logistic regression and RCS showed that there exists a nonlinear positive correlation between 25(OH)D concentration and pregnancy outcome (CPR and LBR) (nonlinear < 0.001, respectively). The women with a sufficient 25(OH)D concentration (30ng/ml) had 1.07 (clinical pregnancy) and 1.05 (live birth) times higher successful birth outcomes compared to women with an insufficient 25(OH)D concentration (25ng/ml). (OR25ng/ml,ref=30ng/ml [95%CI] = 0.935[0.932-0.938] and 0.947[0.945-0.950],PConclusionIn Chinese women receiving IVF/ICSI-ET, the serum level of 25(OH)D demonstrated a non-linear positive correlation with pregnancy outcome（CPR&LBR, with stronger correlations above 25 ng/ml and worse yields below 30 ng/ml. However, it could not yet be considered different in distinct ages.

IntroductionMorphological evaluation is used to select embryos for in vitro fertilisation. However, it does not fully reflect the implantation potential. Preimplantation genetic testing for aneuploidies (PGT-A) can detect embryonic aneuploidy, but biopsy procedure is invasive. Currently, a non-invasive PGT (ni-PGT) approach using spent medium is being evaluated. However, the clinical benefit of ni-PGT has not been clearly demonstrated. A multicentre randomised trial is needed to verify whether ni-PGT can be an new effective tool for evaluating embryos.Methods and analysisOverall, 1148 couples aged 35~42 (women) receiving in vitro fertilization–intracytoplasmic sperm injection are planned to be enrolled. Couples will be digitally randomised to (1) ni-PGT and (2) conventional morphology groups at a 1:1 treatment ratio. The primary outcome will be the ongoing pregnancy rate related to the first transfer cycle within 6 months after oocyte retrieval.Ethics and disseminationThe study protocol is approved by the Ethics Committee of Peking University Third Hospital and the participating hospitals. The results will be disseminated through international conferences and scientific journals.Trial registration numberNCT04339166.

The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas (CRISPR-associated) nucleases have been widely applied for genome engineering. Cas9 (Streptococcus pyogenes Cas9 [SpCas9] and Staphylococcus aureus Cas9 [SaCas9]) and Cpf1 (i.e., Francisella novicida U112 Cpf1 [FnCpf1], also named FnCas12a) were harnessed to perform gene editing in human cells. Precise genetic modification by homology-directed repair (HDR) is an attractive approach for in situ gene correction. However, so far, the comparative efficiencies of HDR mediated by different CRISPR orthologs remain unknown. To address this question, in this study, we developed a reporter system to investigate HDR efficiencies triggered by various CRISPR orthologs. We found that SpCas9 and SaCas9, the two most commonly used Cas9 enzymes, possessed a similar ability to induce HDR. Interestingly, with the increasing amount of coding plasmids or additional nuclear localization sequences, FnCpf1 could improve the HDR efficacy. Collectively, our study provides insights for the rational selection of appropriate tools for human genome manipulation.

Since the reproductive toxicity of COVID-19 vaccines have not been assessed in previous clinical trials, and studies have shown that SARS-CoV-2 is associated with a decrease in sperm parameters. Although it has been reported that the mRNA SARS-CoV-2 vaccines do not adversely affect semen parameters, whether this conclusion applies to inactivated vaccines remains unclear. Here, we conducted a study among patients who accepted in vitro fertilization (IVF) at the reproductive centre between June and August of 2021. In the enrolled cases, men who have completed two doses of COVID-19 inactivated vaccine were included in "vaccine group" (N = 105), and those who were not vaccinated were included in "control group" (N = 155). In this study, we compare the sperm parameters and embryo quality between these two groups. Our data showed that the sperm parameters were similar in terms of volume, sperm concentration, sperm count, progressive motility, total motility and total motile sperm count between these two groups. Similarly, no significant differences were observed in IVF outcomes. The mean number of 2PN, cleavage-stage embryos, blastocysts, and good-quality blastocysts was 8.59 ± 4.47, 5.06 ± 3.17 and 2.08 ± 1.79 in vaccine group, 7.75 ± 4.14, 4.34 ± 3.06 and 1.74 ± 1.54 in control group, respectively. The high-quality blastocyst rate was 41.05% (218 of 531) in vaccine group and 40.03% (269 of 672) in control group (p 0.05). In addition, no differences were observed in biochemical and clinical pregnancy rates between the two groups. In summary, our results revealed that COVID-19 inactivated vaccine administration exhibited no negative effect on sperm parameters and embryo quality in IVF.

Background. Deficient spermatozoon motility is one of the main causes of male infertility. However, there are still no accurate and effective treatments in a clinical setting for male asthenospermia. Exploring the genes and mechanism of asthenospermia has become one of the hot topics in reproductive medicine. Our aim is to study the effect of SLRIP on human spermatozoon motility and oxidative stress. Methods. Sperm samples were collected including a normospermia group (60 cases) and an asthenospermia group (50 cases). SLIRP protein expression in spermatozoa was examined by western blotting, and relative mRNA expression of SLIRP in spermatozoa was quantified by reverse transcription polymerase chain reaction. Levels of reactive oxygen species (ROS), adenosine triphosphate (ATP) content, and the activity of manganese superoxide dismutase (MnSOD) in spermatozoa were also measured. Results. The mRNA level and protein expression of SLIRP in the asthenospermia group were significantly reduced compared with those in the normospermia group. The ROS active oxygen level in the asthenospermia group significantly increased; however, the ATP content decreased significantly as well as the activity of MnSOD. Conclusion. SLIRP regulates human male fertility, and SLIRP and sperm progressive motility are positively correlated. The expression of SLIRP is declined, oxidative damage is increased, and energy metabolism is decreased in spermatozoa of asthenospermia patients compared to normospermia participants.

Base editing is a form of genome editing that can directly convert a single base (C or A) to another base (T or G), which is of great potential in biomedical applications. The broad application of base editing is limited by its low activity and specificity, which still needs to be resolved. To address this, a simple and quick method for the determination of its activity/specificity is highly desired. Here, we developed a novel system, which could be harnessed for quick detection of editing activity and specificity of base editors (BEs) in human cells. Specifically, multiple cloning sites (MCS) were inserted into the human genome via lentivirus, and base editing targeting the MCS was performed with BEs. The base editing activities were assessed by specific restriction enzymes. The whole process only includes nucleotide-based targeting the MCS, editing, PCR, and digestion, thus, we named it NOTEPAD. This straightforward approach could be easily accessed by molecular biology laboratories. With this method, we could easily determine the BEs editing efficiency and pattern. The results revealed that BEs triggered more off-target effects in the genome than on plasmids including genomic indels (insertions and deletions). We found that ABEs (adenine base editors) had better fidelity than CBEs (cytosine base editors). Our system could be harnessed as a base editing assessment platform, which would pave the way for the development of next-generation BEs.

To investigate the feasibility of switching from in vitro fertilization (IVF) to in vitro maturation (IVM) combined with all-blastocyst-culture and transfer as a supplementary infertility treatment in patients with ovarian hyperstimulation syndrome (OHSS) tendency METHODS: Retrospective cohort study including 184 patients who switched from IVF and underwent 192 IVM cycles between January 2016 and December 2020. The outcomes were compared between cleavage-stage embryo transfer (group A, n = 74) and blastocyst-stage transfer (group B, n = 52) groups.The OHSS rate is 0%. 66 cycles were canceled for transfer. Among the 126 transfer cycles, number of retrieved oocytes, proportion of metaphase II oocytes, cleavage rate, and proportion of high-quality embryos on day 3 post-fertilization are significantly lower in group A than that in group B. On the contrary, number of transferred embryos is significantly lower in group B than that in group A, whereas the rates of implantation, clinical pregnancy, and live births are significantly higher in group B than that in group A.Timely switching to IVM combined with all-blastocyst-culture and transfer for patients undergoing controlled ovarian hyperstimulation and exhibiting characteristics of OHSS tendency is feasible as a supplementary infertility treatment.

The purpose of this study was to explore the influence of decreased serum estradiol (The clinical data of 300 IVF-ET cycles with patients were analyzed retrospectively. According to the presence of fallingDuration and total dosage of gonadotropin (Gn) used were statistically more in group A than in group B (Reduction of

Objective. The purpose of this study was to explore the influence of decreased serum estradiol (E2) levels during controlled ovarian hyperstimulation (COH) on in vitro fertilization and embryo transfer (IVF). Methods. The clinical data of 300 IVF-ET cycles with patients were analyzed retrospectively. According to the presence of falling E2 level during the COH, we divided all subjects into two groups: the E2 levels fall group (n = 120, group A) and the control group (n = 180, group B). In group A, there were 57 patients with falling E2 with drug dosage reduction. The other 63 patients experienced the decreased E2 level spontaneously. The clinical and laboratory variables in the groups were compared. Receiver operator characteristic (ROC) curve analyses were carried out in order to evaluate the predict value of E2 level on the day of human chorionic gonadotropin (hCG) administration on IVF outcomes. Results. Duration and total dosage of gonadotropin (Gn) used were statistically more in group A than in group B ( P P = 0.048). Women in group A had lower clinical pregnancy rate ( P = 0.029), live birth rate ( P P = 0.001), and higher early abortion rates ( P = 0.008) than group B. Women with spontaneously falling E2 group had a higher BMI index than those in the drug dosage reduction group ( P = 0.001). More dosage and longer duration of Gn in spontaneously falling E2 group than in the drug dosage reduction group ( P 2020 pg/ml on the hCG day might be an index to predict live birth. The sensitivity was 57.0% and the specificity was 61.7%. Conclusions. Reduction of E2 during COH might adversely affect the clinical pregnancy, early abortion, and ongoing pregnancy of IVF-ET.

Clinical and genetic characteristics of ELANE mutation of a 3-year-old male who had a severe congenital neutropenia (SCN) were examined. We then investigated whether CRISPR/Cas9-mediated gene editing could correct the mutation.The proband underwent extensive clinical assessments, such as exome sequencing and bioinformatics analysis, so that pathogenic genes could be identified. Sanger sequencing was also utilized for confirmation. The cell line, 293-ELANE, harboring ELANE mutation was generated, and the mutation was then corrected by CRISPR/Cas9-mediated homology-directed repair (HDR).The ELANE gene test in the proband unveiled a heterozygous de novo missense mutation: c. 248T A (p.V83D), which was not detected in his asymptomatic parents who had provided peripheral blood samples. We found that 46.01% of his father's sperm cells had the same mutation. These results demonstrate that the proband inherited the ELANE mutation from his father, who had an average neutrophil count but had a germline mosaicism. The highest repair efficiency of CRISPR/Cas9-mediated HDR for 293-ELANE is 4.43%.We identified a missense mutation (p.V83D) in ELANE that causes SCN. This is the first report on paternal semen mosaicism of an ELANE mutation. Our study paves the way for preimplantation genetic diagnosis (PGD) based on ELANE mutation prevention and clinical treatment of congenital disabilities.

BACKGROUND: The long protocol has been recognized as the gold standard in controlled ovarian hyperstimulation (COH). However, the full dose of gonadotropin-releasing hormone agonist (GnRH-a) under the prolonged protocol has become increasingly popular in China. This study sought to compare pregnancy outcomes among the following 3 groups: a long protocol group, and 2 types of improved prolonged protocol groups. METHODS: A retrospective cohort study was conducted of 550 patients undergoing fresh embryo transfer (ET). Patients were treated either with the improved prolonged protocol in the follicular phase (Group 1; n=288) or the mid-luteal phase (Group 2; n=143), or the long protocol (Group 3; n=119). The clinical and laboratory outcomes of the 3 groups were compared. RESULTS: The general characteristics of the women in the 3 groups were comparable. On the day on which gonadotropin (Gn) was first administered and on the day on which human chorionic gonadotropin (hCG) was administered, the luteinizing hormone (LH) levels of patients in both Groups 1 and 2 were lower than those of patients in Group 3. The number of oocytes retrieved, fertilized, and cleaved, and the number of high-quality embryos in the 3 procedures were similar. However, the number of transferred embryos, the rate of blastocyst progression, and the rate of implantation differed. The clinical pregnancy rates (CPRs)were significantly higher in the prolonged protocol groups (62.5% and 61.5%) than the long protocol group (48.7%). Further, statistically significant differences in the live-birth rates (LBRs) (56.9% vs. 57.3% vs. 42.9%) were observed. However, no differences in early abortion rates were found. CONCLUSIONS: As a result of pituitary downregulation with GnRH-a, the prolonged groups had better CPRs and LBRs than the long protocol group. The prolonged protocol in the mid-luteal phase was equally effective as that in the early follicular phase in fresh in-vitro fertilization (IVF)/intracytoplasmic sperm injection-embryo transfer (ICSI-ET) cycles. High LH levels on the day of hCG may be a predictor of adverse clinical outcomes.

BackgroundIt is commonly believed that using frozen sperms after TESA was effective for OA patients. Nevertheless, scholars are worried about the prognosis of ICSI with frozen testicular sperm. In this study, we aim to compare the pregnancy and neonatal outcomes of ICSI using cryopreserved versus fresh spermatozoa collected by TESA.MethodsA total of 317 cases of OA patients treated with ICSI in a university affiliated hospital from January 2016 to December 2020 were included in this retrospective study, and they were divided into two groups according to the sperm used for ICSI: Frozen sperm group (N=154) and Fresh sperm group(N=163). The outcomes were measured by the following indicators: Two pronucleus (2PN) fertilization rate, 2PN cleavage rate, high-quality blastocyst rate, average number of embryos transferred, implantation rate, clinical pregnancy rate, multiple pregnancy rate, miscarriage rate, preterm birth rate, live birth rate (LBR) , sex ratio at birth (male) and average newborn birth weight.ResultsThe present data showed no statistically significant difference in 2PN fertilization rates, 2PN cleavage rates, high-quality blastocyst rates and the average number of embryos transferred in the two groups. Similarly, no difference was found in implantation rate, clinical pregnancy rate, multiple pregnancy rate, miscarriage rate, premature delivery rate, LBR and sex ratio at birth (P>0.05). The average newborn birth weight was similar in the two groups (2932.61±728.40 vs 3100.32±515.64) (P>0.05), but there was a higher incidence of Low-Birth-Weight newborn in the frozen group (20.91% vs 8.49%)(PConclusionsAs for men with obstructive azoospermia, the use of frozen testicular sperm by TESA was efficient. There was a similar pregnancy outcome of ICSI using frozen or fresh spermatozoa collected by TESA. However, it may lead to higher incidence of newborns of low birth weight, which needs further research based on larger samples.

Clustered regularly interspaced short palindromic repeats and associated proteins (CRISPR-Cas) of bacterial adaptive immunity have been adopted as a powerful and versatile tool for manipulation of the genome. This paradigm has been widely applied in biological research and treatments of animal or cellular disease models. A critical feature of CRISPR-Cas is the protospacer adjacent motif (PAM), which dictates the DNA target recognition mechanism of Cas proteins. While, direct identifying functional PAM sequences in human cells remains a challenge. Here, we developed a positive screen system termed PAM-DOSE (PAM Definition by Observable Sequence Excision) to delineate the functional PAMs in human cells. Specifically, the PAM libraries for CRISPR-Cas (SpCas9, SpCas9-NG, FnCas12a, AsCas12a, LbCas12a and MbCas12a) were generated and the corresponding CRISPR-Cas mediated cleaved fragments with functional PAM in human cells were harvested for DNA sequencing, which could be tracked and visualized with either florescence microscopy or flow cytometry analysis. With this system, we identified the functional PAMs of CRISPR-Cas members. We also found that spacer sequence affects the PAM preference of Cas proteins. This method will facilitate identification of functional PAMs for Cas-mediated human genome editing applications. Keywords: CRISPR-Cas, PAM, Human, Next-generation Sequencing

Background: Assisted reproductive techniques (ART) have been extensively used to treat infertility. Inaccurate prediction of a couple’s fertility often leads to lowered self-esteem for patients seeking ART treatment and causes fertility distress. Objective: This prospective study aimed to statistically analyze patient data from a single reproductive medical center over a period of 18 months, and to establish mathematical models that might facilitate accurate prediction of successful pregnancy when ART are used. Methods: In the present study, we analyzed clinical data prospectively collected from 760 infertile patients visiting the second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University between June 1, 2016 and December 31, 2017. Various advanced statistical methods, including broken-line regression, were employed to analyze the data. Results: Age remained the most important factor affecting the outcome of IVF/ICSI. Using the broken-line regression model, the fastest clinical pregnancy declining age was between 25 and 32. Female infertility type was found to be a key predictor for the number of good-quality embryos and successful pregnancy, along with the antral follicle count (AFC), total number of embryos, recombinant follicle stimulating hormones (rFSH) dosage, estradiol (E2) on the trigger day, and total number of oocytes retrieved. rFSH dosage was also significantly associated with the number of oocytes retrieved and the number of frozen embryos. Conclusion: The fastest clinical pregnancy declining age is ranged between 25 and 32, and female infertility type is evidenced as another key predictive factor for the cumulative outcome of ART.

To analyze the effects of blastocysts on the 5th day (D5) and 6th day (D6) of frozen–thawed blastocyst transplantation on pregnancy outcome and provide evidence for further improvement of the strategy. This study included transfers from the Reproductive Medicine Center of the Second Affiliated Hospital of Wenzhou Medical University during freeze–thaw cycles from January 2016 to December 2017. They were divided into D5 group (1616 cases) and D6 group (619 cases) according to blastocyst formation and development. Each group was further divided into 5 groups according to the quality of the blastocyst and the number of transplants, making a total of 10 groups. Following the frozen transplantation cycle, the transplanting rate was significantly higher for D5 (41.73%) than for D6 (23.98%) (P < 0.05); the ongoing pregnancy rate (47,40%) was also significantly higher than that of D6 (28.43%) (P < 0.05).In the frozen–thawed blastocyst resuscitation transplantation, compared to D6 blastocysts, D5 blastocysts were more conducive to blastocyst implantation and could be used to achieve better clinical pregnancy outcome. In blastocyst selection, a single D5 excellent blastocyst transplant is preferred. Only at the 6th day of non-excellent D6, 2 blastocysts are recommended for transplantation.

Leydig cells (LCs) play crucial roles in producing testosterone, which is critical in the regulation of male reproduction and development. Low levels of testosterone will lead to male hypogonadism. LC transplantation is a promising alternative therapy for male hypogonadism. However, the source of LCs limits this strategy for clinical applications. Thus far, others have reported that LCs can be derived from stem cells by gene transfection, but the safe and effective induction method has not yet been reported. Here, we report that Leydig-like cells can be derived from human induced pluripotent stem cells (iPSCs) using a novel differentiation protocol based on molecular compounds. The iPSCs-derived Leydig-like cells (iPSC-LCs) acquired testosterone synthesis capabilities, had the similar gene expression profiles with LCs, and positively expressed Leydig cell lineage-specific protein markers LHCGR, STAR, SCARB1, SF-1, CYP11A1, HSD3B1, and HSD17B3 as well as negatively expressed iPSC-specific markers NANOG, OCT4, and SOX2. When iPSC-LCs labeled with lipophilic red dye (PKH26) were transplanted into rat testes that were selectively eliminated endogenous LCs using EDS (75 mg/kg), the transplanted iPSC-LCs could survive and function in the interstitium of testes, and accelerate the recovery of serum testosterone levels and testis weights. Collectively, these findings demonstrated that the iPSCs were able to be differentiated into Leydig-like cells by few defined molecular compounds, which may lay the safer groundwork for further clinical application of iPSC-LCs for hypogonadism.

Summary Background Elective single embryo transfer (eSET) has been increasingly advocated, but concerns about the lower pregnancy rate after reducing the number of embryos transferred have encouraged transfer of multiple embryos. Extended embryo culture combined with electively freezing all embryos and undertaking a deferred frozen embryo transfer might increase pregnancy rate after eSET. We aimed to establish whether elective frozen single blastocyst transfer improved singleton livebirth rate compared with fresh single blastocyst transfer. Methods This multicentre, non-blinded, randomised controlled trial was undertaken in 21 academic fertility centres in China. 1650 women with regular menstrual cycles undergoing their first cycle of in-vitro fertilisation were enrolled from Aug 1, 2016, to June 3, 2017. Eligible women were randomly assigned to either fresh or frozen single blastocyst transfer. The randomisation sequence was computer generated, with block sizes of two, four, or six, stratified by study site. For those assigned to frozen blastocyst transfer, all blastocysts were cryopreserved and a delayed frozen-thawed single blastocyst transfer was done. The primary outcome was singleton livebirth rate. Analysis was by intention to treat. This trial is registered at the Chinese Clinical Trial Registry, number ChiCTR-IOR-14005405. Findings 825 women were assigned to each group and included in analyses. Frozen single blastocyst transfer resulted in higher rates of singleton livebirth than did fresh single blastocyst transfer (416 [50%] vs 329 [40%]; relative risk [RR] 1·26, 95% CI 1·14–1·41, p vs nine of 825 [1·1%] in fresh single blastocyst transfer; p=0·16), pregnancy loss (134 of 583 [23·0%] vs 124 of 481 [25·8%]; p=0·29), other obstetric complications, and neonatal morbidity were similar between the two groups. Frozen single blastocyst transfer was associated with a higher risk of pre-eclampsia (16 of 512 [3·1%] vs four of 401 [1·0%]; RR 3·13, 95% CI 1·06–9·30, p=0·029). Interpretation Frozen single blastocyst transfer resulted in a higher singleton livebirth rate than did fresh single blastocyst transfer in ovulatory women with good prognosis. The increased risk of pre-eclampsia after frozen blastocyst transfer warrants further studies. Funding The National Key Research and Development Program of China.

Ruqing Yang,1 Minghua Jiang,2 Junzhao Zhao,1 Hui Chen,2 Jian Gong,2 Yaying You,3 Laiyue Song,3 Zhen Li,4 Qian Li2 1Reproductive Medicine Center, Department of Gynaecology and Obstetrics, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325000, China; 2Department of Clinical Laboratory, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325000, China; 3The Second Clinical Medical College, Wenzhou Medical University, Wenzhou, Zhejiang 325000, China; 4Department of Traditional Chinese Medicine, The Third Affiliated Hospital of Guangzhou Medical University, Key Laboratory for Major Obstetric Diseases of Guangdong Province, Guangzhou, Guangdong 510150, China Background: Near-triploidy/tetraploidy is rarely found in acute leukemia. Only limited data are available to characterize this condition, and it remains largely unknown. Patients and methods: In our study, we performed karyotype analysis on 1,031 patients diagnosed with acute leukemia from 2006 to 2018. A total of 10 patients of near-triploidy/tetraploidy karyotype were enrolled. Two cases of near-triploidy (66–79 chromosomes) and eight cases of near-tetraploidy (84–100 chromosomes) were identified. Bone marrow samples of these 10 patients were analyzed by fluorescence in situ hybridization with 19 commercially available probes that detected a small portion of gene alterations and large regions of chromosome amplifications. Results: Of the six patients with acute myelocytic leukemia, we detected three cases of double t(8;21)(q22;q22) that have not been previously reported, and one of them demonstrated ins(21;8)(q22;q24q22). We also describe a novel pediatric case carrying double t(15;17)(q22;q21) and receiving targeted treatment with all-trans retinoic acid therapy. To date, this case has responded well to the regimen and has shown continuous complete remission. All patients received chemotherapy. One of them received allogeneic hematopoietic stem cell transplant (HSCT) and survived for 22 months. Eight of the 10 patients died, and the median overall survival was 11 months. Conclusion: Using fluorescence in situ hybridization, we identified the distinct complex karyotype of near-triploidy/tetraploidy and provided further prognostic information. Tetraploidy acute promyelocytic leukemia had favorable prognosis; thus, HSCT was not necessary. The case of insertion t(21;8)(q22;q24q22) in tetraploidy responded poorly to chemotherapy and achieved molecular remission with difficultly. Data from patients in this group indicated that near-triploidy/tetraploidy acute leukemia has poor prognosis and new therapy is urgently needed. Keywords: near-triploidy, tetraploidy karyotype, acute leukemia, fluorescence in situ hybridization, gene

BACKGROUND The aim of this study was to evaluate the efficacy of specific in vitro fertilization (IVF) protocols for patients with polycystic ovary syndrome (PCOS), and therefore, analyze the first-rank intention IVF protocol. MATERIAL AND METHODS In this study, 408 PCOS patients (464 treatment cycles) were enrolled and assigned to one of 3 groups: group 1 [oral contraceptive long-term regimen group (OC-L protocol group, n=91)], group 2 (GnRH antagonist protocol, n=80), and group 3 [follicular phase long-term regimen group, C₁-L protocol group n=293]. The endpoints are the number of eggs, oocyte maturation rate, high-quality embryo rate and clinical pregnancy rate after fresh embryos transfer, the incidence of ovarian hyperstimulation syndrome and abortion rate. RESULTS The number of eggs, oocyte maturation rate, and high-quality embryo rate in the C₁-L protocol group were significantly higher than those in the other 2 groups. The fertilization rate and cleavage rate of the 3 groups were not significantly different. After fresh embryo transplantation, the pregnancy rate of C₁-L protocol group was significantly higher than that of the other groups. CONCLUSIONS This study showed that the super-long downregulation in follicular phase regimen has advantages of simple treatment process, high oocyte maturation rate, high quality embryo rate, and pregnancy rate. It is a good choice for PCOS patients to promote ovulation during IVF.

Cas12a-mediated targeted genome engineering strategies have enabled a broad range of research and clinical applications. However, the limited target-selection spectrum and low activity/fidelity remain a bottleneck for its widespread application in precision site-specific human genome editing. Therefore, there exists an acute need to identify novel Cas12a nucleases with improved features for genome editing. By screening a range of candidate Cas12a nucleases, here we demonstrate that Lb2Cas12a possesses genome editing activity in human cells and it has greater flexibility in PAM (5'-BYYV-3') selection. Furthermore, we engineered Lb2Cas12a to generate variants (Lb2Cas12a-RVR and Lb2Cas12a-RR), which greatly expands the target-selection spectrum. Our study illustrated that Lb2Cas12a could be harnessed as additional genome editing tool for the manipulation of human genome.