Estradiol-to-follicle ratio on human chorionic gonadotropin day is a novel predictor of gestational diabetes mellitus in women receiving fresh embryo transfer.

Aims: To assess the predictive value of estradiol (E2) related parameters on the incidence of gestational diabetes mellitus (GDM) in women undergoing fresh embryo transfer. Materials and methods: A Post-hoc analysis of a prospective cohort study. Results: We identified an optimal E2/follicle (E2/F) ratio threshold of 246.03 pg/ml on the day of human chorionic gonadotropin (hCG) administration. Women with an E2/F ratio exceeding this threshold had significantly lower rates of GDM (12.75% vs. 20.41%, P < 0.001) and ovarian hyperstimulation syndrome (OHSS) (11.75% vs. 15.48%, P = 0.03). Additional E2 parameters were also evaluated: baseline E2, E2 on hCG day, E2 increase, and E2 fold change. Lower GDM rates were observed in women with baseline E2 above 31.50 pg/ml (13.51% vs. 19.42%, P <0.01), E2 on hCG day above 3794.50 pg/ml (12.26% vs. 19.32%, P < 0.001), and E2 increase above 3771.50 pg/ml (12.24% vs. 19.28%, P < 0.001). There were no significant differences in OHSS rates for these additional E2 parameters. After adjusting for confounders, lower E2/F ratio (OR: 1.626, 95% CI: 1.229-2.150, P <0.01), E2 on hCG day (OR: 1.511, 95% CI: 1.133-2.016, P = 0.01), and E2 increase (OR: 1.522, 95% CI: 1.141-2.031, P <0.01) were identified as risk factors for GDM. Conclusion: This study demonstrates that an E2/F ratio over 246.03 pg/ml is significantly associated with a reduced risk of both GDM and OHSS in women undergoing fresh embryo transfer, highlighting the E2/F ratio as a superior predictive biomarker compared to other E2-related parameters.

Diagnosis and management of heterotopic intramural pregnancy after in vitro fertilization: an eight-case series.

PURPOSE: To analyze the ultrasound characteristics, clinical management, and pregnancy outcomes of heterotopic intramural pregnancies (HIMPs) after embryo transfer. METHODS: This was a retrospective observational study of women who were diagnosed with HIMPs. The ultrasound characteristics, clinical treatment, and pregnancy outcomes of patients with HIMPs were evaluated. RESULTS: Eight women with HIMPs were included. Among them, 6 patients were diagnosed by transvaginal sonography, and 2 patients were misdiagnosed with heterotopic interstitial pregnancy. The diagnostic accuracy was 75% (6/8). Five patients with HIMPs were diagnosed at the time of the initial scan (5+6-6+3 weeks). An intramural gestational sac was observed in all 6 patients, and an embryo with cardiac activity was detected in one patient on the follow-up scans. Intrauterine pregnancies (IUPs) were revealed in all 6 patients, and embryo(s) with cardiac activity were observed in 5 patients at the time of the initial diagnosis or later. The patients receiving expectant treatment all presented with bagel signs, while patients with embryos with cardiac activity all underwent surgery. Among the 6 diagnosed women, 1 patient was initially treated medically, 4 were treated expectantly, and 1 was treated surgically. Among the 6 diagnosed patients, the IUPs of 5 patients resulted in live infants. CONCLUSION: Single ET should be recommended to decrease the possibility of HIMP. An accurate diagnosis of HIMP was reached in most cases by detailed ultrasound early in the first trimester. Most IUPs of HIMPs seem to have good outcomes with timely and proper management. Expectant management might be a possible choice for nonviable intramural pregnancies.

Novel PLCZ1 compound heterozygous mutations indicate gene dosage effect involved in total fertilisation failure after ICSI.

In brief: PLCZ1 mutations are related to total fertilisation failure (TFF) after intracytoplasmic sperm injection (ICSI), characterised by abnormal oocyte oscillations. The novel PLCZ1 compound heterozygous mutations reported by this study were associated with TFF after ICSI, with one of the mutations indicating a gene dosage effect. Abstract: Oocyte activation failure is thought to be one of the main factors for total fertilisation failure (TFF) after intracytoplasmic sperm injection (ICSI), which could be induced by abnormal calcium oscillations. Phospholipase C zeta (PLCZ), a sperm factor, is associated with Ca2+ oscillations in mammalian oocytes. To date, some mutations in PLCZ1 (the gene that encodes PLCZ) have been linked to TFF, as demonstrated by the observed reduction in protein levels or activity to induce Ca2+ oscillations. In this study, normozoospermic males whose sperms exhibited TFF after ICSI and their families were recruited. First, mutations in the PLCZ1 sequence were identified by whole exome sequencing and validated using Sanger sequencing. Then, the locations of PLCZ1/PLCZ and the transcript and protein levels in the sperm of the patients were studied. Subsequently, in vitro function analysis and in silico analysis were performed to investigate the function-structure correlation of mutations identified in PLCZ1 using western blotting, immunofluorescence, RT-qPCR, and molecular simulation. Ca2+ oscillations were detected after cRNA microinjection into MII mouse oocytes to investigate calcium oscillations induced by abnormal PLCZ. Five variants with compound heterozygosity were identified, consisting of five new mutations and three previously reported mutations distributed across the main domains of PLCZ, except the EF hands domain. The transcript and protein levels decreased to varying degrees among all detected mutations in PLCZ1 when transfected in HEK293T cells. Among these, mutations in M138V and R391* of PLCZ were unable to trigger typical Ca2+ oscillations. In case 5, aberrant localisation of PLCZ in the sperm head and an increased expression of PLCZ in the sperm were observed. In conclusion, this study enhances the potential for genetic diagnosis of TFF in clinics and elucidates the possible relationship between the function and structure of PLCZ in novel mutations.

Human umbilical cord-derived mesenchymal stem cell transplantation improves the long COVID.

No effective treatments can ameliorate symptoms of long COVID patients. Our study assessed the safety and efficacy of human umbilical cord-derived mesenchymal stem cells (UC-MSCs) in the treatment of long COVID patients. Ten long COVID patients were enrolled and received intravenous infusions of UC-MSCs on Days 0, 7, and 14. Adverse events and clinical symptoms were recorded, and chest-high-resolution CT (HRCT) images and laboratory parameters were analyzed. During UC-MSCs treatment and follow-up, we did not observe serious adverse events, the symptoms of long COVID patients were significantly relieved in a short time, especially sleep difficulty, depression or anxiety, memory issues, and so forth, and the lung lesions were also repaired. The routine laboratory parameters did not exhibit any significant abnormalities following UC-MSCs transplantation (UMSCT). The proportion of regulatory T cells gradually increased, but it was not statistically significant until 12 months. The proportion of naive B cells was elevated, while memory B cells, class-switched B-cells, and nonswitched B-cells decreased at 1 month after infusion. Additionally, we observed a transient elevation in circulating interleukin (IL)-6 after UMSCT, while tumor necrosis factor (TNF)-α, IL-17A, and IL-10 showed no significant changes. The levels of circulating immunoglobulin (Ig) M increased significantly at month 2, while IgA increased significantly at month 6. Furthermore, the SARS-CoV-2 IgG levels remained consistently high in all patients at Month 6, and there was no significant decrease during the subsequent 12-month follow-up. UMSCT was safe and tolerable in long COVID patients. It showed potential in alleviating long COVID symptoms and improving interstitial lung lesions.

Vitamin D binding protein correlate with estrogen increase after administration of human chorionic gonadotropin but do not affect ovulation, embryo, or pregnancy outcomes.

Background: Vitamin D binding protein (DBP) might increase substantially after ovarian stimulation and hence could be associated with IVF/ICSI outcomes because it determines the fraction of free bioavailable 25(OH) vitamin D. In this study, we aim to determine whether DBP is associated with E2 level after ovarian stimulation and IVF/ICSI outcomes. Design: Post-hoc analysis of a prospective observational cohort. Setting: Single-center study. Participants: 2569 women receiving embryo transfer. Intervention: None. Main outcome measures: The main outcomes were oocyte and embryo quality as well as pregnancy outcomes. Results: DBP concentration correlates with E2 on hCG day (=day of inducing ovulation with hCG; correlation coefficient r = 0.118, P<0.001) and E2 x-fold change to baseline level (r = 0.108, P<0.001). DBP is also positively correlated with total 25(OH)D (r = 0.689, R2 = 0.475, P<0.001) and inversely with free 25(OH)D (r=-0.424, R2=0.179, P<0.001), meaning that E2-stimulated DBP synthesis results in a decrease of free 25(OH)D during ovarian stimulation. However, such alteration does not affect IVF/ICSI outcomes when considering confounding factors, such as the number and quality of oocytes nor embryo quality as well as pregnancy outcomes. Conclusion: DBP concentration correlates with the degree of E2 increase after ovarian stimulation. DBP is also positively correlated with total 25(OH)D and inversely with free 25(OH)D, suggesting that the proportion of free 25(OH)D decreases during ovarian stimulation caused by E2-stimulated DBP synthesis. However, such alteration does not affect clinical IVF/ICSI outcomes.

Early GnRH-agonist therapy does not negatively impact the endometrial repair process or live birth rate.

Study objective: To investigate whether different timings of GnRH-a downregulation affected assisted reproductive outcomes in infertile women with moderate-to-severe intrauterine adhesions (IUAs) accompanied by adenomyosis. Design: A retrospective case series. Setting: An assisted reproductive technology center. Patients: The study reviewed 123 infertile women with moderate-to-severe IUAs accompanied by adenomyosis undergoing their first frozen-thawed embryo transfer (FET) cycles between January 2019 and December 2021. Measurements and main results: The majority of patients had moderate IUA (n=116, 94.31%). The average Basal uterine volume was 73.58 ± 36.50 cm3. The mean interval from operation to the first downregulation was 21.07 ± 18.02 days (range, 1-79 days). The mean duration of hormone replacement therapy (HRT) was 16.93 ± 6.29 days. The average endometrial thickness on the day before transfer was 10.83 ± 1.75 mm. A total of 70 women achieved clinical pregnancy (56.91%). Perinatal outcomes included live birth (n=47, 67.14%), early miscarriage (n=18, 25.71%), and late miscarriage (n=5, 7.14%). The time interval between uterine operation and the first downregulation was not a significant variable affecting live birth. Maternal age was the only risk factor associated with live birth (OR:0.89; 95% CI: 0.79-0.99, P=0.041). Conclusions: The earlier initiation of GnRH-a to suppress adenomyosis prior to endometrial preparation for frozen embryo transfer did not negatively impact repair of the endometrium after resection.

The therapeutic effect of anti-CD19 antibody on DHEA-induced PCOS mice.

Immune dysregulation has been summarized as a critical factor in the occurrence and development of Polycystic ovary syndrome (PCOS), but potential mediators and mechanisms remain unclear. Our previous study showed that CD19+ B cells were involved in the pathogenesis of dehydroepiandrosterone (DHEA)-induced PCOS mice. Here, we studied the therapeutic potential of anti-CD19 antibody (aCD19 Ab) on DHEA-induced PCOS mice. The results showed that aCD19 Ab treatment improved ovarian pathological structure and function of PCOS mice, manifested by an increased number of corpus luteum, a decreased number of cystic follicles and atretic follicles, and regular estrus cycles. The aCD19 Ab treatment reduced the proportion of splenic CD21+ CD23low marginal zone B cells as well as the level of serum IgM and decreased the percentage of peripheral blood and splenic neutrophils. In particular, aCD19 Ab treatment reduced the apoptosis of granulosa cells and macrophage infiltration in ovarian secondary follicles of PCOS mice, as well as the expression of TNF-α in ovarian tissue and serum TNF-α levels. Moreover, we confirmed that TNF-α induced the apoptosis of human ovarian granulosa tumor cell line cells in vitro. Thus, our work demonstrates that aCD19 Ab treatment improves ovarian pathological phenotype and function by reducing local and systemic inflammation in PCOS mice, which may provide a novel insight into PCOS therapy.

Toxins in Botanical Drugs and Plant-derived Food and Feed - from Science to Regulation: A Workshop Review.

In September 2022, the 3rd International Workshop on pyrrolizidine alkaloids (PAs) and related phytotoxins was held on-line, entitled 'Toxins in botanical drugs and plant-derived food and feed - from science to regulation'. The workshop focused on new findings about the occurrence, exposure, toxicity, and risk assessment of PAs. In addition, new scientific results related to the risk assessment of alkenylbenzenes, a distinct class of herbal constituents, were presented. The presence of PAs and alkenylbenzenes in plant-derived food, feed, and herbal medicines has raised health concerns with respect to their acute and chronic toxicity but mainly related to the genotoxic and carcinogenic properties of several congeners. The compounds are natural constituents of a variety of plant families and species widely used in medicinal, food, and feed products. Their individual occurrence, levels, and toxic properties, together with the broad range of congeners present in nature, represent a striking challenge to modern toxicology. This review tries to provide an overview of the current knowledge on these compounds and indicates needs and perspectives for future research.

Deleterious variants in X-linked RHOXF1 cause male infertility with oligo- and azoospermia.

Oligozoospermia and azoospermia are two common phenotypes of male infertility characterized by massive sperm defects owing to failure of spermatogenesis. The deleterious impact of candidate variants with male infertility is to be explored. In our study, we identified three hemizygous missense variants (c.388G>A: p.V130M, c.272C>T: p.A91V, and c.467C>T: p.A156V) and one hemizygous nonsense variant (c.478C>T: p.R160X) in the Rhox homeobox family member 1 gene (RHOXF1) in four unrelated cases from a cohort of 1201 infertile Chinese men with oligo- and azoospermia using whole-exome sequencing and Sanger sequencing. RHOXF1 was absent in the testicular biopsy of one patient (c.388G>A: p.V130M) whose histological analysis showed a phenotype of Sertoli cell-only syndrome. In vitro experiments indicated that RHOXF1 mutations significantly reduced the content of RHOXF1 protein in HEK293T cells. Specifically, the p.V130M, p.A156V, and p.R160X mutants of RHOXF1 also led to increased RHOXF1 accumulation in cytoplasmic particles. Luciferase assays revealed that p.V130M and p.R160X mutants may disrupt downstream spermatogenesis by perturbing the regulation of doublesex and mab-3 related transcription factor 1 (DMRT1) promoter activity. Furthermore, ICSI treatment could be beneficial in the context of oligozoospermia caused by RHOXF1 mutations. In conclusion, our findings collectively identified mutated RHOXF1 to be a disease-causing X-linked gene in human oligo- and azoospermia.

Mosaic variegated aneuploidy syndrome with tetraploid, and predisposition to male infertility triggered by mutant CEP192.

In this study, we report on mosaic variegated aneuploidy (MVA) syndrome with tetraploidy and predisposition to infertility in a family. Sequencing analysis identified that the CEP192 biallelic variants (c.1912C>T, p.His638Tyr and c.5750A>G, p.Asn1917Ser) segregated with microcephaly, short stature, limb-extremity dysplasia, and reduced testicular size, while CEP192 monoallelic variants segregated with infertility and/or reduced testicular size in the family. In 1,264 unrelated patients, variant screening for CEP192 identified a same variant (c.5750A>G, p.Asn1917Ser) and other variants significantly associated with infertility. Two lines of Cep192 mice model that are equivalent to human variants were generated. Embryos with Cep192 biallelic variants arrested at E7 because of cell apoptosis mediated by MVA/tetraploidy cell acumination. Mice with heterozygous variants replicated the predisposition to male infertility. Mouse primary embryonic fibroblasts with Cep192 biallelic variants cultured in vitro showed abnormal morphology, mitotic arresting, and disruption of spindle formation. In patient epithelial cells with biallelic variants cultured in vitro, the number of cells arrested during the prophase increased because of the failure of spindle formation. Accordingly, we present mutant CEP192, which is a link for the MVA syndrome with tetraploidy and the predisposition to male infertility.

Acquired aplastic anaemia after SARS-CoV-2 infection in China: a case report.

Since the coronavirus disease 2019 (COVID-19) pandemic began, several research groups in different countries have described cases of aplastic anaemia (AA) after COVID-19 or COVID-19 vaccination. Here, we present the case of a patient with new-onset AA in Changsha, China, that was presumably associated with preceding severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We conducted an epidemiological assessment of the incidence rate of blood system diseases from July 1, 2022, to May 31, 2023, in the haematology department of the Second Xiangya Hospital of Central South University and Hunan Children's Hospital. The detection rates of AA and leukaemia in the first two months after the epidemic outbreak were higher than those before and during the outbreak. However, only the difference in the detection rate of leukaemia was statistically significant.

Trace Sample Proteome Quantification by Data-Dependent Acquisition without Dynamic Exclusion.

Despite continuous technological improvements in sample preparation, mass-spectrometry-based proteomics for trace samples faces the challenges of sensitivity, quantification accuracy, and reproducibility. Herein, we explored the applicability of turboDDA (a method that uses data-dependent acquisition without dynamic exclusion) for quantitative proteomics of trace samples. After systematic optimization of acquisition parameters, we compared the performance of turboDDA with that of data-dependent acquisition with dynamic exclusion (DEDDA). By benchmarking the analysis of trace unlabeled human cell digests, turboDDA showed substantially better sensitivity in comparison with DEDDA, whether for unfractionated or high pH fractionated samples. Furthermore, through designing an iTRAQ-labeled three-proteome model (i.e., tryptic digest of protein lysates from yeast, human, and E. coli) to document the interference effect, we evaluated the quantification interference, accuracy, reproducibility of iTRAQ labeled trace samples, and the impact of PIF (precursor intensity fraction) cutoff for different approaches (turboDDA and DEDDA). The results showed that improved quantification accuracy and reproducibility could be achieved by turboDDA, while a more stringent PIF cutoff resulted in more accurate quantification but less peptide identification for both approaches. Finally, the turboDDA strategy was applied to the differential analysis of limited amounts of human lung cancer cell samples, showing great promise in trace proteomics sample analysis.

Cascade signal amplification strategy by coupling chemical redox-cycling and Fenton-like reaction: Toward an ultrasensitive split-type fluorescent immunoassay.

An ultrasensitive split-type fluorescent immunobiosensor has been reported based on a cascade signal amplification strategy by coupling chemical redox-cycling and Fenton-like reaction. In this strategy, Cu2+ could oxidize chemically o-phenylenediamine (OPD) to generate photosensitive 2, 3-diaminophenazine (DAP) and Cu+/Cu0. On one hand, the generated Cu0 in turn catalyzed the oxidation of OPD. On the other hand, the introduced H2O2 reacted with Cu + ion to produce hydroxyl radicals (·OH) and Cu2+ ion through a Cu + -mediated Fenton-like reaction. The produced ·OH and recycled Cu2+ ion could take turns oxidizing OPD to generate more photoactive DAP, which triggering a self-sustaining chemical redox-cycling reaction and leading to a remarkable fluorescent improvement. It was worth mentioning that the cascade reaction did not stop until OPD molecules were completely consumed. Based on the H2O2-triggered cascade signal amplification, the strategy was exploited for the construction of split-type fluorescent immunoassay by taking interleukin-6 (IL-6) as the model target. It was realized for the ultrasensitive determination of IL-6 in a linear ranging from 20 fg/mL to 10 pg/mL with a limit of detection of 5 fg/mL. The study validated the practicability of the cascade signal amplification on the fluorescent bioanalysis and the superior performance in fluorescent immunoassay. It is expected that the strategy would offer new opportunities to develop ultrasensitive fluorescent methods for biosensor and bioanalysis.

Letrozole-stimulated endometrial preparation protocol is a superior alternative to hormone replacement treatment for frozen embryo transfer in women with polycystic ovary syndrome, a cohort study.

BACKGROUND: The current routine endometrial preparation protocol for women with polycystic ovary syndrome (PCOS) is hormone replacement treatment (HRT). Letrozole is rarely used in frozen embryo cycles. Evidence confirming whether letrozole-stimulated (LS) protocol is suitable for frozen embryo transfer in patients with PCOS and for whom is suitable remains lacking. METHODS: This was a retrospective cohort study involving all frozen embryo transfer cycles with LS and HRT for PCOS during the period from Jan 2019 to December 2020 at a tertiary care center. Multivariate Logistic regression was used to analyze the differences in clinical pregnancy rate, live birth rate, miscarriage rate, the incidence of other pregnancy and obstetric outcomes between LS and HRT protocols after adjusting for possible confounding factors. Subgroup analysis was used to explore the population for which LS protocol was suitable. RESULTS: The results of multivariate logistic regression showed that LS was significantly associated with a higher clinical pregnancy rate (70.9% vs. 64.4%;aOR:1.41, 95%CI: 1.18,1.68), live birth rate (60.5% vs. 51.4% aOR:1.49, 95%CI: 1.27,1.76), and a lower risk of miscarriage (14.7% vs. 20.1% aOR: 0.68, 95%CI: 0.53,0.89), hypertensive disorders of pregnancy (6.7% vs. 8.9% aOR: 0.63, 95%CI: 0.42,0.95), and gestational diabetes mellitus (16.7% vs. 20.7% aOR:0.71, 95%CI: 0.53,0.93) than HRT. There were no significant differences in other outcomes such as preterm birth, cesarean delivery, small for gestational age, or large for gestational age between the two endometrial preparation protocols. Subgroup analysis showed that LS had higher live birth rates than HRT in most of the subgroups; in the three subgroups of maternal age ≥ 35 years, menstrual cycle < 35 days, and no insulin resistance, the live birth rates of the two endometrial preparation protocols were comparable. CONCLUSIONS: LS protocol could improve the live birth rate and reduce the incidence of miscarriage, hypertensive disorders of pregnancy and gestational diabetes mellitus in patients with PCOS. LS protocol is suitable for all types of patients with PCOS. LS should be considered the preferred endometrial preparation protocol for women with PCOS.

Conventional IVF is feasible in preimplantation genetic testing for aneuploidy.

PURPOSE: To investigate the feasibility of the application of conventional in vitro fertilization (cIVF) for couples undergoing preimplantation genetic testing for aneuploidies (PGT-A) with non-male factor infertility. METHODS: To evaluate the efficiency of sperm whole-genome amplification (WGA), spermatozoa were subjected to three WGA protocols: Picoplex, ChromInst, and multiple displacement amplification (MDA). In the clinical studies, 641 couples who underwent PGT-A treatment for frozen embryos between January 2016 and December 2021 were included to retrospectively compare the chromosomal and clinical outcomes of cIVF and intracytoplasmic sperm injection (ICSI). Twenty-six couples were prospectively recruited for cIVF and PGT-A treatment between April 2021 and April 2022; parental contamination was analyzed in biopsied samples; and 12 aneuploid embryos were donated to validate the PGT-A results. RESULTS: Sperm DNA failed to amplify under Picoplex and ChromInst conditions but could be amplified using MDA. In frozen PGT-A cycles, no significant differences in the average rates of euploid, mosaic, and aneuploid embryos per cycle between the cIVF-PGT-A and ICSI-PGT-A groups were observed. The results of the prospective study that recruited couples for cIVF-PGT-A treatment showed no paternal contamination and one case of maternal contamination in 150 biopsied trophectoderm samples. Among the 12 donated embryos with whole-chromosome aneuploidy, 11 (91.7%) presented uniform chromosomal aberrations, which were in agreement with the original biopsy results. CONCLUSIONS: Under the Picoplex and ChromInst WGA protocols, the risk of parental contamination in the cIVF-PGT-A cycles was low. Therefore, applying cIVF to couples with non-male factor infertility who are undergoing PGT-A is feasible.

Live birth after letrozole-stimulated cycles versus hormone replacement treatment cycles for the first frozen embryo transfer in women with polycystic ovary syndrome: protocol for a multicentre randomised controlled trial.

INTRODUCTION: Hormone replacement treatment (HRT) is the most commonly used endometrial preparation protocol for frozen embryo transfer (FET) in patients with polycystic ovary syndrome (PCOS). However, studies have found that HRT may increase the risk of hypertensive disorders of pregnancy and some obstetric complications. Letrozole is a new first-line ovulation induction drug for PCOS and can effectively induce spontaneous ovulation by reducing oestrogen levels. However, letrozole is still rarely used in FET and has only been reported in a few studies in Asian populations. High-quality, well-powered randomised controlled trials (RCTs) comparing HRT and letrozole-stimulated protocols are lacking. The aim of this study is to compare the efficacy and safety of two protocols in patients with PCOS. METHODS AND ANALYSIS: This is a multicentre, open-label RCT in four reproductive medical centres in China. In total, 1078 women with PCOS will be randomised (1:1) to the letrozole-stimulated or HRT group in their first FET cycle and their pregnancy and perinatal outcomes during this cycle will be followed up and analysed. The primary outcome is live birth. Secondary outcomes are cycle cancellation rate, biochemical pregnancy, clinical pregnancy, miscarriage, ectopic pregnancy, obstetric and perinatal complications, neonatal complications and birth weight. ETHICS AND DISSEMINATION: Ethical approval was obtained from the Institutional Review Board of Reproductive and Genetic Hospital of CITIC-XIANGYA (LL-SC-2022-001). Written informed consent will be obtained from each participant. The findings will be disseminated through conference presentations and publication in peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT05227391.

Immune profiling and RNA-seq uncover the cause of partial unexplained recurrent implantation failure.

BACKGROUND: Detailed knowledge of the changes in endometrial immune cells during the window of implantation in unexplained recurrent implantation failure (RIF) patients, the functions performed by immune cells, and the interactions between them is largely lacking. This study aimed to classify RIF patients and explore the mechanism through endometrial immune profiling and RNA-seq analysis. METHODS: This study enrolled a total of 172 patients, comprising 144 women with unexplained RIF and 28 fertile women. Endometrial samples were collected using endometrial scratching at the mid-luteal phase before in vitro fertilization treatment or pregnancy. Transcriptome sequencing and immunohistochemical staining of endometrial immune cells including natural killer (NK) cells, macrophages, T cells, and B cells were performed. MAIN OUTCOME MEASURE(S): Comparison of the percentage of endometrial immune cells and the RNA-seq information between RIF patients and fertile control patients. RESULT(S): The proportions of uterine CD56+ uNK cells, CD57+ NKT cells, CD68+ macrophages, and CD19+ B cells were significantly elevated in RIF patients. In addition, the number of positive CD68 glandular lumens was significantly higher in RIF patients than in the fertile group. In addition, based on this result, we classified RIF patients into three categories. CONCLUSION(S): Hyperactivation of endometrial immune cells may be associated with reduced endometrial tolerance and recurrent implantation failure, affecting pregnancy outcomes in RIF patients.

Histological endometrial dating: a reliable tool for personalized frozen-thawed embryo transfer in patients with repeated implantation failure in natural cycles.

OBJECTIVE: To evaluate the clinical availability and stability of histological endometrial dating as a tool for personalized frozen-thawed embryo transfer (pFET) in patients with repeated implantation failure (RIF) in natural cycles. METHODS: A total of 1245 RIF patients were recruited to the present study. All of the patients received an endometrial dating evaluation on day 7 post-ovulation (PO + 7) to guide their first pFET. The second and third pFETs were executed according to histological examination (again employing biopsy) or by reference to previous results. Subsequent pregnancy outcomes for all of the cycles were ultimately tracked. RESULTS: The out-of-phase rate for RIF patients was 32.4% (404/1245) and the expected dating rate (the probability of the expected endometrial dating aligning with repeat biopsy) for endometrial dating reevaluation was as high as 94.3% (50/53). The clinical pregnancy rates of first, second, and third pFETs were 65.3%, 50.0%, and 44.4%, respectively; and the cumulative clinical pregnancy rate attained 74.9% after three transfers. Endometrial dating reevaluations met expectations with more than a 2-year duration in three cases and elicited favorable clinical outcomes. CONCLUSION: We validated the relatively high stability of the histological endometrial dating platform-including the out-of-phase rate and the expected dating rate of reevaluation in patients with RIF-by expanding the sample size. The pFET, based on histological endometrial dating, was of acceptable clinical value and was worthy of promotion in patients with unexplained RIF.

A 1-kb human CDCA8 promoter directs the spermatogonia-specific luciferase expression in adult testis.

Cell division cycle associated 8 (CDCA8) is a component of the chromosomal passenger complex and plays an essential role in mitosis, meiosis, cancer growth, and undifferentiated state of embryonic stem cells. However, its expression and role in adult tissues remain largely uncharacterized. Here, we studied the CDCA8 transcription in adult tissues by generating a transgenic mouse model, in which the luciferase was driven by a 1-kb human CDCA8 promoter. Our previous study showed that this 1-kb promoter was active enough to dictate reporter expression faithfully reflecting endogenous CDCA8 expression. Two founder mice carrying the transgene were identified. In vivo imaging and luciferase assays in tissue lysates revealed that CDCA8 promoter was highly activated and drove robust luciferase expression in testes. Subsequently, immunohistochemical and immunofluorescent staining showed that in adult transgenic testes, the expression of luciferase was restricted to a subset of spermatogonia that were located along the basement membrane and positive for the expression of GFRA1, a consensus marker for early undifferentiated spermatogonia. These findings for the first time indicate that the CDCA8 was transcriptionally activated in testis and thus may play a role in adult spermatogenesis. Moreover, the 1-kb CDCA8 promoter could be used for spermatogonia-specific gene expression in vivo and the transgenic lines constructed here could also be used for recovery of spermatogonia from adult testes.

Ultra-low-coverage genome-wide association study-insights into gestational age using 17,844 embryo samples with preimplantation genetic testing.

BACKGROUND: Very low-coverage (0.1 to 1×) whole genome sequencing (WGS) has become a promising and affordable approach to discover genomic variants of human populations for genome-wide association study (GWAS). To support genetic screening using preimplantation genetic testing (PGT) in a large population, the sequencing coverage goes below 0.1× to an ultra-low level. However, the feasibility and effectiveness of ultra-low-coverage WGS (ulcWGS) for GWAS remains undetermined. METHODS: We built a pipeline to carry out analysis of ulcWGS data for GWAS. To examine its effectiveness, we benchmarked the accuracy of genotype imputation at the combination of different coverages below 0.1× and sample sizes from 2000 to 16,000, using 17,844 embryo PGT samples with approximately 0.04× average coverage and the standard Chinese sample HG005 with known genotypes. We then applied the imputed genotypes of 1744 transferred embryos who have gestational ages and complete follow-up records to GWAS. RESULTS: The accuracy of genotype imputation under ultra-low coverage can be improved by increasing the sample size and applying a set of filters. From 1744 born embryos, we identified 11 genomic risk loci associated with gestational ages and 166 genes mapped to these loci according to positional, expression quantitative trait locus, and chromatin interaction strategies. Among these mapped genes, CRHBP, ICAM1, and OXTR were more frequently reported as preterm birth related. By joint analysis of gene expression data from previous studies, we constructed interrelationships of mainly CRHBP, ICAM1, PLAGL1, DNMT1, CNTLN, DKK1, and EGR2 with preterm birth, infant disease, and breast cancer. CONCLUSIONS: This study not only demonstrates that ulcWGS could achieve relatively high accuracy of adequate genotype imputation and is capable of GWAS, but also provides insights into the associations between gestational age and genetic variations of the fetal embryos from Chinese population.