Publications by authors named "William S B Yeung"

151 Publications

Effect of serum vitamin D level before ovarian stimulation on the cumulative live birth rate of women undergoing in vitro fertilization: a retrospective analysis.

Endocr Connect 2022 Jan 1. Epub 2022 Jan 1.

E Ng, Department of Obstetrics and Gynecology, The University of Hong Kong, Hong Kong, Hong Kong.

Objective: Vitamin D receptors are present in the female reproductive tract. Studies on the association between serum vitamin D level and pregnancy rate of in vitro fertilization (IVF) showed inconsistent results and focused on a single fresh or frozen embryo transfer cycle. The objective of our study was to evaluate if serum vitamin D level before ovarian stimulation was associated with the cumulative live birth rate (CLBR) of the first IVF cycle.

Design: Retrospective cohort study.

Methods: Women who underwent the first IVF cycle from 2012 to 2016 at a university-affiliated reproductive medicine center were included. Archived serum samples taken before ovarian stimulation were analyzed for 25(OH)D levels using liquid chromatography-mass spectrometry.

Results: 1,113 had pregnancy outcome from the completed IVF cycle. The median age (25th-75th percentile) of the women was 36 (34-38) years and serum 25(OH)D level was 53.4 (41.9-66.6)nmol/L. The prevalence of vitamin D deficiency (less than 50nmol/L) was 42.2%. The CLBR in the vitamin D deficient group was significantly lower compared to the non-deficient group (43.9%,208/474 vs 50.9%,325/639, p=0.021, unadjusted), and after controlling for women's age, body mass index, antral follicle count, type and duration of infertility. There were no differences in the clinical/ongoing pregnancy rate, live birth rate and miscarriage rate in the fresh cycle between the vitamin D deficient and non-deficient groups.

Conclusions: Vitamin D deficiency was prevalent in infertile women in subtropical Hong Kong. The CLBR of the first IVF cycle in the vitamin D deficient group was significantly lower compared to the non-deficient group.
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http://dx.doi.org/10.1530/EC-21-0444DOI Listing
January 2022

Bisphenol A Analogues Suppress Spheroid Attachment on Human Endometrial Epithelial Cells through Modulation of Steroid Hormone Receptors Signaling Pathway.

Cells 2021 10 26;10(11). Epub 2021 Oct 26.

Department of Obstetrics and Gynaecology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China.

Bisphenol A (BPA) is a well-known endocrine disruptor, widely used in various consumer products and ubiquitously found in air, water, food, dust, and sewage leachates. Recently, several countries have restricted the use of BPA and replaced them with bisphenol S (BPS) and bisphenol F (BPF), which have a similar chemical structure to BPA. Compared to BPA, both BPS and BPF have weaker estrogenic effects, but their effects on human reproductive function including endometrial receptivity and embryo implantation still remain largely unknown. We used an in vitro spheroid (blastocyst surrogate) co-culture assay to investigate the effects of BPA, BPS, and BPF on spheroid attachment on human endometrial epithelial cells, and further delineated their role on steroid hormone receptor expression. We also used transcriptomics to investigate the effects of BPA, BPS, and BPF on the transcriptome of human endometrial cells. We found that bisphenol treatment in human endometrial Ishikawa cells altered estrogen receptor alpha (ERα) signaling and upregulated progesterone receptors (PR). Bisphenols suppressed spheroid attachment onto Ishikawa cells, which was reversed by the downregulation of PR through PR siRNA. Overall, we found that bisphenol compounds can affect human endometrial epithelial cell receptivity through the modulation of steroid hormone receptor function leading to impaired embryo implantation.
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http://dx.doi.org/10.3390/cells10112882DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8616109PMC
October 2021

Simulating nature in sperm selection for assisted reproduction.

Nat Rev Urol 2022 01 5;19(1):16-36. Epub 2021 Nov 5.

Department of Obstetrics and Gynaecology, The University of Hong Kong, Queen Mary Hospital, Hong Kong, China.

Sperm selection in the female reproductive tract (FRT) is sophisticated. Only about 1,000 sperm out of millions in an ejaculate reach the fallopian tube and thus have a chance of fertilizing an oocyte. In assisted reproduction techniques, sperm are usually selected using their density or motility, characteristics that do not reflect their fertilization competence and, therefore, might result in failure to fertilize the oocyte. Although sperm processing in in vitro fertilization (IVF) and intrauterine insemination (IUI) bypasses many of the selection processes in the FRT, selection by the cumulus mass and the zona pellucida remain intact. By contrast, the direct injection of a sperm into an oocyte in intracytoplasmic sperm injection (ICSI) bypasses all natural selection barriers and, therefore, increases the risk of transferring paternal defects such as fragmented DNA and genomic abnormalities in sperm to the resulting child. Research into surrogate markers of fertilization potential and into simulating the natural sperm selection processes has progressed. However, methods of sperm isolation - such as hyaluronic acid-based selection and microfluidic isolation based on sperm tactic responses - use only one or two parameters and are not comparable with the multistep sperm selection processes naturally occurring within the FRT. Fertilization-competent sperm require a panel of molecules, including zona pellucida-binding proteins and ion channel proteins, that enable them to progress through the FRT to achieve fertilization. The optimal artificial sperm selection method will, therefore, probably need to use a multiparameter tool that incorporates the molecular signature of sperm with high fertilization potential, and their responses to external cues, within a microfluidic system that can replicate the physiological processes of the FRT in vitro.
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http://dx.doi.org/10.1038/s41585-021-00530-9DOI Listing
January 2022

A retrospective analysis of artificial oocyte activation in patients with low or no fertilisation in intracytoplasmic sperm injection cycles.

J Obstet Gynaecol 2021 Aug 12:1-6. Epub 2021 Aug 12.

Department of Obstetrics and Gynaecology, The University of Hong Kong, Queen Mary Hospital, Hong Kong, Hong Kong.

Intracytoplasmic sperm injection (ICSI) is commonly used to treat severe male factor infertility in assisted reproduction. A small percentage of patients face suboptimal fertilisation rate or even fertilisation failure despite having ICSI. Artificial oocyte activation (AOA) has been proposed as a suitable method to overcome their problem. This is a retrospective cohort analysis of ICSI cycles undergoing AOA. Injected metaphase II oocytes were exposed to either calcium ionophore (A23187) after ICSI or injection of calcium chloride during ICSI followed by incubation with A23187 after ICSI. The previous ICSI cycles of the patients formed the historical control group. Thirty-four AOA cycles were analysed. The normal fertilisation rate (52.1%) was significantly improved in the AOA group. The percentage of failed fertilisation cycles (11.8%) were significantly reduced in the AOA group. The cumulative clinical pregnancy rate (47.1%) and live birth rate (29.4%) were significantly increased when compared to the previous cycles. Subgroup analysis revealed that the performance of the A23187 only protocol and the concomitant injection of calcium chloride protocol were comparable in terms of laboratory parameters and pregnancy outcomes. AOA is an effective method to improve the fertilisation rate and pregnancy outcome of infertile couples with previous fertilisation problem after ICSI.IMPACT STATEMENT A failed and low fertilisation rate after ICSI is not uncommon in assisted reproduction. AOA is normally used to improve fertilisation but there are discrepancies in the efficacy of the treatment. AOA improves the fertilisation rate and pregnancy outcomes of couples with suboptimal fertilisation rate and fertilisation failure in previous ICSI cycles. The efficacies of two AOA protocols were comparable. The A23187 only protocol was recommended because of its simplicity. AOA should be considered as a routine procedure for infertile couples with compromised fertilisation rates in previous ICSI cycles.
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http://dx.doi.org/10.1080/01443615.2021.1922878DOI Listing
August 2021

Identification of Sialyl-Lewis(x)-Interacting Protein on Human Spermatozoa.

Front Cell Dev Biol 2021 20;9:700396. Epub 2021 Jul 20.

Department of Obstetrics and Gynecology, Queen Mary Hospital, The University of Hong Kong, Hong Kong, Hong Kong.

Capacitated spermatozoa initiate fertilization by binding to the zona pellucida (ZP). Defective spermatozoa-ZP binding causes infertility. The sialyl-Lewis(x) (SLeX) sequence is the most abundant terminal sequence on the glycans of human ZP glycoproteins involving in spermatozoa-ZP binding. This study aimed to identify and characterize the SLeX-binding proteins on human spermatozoa. By using affinity chromatography followed by mass spectrometric analysis, chromosome 1 open reading frame 56 (C1orf56) was identified to be a SLeX-binding protein of capacitated spermatozoa. The acrosomal region of spermatozoa possessed C1orf56 immunoreactive signals with intensities that increased after capacitation indicating translocation of C1orf56 to the cell surface during capacitation. Treatment with antibody against C1orf56 inhibited spermatozoa-ZP binding and ZP-induced acrosome reaction. Purified C1orf56 from capacitated spermatozoa bound to human ZP. A pilot clinical study was conducted and found no association between the percentage of capacitated spermatozoa with C1orf56 expression and fertilization (IVF) rate in assisted reproduction treatment. However, the percentage of C1orf56 positive spermatozoa in the acrosome-reacted population was significantly ( < 0.05) lower in cycles with a fertilization rate < 60% when compared to those with a higher fertilization rate, suggesting that C1orf56 may have functions after ZP-binding and acrosome reaction. A larger clinical trial is needed to determine the possible use of sperm C1orf56 content for the prediction of fertilization potential of sperm samples.
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http://dx.doi.org/10.3389/fcell.2021.700396DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8329450PMC
July 2021

Current Understandings of Core Pathways for the Activation of Mammalian Primordial Follicles.

Cells 2021 06 13;10(6). Epub 2021 Jun 13.

Shenzhen Key Laboratory of Fertility Regulation, Center of Assisted Reproduction and Embryology, The University of Hong Kong-Shenzhen Hospital, Haiyuan First Road 1, Shenzhen 518053, China.

The mammalian ovary has two main functions-producing mature oocytes for fertilization and secreting hormones for maintaining the ovarian endocrine functions. Both functions are vital for female reproduction. Primordial follicles are composed of flattened pre-granulosa cells and a primary oocyte, and activation of primordial follicles is the first step in follicular development and is the key factor in determining the reproductive capacity of females. The recent identification of the phosphatidylinositol 3 kinase (PI3K)/phosphatase and tensin homolog deleted on chromosome 10 (PTEN) signaling pathway as the key controller for follicular activation has made the study of primordial follicle activation a hot research topic in the field of reproduction. This review systematically summarizes the roles of the PI3K/PTEN signaling pathway in primordial follicle activation and discusses how the pathway interacts with various other molecular networks to control follicular activation. Studies on the activation of primordial follicles have led to the development of methods for the in vitro activation of primordial follicles as a treatment for infertility in women with premature ovarian insufficiency or poor ovarian response, and these are also discussed along with some practical applications of our current knowledge of follicular activation.
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http://dx.doi.org/10.3390/cells10061491DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8231864PMC
June 2021

Single-cell RNA sequencing of cultured human endometrial CD140bCD146 perivascular cells highlights the importance of in vivo microenvironment.

Stem Cell Res Ther 2021 05 29;12(1):306. Epub 2021 May 29.

Shenzhen Key Laboratory of Fertility Regulation, Reproductive Medicine Center, The University of Hong Kong - Shenzhen Hospital, Shenzhen, China.

Background: Endometrial mesenchymal-like stromal/stem cells (eMSCs) have been proposed as adult stem cells contributing to endometrial regeneration. One set of perivascular markers (CD140b&CD146) has been widely used to enrich eMSCs. Although eMSCs are easily accessible for regenerative medicine and have long been studied, their cellular heterogeneity, relationship to primary counterpart, remains largely unclear.

Methods: In this study, we applied 10X genomics single-cell RNA sequencing (scRNA-seq) to cultured human CD140bCD146 endometrial perivascular cells (ePCs) from menstrual and secretory endometrium. We also analyzed publicly available scRNA-seq data of primary endometrium and performed transcriptome comparison between cultured ePCs and primary ePCs at single-cell level.

Results: Transcriptomic expression-based clustering revealed limited heterogeneity within cultured menstrual and secretory ePCs. A main subpopulation and a small stress-induced subpopulation were identified in secretory and menstrual ePCs. Cell identity analysis demonstrated the similar cellular composition in secretory and menstrual ePCs. Marker gene expression analysis showed that the main subpopulations identified from cultured secretory and menstrual ePCs simultaneously expressed genes marking mesenchymal stem cell (MSC), perivascular cell, smooth muscle cell, and stromal fibroblast. GO enrichment analysis revealed that genes upregulated in the main subpopulation enriched in actin filament organization, cellular division, etc., while genes upregulated in the small subpopulation enriched in extracellular matrix disassembly, stress response, etc. By comparing subpopulations of cultured ePCs to the publicly available primary endometrial cells, it was found that the main subpopulation identified from cultured ePCs was culture-unique which was unlike primary ePCs or primary endometrial stromal fibroblast cells.

Conclusion: In summary, these data for the first time provides a single-cell atlas of the cultured human CD140bCD146 ePCs. The identification of culture-unique relatively homogenous cell population of CD140bCD146 ePCs underscores the importance of in vivo microenvironment in maintaining cellular identity.
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http://dx.doi.org/10.1186/s13287-021-02354-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8164319PMC
May 2021

Placenta-Derived Exosomes as a Modulator in Maternal Immune Tolerance During Pregnancy.

Front Immunol 2021 11;12:671093. Epub 2021 May 11.

Department of Obstetrics and Gynaecology, Li Ka Shing (LKS) Faculty of Medicine, The University of Hong Kong, Hong Kong, Hong Kong.

Exosomes are a subset of extracellular vesicles with an average diameter of ~100nm. Exosomes are released by all cells through an endosome-dependent pathway and carry nucleic acids, proteins, lipids, cytokines and metabolites, mirroring the state of the originating cells. The function of exosomes has been implicated in various reproduction processes, such as embryo development, implantation, decidualization and placentation. Placenta-derived exosomes (pEXO) can be detected in the maternal blood as early as 6 weeks after conception and their levels increase with gestational age. Importantly, alternations in the molecular signatures of pEXO are observed in pregnancy-related complications. Thus, these differentially expressed molecules could be the potential biomarkers for diagnosis of the pregnancy-associated diseases. Recent studies have demonstrated that pEXO play a key role in the establishment of maternal immune tolerance, which is critical for a successful pregnancy. To gain a better understanding of the underlying mechanism, we highlighted the advanced studies of pEXO on immune cells in pregnancy.
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http://dx.doi.org/10.3389/fimmu.2021.671093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8144714PMC
November 2021

Embryo-endometrium crosstalk: a new understanding from in vitro model.

Fertil Steril 2021 04 6;115(4):907-908. Epub 2021 Mar 6.

The University of Hong Kong-Shenzhen Hospital, Center for Reproductive Medicine, Shenzhen, People's Republic of China.

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http://dx.doi.org/10.1016/j.fertnstert.2021.01.034DOI Listing
April 2021

The male germline-specific protein MAPS is indispensable for pachynema progression and fertility.

Proc Natl Acad Sci U S A 2021 02;118(8)

Shenzhen Key Laboratory of Fertility Regulation, Center of Assisted Reproduction and Embryology, The University of Hong Kong-Shenzhen Hospital, 518053 Shenzhen, Guangdong, China;

Meiosis is a specialized cell division that creates haploid germ cells from diploid progenitors. Through differential RNA expression analyses, we previously identified a number of mouse genes that were dramatically elevated in spermatocytes, relative to their very low expression in spermatogonia and somatic organs. Here, we investigated in detail one of these genes, and independently conclude that it encodes a male germline-specific protein, in agreement with a recent report. We demonstrated that it is essential for pachynema progression in spermatocytes and named it male pachynema-specific (MAPS) protein. Mice lacking ( ) suffered from pachytene arrest and spermatocyte death, leading to male infertility, whereas female fertility was not affected. Interestingly, pubertal spermatocytes were arrested at early pachytene stage, accompanied by defects in DNA double-strand break (DSB) repair, crossover formation, and XY body formation. In contrast, adult spermatocytes only exhibited partially defective crossover but nonetheless were delayed or failed in progression from early to mid- and late pachytene stage, resulting in cell death. Furthermore, we report a significant transcriptional dysregulation in autosomes and XY chromosomes in both pubertal and adult pachytene spermatocytes, including failed meiotic sex chromosome inactivation (MSCI). Further experiments revealed that MAPS overexpression in vitro dramatically decreased the ubiquitination levels of cellular proteins. Conversely, in pachytene cells, protein ubiquitination was dramatically increased, likely contributing to the large-scale disruption in gene expression in pachytene cells. Thus, MAPS is a protein essential for pachynema progression in male mice, possibly in mammals in general.
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http://dx.doi.org/10.1073/pnas.2025421118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7923350PMC
February 2021

The fungicide Mancozeb reduces spheroid attachment onto endometrial epithelial cells through downregulation of estrogen receptor β and integrin β3 in Ishikawa cells.

Ecotoxicol Environ Saf 2021 Jan 13;208:111606. Epub 2020 Nov 13.

Department of Obstetrics and Gynaecology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong, China; Shenzhen Key Laboratory of Fertility Regulation, The University of Hong Kong-Shenzhen Hospital, Futian District, Shenzhen, China. Electronic address:

Mancozeb is a metal-containing ethylene bis-dithiocarbamate fungicide widely used in agriculture. Ethylene thiourea (ETU) is the primary metabolite of Mancozeb. Mancozeb has been associated with spontaneous abortions and abnormal menstruation in women. However, the effects of Mancozeb and ETU on embryo attachment remain unknown. The human blastocyst surrogate trophoblastic spheroids (JEG-3), endometrial epithelial surrogate adenocarcinoma cells (Ishikawa), or human primary endometrial epithelial cells (EECs) monolayer were used in the spheroid attachment models. Ishikawa and EECs were pretreated with different concentrations of Mancozeb or ETU for 48 h before the attachment assay. Gene expression profiles of Ishikawa cells were examined to understand how Mancozeb modulates endometrial receptivity with Microarray. The genes altered by Mancozeb were confirmed by qPCR and compared with the ETU treated groups. Mancozeb and ETU treatment inhibited cell viability at 10 μg/mL and 5000 µg/mL, respectively. At non-cytotoxic concentrations, Mancozeb at 3 μg/mL and ETU at 300 μg/mL reduced JEG-3 spheroid attachment onto Ishikawa cells. A similar result was observed with human primary endometrial epithelial cells. Mancozeb at 3 μg/mL modified the transcription of 158 genes by at least 1.5-fold in Microarray analysis. The expression of 10 differentially expressed genes were confirmed by qPCR. Furthermore, Mancozeb decreased spheroid attachment possibly through downregulating the expression of endometrial estrogen receptor β and integrin β3, but not mucin 1. These results were confirmed in both overexpression and knockdown experiments and co-culture assay. Mancozeb but not its metabolite ETU reduced spheroid attachment through modulating gene expression profile and decreasing estrogen receptor β and integrin β3 expression of endometrial epithelial cells.
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http://dx.doi.org/10.1016/j.ecoenv.2020.111606DOI Listing
January 2021

The novel male meiosis recombination regulator coordinates the progression of meiosis prophase I.

J Genet Genomics 2020 08 26;47(8):451-465. Epub 2020 Aug 26.

Shenzhen Key Laboratory of Fertility Regulation, Center of Assisted Reproduction and Embryology, The University of Hong Kong - Shenzhen Hospital, Shenzhen, 518000, China; Department of Obstetrics and Gynecology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, 999077, China. Electronic address:

Meiosis is a specialized cell division for producing haploid gametes in sexually reproducing organisms. In this study, we have independently identified a novel meiosis protein male meiosis recombination regulator (MAMERR)/4930432K21Rik and showed that it is indispensable for meiosis prophase I progression in male mice. Using super-resolution structured illumination microscopy, we found that MAMERR functions at the same double-strand breaks as the replication protein A and meiosis-specific with OB domains/spermatogenesis associated 22 complex. We generated a Mamerr-deficient mouse model by deleting exons 3-6 and found that most of Mamerr spermatocytes were arrested at pachynema and failed to progress to diplonema, although they exhibited almost intact synapsis and progression to the pachytene stage along with XY body formation. Further mechanistic studies revealed that the recruitment of DMC1/RAD51 and heat shock factor 2-binding protein in Mamerr spermatocytes was only mildly impaired with a partial reduction in double-strand break repair, whereas a substantial reduction in ubiquitination on the autosomal axes and on the XY body appeared as a major phenotype in Mamerr spermatocytes. We propose that MAMERR may participate in meiotic prophase I progression by regulating the ubiquitination of key meiotic proteins on autosomes and XY chromosomes, and in the absence of MAMERR, the repressed ubiquitination of key meiotic proteins leads to pachytene arrest and cell death.
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http://dx.doi.org/10.1016/j.jgg.2020.08.001DOI Listing
August 2020

Natural killer and NKT cells in the male reproductive tract.

J Reprod Immunol 2020 11 12;142:103178. Epub 2020 Jul 12.

Department of Dermatology and Allergy / Andrology Unit, University of Bonn, 53105 Bonn, Germany. Electronic address:

Natural killer (NK) cells are important effector lymphocytes that play a pivotal role in the innate and adaptive immune responses to tumors and viral infection. NKT cells are a heterogeneous group of T cells that share properties with both T cells and NK cells. They display immunoregulatory properties as they facilitate the cell-mediated immune response to tumors and infectious diseases, and inhibit cell-mediated immunity associated with autoimmune diseases and allograft rejection. However, the roles of NK and NKT cells in the male reproductive tract remain largely unexplored, in particular, NKT cells, tissue distribution, and state of health or disease. Infection and inflammation of the male genital tract are thought to be the primary etiological factors of male infertility. In this review, we considered this complex and rapidly growing field. We summarize the recent findings and the characterization and roles of NK and NKT cells in the male reproductive tract, including the testis, epididymis, prostate, seminal vesicle, and semen, to enhance our understanding of the immunological mechanisms of male infertility and for the design effective vaccines for male reproductive health in the future.
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http://dx.doi.org/10.1016/j.jri.2020.103178DOI Listing
November 2020

Understanding the regulatory mechanisms of endometrial cells on activities of endometrial mesenchymal stem-like cells during menstruation.

Stem Cell Res Ther 2020 06 17;11(1):239. Epub 2020 Jun 17.

Shenzhen Key Laboratory of Fertility Regulation, Reproductive Medicine Centre, The University of Hong Kong Shenzhen Hospital, Shenzhen, Guangdong, China.

Background: The identification of endometrial stem/progenitor cells in a high turnover rate tissue suggests that a well-orchestrated underlying network controls the behaviour of these stem cells. The thickness of the endometrium can grow from 0.5-1 mm to 5-7 mm within a week indicating the need of stem cells for self-renewal and differentiation during this period. The cyclical regeneration of the endometrium suggests specific signals can activate the stem cells during or shortly after menstruation.

Methods: Endometrial mesenchymal stem-like cells (eMSCs) were cocultured with endometrial epithelial or stromal cells from different phases of the menstrual cycle; the clonogenicity and the phenotypic expression of eMSC markers (CD140b and CD146) were assessed. The functional role of WNT/β-catenin signalling on eMSC was determined by western blot analysis, immunofluorescent staining, flow cytometry, quantitative real-time PCR and small interfering RNA. The cytokine levels in the conditioned medium of epithelial or stromal cells cocultured with eMSCs were evaluated by enzyme-linked immunosorbent assays.

Results: Coculture of endometrial cells (epithelial or stromal) from the menstrual phase enhanced the clonogenicity and self-renewal activities of eMSCs. Such phenomenon was not observed in niche cells from the proliferative phase. Coculture with endometrial cells from the menstrual phase confirmed an increase in expression of active β-catenin in the eMSCs. Treatment with IWP-2, a WNT inhibitor, suppressed the observed effects. Anti-R-spondin-1 antibody reduced the stimulatory action of endometrial niche cells on WNT/β-catenin activation in the T cell factor/lymphoid enhancer-binding factor luciferase reporter assay. Moreover, the mRNA level and protein immunoreactivities of leucine-rich repeat-containing G-protein coupled receptor 5 were higher in eMSCs than unfractionated stromal cells. Conditioned media of endometrial niche cells cocultured with eMSCs contained increased levels of C-X-C motif ligand 1 (CXCL1), CXCL5 and interleukin 6. Treatment with these cytokines increased the clonogenic activity and phenotypic expression of eMSCs.

Conclusions: Our findings indicate a role of WNT/β-catenin signalling in regulating activities of endometrial stem/progenitor cells during menstruation. Certain cytokines at menstruation can stimulate the proliferation and self-renewal activities of eMSCs. Understanding the mechanism in the regulation of eMSCs may contribute to treatments of endometrial proliferative disorders such as Asherman's syndrome.
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http://dx.doi.org/10.1186/s13287-020-01750-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7302161PMC
June 2020

Decidual glycodelin-A polarizes human monocytes into a decidual macrophage-like phenotype through Siglec-7.

J Cell Sci 2020 07 23;133(14). Epub 2020 Jul 23.

Department of Obstetrics and Gynaecology, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong S.A.R

Decidual macrophages constitute 20-30% of the total leukocytes in the uterus of pregnant women, regulating the maternal immune tolerance and placenta development. Abnormal number or activities of decidual macrophages (dMs) are associated with fetal loss and pregnancy complications, such as preeclampsia. Monocytes differentiate into dMs in a decidua-specific microenvironment. Despite their important roles in pregnancy, the exact factors that regulate the differentiation into dMs remain unclear. Glycodelin-A (PAEP, hereafter referred to as GdA) is a glycoprotein that is abundantly present in the decidua, and plays an important role in fetomaternal defense and placental development. It modulates the differentiation and activity of several immune cell types residing in the decidua. In this study, we demonstrated that GdA induces the differentiation of human monocytes into dM-like phenotypes in terms of transcriptome, cell surface marker expression, secretome, and regulation of trophoblast and endothelial cell functions. We found that Sialic acid-binding Ig-like lectin 7 (Siglec-7) mediates the binding and biological actions of GdA in a sialic acid-dependent manner. We, therefore, suggest that GdA, induces the polarization of monocytes into dMs to regulate fetomaternal tolerance and placental development.
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http://dx.doi.org/10.1242/jcs.244400DOI Listing
July 2020

A randomised trial comparing conventional semen parameters, sperm DNA fragmentation levels and satisfaction levels between semen collection at home and at the clinic.

Andrologia 2020 Aug 26;52(7):e13628. Epub 2020 May 26.

Center of Assisted Reproduction and Embryology, The University of Hong Kong - Shenzhen Hospital, Shenzhen, China.

The aim of the randomised trial was to compare conventional semen parameters, sperm DNA fragmentation levels and satisfaction levels between semen samples collected at home and at the clinic. We recruited 110 men with a history of infertility for at least 1 year from the outpatient andrology clinic. Each man collected two semen samples, one at home and one at the clinic. Men were randomly assigned into the home first (n = 55) or clinic first (n = 55) groups. The primary outcome was sperm concentration. There was no significant difference in sperm concentration, sperm DNA fragmentation levels or other conventional semen parameters between home first and clinic first samples (p > .05), while satisfaction levels were significantly higher for home first samples (p < .01). Consistent results were obtained when comparing home-collected and clinic-collected samples within individuals. Men can be offered the option to collect semen samples at home for examination or assisted reproduction without compromising semen quality, especially for those with difficulty in producing semen samples at the clinic.
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http://dx.doi.org/10.1111/and.13628DOI Listing
August 2020

MicroRNA-135a-induced formation of CD133+ subpopulation with cancer stem cell properties in cervical cancer.

Carcinogenesis 2020 11;41(11):1592-1604

Shenzhen Key Laboratory of Fertility Regulation, Shenzhen, People's Republic of China.

Cancer stem cells (CSCs) play significant roles in tumor initiation. MicroRNA-135a (miR-135a) induced the formation of a CD133+ subpopulation from a human papillomavirus-immortalized cervical epithelial cell line. Compared with the CD133- cells, the CD133+ cells expressed higher levels of miR-135a and OCT4, exhibited significantly higher tumorsphere forming capacity and the time required for tumorsphere formation was shortened in the second generation. Serum induction suppressed the expression of CD133, OCT4 and miR-135a, but increased expression of involucrin in the miR-135a-induced CD133+ cells. The miR-135a-induced CD133+ cells were tumorigenic in a limiting dilution approach in vivo. The cells expressed significantly higher level of active β-catenin and OCT4 than the CD133- counterpart. Wnt3a enhanced the expression of OCT4 and CD133 in cervical cancer cells but failed to enhance CD133 transcription in normal cervical cells. Wnt3a stimulation also increased tumorsphere size and self-renewal of miR-135a-induced CD133+ subpopulation. Wnt/β-catenin inhibition suppressed tumorsphere formation while Wnt3a partially nullified the inhibitory effect. Taken together, miR-135a induced the formation of a subpopulation of cells with CSC properties both in vitro and in vivo and the Wnt/β-catenin signaling pathway is essential to maintain its tumorigenicity.
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http://dx.doi.org/10.1093/carcin/bgaa025DOI Listing
November 2020

Altered glycosylation of glycodelin in endometrial carcinoma.

Lab Invest 2020 07 23;100(7):1014-1025. Epub 2020 Mar 23.

Department of Clinical Chemistry, University of Helsinki and Helsinki University Hospital, Helsinki, Finland.

Glycodelin is a major glycoprotein expressed in reproductive tissues, like secretory and decidualized endometrium. It has several reproduction related functions that are dependent on specific glycosylation, but it has also been found to drive differentiation of endometrial carcinoma cells toward a less malignant phenotype. Here we aimed to elucidate whether the glycosylation and function of glycodelin is altered in endometrial carcinoma as compared with a normal endometrium. We carried out glycan structure analysis of glycodelin expressed in HEC-1B human endometrial carcinoma cells (HEC-1B Gd) by mass spectrometry glycomics strategies. Glycans of HEC-1B Gd were found to comprise a typical mixture of high-mannose, hybrid, and complex-type N-glycans, often containing undecorated LacNAc (Galβ1-4GlcNAc) antennae. However, several differences, as compared with previously reported glycan structures of normal human decidualized endometrium-derived glycodelin isoform, glycodelin-A (GdA), were also found. These included a lower level of sialylation and more abundant poly-LacNAc antennae, some of which are fucosylated. This allowed us to select lectins that showed different binding to these classes of glycodelin. Despite the differences in glycosylation between HEC-1B Gd and GdA, both showed similar inhibitory activity on trophoblast cell invasion and peripheral blood mononuclear cell proliferation. For the detection of cancer associated glycodelin, we established a novel in situ proximity-ligation based histochemical staining method using a specific glycodelin antibody and UEAI lectin. We found that the UEAI reactive glycodelin was abundant in endometrial carcinoma, but virtually absent in normal endometrial tissue even when glycodelin was strongly expressed. In conclusion, we established a histochemical staining method for the detection of endometrial carcinoma-associated glycodelin and showed that this specific glycodelin is exclusively expressed in cancer, not in normal endometrium. Similar methods can be used for studies of other glycoproteins.
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http://dx.doi.org/10.1038/s41374-020-0411-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7312397PMC
July 2020

Adrenomedullin insufficiency alters macrophage activities in fallopian tube: a pathophysiologic explanation of tubal ectopic pregnancy.

Mucosal Immunol 2020 09 13;13(5):743-752. Epub 2020 Mar 13.

Shenzhen Key Laboratory of Fertility Regulation, The University of Hong Kong-Shenzhen Hospital, Shenzhen, 1, Haiyuan 1st Road, Futian District, Shenzhen, Guangdong, China.

Ectopic pregnancy is the major cause of maternal morbidity and mortality in the first trimester of pregnancy. Tubal ectopic pregnancy (TEP) accounts for nearly 98% of all ectopic pregnancies. TEP is usually associated with salpingitis but the underlying mechanism in salpingitis leading to TEP remains unclear. Adrenomedullin (ADM) is a peptide hormone abundantly expressed in the fallopian tube with potent anti-inflammatory activities. Its expression peaks at the early luteal phase when the developing embryo is being transported through the fallopian tube. In the present study, we demonstrated reduced expression of ADM in fallopian tubes of patients with salpingitis and TEP. Using macrophages isolated from the fallopian tubes of these women, our data revealed that the salpingistis-associated ADM reduction contributed to aggravated pro-inflammatory responses of the tubal macrophages resulting in production of pro-inflammatory and pro-implantation cytokines IL-6 and IL-8. These cytokines activated the expression of implantation-associated molecules and Wnt signaling pathway predisposing the tubal epithelium to an adhesive and receptive state for embryo implantation. In conclusion, this study provided evidence for the role of ADM in the pathogenesis of TEP through regulating the functions of tubal macrophages.
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http://dx.doi.org/10.1038/s41385-020-0278-6DOI Listing
September 2020

Mir-let-7a/g Enhances Uterine Receptivity via Suppressing Wnt/β-Catenin Under the Modulation of Ovarian Hormones.

Reprod Sci 2020 05 15;27(5):1164-1174. Epub 2020 Jan 15.

Department of Obstetrics and Gynaecology, The University of Hong Kong, Hong Kong, China.

Microarray has indicated a huge number of miRNAs exist in reproductive tissues and cells. Moreover, the expression of miRNA in the reproductive system varies under the strict monitoring of different regulations. To understand the role of miRNA-mediated post-transcriptional gene regulation in female reproduction, we investigated the level and function of a mir-let-7 family member in both mice and human uterine receptivity. As we observed, mir-let-7 a/g had a higher expression in mouse and human receptive uterine epithelium; the level of mir-let-7a was under the inverse regulation of estrogen and progesterone; upregulated mir-let-7a/g in mouse and human uterine epithelium increased uterine receptivity, thus improved implantation-related embryo attachment and outgrowth ability; the let-7a/g enhanced uterine receptivity through suppressing canonical Wnt signaling. In summary, our findings suggest that mir-let-7 a/g increases uterine receptivity via inhibiting Wnt signaling and under the modulation of ovarian hormones.
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http://dx.doi.org/10.1007/s43032-019-00115-3DOI Listing
May 2020

High-resolution mapping of reciprocal translocation breakpoints using long-read sequencing.

MethodsX 2019 31;6:2499-2503. Epub 2019 Oct 31.

Department of Obstetrics and Gynecology, The University of Hong Kong, Queen Mary Hospital, Pokfulam, Hong Kong.

Long-read nanopore sequencing enables direct high-resolution breakpoint mapping on balanced carriers of reciprocal translocation. The mean sequencing depth on the translocated chromosomes to achieve accurate mapping of breakpoints ranged from 2.5-fold to 6.2-fold. To speed up determination of the breakpoints from long-read sequencing data, alignment reads on the translocated chromosomes were extracted before piped into NanoSV. Checking the position of breakpoints on Interactive Genomics Viewer (IGV) was crucial to successful design of breakpoint PCR primers, especially when large deletion was involved at the breakpoints. •Long-read sequencing enables accurate breakpoint mapping with base-pair resolution•Splitting bam files by translocated chromosomes drastically speeded up the breakpoint determination•IGV helps to identify the breakpoint positions and facilitate the design of breakpoint PCR primers.
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http://dx.doi.org/10.1016/j.mex.2019.10.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6939040PMC
October 2019

Regulation of human trophoblast surrogate Jeg-3 spheroids implantation potential by Wnt/β-catenin pathway and lin28a/let-7a axis.

Exp Cell Res 2020 03 24;388(1):111718. Epub 2019 Dec 24.

Department of Obstetrics and Gynaecology, The University of Hong Kong, Pokfulam, Hong Kong, China; Centre of Reproduction, Development of Growth, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, China. Electronic address:

Successful implantation happens only when the development of a competent blastocyst synchronized with the differentiation of a receptive uterus. The exact mechanism affecting embryo implantation competency is still unclear. Previous data from our laboratory showed that several members of the let-7 family were up-regulated in the implanting dormant blastocysts and prohibited embryo activation through down-regulation integrin-β3. However, how the mir-let-7 family is regulated is still a question. In this study, the in vitro co-culture model was applied to imitate implantation. Human embryo surrogate Jeg-3 spheroids and endometrium epithelial cells Ishikawa were used. The following views were demonstrated. Firstly,Wnt/β-catenin signaling is essential for Jeg-3 spheroids implantation. Secondly, mir-let-7a is repressed by Wnt signaling, and low let-7a is beneficial for spheroids attachment and outgrowth. Third, in contrast with let-7a, lin28a is up-regulated by Wnt and promotes attachment and outgrowth. Lastly, the function of Wnt in embryo surrogate spheroids in implantation is mediated through lin28a/let-7a axis. In summary, our findings suggest Wnt/β-catenin signaling strength human embryo surrogate spheroids implanting potential through regulation lin28a/let-7a axis.
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http://dx.doi.org/10.1016/j.yexcr.2019.111718DOI Listing
March 2020

Bisphenol compounds regulate decidualized stromal cells in modulating trophoblastic spheroid outgrowth and invasion in vitro†.

Biol Reprod 2020 03;102(3):693-704

Department of Obstetrics and Gynaecology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China.

Bisphenol A (BPA) is commonly found in epoxy resins used in the manufacture of plastic coatings in food packaging and beverage cans. There is a growing concern about BPA as a weak estrogenic compound that can affect human endocrine function. Chemicals structurally similar to BPA, such as bisphenol F (BPF) and bisphenol S (BPS), have been developed as substitutes in the manufacturing industry. Whether these bisphenol substitutes have adverse effects on human endocrine and reproductive systems remains largely unknown. This study investigated the effects of BPA, BPF, and BPS on regulating the function of decidualized human primary endometrial stromal cells on trophoblast outgrowth and invasion by indirect and direct co-culture models. All three bisphenols did not affect the stromal cell decidualization process. However, BPA- and BPF-treated decidualized stromal cells stimulated trophoblastic spheroid invasion in the indirect coculture model. The BPA-treated decidualized stromal cells had upregulated expressions of several invasion-related molecules including leukemia inhibitory factor (LIF), whereas both BPA- and BPF-treated decidualized stromal cells had downregulated expressions of anti-invasion molecules including plasminogen activator inhibitor type 1 (PAI-1) and tumor necrosis factor (TNFα) . Taken together, BPA and BPF altered the expression of invasive and anti-invasive molecules in decidualized stromal cells modulating its function on trophoblast outgrowth and invasion, which could affect the implantation process and subsequent pregnancy outcome.
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http://dx.doi.org/10.1093/biolre/ioz212DOI Listing
March 2020

Myometrial Cells Stimulate Self-Renewal of Endometrial Mesenchymal Stem-Like Cells Through WNT5A/β-Catenin Signaling.

Stem Cells 2019 11 8;37(11):1455-1466. Epub 2019 Oct 8.

Shenzhen Key Laboratory Fertility Regulation, The University of Hong Kong Shenzhen Hospital, Shenzhen, People's Republic of China.

Human endometrium undergoes cycles of proliferation and differentiation throughout the reproductive years of women. The endometrial stem/progenitor cells contribute to this regenerative process. They lie in the basalis layer of the endometrium next to the myometrium. We hypothesized that human myometrial cells provide niche signals regulating the activities of endometrial mesenchymal stem-like cells (eMSCs). In vitro coculture of myometrial cells enhanced the colony-forming and self-renewal ability of eMSCs. The cocultured eMSCs retained their multipotent characteristic and exhibited a greater total cell output when compared with medium alone culture. The expression of active β-catenin in eMSCs increased significantly after coculture with myometrial cells, suggesting activation of WNT/β-catenin signaling. Secretory factors in spent medium from myometrial cell culture produced the same stimulatory effects on eMSCs. The involvement of WNT/β-catenin signaling in self-renewal of eMSCs was confirmed with the use of WNT activator (Wnt3A conditioned medium) and WNT inhibitors (XAV939 and inhibitor of Wnt Production-2 [IWP-2]). The myometrial cells expressed more WNT5A than other WNT ligands. Recombinant WNT5A stimulated whereas anti-WNT5A antibody suppressed the colony formation, self-renewal, and T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) transcriptional activities of eMSCs. Moreover, eMSCs expressed FZD4 and LRP5. WNT5A is known to activate the canonical WNT signaling in the presence of these receptor components. WNT antagonist, DKK1, binds to LRP5/6. Consistently, DKK1 treatment nullified the stimulatory effect of myometrial cell coculture. In conclusion, our findings show that the myometrial cells are niche components of eMSCs, modulating the self-renewal activity of eMSCs by WNT5A-dependent activation of WNT/β-catenin signaling. Stem Cells 2019;37:1455-1466.
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http://dx.doi.org/10.1002/stem.3070DOI Listing
November 2019

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the differentiation of embryonic stem cells towards pancreatic lineage and pancreatic beta cell function.

Environ Int 2019 09 10;130:104885. Epub 2019 Jun 10.

Department of Obstetrics and Gynaecology, The University of Hong Kong, Hong Kong, China; Shenzhen Key Laboratory of Fertility Regulation, The University of Hong Kong Shenzhen Hospital, Shenzhen, China. Electronic address:

Animal and epidemiological studies demonstrated association of persistent exposure of TCDD, an endocrine disrupting chemical, to susceptibility of type 2 diabetes (T2D). High doses of TCDD were commonly employed in experimental animals to illustrate its diabetogenic effects. Data linking the epigenetic effects of low doses of TCDD on embryonic cells to T2D susceptibility risks is very limited. To address whether low dose exposure to TCDD would affect pancreatic development, hESCs pretreated with TCDD at concentrations similar to human exposure were differentiated towards pancreatic lineage cells, and their global DNA methylation patterns were determined. Our results showed that TCDD-treated hESCs had impaired pancreatic lineage differentiation potentials and altered global DNA methylation patterns. Four of the hypermethylated genes (PRKAG1, CAPN10, HNF-1B and MAFA) were validated by DNA bisulfite sequencing. PRKAG1, a regulator in the AMPK signaling pathway critical for insulin secretion, was selected for further functional study in the rat insulinoma cell line, INS-1E cells. TCDD treatment induced PRKAG1 hypermethylation in hESCs, and the hypermethylation was maintained after pancreatic progenitor cells differentiation. Transient Prkag1 knockdown in the INS-1E cells elevated glucose stimulated insulin secretions (GSIS), possibly through mTOR signaling pathway. The current study suggested that early embryonic exposure to TCDD might alter pancreatogenesis, increasing the risk of T2D.
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http://dx.doi.org/10.1016/j.envint.2019.05.079DOI Listing
September 2019

The role of galectin-3 in spermatozoa-zona pellucida binding and its association with fertilization in vitro.

Mol Hum Reprod 2019 08;25(8):458-470

Department of Obstetrics and Gynaecology, The University of Hong Kong, Queen Mary Hospital, Hong Kong, Hong Kong SAR.

Human spermatozoa can fertilize an oocyte only after post-testicular maturation and capacitation. These processes involve dynamic modification and reorganization of the sperm plasma membrane, which allow them to bind to the zona pellucida (ZP) of the oocyte. Defective sperm-ZP binding is one of the major causes of male subfertility. Galectin-3 is a secretory lectin in human seminal plasma well known for its action on cell adhesion. The aim of this study was to determine the role of galectin-3 in spermatozoa-ZP interaction and its association with fertilization rate in clinical assisted reproduction. Our studies revealed that the acrosomal region of ejaculated and capacitated spermatozoa possess strong galectin-3 immunoreactivity, which is much stronger than that of epididymal spermatozoa. Expression of galectin-3 can also be detected on seminal plasma-derived extracellular vesicles (EVs) and can be transferred to the sperm surface. Blocking of sperm surface galectin-3 function by antibody or carbohydrate substrate reduced the ZP-binding capacity of spermatozoa. Purified galectin-3 is capable of binding to ZP, indicating that galectin-3 may serve as a cross-linking bridge between ZP glycans and sperm surface glycoproteins. Galectin-3 levels in seminal plasma-derived EVs were positively associated with fertilization rates. These results suggest that galectin-3 in EVs is transferred to the sperm surface during post-testicular maturation and plays a crucial role in spermatozoa-ZP binding after capacitation. Reduced galectin-3 expression in seminal plasma-derived EVs may be a cause behind a low fertilization rate. Further studies with more clinical samples are required to confirm the relationship between galectin-3 levels and IVF outcomes.
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http://dx.doi.org/10.1093/molehr/gaz030DOI Listing
August 2019

Distinguishing between carrier and noncarrier embryos with the use of long-read sequencing in preimplantation genetic testing for reciprocal translocations.

Genomics 2020 01 1;112(1):494-500. Epub 2019 Apr 1.

Department of Obstetrics and Gynecology, The University of Hong Kong, Queen Mary Hospital, Pokfulam, Hong Kong.

Balanced reciprocal translocation carriers are usually phenotypically normal but are at an increased risk of infertility, recurrent miscarriage or having affected children. Preimplantation genetic testing on chromosomal structural rearrangement (PGT-SR) offers a way to screen against unbalanced embryos. Here, we demonstrated a new method to distinguish carrier from noncarrier embryos. Translocation breakpoints were first delineated by nanopore sequencing followed by polymerase chain reaction (PCR) across breakpoints. High-resolution breakpoint mapping was successful in all (9/9) balanced reciprocal translocation carriers. Retrospective analysis of their embryo biopsies with breakpoint PCR showed 100% concordant results with PGT-SR on trophectoderm biopsies (40/40) and 53% concordance on blastomere biopsies (8/15). The low concordant rate in blastomeres was due to failure in the amplification of derivative chromosomes involving large deletions. Breakpoint PCR also showed 100% concordant results with prenatal/postnatal outcomes on 5 pregnancies, indicating that our new method can accurately distinguish carrier from noncarrier embryos.
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http://dx.doi.org/10.1016/j.ygeno.2019.04.001DOI Listing
January 2020

Alteration of the immune cell profiles in the pathophysiology of tubal ectopic pregnancy.

Am J Reprod Immunol 2019 04 18;81(4):e13093. Epub 2019 Feb 18.

Department of Obstetrics & Gynaecology, The University of Hong Kong, Hong Kong, SAR, China.

Tubal ectopic pregnancy (TEP) refers to implantation of conceptus in the fallopian tube. It makes up over 98% of ectopic pregnancy (EP), which is the leading cause of maternal morbidity and mortality in the first trimester of pregnancy. Immune cells at the maternal-fetal interface play important roles in the process of embryo implantation, stroma decidualization, and early placental development. Alterations in the composition, phenotype, and activity of the immune cells in the fallopian tubes contribute toward the onset of TEP. In this review, we compare the leukocytic proportions in decidua of normal pregnancy, and in decidua and fallopian tubes of TEP. The possible functions of these immune cells in the pathophysiology of TEP are also discussed.
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http://dx.doi.org/10.1111/aji.13093DOI Listing
April 2019

Glycodelin-A stimulates the conversion of human peripheral blood CD16-CD56bright NK cell to a decidual NK cell-like phenotype.

Hum Reprod 2019 04;34(4):689-701

Department of Obstetrics and Gynaecology, LKS Faculty of Medicine, The University of Hong Kong, 7/F, Laboratory Block, 21 Sassoon Road, Pokfulam, Hong Kong.

Study Question: Does glycodelin-A (GdA) induce conversion of human peripheral blood CD16-CD56bright natural killer (NK) cells to decidual NK (dNK) cells to facilitate placentation?

Summary Answer: GdA binds to blood CD16-CD56bright NK cells via its sialylated glycans and converts them to a dNK-like cells, which in turn regulate endothelial cell angiogenesis and trophoblast invasion via vascular endothelial growth factor (VEGF) and insulin-like growth factor-binding protein 1 (IGFBP-1) secretion, respectively.

What Is Known Already: dNK cells are the most abundant leucocyte population in the decidua. These cells express CD16-CD56bright phenotype. Peripheral blood CD16-CD56bright NK cells and hematopoietic precursors have been suggested to be capable of differentiating towards dNK cells upon exposure to the decidual microenvironment. These cells regulate trophoblast invasion during spiral arteries remodelling and mediate homoeostasis and functions of the endothelial cells. GdA is an abundant glycoprotein in the human decidua with peak expression between the 6th and 12th week of gestation, suggesting a role in early pregnancy. Indeed, GdA interacts with and modulates functions and differentiation of trophoblast and immune cells in the human feto-maternal interface. Aberrant GdA expression during pregnancy is associated with unexplained infertility, pregnancy loss and pre-eclampsia.

Study Design, Size, Duration: CD16+CD56dim, CD16-CD56bright and dNK cells were isolated from human peripheral blood and decidua tissue, respectively, by immuno-magnetic beads or fluorescence-activated cell sorting. Human extravillous trophoblasts were isolated from first trimester placental tissue after termination of pregnancy. Biological activities of the cells were studied after treatment with GdA at a physiological dose of 5 μg/mL. GdA was purified from human amniotic fluid by immuno-affinity chromatography.

Participants/materials, Setting, Methods: Expression of VEGF, CD9, CD49a, CD151 and CD158a in the cells were determined by flow cytometry. Angiogenic proteins in the spent media of NK cells were determined by cytokine array and ELISA. Blocking antibodies were used to study the functions of the identified angiogenic proteins. Endothelial cell angiogenesis was determined by tube formation and trans-well migration assays. Cell invasion and migration were determined by trans-well invasion/migration assay. Binding of normal and de-sialylated GdA, and expression of L-selectin and siglec-7 on the NK cells were analysed by flow cytometry. The association between GdA and L-selectin on NK cells was confirmed by immunoprecipitation. Extracellular signal-regulated protein kinases (ERK) activation was determined by Western blotting and functional assays.

Main Results And The Role Of Chance: GdA treatment enhanced the expression of dNK cell markers CD9 and CD49a and the production of the functional dNK secretory product VEGF in the peripheral blood CD16-CD56bright NK cells. The spent media of GdA-treated CD16-CD56bright NK cells promoted tube formation of human umbilical vein endothelial cells and invasiveness of trophoblasts. These stimulatory effects were mediated by the stimulatory activities of GdA on an ERK-activation dependent production of VEGF and IGFBP-1 by the NK cells. GdA had a stronger binding affinity to the CD16-CD56bright NK cells as compared to the CD16+CD56dim NK cells. This GdA-NK cell interaction was reduced by de-sialylation. GdA interacted with L-selectin, expressed only in the CD16-CD56bright NK cells, but not in the CD16+CD56dim NK cells. Anti-L-selectin functional blocking antibody suppressed the binding and biological activities of GdA on the NK cells.

Large Scale Data: N/A.

Limitations, Reasons For Caution: Some of the above findings are based on a small sample size of peripheral blood CD16-CD56bright NK cells. These results need to be confirmed with human primary dNK cells.

Wider Implications Of The Findings: This is the first study on the biological role of GdA on conversion of CD16-CD56bright NK cells to dNK-like cells. Further investigation on the glycosylation and functions of GdA will enhance our understanding on human placentation and placenta-associated complications with altered NK cell biology.

Study Funding/competing Interest(s): This work was supported by the Hong Kong Research Grant Council Grant 17122415, Sanming Project of Medicine in Shenzhen, the Finnish Cancer Foundation, Sigrid Jusélius Foundation and the Finnish Society of Clinical Chemistry. The authors have no competing interests to declare.
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http://dx.doi.org/10.1093/humrep/dey378DOI Listing
April 2019

Meiotic chromatid recombination and segregation assessed with human single cell genome sequencing data.

J Med Genet 2019 03 4;56(3):156-163. Epub 2018 Dec 4.

Center for Reproductive Medicine, Third Hospital, Peking University, Beijing, China.

Background: The human oocyte transmits one set of haploid genome into female pronucleus (FPN) while discards the remaining genome into the first polar body (PB1) and the second polar body (PB2). The FPN genome carries an assembly of maternal and paternal genome that resulted from homologous recombination during the prophase of the first meiosis. However, how parental genome has been shuffled and transmitted is difficult to assess by analysing only the progeny's genome.

Objective: To assess meiotic chromatid recombination and segregation in human oocytes.

Methods: Single cell genome sequencing data of PB1, PB2 and FPN that originated from the same oocyte were used to analyse the human oocyte homologous chromosome interaction and segregation. To analyse whether chromosomes were non-randomly segregated into polar bodies or pronucleus, we analysed the ratio of crossover in PB2 and FPN, and constructed a model to detect the randomness of oocyte chromosome segregation.

Results: We found that during oocyte meiosis, in addition to homologous chromosome recombination, there was also a genome conversion phenomenon which generated a non-reciprocal genetic information transmission between homologous chromosomes. We also inferred that during meiosis, DNA breaks and repairs frequently occurred at centromere-adjacent regions. From our data we did not find obvious evidence supporting the crossover number-based or SNP-based meiotic drive in oocytes.

Conclusion: In addition to the crossover-based recombination, during human oocyte meiosis, a direct genome conversion between homologous chromosomes is used in some oocytes. Our findings are helpful in understanding the specific features of meiotic chromatid recombination and segregation in human oocytes.
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http://dx.doi.org/10.1136/jmedgenet-2018-105612DOI Listing
March 2019
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