Gene sequencing and result analysis of balanced translocation carriers by third-generation gene sequencing technology.

Because the total gene copy number remains constant and all genes are normally expressed, carriers of balanced chromosomal translocations usually have a normal phenotype but are able to produce many different types of gametes during meiosis, and unbalanced gametes lead to increased risks of infertility, recurrent spontaneous abortion, stillbirth, neonatal death or malformations and intellectual abnormalities in offspring. The key to balanced translocations lies in finding the breakpoints, but current genetic testing techniques are all short-read sequencing, with the disadvantage of procedural complexity and imprecision for precisely identifying the breakpoints. The latest third-generation sequencing technology overcomes these drawbacks and uses robust long-read sequencing to accurately and rapidly detect genome-wide information and identify breakpoint locations. In this paper, we performed whole genome long-read sequencing using an Oxford Nanopore sequencer to detect the breakpoints of 4 balanced chromosomal translocation carriers. The results showed that employing about ~ 10× coverage confirmed 6 of the 8 breakpoints, of which, 2 had microdeletions/insertions identified near the breakpoints and 4 had breakpoints that disrupted the normal gene structure and were simultaneously tested for genome-wide structural variation (SV). The results show that whole genome long-read sequencing is an efficient method for pinpointing translocation breakpoints and providing genome-wide information, which is essential for medical genetics and preimplantation genetic testing.

GOLPH3 modulates expression and alternative splicing of transcription factors associated with endometrial decidualization in human endometrial stromal cells.

Endometrial decidualization is a decidual tissue formed by the proliferation and re-differentiation of endometrial stroma stimulated by decidualization inducing factors. It is very important for the proper maintenance of pregnancy. Previous studies speculated that Golgi phosphoprotein 3 (GOLPH3) may have a regulatory role in the process of endometrial decidualization, while the specific molecular mechanisms of GOLPH3 is unclear. In this part, GOLPH3 was silenced in human endometrial stromal cells (hESCs), and the transcriptome data (RNA-seq) by GOLPH3 knockdown (siGOLPH3) was obtained by high-throughput sequencing technology so as to analyze the potential targets of GOLPH3 at expression and alternative splicing levels in hESCs. Through bioinformatics analysis, we found that siGOLPH3 can significantly affect the overall transcriptional level of hESCs. A total of 6,025 differentially expressed genes (DEGs) and 4,131 differentially alternative splicing events (DASEs) were identified. Through functional cluster analysis of these DEGs and genes where differential alternative splicing events are located, it is found that they are enriched in the PI3K/Akt signaling pathway, RNA splicing and processing, transcription factors and other pathways related to endometrial decidualization and important biological processes, indicating the important biological function of GOLPH3. At the same time, we focused on the analysis of the transcription factors regulated by GOLPH3, including gene expression regulation and the regulation of variable splicing. We found that GOLPH3can regulate the expression of transcription factors such as LD1, FOSL2, GATA2, CSDC2 and CREB3L1. At the same time, it affects the variable splicing mode of FOXM1 and TCF3. The function of these transcription factors is directly related to decidualization of endometrium. Therefore, we infer that GOLPH3 may participate in endometrial de membrane by regulating expression and alternative splicing levels of transcription factors. We further identified the role of GOLPH3 in the transcriptional mechanism. At the same time, it also expands the function mode of GOLPH3 protein molecule, and provides a theoretical basis for downstream targeted drug research and development and clinical application.

Evaluating an external quality assurance program for semen analysis in China during 2009-2020.

INTRODUCTION: Semen analysis (SA) plays a key role in guiding treatments of male reproductive diseases and infertility due to male factors; however, it remains challenging to conduct an accurate SA due to lack of standardization, highly subjective assessments, and problems with automated procedures. Therefore, quality assurance (QA) and teaching courses are essential for making the laboratory results more consistent. MATERIALS AND METHODS: The external quality assurance (EQA) scheme was organized by national human sperm bank technology training bases in Guangdong province in China between 2009 and 2020. Until 2020, 124 laboratories from China participated in the EQA program. The EQA scheme per year has been organized involving two semen aliquots for sperm concentration, two video recordings for motility, and two smears for sperm morphology. All samples used in the EQA scheme were obtained from different healthy donors or patients. RESULTS: We estimated that the median coefficient of variation (CV) of sperm concentration, ignoring the method used, was 26.6%. Using a 100 µm deep counting chamber led to a decreasing CV of 13.6%. For sperm motility, the median CV of nonprogressive motility was high (50.8%), but the CV of progressive motility (13.2%), immotile sperm (14.3%), and total motility (11.8%) were acceptable. The morphology assessment revealed large variability (44.4%) irrespective of the classification criteria. DISCUSSION: The reduction of interlaboratory variability is still a challenge during SA in China. Therefore, it is critical to increase awareness of joining EQA schemes and establish standardized training centers to follow WHO-recommended procedures toward Chinese standards.

Tripronuclear Zygotes in IVF Laboratory Quality Control: Experimental Evaluation and Potential Applications.

This study set out to evaluate quality control within a new in vitro fertilization (IVF) laboratory environment and of new incubators based on the culture results of tripronuclear zygotes. The representative environmental indicators within new and old IVF laboratories were monitored, and tripronuclear zygotes were cultured in the two laboratories; the results were analyzed and compared. Subsequently, tripronuclear (3PN) zygotes were cultured in both new and old incubators and the culture results were compared. No differences were found in embryo development between 3PN zygotes in the old and new laboratories. However, in the quality control test, the degeneration rate and developmental arrest rate in the new incubator early phase group were significantly increased when compared with the old incubators. Moreover, the grade I embryo rate also decreased significantly. Nevertheless, all the above comparisons in the new incubator later phase group showed no statistical significance as compared to those observed in old incubators. Tripronuclear zygotes are sensitive to the environment in IVF laboratories and can be considered useful during quality control trials of new IVF laboratories and new equipment including incubators.

PTEN Expression in Human Granulosa Cells Is Associated with Ovarian Responses and Clinical Outcomes in IVF.

The ovarian reserve determines the success of in vitro fertilization (IVF) and embryo transfer treatment. It predicts the ovarian response in controlled ovarian hyperstimulation cycles. Apoptosis in granulosa cells surrounding oocytes is important for ovarian function and has been closely associated with follicular atresia. PTEN (encoding phosphatase and tensin homolog) is a well-known tumor suppressor gene that functions as a mediator of apoptosis and is crucial for mammal reproduction. In the present study, we analyzed the expression level of PTEN in human granulosa cells and aimed to investigate its association with the ovarian response and clinical outcomes in IVF. Apoptosis in granulosa cells were analyzed using Annexin V-Allophycocyanin staining after PTEN short hairpin RNA lentivirus transfection. Real-time fluorescent quantitative PCR analysis showed that the PTEN transcript level was significantly higher in poor responders and significantly lower in high responders, compared with that in normal responders. However, PTEN expression in the pregnancy group decreased slightly, but not significantly, compared with that in the non-pregnancy group. The apoptosis rate of granulosa cells declined significantly after 24-h transfection of the PTEN-shRNA lentivirus. These results suggest a fundamental role of PTEN in the regulation of follicular development, and that it might be involved in the pathogenesis of follicular dysplasia and ovarian dysfunction.

A Hyperandrogenic Environment Causes Intrinsic Defects That Are Detrimental to Follicular Dynamics in a PCOS Mouse Model.

Polycystic ovary syndrome (PCOS) is a common cause of female infertility. Hyperandrogenism is both a major symptom and key diagnostic trait of PCOS; however, the direct impact of this androgen excess on ovarian dynamics is unclear. By combining a DHT-induced PCOS mouse model with an ex vivo follicle culture system, we investigated the impact of hyperandrogenism on ovarian function. Ovaries from PCOS mice exhibited the characteristic polycystic ovary morphology with numerous large cystic follicles and no corpora lutea present. Isolation and individual culture of preantral and antral follicles from PCOS mice resulted in slower growth rates during 5 days compared with the follicles isolated from control mice (P < 0.01). In contrast, preovulatory follicles from PCOS mice exhibited a significant increase in growth rate compared with controls (P < 0.01). Preantral follicles from PCOS ovaries maintained comparable follicular health as control follicles, but antral and preovulatory PCOS follicles exhibited reduced follicle health (P < 0.01) and survival rates (P < 0.01). Compared with controls, PCOS females also exhibited a poorer response to hyperstimulation (P < 0.01), impaired oocyte function evident by increased levels of reactive oxygen species (P < 0.01), and a reduction in on-time embryo development (P < 0.01). These results demonstrate that prolonged exposure to androgen excess leads to aberrant follicle development, which persists even after removal from the hyperandrogenic environment, causing perturbed follicular developmental trajectories. These findings indicate that an in vivo hyperandrogenic environment in patients with PCOS may intrinsically induce detrimental effects on follicles and oocytes.

[Impact of GOLPH3 expression in cumulus granulosa cells on outcomes of intracytoplasmic sperm injection].

OBJECTIVE: To evaluate the impact of GOLPH3 expression in cumulus granulosa cells on the outcomes of intracytoplasmic sperm injection (ICSI). METHODS: A total of 119 women receiving ICSI due to male infertility at our center between April, 2012 and June, 2014 were enrolled in the study. Cumulus granulosa cells were collected from the women for detection of GOLPH3 expressions using immunocytochemistry, Western blotting, and real-time PCR. GOLPH3 expression rate was compared between women with and without clinical pregnancy following ICSI, and the associations of GOLPH3 expression with the laboratory indicators of ICSI outcomes were assessed. RESULTS: Immunocytochemistry showed that GOLPH3 expression was located mainly in in the plasma of the cumulus granulosa cells. The rate and intensity of GOLPH3 expression in the cumulus granulosa cells differed significantly between women with and without clinical pregnancy following ICSI (P<0.05). GOLPH3 expression was found to positively correlate with the numbers of punctured follicles, grade III oocyte cumulus complex, ICSI oocytes, fertilized oocytes, cleavage, high quality embryos, blastocysts, high quality blastocysts, and frozen embryos (all P<0.01). The results of RTPCR and Western blotting revealed significant differences in GOLPH3 expressions at both the mRNA and protein levels in the cumulus granulosa cells between the pregnant and non-pregnant groups after ICSI (t=14.560, P=0.000). Western blot analysis revealed significant difference of GOLPH3 protein expression in cumulus granulosa cells between women with and without clinical pregnancy following ICSI. CONCLUSION: GOLPH3 expression in the cumulus granulosa cells plays an important role in the development of oocytes and promotion of conception to affect the outcomes of ICSI.

Effect of GOLPH3 on cumulus granulosa cell apoptosis and ICSI pregnancy outcomes.

Previous studies have shown that GOLPH3 mediates cell growth, proliferation and differentiation and inhibits cell apoptosis; however, the role of GOLPH3 in cumulus granulosa cells and the value of GOLPH3 in predicting ICSI pregnancy outcomes remain unknown until now. Our findings showed higher positive expression rate, score of staining intensity, and immunohistochemical score of GOLPH3 in the cumulus granulosa cells of the pregnant women relative to non-pregnant women, and a higher apoptotic rate of cumulus granulosa cells was detected in non-pregnant women than in pregnant women. Pearson correlation analyses revealed that pregnancy correlated negatively with GOLPH3 expression and apoptosis of cumulus granulosa cells, and positively with the number of follicles punctured, number of grade III oocytes, number of eggs retrieved for ICSI, number of zygotes, number of cleavage-stage embryos, number of top-quality embryos, number of blastocysts, number of top-quality blastocysts, and number of frozen embryos. GOLPH3 may be involved in the apoptosis of cumulus granulosa cells, which may correlate with oocyte maturation and egg development. GOLPH3 expression in cumulus granulosa cells may facilitate the selection of top-quality eggs and embryos, the prediction of the clinical pregnancy outcomes of ICSI, and the increase of the pregnancy rate.

ZEB1 promotes epithelial-mesenchymal transition in cervical cancer metastasis.

OBJECTIVE: To investigate role of Zinc finger E-box binding homeobox 1 (ZEB1) in cervical cancer tissue (squamous cell carcinoma, SCC). DESIGN: Exploratory study. SETTING: University hospital. PATIENT(S): Sixty patients with SCC, including stage CINIII (n = 10), IB1 (n = 10), IB2 (n = 10), IIA1 (n = 10), IIA2 (n = 10), and IIB (n = 10) were studied. INTERVENTION(S): Caski cells were transfected with recombinant shZEB1 lentivirus or shCtrl lentivirus to generate stable ZEB1-knockdown Caski cells. MAIN OUTCOME MEASURE(S): ZEB1 expression was analyzed by quantitative real-time polymerase chain reaction and immunohistochemistry in cervical cancer tissues. ZEB1 expression in Caski cells was down-regulated by short-hairpin RNA (shRNA) interference, and changes in ZEB1 expression corresponded with changes in the proliferation and migratory ability of Caski cells. RESULT(S): Quantitative real-time polymerase chain reaction and immunohistochemistry results revealed that ZEB1 expression and the ratio of Vimentin to E-cadherin were high in 27 of 50 SCC patients and correlated with advanced International Federation of Gynecology and Obstetrics stage, tumor size >4 cm, and parametrial invasion. However, the expression of ZEB1 in cervical cancer tissue was independent of age and SCC antigen level. Transfection of ZEB1 shRNA in Caski cells significantly decreased the messenger RNA and protein expression of ZEB1, parallel with increased expression of the epithelial marker E-cadherin and decreased expression of the mesenchymal marker Vimentin. Furthermore, the proliferation and migratory ability of Caski cells were significantly lower in the transfected group than in the nontransfected control group. CONCLUSION(S): Down-regulation of ZEB1 expression may protect the invasive front of the tumors from converting to a mesenchymal phenotype by reducing the proliferation and motility of cervical cancer cells, suggesting that ZEB1 might be a potential therapeutic target for SCC.