PEG10 amplification at 7q21.3 potentiates large-cell transformation in cutaneous T-cell lymphoma.

Mycosis fungoides (MF), the most common form of cutaneous T-cell lymphoma, undergo large-cell transformation (LCT) in the late stage, manifesting aggressive behavior, resistance to treatments, and poor prognosis, but the mechanisms involved remain unclear. To identify the molecular driver of LCT, we collected tumor samples from 133 MF patients and performed whole-transcriptome sequencing on 49 advanced-stage MF patients, followed by integrated copy number inference and genomic hybridization. Tumors with LCT showed unique transcriptional programs and enriched expressions of genes at chr7q. Paternally expressed gene 10 (PEG10), an imprinted gene at 7q21.3, was ectopically expressed in malignant T cells from LCT, driven by 7q21.3 amplification. Mechanistically, aberrant PEG10 expression increased cell size, promoted cell proliferation, and conferred treatment resistance by a PEG10/KLF2/NF-κB axis in in vitro and in vivo models. Pharmacologically targeting PEG10 reversed the phenotypes of proliferation and treatment resistance in LCT. Our findings reveal new molecular mechanisms underlying LCT and suggest that PEG10 inhibition may serve as a promising therapeutic approach in late-stage aggressive T-cell lymphoma.

Bioactive Injectable and Self-Healing Hydrogel Via Cell-Free Fat Extract for Endometrial Regeneration.

The damaged endometrium and the formation of fibrosis are key barriers to pregnancy and further lead to infertility. However, how to promote endometrium repair is always a challenge. Here, a bioactive injectable and self-healing hydrogel is developed by physically combination of thiolated polyethylene (PEG), Cu2+ and cell-free fat extract (CEFFE, CF) for endometrial regeneration and fertility. By inheriting the advantages of various active proteins contained in CEFFE, it could induce the overall repair of endometrial microenvironment for intrauterine adhesion (IUA). In vitro, CF@Cu-PEG reduces endometrial cell apoptosis by more than 50%, and increases angiogenesis by 92.8%. In the IUA mouse, injection of CF@Cu-PEG significantly reduces the rate of uterine hydrometra and prevents the formation of endometrial fibrosis. Remarkably, CF@Cu-PEG contributes to the repair of endometrial microstructure, especially increases the number of endometrial pinopodes, significantly improves endometrial receptivity, and increases the pregnancy rate of IUA mice from 7.14% to 66.67%. In summary, through the physically combination of CEFFE and Cu-PEG, the construction of loaded bioactive injectable hydrogel not only inhibits the IUA, but also induces the self-repair of endometrial cells in situ and improves fertility, providing a new strategy for IUA repair in clinical application.

Downregulation of SEPTIN11 inhibits endometrial epithelial cell adhesive function in patients with elevated peripheral blood natural killer cell counts.

RESEARCH QUESTION: What is the underlying mechanism of IVF and embryo transfer (IVF-ET) failure in patients with elevated peripheral blood natural killer cell (pNK) counts? DESIGN: Patients undergoing IVF-ET cycles for tubal obstruction or pelvic adhesion (n = 486) were assigned to three groups: high (CD56+CD16+pNK >30% [n = 49]); medium (15< CD56+CD16+pNK ≤30% [n = 211]); and normal pNK groups (5≤ CD56+CD16+pNK ≤15% [n = 226]). Their general condition, previous pregnancy history and IVF outcomes were compared. Uterine fluid and endometrial tissue from patients in the high and normal pNK groups were collected during the mid-secretory phase and studied to elucidate the molecular mechanism underlying impaired endometrial receptivity. RESULTS: The highest incidence of IVF-ET cycles (P < 0.0001) and biochemical pregnancy losses (P < 0.0001), and lowest implantation and clinical pregnancy rates (both P < 0.0001), were observed in patients with pNK over 30%. No significant difference was found in the number of previous miscarriages and rate of spontaneous miscarriage in IVF outcomes. Lower Septin11 (SEPT11) expression in the uterine fluid and endometrial epithelial cells (EEC), and higher endometrial IFN-γ, was observed in patients with high pNK. Ishikawa cell and human endometrial epithelial cell (HEEC) adhesion was inhibited after SEPT11 knock-down. Elevated IFN-γ decreased the SEPT11 protein levels in Ishikawa cells and HEECs. CONCLUSIONS: CD56+CD16+pNK above 30% may be a threshold for adverse IVF-ET outcomes. Low SEPT11 expression in EEC inhibits cell adhesion, which may cause impaired endometrial receptivity in patients with elevated pNK. The level of SEPT11 in mid-secretory uterine fluid could serve as a non-invasive marker to assess endometrial receptivity in these patients.

Decreased CD44v3 expression impairs endometrial stromal cell proliferation and decidualization in women with recurrent implantation failure.

BACKGROUND: The precise pathogenesis of poor endometrial receptivity in recurrent implantation failure (RIF) remains unclear. This study was aimed at exploring the effects of different CD44 isoforms in the mid-secretory phase endometrium on endometrial receptivity in women with RIF. METHODS: Mid-secretory phase endometrial tissue samples were obtained from the following two groups of women who had undergone IVF: (a) 24 patients with RIF and (b) 18 patients with infertility due to tubal obstruction, who had achieved a successful clinical pregnancy after the first embryo transfer in IVF (control group). Identification of differentially expressed CD44 isoforms in endometrial tissues was assessed using immunohistochemistry, qPCR, and western blotting. Effects of overexpression and knockdown of CD44v3 on proliferation and decidualization of immortalized human endometrial stromal cells (T-HESCs) and primary HESCs were investigated by qPCR and western blot analysis. A heterologous coculture system of embryo implantation was constructed to mimic the process of trophoblast invasion during implantation. RESULTS: The expression of CD44v3 was significantly higher in the mid-secretory phase of endometrial stromal cells than in the proliferation phase, but was notably lower in RIF patients. Knockdown of CD44v3 significantly downregulated cell proliferation both in T-HESCs and HESCs. The expression of decidualization markers, prolactin (PRL) and insulin like growth factor binding protein-1 (IGFBP1), was notably decreased following the knockdown of CD44v3, whereas the expression of both PRL and IGFBP1 increased after its overexpression in HESCs. Furthermore, the CD44v3-knockdown HESCs displayed significant deficiency in supporting trophoblast outgrowth in a coculture system of embryo implantation; however, overexpression of CD44v3 in HESCs promoted trophoblast outgrowth. CONCLUSION: The reduced expression of CD44v3 suppresses the proliferation and decidualization of HESCs, which might play a pivotal role in poor endometrial receptivity in women with RIF.

Increased expression of HMGB1 in the implantation phase endometrium is related to recurrent implantation failure.

BACKGROUND: Impaired endometrial receptivity was the main cause of recurrent implantation failure (RIF); however, its underlying mechanisms had not been elucidated. This study aimed to determine the expression level of high-mobility group box protein 1 (HMGB1) in the endometrium with RIF and its effect on endometrial receptivity. METHODS AND RESULTS: Genome-wide expression profiling, real-time reverse transcription PCR, immunohistochemical staining, western blot, and in vitro assays were performed in this study. We found that HMGB1 expression was significantly decreased in the implantation phase endometrium in the control group (patients with tubal infertility and successfully achieve conception after the first embryo transfer) (P = 0.006). However, the expression levels of HMGB1 mRNA and protein were significantly upregulated during the implantation phase in endometrial tissues obtained from patients with RIF compared to that in the control group (P = 0.001), consistent with the results of the genome-wide expression profiling. Moreover, in vitro assays showed that increased expression of HMGB1 in human endometrial epithelial cells dramatically displayed a marked deficiency in supporting blastocysts and human embryonic JAR cells adhesion, which mimic the process of embryo adhesion. CONCLUSION: These findings strongly indicated that increased HMGB1 levels suppressed the epithelial cell adhesion capability, therefore contributing to impaired endometrial receptivity in patients with recurrent implantation failure, which can be used as a target for the recognition and treatment of recurrent implantation failure in clinical practice.

Down-regulation of S100P induces apoptosis in endometrial epithelial cell during GnRH antagonist protocol.

BACKGROUND: The gonadotropin-releasing hormone (GnRH) antagonist protocol for in vitro fertilization (IVF) often leads to lower pregnancy rates compared to the GnRH agonist protocol. Decreased endometrial receptivity is one reason for the lower success rate, but the mechanisms underlying this phenomenon remain poorly understood. The S100 calcium protein P (S100P) is a biomarker for endometrial receptivity. Both GnRH antagonist and S100P are involved in mediating cell apoptosis. However, the involvement of S100P in reduced endometrial receptivity during the GnRH antagonist protocol remains unclear. METHODS: Endometrial tissue was collected at the time of implantation window from patients undergoing the GnRH agonist (GnRH-a) or GnRH antagonist (GnRH-ant) protocols, as well as from patients on their natural cycles. Endometrial cell apoptosis and expression levels of S100P, HOXA10, Bax, and Bcl-2 were assessed. Ishikawa cells were cultured to evaluate the effects that GnRH antagonist exposure or S100P up- or down- regulation had on apoptosis. RESULTS: Endometrial tissue from patients in the GnRH-ant group showed elevated apoptosis and decreased expression of the anti-apoptotic marker Bcl-2. In addition, endometrial expression of S100P was significantly reduced in the GnRH-ant group, and expression of HOXA10 was lower. Immunofluorescence colocalization analysis revealed that S100P was mainly distributed in the epithelium. In vitro experiments showed that knockdown of S100P in Ishikawa cells induced apoptosis, decreased expression of Bcl-2, while overexpression of S100P caused the opposite effects and decreased expression of Bax. Furthermore, endometrial epithelial cells exposed to GnRH antagonist expressed lower levels of S100P and Bcl-2, increased expression of Bax, and had higher rates of apoptosis. The increased apoptosis induced by GnRH antagonist treatment could be rescued by overexpression of S100P. CONCLUSIONS: We found that GnRH antagonist treatment induced endometrial epithelial cell apoptosis by down-regulating S100P, which was detrimental to endometrial receptivity. These results further define a mechanistic role for S100P in contributing to endometrial apoptosis during GnRH antagonist treatment, and suggest that S100P is a potential clinical target to improve the success of IVF using the GnRH antagonist protocol.

Exosomal miR-146a-5p from Treponema pallidum-stimulated macrophages reduces endothelial cells permeability and monocyte transendothelial migration by targeting JAM-C.

Exosomal microRNAs (miRNAs) transferred between cells have been implicated in modulating the host immune response in microbial infections. In this study, we isolated exosomes from Treponema pallidum (T. pallidum)-stimulated macrophages and detected differential exosomal miRNA expression using both microarrays, and RT-qPCR. A total of 65 differentially expressed miRNAs (35 upregulated and 30 downregulated) were identified. Of all identified miRNAs, miR-146a-5p was one of the most significantly changed miRNAs with high expression in exosomes from T. pallidum-stimulated macrophages. Furthermore, we isolated plasma exosomes from early syphilis patients and healthy controls, and confirmed miR-146a-5p upregulation in the former group. We also show that exosomal miR-146a-5p is efficiently transported into endothelial cells, reducing monocyte transendothelial migration and endothelial permeability by targeting junctional adhesion molecule C (JAM-C). Luciferase reporter assays confirmed binding of exosomal miR-146a-5p to the 3'untranslated region (3'UTR) of JAM-C. We then demonstrated that also exosomes derived from macrophages stimulated by T. pallidum expressed high levels of miR-146a-5p which could be delivered to endothelial cells, and decreased monocyte transendothelial migration by targeting JAM-C. Overall, this work provides novel insights into the mechanism by which T. pallidum hampers inflammatory reactions of the host via a blockade of leukocytes transendothelial migration and endothelial permeability.

High Expression of IKZF2 in Malignant T Cells Promotes Disease Progression in Cutaneous T Cell Lymphoma.

Cutaneous T cell lymphoma is a generally indolent disease derived from skin-homing mature T cells. However, in advanced stages, cutaneous T cell lymphoma may manifest aggressive clinical behaviour and lead to a poor prognosis. The mechanism of disease progression in cutaneous T cell lymphoma remains unknown. This study, based on a large clinical cohort, found that IKZF2, an essential transcription factor during T cell development and differentiation, showed stage- dependent overexpression in the malignant T cells in mycosis fungoides lesions. IKZF2 is specifically over- expressed in advanced-stage mycosis fungoides lesions, and correlates with poor prognosis. Mechanistically, overexpression of IKZF2 promotes cutaneous T cell lymphoma progression via inhibiting malignant cell apoptosis and may contribute to tumour immune escape by downregulating major histocompatibility complex II molecules and up-regulating the production of anti-inflammatory cytokine interleukin-10 by malignant T cells. These results demonstrate the important role of IKZF2 in high-risk cutaneous T cell lymphoma and pave the way for future targeted therapy.

Transvaginal ovarian drilling followed by controlled ovarian stimulation from the next day improves ovarian response for the poor responders with polycystic ovary syndrome during IVF treatment: a pilot study.

BACKGROUND: Poor response patients with PCOS who are not susceptible to gonadotropin stimulation are more likely to have canceled cycles or poor clinical outcomes during IVF treatment. However, some limitations exist in the present therapies. In this study, we evaluated the effects of using the transvaginal ovarian drilling (TVOD) followed by controlled ovarian stimulation (COS) from the second day of these poor responders. METHODS: During IVF, 7 poor responders with PCOS and 28 PCOS patients (14 normal and 14 high responders) were recruited. All patients received COS with the gonadotropin-releasing hormone antagonist protocol. For the poor responders, after undergoing 10 to 14 days of ovulation induction with no response, the TVOD was applied and then ovarian stimulation was performed from the next day at the same gonadotropin dose. Serum samples during COS and follicular fluid samples from the dominant follicles on the oocyte pick-up (OPU) day in all three groups were collected. Besides, follicular fluid from small follicles (diameter < 1 cm) in the normal and high responders on the OPU day and those in the poor responders on the TVOD day were gathered. Hormonal levels were examined in all samples using immunometric assays. RESULTS: All the poor responders restored ovary response after receiving TVOD. There was no significant difference in the stimulation duration, total gonadotrophin dose used and the clinical outcomes among the three groups. The body mass index, serum and follicular levels of anti-Müllerian hormone (AMH) and testosterone in poor responders were higher than those in the other two groups, and the application of TVOD significantly decreased the levels of AMH and testosterone in both serum and follicular fluid. CONCLUSIONS: TVOD followed by ovulation induction from the next day is effective and convenient for poor responders with PCOS. The decline of AMH and testosterone resulted from TVOD may be the main reason resulting in the recovery of ovary sensitivity to gonadotropins. The small sample size is the primary limitation of this study, future studies using a large population cohort and monitoring the long-term outcomes of this strategy will be required. TRIAL REGISTRATION: ChiCTR1900023612. Registered 04 June 2019-Retrospectively registered.

Promotion of the occurrence of endometrioid carcinoma by S100 calcium binding protein P.

BACKGROUND: Endometrial cancer, one of the most common malignant tumors, is a serious threat to women's health. Endometrial hyperplasia is a precursor of endometrial cancer. S100 calcium binding protein P (S100P) has been found to play important roles in many types of cancer. The present study aimed to investigate the expression of S100P in endometrial cancer and its precursor lesions, and to explore the possible mechanisms. METHODS: We collected paraffin sections of normal endometrium, simple and complex non-atypical hyperplasia, atypical hyperplasia, and endometrioid carcinoma. The expression of S100P in endometrial cancer and its precancerous lesions was observed using immunohistochemistry. We also cultured primary endometrial cells and endometrial cancer cell lines (Ishikawa and RL95-2), and observed the expression of S100P in these cells. Laser confocal microscopy was used to observe the co-localization of S100P and its interacting protein Ezrin in RL95-2 cells. We employed lentiviruses to knockdown and overexpress S100P and then detected the F-actin distribution and cell invasion using phalloidin staining and Transwell assays. RESULTS: There was a gradual increase in the S100P signal as the disease progressed from normal endometrium and simple non-atypical hyperplasia, to complex non-atypical hyperplasia, atypical hyperplasia, and then to endometrial cancer. S100P was mainly distributed in the cytoplasm and co-localized with Ezrin in endometrial cancer cells. After knocking down S100P, F-actin aggregated in the nucleus or to the local cell membrane. Furthermore, knockdown of S100P in Ishikawa cells decreased their cell invasion capability. Meanwhile, S100P overexpression in endometrial stromal cells increased cell invasion. CONCLUSIONS: These data suggested that S100P might be involved in the occurrence and development of endometrial cancer via interaction with Ezrin and re-organization of F-actin to promote cell invasion.

Evaluation of IL-17A, IL-17F, IL-23R, VDR, CCL2, CCL5, CCR2, and CCR5 gene polymorphisms and expression in Chinese individuals with syphilis.

Syphilis is a sexually transmitted disease caused by the infection of Treponema pallidum subspecies pallidum. T-helper type 17-related genes, vitamin D receptor (VDR) gene, and chemokine/chemokine receptor genes are crucial in microbial infection. A total of 16 single-nucleotide polymorphisms (SNPs) in eight genes (interleukin [IL]-17A, IL-17F, IL-23R, VDR, C-C motif chemokine ligand [CCL] 2, CCL5, C-C chemokine receptor [CCR] 2, and CCR5) were analyzed in 188 patients with syphilis and 216 healthy controls. The results showed a strong correlation of IL-17A rs2275913 (AA vs AG + GG: odds ratio [OR], 1.78; 95% confidence interval [CI], 1.09 to 2.92; P = 0.020; A vs G: OR, 1.33; 95% CI, 1.01 to 1.76; P = 0.043) and rs3819024 (GG vs AA + GA: OR, 1.76; 95% CI, 1.06 to 2.91; P = 0.028; G vs A: OR, 1.36; 95% CI, 1.03 to 1.80; P = 0.030) with syphilis. In haplotype analysis, IL-17A rs2275913A/rs3819024G showed a risk effect (OR, 1.38; 95% CI, 1.04 to 1.82; P = 0.026), whereas IL-17A rs2275913G/rs3819024A showed a protective effect (OR, 0.76; 95% CI, 0.57 to 0.998; P = 0.048). The expression levels of IL-17A messenger RNA (mRNA) in peripheral blood mononuclear cells and IL-17A secretion in plasma were further examined. No significant differences were found between patients with syphilis and healthy controls. The study also explored whether IL-17A rs2275913 and rs3819024 were associated with the expression of IL-17A mRNA and IL-17A secretion in patients with syphilis. Similar negative results were found. In conclusion, the polymorphisms of IL-17A rs2275913 and rs3819024 and the haplotype containing these two SNPs influenced the susceptibility to syphilis in a Han Chinese population.

Decreased PECAM1-mediated TGF-ß1 expression in the mid-secretory endometrium in women with recurrent implantation failure.

STUDY QUESTION: Is recurrent implantation failure (RIF) associated with decreased expression of platelet and endothelial cell adhesion molecule 1 (PECAM1) and transforming growth factor ß1 (TGF-ß1) in the endometrium during the implantation window? SUMMARY ANSWER: The present study demonstrates that the expression of PECAM1 and TGF-ß1 is significantly decreased in the mid-secretory endometrium in women with RIF, which may account for embryo implantation failure. WHAT IS KNOWN ALREADY: RIF has become a bottleneck issue that hampers the improvement of pregnancy rates in IVF-embryo transfer (IVF-ET). The causes of RIF are complex and may involve the dysregulation of various growth factors, metabolites, and inflammatory cytokines. At present, the precise pathogenesis of RIF has not been elucidated. STUDY DESIGN, SIZE, DURATION: This was a prospective case-control study. Endometrial tissue samples were obtained from January 2014 to December 2016 from two groups of women who had undergone IVF (RIF group, 22 women who underwent ≥3 ETs including a total of ≥4 good-quality embryos without pregnancy, control group, 18 women who conceived in their first treatment cycle). At the same time, samples were obtained from 18 women with infertility secondary to tubal factor in the early proliferative, late proliferative and mid-secretory phases of the menstrual cycle (n = 6 per group). Samples used for isolation of primary human endometrial epithelial cells and stromal cells (HEECs and HESCs) were collected in December 2017 from six women with infertility secondary to tubal factor. PARTICIPANTS/MATERIALS, SETTING, METHODS: We investigated gene expression using integrative whole genome expression microarray analysis, including differentially expressed gene screening, principal component analysis, and functional enrichment analysis. RT-qPCR, western blotting, immunohistochemistry, immunofluorescence co-localization analysis and short hairpin RNA (shRNA) plasmid transfection in Ishikawa cell line, HEECs and HESCs were used to investigate the expression of PECAM1 and TGF-ß1. MAIN RESULTS AND THE ROLE OF CHANCE: Integrative data mining of whole-genome expression profiles identified cell adhesion as a key regulator in RIF. Database retrieval and literature review screened several novel cell adhesion-related genes that might participate in embryo implantation, which include PECAM1, intercellular adhesion molecule 2 (ICAM2), integrin subunit ß2 (ITGB2), selectin P (SELP) and TEK receptor tyrosine kinase (TEK). Among these targets, the mRNA and protein levels of PECAM1 were significantly lower in the RIF group than those in the control group. During the menstrual cycles of women with secondary infertility, the protein expression level of PECAM1 was the lowest in early proliferative phase, slightly increased in late proliferative phase and was the highest in mid-secretory phase. While the expression level of HOXA10, an endometrial receptivity marker, kept at a low level in early proliferative phase and increased in late proliferative phase, then maintained at a high level in the mid-secretory phase. Furthermore, TGF-ß1, mediated by PECAM1, was also decreased significantly in the RIF group. Using shRNA-based approach, we demonstrated that the depletion of PECAM1 significantly decreased the expression of TGF-ß1 in Ishikawa cells, as well as in primary HEECs and HESCs. These results indicated that PECAM1 and TGF-ß1 might play a pivotal role in modulating endometrial receptivity. LIMITATIONS REASONS FOR CAUTION: Although we have shown that PECAM1 and TGF-ß1 were down-regulated in the women with RIF, the molecular mechanism of the effect of the factors on the endometrial receptivity remain unclear. WIDER IMPLICATIONS OF THE FINDINGS: Our findings provide insight into the contribution of PECAM1 and TGF-ß1 in regulating implantation, which could be used to develop potential therapeutic methods for RIF. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by grants from the National Natural Science Foundation of China (Nos. 81771656 and 81370763), Special fund for clinical research of the Chinese Medical Association (No. 16020480664), and the Merck Serono China Research Fund for Fertility Agreement. The authors have no competing interests.

Increased AIF-1-mediated TNF-α expression during implantation phase in IVF cycles with GnRH antagonist protocol.

STUDY QUESTION: Is allograft inflammatory factor-1 (AIF-1), a cytokine associated with inflammation and allograft rejection, aberrantly elevated in in vitro fertilization (IVF) cycles with gonadotropin-releasing hormone (GnRH) antagonist protocol with potential effects on endometrial receptivity? SUMMARY ANSWER: Our findings indicated AIF-1 is increased in IVF cycles with GnRH antagonist protocol and mediates greater TNF-α expression during implantation phase, which may be unfavorable for embryo implantation. WHAT IS KNOWN ALREADY: Studies have shown that GnRH antagonist protocol cycles have lower implantation and clinical pregnancy rates than GnRH agonist long protocol cycles. Endometrial receptivity but not embryo quality is a key factor contributing to this phenomenon; however, the mechanism is still unknown. STUDY DESIGN, SIZE, DURATION: Implantation and pregnancy rates were studied in 238 patients undergoing their first cycle of IVF/ICSI between 2012 and 2014. Forty of these patients opted to have no fresh embryo replacement and were divided into two equal groups: (i) GnRH antagonist protocol and (ii) GnRH agonist long protocol, group 3 included 20 infertile women with a tubal factor in untreated cycles. During the same interval, endometrial tissues were taken from 18 infertile women with a tubal factor in the early proliferative phase, late proliferative phase, and mid-secretory phase of the menstrual cycle (n = 6/group). PARTICIPANTS/MATERIALS, SETTING, METHODS: Microarray analysis, RT-qPCR, Western blot analysis, immunohistochemistry were used to investigate the expression levels of AIF-1 and the related cytokines (TNF-α, IL1ß, IL1RA, IL6, IL12, IL15 and IL18). The effect of AIF-1 on uterine receptivity was modeled using in vitro adhesion experiments (coculture of JAR cells and Ishikawa cells). MAIN RESULTS AND THE ROLE OF CHANCE: The expression of AIF-1 was the highest in early proliferative phase, decreasing thereafter in the late proliferative phase, and almost disappearing in the mid-secretory phase, indicating that low AIF-1 expression might be important for embryo implantation during implantation phase. Microarray results revealed that AIF-1 was upregulated in the antagonist group compared with the control group (fold change [FC] = 3.75) and the agonist (FC = 2.20) group. The raw microarray data and complete gene expression table were uploaded to GEO under the accession number of GSE107914. Both the mRNA and protein expression levels of AIF-1 and TNF-α were the higher in the antagonist group than in the other two groups (P < 0.05) which did not differ significantly (P > 0.05). The protein levels of TNF-α in both Ishikawa cells and primary endometrial cells were significantly increased (P < 0.05) at 96 h after transfection with the AIF-1 expression vector, indicating that TNF-α was mediated by AIF-1 in endometrial cells. Overexpression of AIF-1 in Ishikawa cells inhibited adhesion of JAR cells to them. Thus, increased AIF-1 might inhibit adhesion during implantation via raised TNF-α. LIMITATIONS REASONS FOR CAUTION: The sample size of the microarray was small, which might weaken the accuracy of our results; however, the sample size of RT-qPCR and the Western blotting assays were sufficient to compensate for this deficiency in our study. In addition, the aberrant AIF-1 and thus TNF-α expression is one of many factors that may contribute to limiting implantation success. Therefore, further extensive in vitro mechanistic and in vivo animal studies are needed to assess the actual functional impact of this pathway. WIDER IMPLICATIONS OF THE FINDINGS: Anti-TNF-α therapy might mitigate the adverse effects of GnRH antagonist on endometrial receptivity and improve the implantation rate in GnRH antagonist protocols in IVF. STUDY FUNDING/COMPETING INTERESTS: This work was supported by grants from the National Natural Science Foundation of China, Grant numbers 81771656 and 81370763; Clinical research special fund of Chinese Medical Association, Grant number 16020480664; Shanghai Jiao Tong University Medicine-Engineering Fund, Grant number YG2017ZD11 and YG2017MS57; and the Merck-Serono China Research Fund for Fertility Agreement. P.C.K.L. is supported by a Canadian Institutes of Health Research Foundation Scheme Grant 143317. None of the authors has any competing interests.

Comparison of clinical outcomes among dual ovarian stimulation, mild stimulation and luteal phase stimulation protocols in women with poor ovarian response.

This study aimed to determine whether consecutive ovarian stimulation in follicular and luteal phases within a single menstrual cycle (dual stimulation) is achievable and superior to conventional stimulation for poor ovarian responders (PORs). Data of 260 PORs were retrospectively collected and divided into three groups. Group A comprised of cycles with dual ovarian stimulation (n = 76), which were divided into two subgroups (follicular [group A-F] and luteal phase stimulation [group A-L]); group B comprised of cycles with ovarian stimulation that was performed only in the luteal phase (n = 52). Group C comprised of mild ovarian stimulation cycles (n = 132). Baseline parameters were not different among the three groups. The numbers of oocytes and embryo obtained were less in group A-F than group B and C, while group A overall had significantly more oocytes and viable embryo retrieved than did group B and C. Group A-L consumed significantly less gonadotropin than group B, without compromising the number of retrieved oocytes and embryo. The pregnancy outcomes of transfer of embryo from different stimulation phases were similar. We conclude that dual ovarian stimulation protocol is effective and potentially optimal for PORs.

The serum follicle stimulating hormone-to-luteinizing hormone ratios can predict assisted reproductive technology outcomes in women undergoing gonadotropin releasing hormone antagonist protocol.

Background: The basal follicle stimulating hormone (FSH)/luteinizing hormone (LH) ratio is a useful predictor of ovarian response. In this study, we investigated whether the FSH/LH ratios during the entire controlled ovarian stimulation (COS) can be used as effective predictors of outcomes in women undergoing in vitro fertilization (IVF) treatment using the gonadotropin releasing hormone antagonist (GnRH-ant) protocol. Methods: A total of 1,681 women undergoing their first GnRH-ant protocol were enrolled in this retrospective cohort study. A Poisson regression model was used to analyze the association between the FSH/LH ratios during COS and embryological outcomes. Receiver operating characteristic analysis was performed to determine the optimal cutoff values for poor responders (≤ 5 oocytes) or poor reproductive potential (≤ 3 available embryos). A nomogram model was constructed to provide a tool for predicting the cycle outcomes of individual IVF treatments. Results: The FSH/LH ratios (at the basal day, stimulation day 6 (SD6) and trigger day) were significantly correlated with the embryological outcomes. The basal FSH/LH ratio was the most reliable predictor of poor responders with a cutoff value of 1.875 (area under the curve (AUC) = 72.3%, P < 0.05), or of poor reproductive potential with a cutoff value of 2.515 (AUC = 66.3%, P < 0.05). The SD6 FSH/LH ratio predicted poor reproductive potential with a cutoff value of 4.14 (AUC = 63.8%, P < 0.05). The trigger day FSH/LH ratio predicted poor responders with a cutoff value of 9.665 (AUC = 63.1%, P < 0.05). The basal FSH/LH ratio, combined with the SD6 and trigger day FSH/LH ratios, slightly increased these AUC values and improved the prediction sensitivity. The nomogram provides a reliable model with which to assess the risk of poor response or poor reproductive potential directly based on the combined indicators. Conclusions: FSH/LH ratios are useful predictors of poor ovarian response or reproductive potential throughout the entire COS with the GnRH antagonist protocol. Our findings also provide insights into the potential for LH supplementation and regimen adjustment during COS to achieve improved outcomes.

The effect of human cumulus cells on the maturation and developmental potential of immature oocytes in ICSI cycles.

PURPOSE: To investigate the effect of human cumulus cells on the maturation and developmental potential of immature oocytes in ICSI cycles. METHODS: Immature oocytes were randomly divided into two groups: the cumulus-denuded oocyte group (group A) and the cumulus-intact oocyte group (group B). Only oocytes that reached metaphase II (MII) stage after in vitro maturation were used in the ICSI procedure. In vivo mature sibling MII oocytes served as the control group. Maturation rate, fertilization rate, embryo quality and developmental potential were examined. RESULTS: There was no significant difference in maturation rate between group A (68.16%) and group B (70.49%; P > 0.05). The total fertilization rate among the three groups was comparable (P > 0.05), while the zygotes with two pronuclei in group A (74.59%) or group B (75.97%) were significantly lower than those in control group (84.29%; P < 0.05). The available embryo rate in group A (11.49%) was markedly lower than that in group B (27.66%; P < 0.05), and both of them were significantly lower than that in control group (62.38%; P < 0.05). The proportion of ≥6-cell embryos in group B (45.74%) was notably higher than in group A (26.44%; P < 0.05), and both were markedly lower than in control group (65.92%; P < 0.05). The proportion of embryos with <10% fragmentation in group A (13.79%) was significantly lower than in group B (29.79%; P < 0.05), and both were notably lower than in control group (42.98%; P < 0.05). CONCLUSIONS: The presence of cumulus cells surrounding the immature oocytes during IVM before ICSI had no influence on nuclear maturation and fertilization, but leads to better subsequent embryonic development. This is perhaps mediated by an improvement in cytoplasmic maturation.

Ectopic pregnancy and failed oocyte retrieval during in vitro fertilization stimulation: Two case reports.

BACKGROUND: Due to a slight rise in beta-human chorionic (ß-hCG) levels that are undetectable, and vaginal bleeding that is similar to regular menstruation, ectopic pregnancy (EP) that occurs during the expected menstrual cycle prior to ovulation induction as part of in vitro fertilization (IVF) is likely to be undiagnosed. We present two cases of unexpected EP and emphasize the importance of the ß-hCG assay when an unexplained increase in progesterone is present prior to the triggering of ovulation during controlled ovarian stimulation (COS). CASE SUMMARY: A 26-year-old woman with primary infertility and a 31-year-old woman with secondary infertility. Both patients sought IVF treatment due to fallopian tube obstruction and underwent COS using the gonadotropin-releasing-hormone (GnRH)-antagonist protocol. In the late stage of COS, progesterone levels in both patients significantly increased, and luteinizing hormone levels decreased, followed by oocyte retrieval failure. A right salpingectomy was performed and tubal ectopic pregnancy was diagnosed by pathology in the first patient, and the second patients was diagnosed with a suspected EP abortion because her ß-hCG levels declined to 12.5 mIU/mL. After full recovery for 2 mo, the first patient entered a new IVF treatment cycle with a GnRH-antagonist regimen and successfully achieved eight oocytes and three viable embryos. After 6 mo, the second patient received another COS treatment with a progestin-primed ovarian stimulation protocol and successfully achieved nine oocytes and five viable embryos. CONCLUSION: ß-hCG levels in the initial and midterm phases of COS must be considered in patients with unusual hormone dynamics.

Cell-Free Fat Extract Improves Ovarian Function and Fertility in Mice With Advanced Age.

The reduction in the quantity and quality of oocytes is the major factor affecting fertility in women with advanced age, who tend to experience delayed childbearing and declined fertility rate. However, effective therapeutic strategies to combat this decrease in ovarian function are lacking in clinical practice. Thus, identifying a new method to rescue ovarian function and improve reproduction in natural age-related decline in fertility is necessary. Cell-free fat extract (CEFFE) has been verified to possess diverse active proteins exerting anti-aging and proliferation-promoting effects. Nonetheless, whether CEFFE can rescue the decline in aged-related ovarian function and improve the fertility of females with advanced age remains unclear. In this study, a natural aging mouse model, exhibiting similarities to the physiological changes of ovarian senescence, was used to observe the anti-aging effect of CEFFE on ovarian functions. We found that CEFFE, injected via the veins, could recover the levels of the sex hormone, increase angiogenesis and the number of growth follicles in the natural aging mice model. Moreover, CEFFE promoted the development of embryos and increased the litter size of aged mice. Transcriptome analysis of the aged mouse ovaries revealed that CEFFE treatment upregulated the expression of genes involved in the repair of DNA damage. And both in vivo and in vitro experiment proved that CEFFE improved the function of granulosa cells, including promoting proliferation, alleviating senescence, and rescuing DNA damage in aged granulosa cells. Collectively, our study implied that CEFFE improved the ovarian function and fertility of naturally aging mice by ameliorating the overall microenvironment of ovary, which provided a theoretical basis for new anti-aging therapeutic strategies for cell-free therapy in ovaries.