A Proteomic Analysis of Human Follicular Fluid: Proteomic Profile Associated with Embryo Quality.

Embryo selection is a key point of in vitro fertilization (IVF). The most commonly used method for embryo selection is morphological assessment. However, it is sometimes inaccurate. Follicular fluid (FF) contains a complex mixture of proteins that are essential for follicle development and oocyte maturation. Analyzing human FF proteomic profiles and identifying predictive biomarkers might be helpful for evaluating embryo quality. A total of 22 human FF samples were collected from 19 infertile women who underwent IVF/intracytoplasmic sperm injection (ICSI) treatment between October 2021 and November 2021. FFs were grouped into two categories on the basis of the day 3 embryo quality, grade I or II in the hqFF group and grade III in the nhqFF group. FF was analyzed by liquid chromatography-tandem mass spectrometry (LC/MS/MS). The key differentially expressed proteins (DEPs) were validated by parallel reaction monitoring (PRM) and enzyme-linked immunosorbent assay (ELISA). Differentially expressed proteins were further analyzed using DAVID software. A total of 558 proteins were identified, of which 50 proteins were differentially expressed in the hqFF versus nhqFF group, including 32 upregulated proteins (> 1.20-fold, P < 0.05) and 18 downregulated proteins (< 0.67-fold, P < 0.05). Bioinformatics analyses showed that the upregulated DEPs were enriched in components of the coagulation and complement systems and negative regulation of peptidase activity, while the downregulated DEPs were enriched in molecular function of extracellular matrix structural and constituent collagen binding. Our results suggested that a number of protein biomarkers in FF were associated with embryo quality. It may help develop an effective and noninvasive method for embryo selection.

Novel mutations in NLRP5 and PATL2 cause female infertility characterized by primarily oocyte maturation abnormality and consequent early embryonic arrest.

PURPOSE: This study aims to identify the genetic causes of 12 women with primary infertility characterized by primarily oocyte maturation abnormality and consequent early embryonic arrest. METHODS: Genomic DNA was isolated from peripheral blood samples. Whole-exome sequencing was performed on the probands, and the identified variants were confirmed by Sanger sequencing. The pathogenicity of the identified variants on the protein was accessed in silico. And we used qRT-PCR to detect the possible effects of the novel mutation on the mRNA level of NLRP5. RESULTS: A novel homozygous frameshift variant (p.V429Efs*30) in NLRP5 and compound heterozygous variants with a novel frameshift variant (p.A297Efs*20) and a recurrent variant (c. 223-14_223-2delCCCTCCTGTTCCA) in PATL2 were identified in two unrelated affected individuals. qRT-PCR showed an obvious decrease of the mutant NLRP5 mRNA. In addition, the truncated proteins of NLRP5 and PATL2 were predicted to be non-functional due to the deletion of the most or the whole region of the critical functional domain(s) respectively. CONCLUSIONS: This study identified novel mutations in NLRP5 and PATL2, further expanding the mutational and phenotypic spectrum of both genes. This is the first report of the NLRP5 mutations that associates with oocyte maturation abnormality in humans.

Novel Mutations in CDC20 Are Associated with Female Infertility Due to Oocyte Maturation Abnormality and Early Embryonic Arrest.

The cell division cycle 20 (CDC20) protein is a co-activator of anaphase-promoting complex/cyclosome (APC/C), required for mitotic exit and also meiotic exit, containing seven WD40 repeats in the C-terminus responsible for protein-protein interactions. Recently, a previous study has shown that biallelic mutations in CDC20 are causative for female infertility with abnormalities in oocyte maturation and embryonic development. This study is to further identify new mutations of CDC20 and the prevalence of variants in our cohort. A cohort of 50 primary infertile females with oocyte maturation abnormality and early embryonic arrest were recruited. Genomic DNA was isolated from peripheral blood samples. Mutation screening of all the coding regions of CDC20 was performed by Sanger sequencing. The pathogenicity of the identified variants on the CDC20 protein was accessed in silico. Two CDC20 variants, a nonsense mutation p.R262* and a missense mutation p.A211T, identified in one female of 50 unrelated affected individuals, accounting for a relative small proportion of this cohort (2%). In silico analysis revealed that the p.R262* would cause no production of protein or a truncated protein lacking five WD40 repeats in the C-terminus; and that p.A211T may interfere with the formation of a deep hydrophobic pocket and thus disturb the binding of CDC20 protein to the substrates of APC/C. This study identified two novel mutations in CDC20, further expanding the mutation spectrum of this gene. Our findings further confirm that biallelic mutations in CDC20 occur in a proportion of infertile females with oocyte maturation abnormality and early embryonic arrest.

MicroRNA­155 targets myosin light chain kinase to inhibit the migration of human bone marrow­derived mesenchymal stem cells.

Toll­like receptors (TLRs) are expressed in human bone marrow­derived mesenchymal stromal cells (BM­MSCs). The activation of TLRs is important in the proliferation, differ-entiation, migration and hematopoiesis­supporting functions of BM­MSCs. MicroRNAs (miRNAs) are involved in various biological functions by mediating mRNA degradation or inhibiting the translation of target genes. Our previous study confirmed that TLRs regulate the migration ability of BM­MSCs. It was also identified that multiple miRNAs were regulated by TLRs. In view of this, it was hypothesized that TLR­regulated miRNAs may be important in regulating the migration of BM­MSCs. The migration ability of BM­MSCs was evaluated following transfection of the cells with the mimics or antagonists of miRNA (miR)­27b, miR­146a, miR­155 and miR­154. miR­155 significantly inhibited cell migration. Myosin light chain kinase (MYLK) was identified as the direct target of miR­155 in BM­MSCs, which was further investigated using the luciferase reporter assay. However, miR­155 did not affect the expression of upstream proteins of the RhoA pathway controlling the activity of MYLK, suggesting that miR­155 directly suppressed the expression of MYLK without affecting the RhoA pathway. These results may facilitate the development and clinical use of BM­MSCs in terms of their migration.

A retrospective study on IVF/ICSI outcome in patients with anti-nuclear antibodies: the effects of prednisone plus low-dose aspirin adjuvant treatment.

BACKGROUND: Anti-nuclear antibodies (ANA) are suspected of having relevance to adverse reproductive events. METHODS: This study aims to investigate the potential effect of ANA on IVF/ICSI outcome and the therapeutic role of prednisone plus low-dose aspirin (P + A) adjuvant treatment in ANA + patients. The first IVF/ICSI cycles without P + A of sixty-six ANA + women were enrolled as the ANA + group, and the 233 first IVF/ICSI cycles of matched ANA- women served as the ANA- group. The ANA + group was divided into the Titre < =1:320 subgroup and the Titre > 1:320 subgroup. Twenty-one ANA + women with adverse outcomes in their first cycles (ANA + cycles without P + A) received P + A adjuvant treatment for three months before the second IVF/ICSI cycle (ANA + cycles with P + A). The clinical characteristics and the IVF/ICSI outcomes were compared, respectively, between 1) the ANA + group and the ANA- group, 2) the Titre < =1:320 subgroup and the Titre > 1:320 subgroup, and 3) the ANA + cycles without P + A and the ANA + cycles with P + A. RESULTS: No significant differences were observed between each of the two-group pairs in the clinical characteristics. The ANA + group exhibited significantly lower MII oocytes rate, normal fertilisation, pregnancy and implantation rates, as well as remarkably higher abnormal fertilisation and early miscarriage rates. The Titre < =1:320 subgroup's IVF/ICSI outcomes were as poor as those of the Titre > 1:320 subgroup. After the P + A adjuvant treatment, the number of two pro-nuclei, perfect embryos and available embryos, and the implantation rate increased significantly. CONCLUSIONS: These observations suggest that ANA could exert a detrimental effect on IVF/ICSI outcome that might not be titre-dependent, and P + A adjuvant treatment could be useful for ANA + patients. This hypothesis should be verified in further prospective randomised studies.

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).

[The measurement of proportion and function of regulatory T cells in unexplained recurrent spontaneous abortion].

OBJECTIVE: To investigate the proportion and function of CD(4)(+)CD(25)(+) regulatory T cells (CD(4)(+)CD(25)(+) Tr) in unexplained recurrent spontaneous abortion (URSA). METHODS: (1) Proportion measurement: the proportion of CD(4)(+)CD(25)(+) Tr cells in peripheral blood was measured by double-label flow cytometric analysis. The samples were taken from 15 URSA women, 15 normal non-pregnancy women and 13 normal pregnancy women. (2) Function measurement: CD(4)(+)CD(25)(+) Tr cells and CD(4)(+)CD(25)(-) T cells were extracted from peripheral blood lymphocytes by the microbeads separation. The purity of CD(4)(+)CD(25)(+) Tr cells and CD(4)(+)CD(25)(-) T cells was measured by flow cytometry. The growth inhibitory effect of CD(4)(+)CD(25)(+) Tr cells on CD(4)(+)CD(25)(-) T cells was assessed in vitro. RESULTS: The proportion of CD(4)(+)CD(25)(+) Tr cells was decreased significantly in URSA women (6.9 +/- 1.8)% than that in normal non-pregnancy women [(10.8 +/- 1.1)%] (P<0.05) and normal pregnancy women [(11.2 +/- 1.4)%] (P<0.01). Moreover, the suppressive rate of CD(4)(+)CD(25)(+) Tr cells was decreased significantly in URSA women (75 +/- 6)% than that in normal non-pregnancy women [(89 +/- 4)%] (P<0.05) and normal pregnancy women [(90 +/- 4)%] (P<0.01). However, with respect to the proportion and function of CD(4)(+)CD(25)(+) Tr cells, there was no significant difference between normal non-pregnancy women and normal pregnancy women (P > 0.05). CONCLUSION: The results suggest that decrease in proportion and function of CD(4)(+)CD(25)(+) Tr cells may be associated with URSA.