POI-associated EIF4ENIF1 mutations exhibit impaired translation regulation abilities.

Various genetic variants have been found to be associated with the clinical onset of premature ovarian insufficiency (POI). However, when measured in vitro, the functional influence of the variants can be difficult to determine. By whole-exome sequencing (WES) of 93 patients with sporadic POI, we found a missense variant c.623G > A;p.R208H in the EIF4ENIF1 gene. In silico prediction of the variant using different algorithms suggested it might be a damaging variant. We compared the property of EIF4ENIF1 R208H and Q842P, a POI-related mutant that we reported previously, with wildtype (WT) protein using 293FT cells in vitro. Surprisingly, a change in subcellular distribution and granule forming ability (Q842P) and nuclear import capacity (R208H) was not observed, despite domain prediction evidences. Since EIF4ENIF1 was reported to inhibit translation, we employed T&T-seq, a translation-transcription dual-omics sequencing method, to profile gene expression upon overexpression of EIF4ENIF1 WT and mutants. EIF4ENIF1 WT overexpression group exhibited significantly (P < 0.0001) lower translation efficiency (TE) than empty vector or GFP overexpression control group. Surprisingly, EIF4ENIF1 Q842P overexpression failed to repress global translation, showing an overall TE significantly higher than WT group. Overexpression R208H significantly (P < 0.0001) lowered the overall TE, whereas exhibiting a reduced translation inhibitory effect on high-TE genes (TE > 2 in GFP control group). Several fertility-associated genes, such as AMH in Q842P group and SERPINE1 and THBS1 in R208H group, was translationally up-regulated in mutant groups versus WT control, suggesting a potential mechanism of mutated EIF4ENIF1 causing POI via impaired translation repression. It is further proposed that T&T-seq can be a sensitive evaluation tool for the measurement of functional alteration by variants in many other translational regulator genes, not only EIF4ENIF1, helping to eliminate misinterpretation of clinical significance of genetic variants.

TP63 truncating mutation causes increased cell apoptosis and premature ovarian insufficiency by enhanced transcriptional activation of CLCA2.

BACKGROUND: Premature ovarian insufficiency (POI) is a severe disorder leading to female infertility. Genetic mutations are important factors causing POI. TP63-truncating mutation has been reported to cause POI by increasing germ cell apoptosis, however what factors mediate this apoptosis remains unclear. METHODS: Ninety-three patients with POI were recruited from Beijing Obstetrics and Gynecology Hospital, Capital Medical University. Whole-exome sequencing (WES) was performed for each patient. Sanger sequencing was used to confirm potential causative genetic variants. A minigene assay was performed to determine splicing effects of TP63 variants. A TP63-truncating plasmid was constructed. Real-time quantitative PCR, western blot analyses, dual luciferase reporter assays, immunofluorescence staining, and cell apoptosis assays were used to study the underlying mechanism of a TP63-truncating mutation causing POI. RESULTS: By WES of 93 sporadic patients with POI, we found a 14-bp deletion covering the splice site in the TP63 gene. A minigene assay demonstrated that the 14-bp deletion variant led to exon 13 skipping during TP63 mRNA splicing, resulting in the generation of a truncated TP63 protein (TP63-mut). Overexpression of TP63-mut accelerated cell apoptosis. Mechanistically, the TP63-mut protein could bind to the promoter region of CLCA2 and activate the transcription of CLCA2 several times compared to that of the TP63 wild-type protein. Silencing CLCA2 using a specific small interfering RNA (siRNA) or inhibiting the Ataxia Telangiectasia Mutated (ATM) pathway using the KU55933 inhibitor attenuated cell apoptosis caused by TP63-mut protein expression. CONCLUSION: Our findings revealed a crucial role for CLCA2 in mediating apoptosis in POI pathogenesis, and suggested that CLCA2 is a potential therapeutic target for POI.

Divergent Associations Between Serum Androgens and Ovarian Reserve Markers Revealed in Patients With Polycystic Ovary Syndrome.

Background: The role of excess androgen in ovarian reserve remains unclear in patients with polycystic ovary syndrome (PCOS). Our study highlights the associations of serum androgen levels and ovarian reserve markers in PCOS and non-PCOS women. Methods: Totally 584 menstrual abnormalities women of 20-45 years were retrospectively evaluated at the Beijing Obstetrics and Gynecology Hospital between January 2021 to October 2021. The enrolled patients were classified into two groups: the PCOS group (n=288) and the non-PCOS group (n=296) based on the Rotterdam consensus for PCOS diagnosis. The serum androgens, including testosterone (T), free testosterone (FT, calculated), bioavailable testosterone (Bio-T, calculated), androstenedione (A2), dihydrotestosterone (DHT), dehydroepiandrosterone (DHEA), and dehydroepiandrosterone sulfate (DHEAS) were assessed with an in-house developed liquid chromatography tandem mass spectrometry (LC-MS/MS) method. The associations between the serum androgens and the hormone markers commonly used for evaluating ovarian reserve function, such as anti-mullerian hormone (AMH) and the ratio of luteinizing hormone (LH)/follicle stimulating hormone (FSH) were explored. Results: The serum T, FT, Bio-T, A2, DHT, DHEA, DHEAS, AMH and LH/FSH of the PCOS group were 51.7 ± 23.2 ng/dL/mL, 8.5 ± 5.0 pg/mL, 210.1 ± 127.7 pg/mL, 1.9 ± 0.8 ng/mL, 0.2 ± 0.1 ng/mL, 6.4 ± 4.2 ng/mL, 2431.0 ± 1030.7 ng/mL, 6.7 ± 3.8 ng/mL, and 1.8 ± 1.4 respectively, which were significantly higher than those in the non-PCOS group (p<0.05). In the group of PCOS patients, T and A2 levels were positively associated with AMH in both multivariate linear regression analysis and Pearson's correlation analysis. Similar but weaker associations were observed in the non-PCOS patients. In the PCOS patients with hyperandrogenemia (HA), the AMH level was significantly higher in the subjects with T increased than in the subjects with non-T androgen(s) increased (A2, DHT, DHEA or DHEAS). Conclusions: The serum androgen levels are positively associated with ovarian reserve markers in both of the PCOS and the non-PCOS patients in our study. In the PCOS group, the highest AMH level was observed in the subjects with the T elevation subgroup, suggesting that T is more closely related with the increase of AMH when compared with other androgens investigated.

Extensive serum biomarker analysis in the prethrombotic state of recurrent spontaneous abortion.

The prethrombotic state (PTS) is a possible cause of recurrent spontaneous abortion (RSA). The aim of this study was to identify serum biomarkers for the detection of RSA with PTS (PSRSA). A Quantibody array 440 was used to screen novel serum-based biomarkers for PSRSA/NRSA (RSA without PTS). Proteins differentially expressed in PSRSA were analysed using bioinformatics methods and subjected to a customized array and enzyme-linked immunosorbent assay (ELISA) validation. We used receiver operating characteristic to calculate diagnostic accuracy, and machine learning methods to establish a biomarker model for evaluation of the identified targets. 20 targets were selected for validation using a customized array, and seven targets via ELISA. The decision tree model showed that IL-24 was the first node and eotaxin-3 was the second node distinguishing the PSRSA and NRSA groups (an accuracy rate of 100% and an AUC of 1). Epidermal growth factor (EGF) as the node distinguished the PSRSA and NC groups (an accuracy rate of 100% and an AUC of 1). EGF as the node distinguished the NRSA and NC groups (an accuracy rate of 96.5% and an AUC of 0.998). Serum DNAM-1, BAFF, CNTF, LAG-3, IL-24, Eotaxin-3 and EGF represent a panel of promising diagnostic biomarkers to detect the PSRSA.

Chinese herbal decoction of Wenshen Yangxue formula improved fertility and pregnancy rate in mice through PI3K/Akt signaling.

OBJECTIVE: Traditional Chinese medicine (TCM) is an effective management to infertility. The association between TCM-mediated fertility and inhibition of phosphatidylinositol-3-kinase (PI3K) would be investigated. METHODS: Institute of Cancer Research mice were treated with three herbal decoctions, named Wenshen Yangxue formula, Wenshen formula, and Yangxue formula, plus with human gonadotropins. PI3K inhibitor wortmannin was administrated to half of mice. Some index such as body weight, fertility ability would be investigated. The expression of P13K/Akt signaling was detected by using Western blot analysis. RESULTS: No difference was observed in body weight among groups. Mice receiving the administration of human gonadotropins and herbal decoctions showed increased follicle numbers, percentage of fertilization, and promoted embryonic development. The treatment of Wenshen Yangxue formula decoction showed the highest efficiency, significant higher than Wenshen and Yangxue formulas. And increased the expression of p-PI3K and p-Akt proteins. CONCLUSION: These results suggested the herbal decoctions promoted the fertilization of mice, which was related to the charge of PI3K/Akt activation.

Wenshen Yangxue decoction improves endometrial receptivity recovery and promotes endometrial angiogenesis in a rat model.

CONTEXT: Wenshen Yangxue decoction (WSYXD) is a famous traditional Chinese medicine (TCM) formula and has been used in infertility treatment, but the exact mechanism is still unknown. OBJECTIVES: To determine if WSYXD improves endometrial receptivity recovery and promotes endometrial angiogenesis in a rat model. MATERIALS AND METHODS: A total of 100 proestrus female SPF Wistar rats were randomly assigned into five groups: control (saline), model (saline and hydroxyurea solution), high (5.2/100 g), middle (2.6/100 g) and low (1.3/100 g) WSYXD dose groups for 10 d. The microvessel densities, endometrial microstructure, as well as blastocysts number, were observed, followed by detection of angiogenesis-related gene/protein expression by immunohistochemistry, western blot and quantitative real-time polymerase chain reaction (RT-PCR), respectively. RESULTS: Compared with the model group, the blastocyst number in WSYXD middle and high groups were significantly increased (4.50 ± 3.11 vs. 13.00 ± 2.12, 14.00 ± 1.83, p < 0.01). Lower MVD can be found in the model group (4.7) when compared with the normal control (13.7), middle (8.4) and high (9.7) dose groups. Additionally, significant differences were observed in VEGF, HIF-1α, p-AKT, p-PI3K, Ang1 and Ang2 (all p < 0.01) among different groups. DISCUSSION AND CONCLUSIONS: In conclusion, WSYXD could help endometrial receptivity recovery and promote endometrial angiogenesis through PI3K, HIF-1α signalling and VEGF expression regulation. This study provides molecular evidence for application of WSYXD in the clinic and promotes new drug development from TCM.

Serum biomarker analysis in patients with recurrent spontaneous abortion.

Recurrent spontaneous abortion (RSA) occurs in 1­5% of parturients. The sustained therapy and research for RSA is expensive, which is a serious issue faced by both patients and doctors. The aim of the present study was to detect protein expression profiles in the serum of RSA patients and healthy controls, and to identify potential biomarkers for this disease. A 1,000­protein microarray consisting of a combination of Human L­507 and L­493 was used. The microarray data revealed that eight serum protein expression levels were significantly upregulated and 143 proteins were downregulated in RSA patients compared with the healthy controls. ELISA individually validated 5 of these 151 proteins in a larger cohort of patients and control samples, demonstrating a significant decrease in insulin­like growth factor­binding protein­related protein 1 (IFGBP­rp1)/IGFBP­7, Dickkopf­related protein 3 (Dkk3), receptor for advanced glycation end products (RAGE) and angiopoietin­2 levels in patients with RSA. Sensitivity and specificity analyses were calculated by a receiver operating characteristics curve, and were revealed to be 0.881, 0.823, 0.79 and 0.814, with diagnostic cut­off points of 95.44 ng/ml for IFGBP­rp1, 32.84 ng/ml for Dkk3, 147.27 ng/ml for RAGE and 441.40 ng/ml for angiopoietin­2. The present study indicated that these four proteins were downregulated in RSA samples and may be useful as biomarkers for the prediction and diagnosis of RSA. Subsequent studies in larger­scale cohorts are required to further validate the diagnostic value of these markers.

[Effect of Wenshen Yangxue Recipe on Inhibin-Activin-Follistatin System in Anovulatory Rats].

Objective To explore the effect of Wenshen Yangxue Recipe (WYR) on inhibin-ac- tivin-follistatin (INH-ACT-FS) system and gonadal hormone level in anovulatory rats. Methods Anovula- tory rat model was established in 76 rats (9 days old) by subcutaneous injecting testosterone propionate (1. 25 mg/0. 05 mL for each rat) from the nape. Totally 58 successfully modeled rats were divided into 5 groups according to random digit table, i.e., the model group (n =10), the Western medicine (WM) group (n =12), high, middle, and low dose WYR groups (n =12). Besides, another ten 22-day old rats were recruited as a normal group. Distilled water was daily administered to rats in the normal group and the model group by gastrogavage. Clomiphene citrate (0. 58 mg/100 g) was daily administered to rats in the WM group for 5 successive days. WYR at 5. 2, 2. 6, 1. 3 mg/100 g was daily administered to rats in high, middle, and low dose WYR groups for 21 successive days. Levels of follicular stimulating hormone (FSH) , luteinizing hormone (LH) , estradiol (E2) , progesterone (P) , and prolactin (PRL) were detected using radioimmunoassay. Contents of inhibin (INH) , activin (ACT) , and follistatin (FS) were measured using ELISA. Results Compared with the normal group, serum levels of FSH and LH increased, and P level decreased in the model group (P <0. 05) ; INH level decreased and FS level increased in the model group (P<0. 05). Compared with the model group, serum FSH level decreased in the WM group and 3 WYR groups, P level decreased in the WM group (P <0. 05); INH increased and FS levels decreased in the WM group and 3 WYR groups; ACT level increased in the high dose WYR group, with statistical differ- ence (P <0. 05). Conclusion WYR promoted follicular development possibly through regulating INH- ACT-FS system and gonadal hormone level.

Regulation and control of wenshen yangxue granule combined with clomifene citrate on INH-ACT-FS system in patients with follicular maldevelopment infertility / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To observe the clinical curative effect of Wenshen Yangxue Granule (WSYXG) combined with clomifene citrate (CC) in treating follicular maldevelopment (FM) infertility, and to explore its possible action channels.</p><p><b>METHODS</b>Ninety patients with FM of Shen-deficiency blood stasis syndrome were randomly assigned to 3 groups, i.e., the Chinese medicine group (CMG, treated with WXYXG), the Western medicine group (WMG, treated with CC), and the combination group of Chinese medicine and Western medicine (CG, treated with both WSYXG and CC), 30 cases in each group. Three menstrual cycles were totally observed. Serum levels of luteinizing hormone (LH), follicle stimulating hormone (FSH), estradiol (E2 ), inhibin B (INHB), activin A (ACTA), and follistatin (FS) were tested before and after treatment, and the ovulation was monitored and their basic body temperature measured.</p><p><b>RESULTS</b>There was no statistical difference in clinical efficacy among the three groups (P> 0.05). Better effects on the Chinese medicine syndrome efficacy, the ovulation rate, and the endometrium thickness on the ovulation day were shown in CMG and CG than in WMG, showing statistical difference (P < 0.05). The E2 level increased on the third day of the first menstrual cycle in CG when compared with before treatment. On the 10th day of the 1st menstrual cycle, the INHB and FS increased and the ACTA decreased, showing statistical difference (P < 0.05). On the 10th day of the 3rd menstrual cycle the serum LH level decreased more obviously in CG than in WMG, showing statistical difference (P < 0.05). On the 3rd day of the 3rd menstrual cycle in CG, the INHB was negatively correlated with FSH (r = -0.492,P < 0.01), and INHB on the 10th day was positively correlated with E2 and FS (r = 0.682, 0.772, P < 0.01), and negatively correlated with ACTA on the 10th day (r = -0.635, P < 0.01).</p><p><b>CONCLUSION</b>WSYXG combined with CC could improve Chinese medicine syndrome, regulate the expressions of FM patients' ovary local factors INHB, ACTA and FS, improve the condition of ovary functions, and control the follicle development.</p>