Yu-Sheng Liu

Study on the relationship between Th17 cells and unexplained recurrent spontaneous abortion.

Citation Liu Y‐S, Wu L, Tong X‐H, Wu L‐M, He G‐P, Zhou G‐X, Luo L‐H, Luan H‐B. Study on the relationship between Th17 cells and unexplained recurrent spontaneous abortion. Am J Reprod Immunol 2011; 65: 503–511

The optimum number of oocytes in IVF treatment: an analysis of 2455 cycles in China

STUDY QUESTION What is the association between the number of oocytes retrieved and the live birth rate (LBR) following first IVF treatment cycles in China? SUMMARY ANSWER In first IVF treatment cycles, the LBR after fresh transfer was maximal in the groups with 6-10 or 11-15 oocytes and reduced in the groups with 0-5 or >15 oocytes. Despite this, the cumulative LBR after including frozen embryo transfer cycles increased with an ovarian response. WHAT IS KNOWN ALREADY There is a strong association between oocyte number and IVF outcome; however, this is a comprehensive analysis conducted to investigate the relationship between oocyte number and fresh cycle as well as cumulative LBRs in first IVF treatment cycles. STUDY DESIGN, SIZE AND DURATION This is a large retrospective cohort study (n = 2455); patients were categorized into four groups according to the number of oocytes retrieved (0-5, 6-10, 11-15 and >15 oocytes). The fresh embryo transfer LBR and cumulative LBR were evaluated by group. Univariate analysis was performed to identify factors that predict the chance of LBR. Multivariate logistic regression was used to assess the association between oocyte number and LBR after adjusting for confounding factors that were identified as significant in the univariate analysis. PARTICIPANTS/MATERIALS, SETTING AND METHODS A total of 2455 women who were undergoing their first IVF treatment cycle at the Reproductive Medicine Center of Anhui Provincial Hospital, P.R. China from April 2007 to December 2011 were identified and reviewed. All patients had normal menstrual cycles and were stimulated with a long GnRH agonist protocol. Associations between oocyte number and LBR and miscarriage rate as well as the rate of moderate-severe ovarian hyperstimulation syndrome (OHSS) were analyzed. MAIN RESULTS AND ROLE OF CHANCE The fresh embryo LBR per started cycle increased with the number of retrieved oocytes up to Groups 2 and 3 (6-10 and 11-15 oocytes) and then decreased, because of the high number of cycles with all embryos being cryopreserved, in order to avoid moderate-severe OHSS in group 4 (>15 oocytes). However, the cumulative LBR per started cycle continued to increase with oocyte number, as did the incidence of moderate-severe OHSS. There was no significant difference in the miscarriage rates among the patient groups. LIMITATIONS, REASONS FOR CAUTION As a retrospective study, our analysis depends on previously recorded data; therefore, certain variables could not be collected. Our findings may be influenced by our young and thin patient population and the inability to control for certain markers of ovarian reserve such as historical maximum serum FSH, antral follicle count and serum anti-Mullerian hormone. WIDER IMPLICATIONS OF THE FINDINGS Our data suggest that in IVF cycles stimulated with a long protocol, the optimal number of oocytes for achieving a live birth is somewhere between 6 and 15. The balance between maximum treatment success and minimum risk of complications, especially OHSS, should be further investigated. STUDY FUNDING/COMPETING INTERESTS This study was supported by the Medical Scientific Research Plan Project of Anhui Provincial Department of Health (13ZC014) and the Natural Science Foundation of Anhui Higher Education Institutions (KJ2013Z132).

Characterization of microRNA profile in human cumulus granulosa cells: Identification of microRNAs that regulate Notch signaling and are associated with PCOS

CONTEXT Polycystic ovary syndrome (PCOS), a complex and heterogeneous endocrine condition, is characterized by polycystic ovaries, hyperandrogenism, insulin resistance and chronic anovulation. Cumulus granulosa cells surrounding the oocyte are involved in different aspects of PCOS pathology. Several studies suggested that miRNAs play an important regulatory role at the post-transcriptional level in cumulus granulosa cells. OBJECTIVE Our objective was to describe the altered miRNA expression profiles and miRNA targeted signaling pathways in PCOS. DESIGN Case-control study that involved 21 women with PCOS and 20 women without the disease (controls). The miRNA expression profiles of human cumulus granulosa cells were determined using next generation sequencing by Illumina Hiseq 2000. The differentially expressed miRNAs and novel miRNAs were validated by quantitative real-time PCR. The Notch3 and MAPK3 were demonstrated to be targeted by miR-483-5p based on quantitative real-time PCR, western blot and luciferase activity assay. RESULTS Compared with controls, a total of 59 known miRNA were identified that differentially expressed in PCOS cumulus granulosa cells, including 21 miRNAs increase and 38 miRNAs decrease. Moreover, the novel miRNAs were predicted in PCOS and control cumulus granulosa cells. The potential regulating roles of miRNA in pathophysiology of PCOS were analyzed by GO and KEGG pathway annotation, and several important processes were identified to be targeted by the differentially expressed miRNAs, such as Notch signaling, regulation of hormone, and energy metabolism. Furthermore, Notch3 and MAPK3, the members of Notch signaling and ERK-MAPK pathway, were demonstrated to be regulated by miR-483-5p based on negative expression correlation validation and detection of Notch3/MAPK3 expression after miR-483-5p mimics transfection. Dual luciferase activity assay suggested that Notch3 and MAPK3 were directly targeted by miR-483-5p. CONCLUSION Our data suggested that miRNAs and their targeted pathways (e.g. Notch signaling pathway) play important roles in the etiology and pathophysiology of PCOS, and provides novel candidates for molecular biomarkers or treatment targets in the research of female infertility associated with PCOS.

Alteration of Th17 and Treg cells in patients with unexplained recurrent spontaneous abortion before and after lymphocyte immunization therapy

BackgroundSeveral types of T cells have been associated with the pathogenesis of unexplained recurrent spontaneous abortion (URSA), including Th1/Th2/Th17/Tregs cell. It has been appreciated that immunotherapy with paternal or third party lymphocytes is an effective method of treatment for URSA patients. The balance of Th1/Th2 cells could be maintained and an increase of Treg cells would be beneficial after immunotherapy; however, the mechanism by which the Th17/Treg balance affects URSA has not yet been fully elucidated.MethodsHere, we used flow cytometry, liquid chip technology and quantitative real-time PCR (qPCR) methods to characterize Th17/Treg cell populations after immunotherapy. We found that after immunotherapy in URSA patients, the percentage of Th17 cells decreased and the percentage of Treg cells in peripheral blood mononuclear cells (PBMC) increased, as detected by flow cytometry.ResultsImmunotherapy may induce a decrease in the Th17/Treg ratio and the Treg bias, which may be beneficial for the maintenance of pregnancy. The expression level of ROR gamma t, a transcription factor found in Th17 cells, decreased and the expression of the Treg-specific transcription factor Foxp3 increased in peripheral blood as detected by qPCR. Immunotherapy may induce a decrease in the ratio of ROR gamma t to Foxp3 and a Treg cell bias, which would be beneficial for pregnancy maintenance. The secretion of the Treg-associated cytokine TGF-beta, as well as Th2 cytokines, was increased in serum, while the secretion of Th17-associated cytokine IL-17A and Th1 cytokine production was decreased. The Th1/Th2 cytokine ratio significantly decreased. Similarly, the Th17/Treg ratio significantly decreased in the total patient after immunotherapy.ConclusionsThese results indicate that in patients with URSA, immunotherapy with mononuclear cells derived from the baby’s father could affect both Th1/Th2 and Th17/Treg balance, and we found that the Th2 and Treg bias would be beneficial for pregnancy, which may lead to a balancing of the Th17/Treg ratio in URSA patients after immunotherapy.

Oocyte quality is decreased in women with minimal or mild endometriosis

Endometriosis, a pathological condition in which the endometrium grows outside the uterus, is one of the most common causes of female infertility; it is diagnosed in 25–40% of infertile women. The mechanism by which endometriosis affects the fertility of females remains largely unknown. We examined the ultrastructure of oocytes from patients with minimal or mild endometriosis and control females undergoing in vitro fertilization (IVF) treatment by transmission electron microscopy (TEM) to investigate the physiological significance of oocyte quality for patients with minimal or mild endometriosis. The TEM results revealed that the oocytes from women with minimal or mild endometriosis exhibited abnormal mitochondrial structure and decreased mitochondria mass. Quantitative real time PCR analysis revealed that the mitochondrial DNA copy number was significantly reduced in the oocytes from women with minimal or mild endometriosis compared with those of the control subjects. Our results suggest that decreased oocyte quality because of impaired mitochondrial structure and functions probably an important factor affecting the fertility of endometriosis patients.

Chronic Unpredictable Stress Decreases Expression of Brain-Derived Neurotrophic Factor (BDNF) in Mouse Ovaries: Relationship to Oocytes Developmental Potential

Background Brain-derived neurotropic factor (BDNF) was originally described in the nervous system but has been shown to be expressed in ovary tissues recently, acting as a paracrine/autocrine regulator required for developments of follicles and oocytes. Although it is generally accepted that chronic stress impairs female reproduction and decreases the expression of BDNF in limbic structures of central nervous system, which contributes to mood disorder. However, it is not known whether chronic stress affects oocytes developments, nor whether it affects expression of BDNF in ovary. Methods Mice were randomly assigned into control group, stressed group, BDNF-treated group and BDNF-treated stressed group. The chronic unpredictable mild stress model was used to produce psychosocial stress in mice, and the model was verified by open field test and hypothalamic-pituitary-adrenal (HPA) axis activity. The methods of immunohistochemistry and western blotting were used to detect BDNF protein level and distribution. The number of retrieved oocytes, oocyte maturation, embryo cleavage and the rates of blastocyst formation after parthenogenetic activation were evaluated. Results Chronic unpredictable stress decreased the BDNF expression in antral follicles, but didn’t affect the BDNF expression in primordial, primary and secondary follicles. Chronic unpredictable stress also decreased the number of retrieved oocytes and the rate of blastocyst formation, which was rescued by exogenous BDNF treatment. Conclusion BDNF in mouse ovaries may be related to the decreased number of retrieved oocytes and impaired oocytes developmental potential induced by chronic unpredictable stress.

Oocytes with a dark zona pellucida demonstrate lower fertilization, implantation and clinical pregnancy rates in IVF/ICSI cycles.

The morphological assessment of oocytes is important for embryologists to identify and select MII oocytes in IVF/ICSI cycles. Dysmorphism of oocytes decreases viability and the developmental potential of oocytes as well as the clinical pregnancy rate. Several reports have suggested that oocytes with a dark zona pellucida (DZP) correlate with the outcome of IVF treatment. However, the effect of DZP on oocyte quality, fertilization, implantation, and pregnancy outcome were not investigated in detail. In this study, a retrospective analysis was performed in 268 infertile patients with fallopian tube obstruction and/or male factor infertility. In 204 of these patients, all oocytes were surrounded by a normal zona pellucida (NZP, control group), whereas 46 patients were found to have part of their retrieved oocytes enclosed by NZP and the other by DZP (Group A). In addition, all oocytes enclosed by DZP were retrieved from 18 patients (Group B). No differences were detected between the control and group A. Compared to the control group, the rates of fertilization, good quality embryos, implantation and clinical pregnancy were significantly decreased in group B. Furthermore, mitochondria in oocytes with a DZP in both of the two study groups (A and B) were severely damaged with several ultrastructural alterations, which were associated with an increased density of the zona pellucida and vacuolization. Briefly, oocytes with a DZP affected the clinical outcome in IVF/ICSI cycles and appeared to contain more ultrastructural alterations. Thus, DZP could be used as a potential selective marker for embryologists during daily laboratory work.

Inhibition of Follicular Development Induced by Chronic Unpredictable Stress Is Associated with Growth and Differentiation Factor 9 and Gonadotropin in Mice

ABSTRACT Chronic psychosocial stress negatively affects ovarian function. Ovarian follicular development is regulated by both pituitary-derived gonadotropins and intraovarian regulatory factors. To date, the suppressive effects of chronic stress on the ovary have been observed to be manifested mainly as an inhibition of gonadotropin release. It is not clear whether there are any other intraovarian regulatory mechanisms involved in this process. Growth and differentiation factor 9 (GDF9) is an important, oocyte-specific paracrine regulator required for follicular development. In this study, the chronic unpredictable mild stress model was used to produce psychosocial stress in mice. The number of different developmental stages of follicles was counted on ovarian sections stained with hematoxylin and eosin. Real-time PCR and Western blotting were used to detect the mRNA and protein levels, respectively, of GDF9. The results show that chronic unpredictable stress inhibits follicular development, increases follicular atresia, and suppresses GDF9 expression. Exogenous gonadotropin treatment partly restores the repressed antral follicular development, but has no effect on the repressed secondary follicular development associated with chronic stress. Treatment with recombinant GDF9 restores secondary follicular development. Cotreatments with GDF9 and gonadotropins restore both secondary and antral follicular development in stressed mice. These findings demonstrate that inhibition of follicular development induced by chronic unpredictable stress is associated with GDF9 and gonadotropin.

Research resources: comparative microRNA profiles in human corona radiata cells and cumulus oophorus cells detected by next-generation small RNA sequencing.

During folliculogenesis, cumulus cells surrounding the oocyte differentiate into corona radiata cells (CRCs) and cumulus oophorus cells (COCs), which are involved in gonadal steroidogenesis and the development of germ cells. Several studies suggested that microRNAs (miRNAs) play an important regulatory role at the post-transcriptional level in cumulus cells. However, comparative miRNA profiles and associated processes in human CRCs and COCs have not been reported before. In this study, miRNA profiles were obtained from CRCs and COCs using next generation sequencing in women undergoing controlled ovarian stimulation for IVF. A total of 785 and 799 annotated miRNAs were identified in CRCs and COCs, while high expression levels of six novel miRNAs were detected both in CRCs and in COCs. In addition, different expression patterns in CRCs and COCs were detected in 72 annotated miRNAs. To confirm the miRNA profile in COCs and CRCs, quantitative real-time PCR was used to validate the expression of annotated miRNAs, differentially expressed miRNAs, and novel miRNAs. The miRNAs in the let-7 family were found to be involved in the regulation of a broad range of biological processes in both cumulus cell populations, which was accompanied by a large amount of miRNA editing. Bioinformatics analysis showed that amino acid and energy metabolism were targeted significantly by miRNAs that were differentially expressed between CRCs and COCs. Our work extends the current knowledge of the regulatory role of miRNAs and their targeted pathways in folliculogenesis, and provides novel candidates for molecular biomarkers in the research of female infertility.

Fertilization rates are improved after IVF if the corona radiata is left intact in vitrified-warmed human oocytes

BACKGROUND Before human MII oocytes are vitrified they are usually denuded from their cumulus cells. In this study we wanted to investigate the effects of an intact corona radiata on the vitrification and fertilization of human oocytes. METHODS The study comprised two different parts. In Part 1, 36 MII stage oocytes, from 6 patients, were randomly assigned into a control group, a group of vitrified-warmed oocytes without a corona radiata and a group of vitrified-warmed oocytes with an intact corona radiata. In each group of 12, 6 oocytes were used for evaluation of the zona pellucida solubility (hardening) and another 6 oocytes were used for the analysis of their ultrastructure. In addition, six polyspermically fertilized oocytes were used as positive controls for zona pellucida hardening. In Part 2, 16 patients in total produced 107 fresh and 98 vitrified-warmed oocytes, with or without an intact corona radiata. All oocytes were fertilized via conventional IVF and embryos were transferred according to our standard ET routines. The oocyte survival and fertilization rates, embryo quality and pregnancy and implantation rates were evaluated. RESULTS There were no differences in oocyte survival, zona pellucida solubility (hardening) or the number of cortical granules between the vitrified-warmed and fresh oocytes. There were also no differences in the zona pellucida solubility and the number of cortical granules between vitrified-warmed oocytes with or without an intact corona radiata. However, the oocytes with an intact corona radiata had a higher fertilization rate after conventional IVF insemination. No differences were seen in the survival and cleavage rates, the percentage of high-quality embryos or the clinical outcome. CONCLUSIONS Zona hardening and ultrastructural damage do not seem to occur in vitrified human oocytes. An intact corona radiata in vitrified-warmed oocytes retains their fertilization capacity in conventional IVF, but does not improve the embryo quality. Poor fertilizing capacities of vitrified-warmed oocytes without an intact corona radiata seem to have been due to the complete removal of the cumulus cells.