Yingyu Liu

Adipose-derived stem cells from pregnant women show higher proliferation rate unrelated to estrogen

BACKGROUND Adipose tissue contains an abundant population of multipotent adipose-derived stem cells (ASCs) and has been an excellent source of mesenchymal stem cells for cell therapy and tissue engineering. To ensure successful cell therapies, consistency of stem cell performance across donors is critical. However, the effect of the donors reproductive status on ASC proliferation rate and differentiation capacity is undefined. METHODS We investigated whether the yield and function of ASCs are affected by the womans reproductive status: pregnancy, premenopause or menopause. ASCs were isolated from the abdomen of 15 women and their proliferation rates and differentiation capacities were compared by cell count. The capacity of ASCs to differentiate into the chondrogenic lineage was investigated by quantitative real-time polymerase chain reaction and immunohistochemistry. RESULTS There was no significant difference in the differentiation capacity between the three groups, whereas the proliferation rate of ASCs from pregnant women was significantly higher than from the other two groups (P < 0.05). The proliferation rate of ASCs after estrogen treatment remained unchanged. CONCLUSIONS Despite the higher proliferation rate in pregnant women, ASCs showed consistency in cell differentiation capacity and were unaffected by donor status. This suggests that factors other than estrogen are responsible for the difference in proliferation.

High isoprostane level in cardinal ligament-derived fibroblasts and urine sample of women with uterine prolapse

We studied the isoprostane level, a well‐recognised biomarker of oxidative stress, from women with uterine prolapse and age‐matched female controls without prolapse. Cardinal ligament‐derived fibroblasts explanted from women with prolapse showed a significant increased level of isoprostane production (P < 0.05) compared with those derived from controls. This concurs with elevated urinary isoprostane levels identified among women with prolapse (P < 0.001) compared with controls. In addition, the matrix metalloproteinase 2 mRNA was significantly increased (P= 0.004) among women with uterine prolapse. Parallel findings of increased isoprostane in cardinal ligament and urine sample among women with prolapse suggest that oxidative stress might be involved in the development of uterine prolapse.

The regulations and role of circadian clock and melatonin in uterine receptivity and pregnancy—An immunological perspective

During normal pregnancy, the mechanism by which the fetus escapes immunological rejection by the maternal womb remains elusive. Given the biological complexities, the immunological mechanism is unlikely to be simply an allograft response in acceptance or rejection of the early pregnancy. Circadian clock responsible for the mammalian circadian rhythm is an endogenously generated rhythm associated with almost all physiological processes including reproduction. There is now growing evidence to suggest that the circadian clocks are intricately linked to the immune system and pregnancy. When perturbed, the role of immune cells can be affected on maintaining the enriched vascular system needed for placentation. This alteration can be triggered by the irregular production of maternal and placental melatonin. Hence, the role of circadian rhythm modulators such as melatonin offers intriguing opportunities for therapy. In this review, we evaluate the complex interaction between the circadian clock and melatonin within the immune system and their roles in the circadian regulation and maintenance of normal pregnancy.

Measurement of uterine natural killer cell percentage in the periimplantation endometrium from fertile women and women with recurrent reproductive failure: establishment of a reference range

Background Uterine natural killer cells are the major leukocytes present in the periimplantation endometrium. Previous studies have found controversial differences in uterine natural killer cell percentage in women with recurrent reproductive failure compared with fertile controls. Objective We sought to compare the uterine natural killer cell percentage in women with recurrent reproductive failure and fertile controls. Study Design This was a retrospective study carried out in university hospitals. A total of 215 women from 3 university centers participated in the study, including 97 women with recurrent miscarriage, 34 women with recurrent implantation failure, and 84 fertile controls. Endometrial biopsy samples were obtained precisely 7 days after luteinization hormone surge in a natural cycle. Endometrial sections were immunostained for CD56 and cell counting was performed by a standardized protocol. Results were expressed as percentage of positive uterine natural killer cell/total stromal cells. Results The median uterine natural killer cell percentage in Chinese ovulatory fertile controls in natural cycles was 2.5% (range 0.9‐5.3%). Using 5th and 95th percentile to define the lower and upper limits of uterine natural killer cell percentage, the reference range was 1.2‐4.5%. Overall, the groups with recurrent reproductive failure had significantly higher uterine natural killer cell percentage than the controls (recurrent miscarriage: median 3.2%, range 0.6‐8.8%; recurrent implantation failure: median 3.1%, range 0.8‐8.3%). However, there was a subset of both groups (recurrent miscarriage: 16/97; recurrent implantation failure: 6/34) that had lower uterine natural killer cell percentage compared to fertile controls. Conclusion A reference range for uterine natural killer cell percentage in fertile women was established. Women with recurrent reproductive failure had uterine natural killer cell percentages both above and below the reference range.

Physiological and pathological angiogenesis in endometrium at the time of embryo implantation

Embryo establishes contact with the endometrium during implantation. Proper endometrial vascular development and maintenance at the time of embryo implantation is crucial for successful pregnancy. Vascular development at the maternal‐embryo interface can be regulated by various cell types, of which uterine natural killer (uNK) cells play an important role. Abnormal angiogenesis and uNK cell number/function may lead to reproductive failure, particularly in women with recurrent miscarriage (RM) and women with recurrent implantation failure (RIF) after IVF‐ET treatment, which are the important clinical hurdles in reproductive medicine to overcome. In this review, we aim to discuss the current knowledge of physiological angiogenic processes and the pathological angiogenesis at the time of implantation, as well as the possible mechanism and potential treatment.

Increased expression of angiogenic cytokines in CD56+ uterine natural killer cells from women with recurrent miscarriage

HighlightsCD56+ uNK cells produce angiogenic factors around the time of implantation.An increased expression of angiogenic factors in uNK cells was observed in RM.The altered expression may associate with underlying endometrial pathology in RM. Objective: To compare the expression pattern of angiogenic cytokines in CD56+ uNK cells from peri‐implantation endometrium in women with a history of recurrent miscarriage and fertile controls. Methods: 28 women were recruited, from which 18 women were diagnosed as recurrent miscarriage and 10 women were of proven fertility. Endometrial biopsy samples were obtained precisely 7 days after luteinization hormone surge in a natural cycle. The angiogenic profile of isolated CD56+ uNK cells was determined by RayBio human angiogenesis antibody array G Series 1000. Differentially expressed angiogenic cytokines between groups were validated by ELISA kits. Results: CD56+ uNK cells freshly isolated from peri‐implantation endometrium were determined to be >90% pure. Angiogenic cytokine array demonstrated that CD56+ uNK cells are one of the angiogenic factors producers in endometrium around the time of embryo implantation. A differential angiogenic cytokine expression profile was found between two groups, with significantly higher expressions of angiogenin, VEGF‐A and bFGF in CD56+ uNK cells from women with recurrent miscarriage, compared with fertile controls. Conclusions: Differential angiogenic cytokine profile of isolated CD56+ uNK cells suggested the role of uNK cells in the altered endometrial vascularity at the time of implantation, which may account for the endometrial contribution to recurrent miscarriage.

Systematic Comparison of Bacterial Colonization of Endometrial Tissue and Fluid Samples in Recurrent Miscarriage Patients: Implications for Future Endometrial Microbiome Studies

BACKGROUND A recent study has reported that the microbiota in endometrial fluid of patients receiving in vitro fertilization and embryo transfer (IVF-ET) may predict implantation and pregnancy rates. However, studies are lacking that simultaneously compare the microbiota between endometrial fluid and tissue samples. Whether the microbiota composition in endometrial fluid reflects that in the endometrial tissue remains unclear. METHODS We systematically profiled the microbiota in endometrial fluid and tissue samples of IVF-ET patients using massively parallel sequencing. The bacterial 16S ribosomal RNA gene (V4 region) was PCR-amplified. Sequencing reads with >98% nucleotide identity were clustered as a bacterial taxon. To account for the different number of reads per sample, we normalized the read counts of each taxon before comparing its relative abundances across samples. RESULTS Thirteen taxa, including Verrucomicrobiaceae, Brevundimonas, Achromobacter, Exiguobacterium, and Flavobacterium, were consistently detected only in endometrial tissue samples but not fluid samples. Eight taxa were detected in fluid but not tissue. Twenty-two taxa were differentially abundant between fluid and tissue samples (adjusted P values, 4.1 × 10-25 to 0.025). The numbers of taxa identified per 1000 sequencing reads, diversity, and evenness in fluid samples were smaller than those in tissue samples. CONCLUSIONS Our data suggest that the microbiota composition in endometrial fluid does not fully reflect that in endometrial tissue. Sampling from both endometrial fluid and biopsy allows a more comprehensive view of microbial colonization. Further efforts are needed to identify the preanalytical effects, including sampling sites, methods, and sequencing depth, on profiling endometrial microbiota.

Transcriptomic profiles in peripheral blood between women with unexplained recurrent implantation failure and recurrent miscarriage and the correlation with endometrium: A pilot study

Aim To study the transcriptome profiles in the blood of recurrent implantation failure (RIF), recurrent miscarriage (RM) and fertile women during the window of implantation, and further analysis the correlation of transcriptome profiles between blood and endometrium. Methods This is an observational prospective study. In total 9 subjects were recruited, 3 RIF, 3 RM, and 3 controls. Paired samples (endometrium and peripheral blood) from the same subjects were precisely timed on the 7th days after luteal hormone surge (LH+7). RNA sequencing was applied to investigate the transcriptome profiles. Results The results of transcriptome in peripheral blood cannot be used to characterize women with RIF and unexplained RM. There was a medium level correlation between transcriptome in peripheral blood and endometrium during the window of implantation. Conclusion The differential transcriptome patterns in blood are not representative of those in endometrium, and the blood transcriptome cannot differentiate among the women with RIF, RM or fertile.

Correlation between three-dimensional power Doppler and morphometric measurement of endometrial vascularity at the time of embryo implantation in women with unexplained recurrent miscarriage

Power Doppler in combination with three-dimensional (3D-PD) ultrasonography has been used as a noninvasive tool to evaluate the vascularity. However, it is unclear whether 3D-PD can accurately reflect endometrial vascularization and replace the invasive endometrial biopsy. This study aims to investigate the correlation between 3D-PD and micro vessel morphometric measurement of endometrial vascularity. Twenty-five women with unexplained recurrent miscarriage were recruited for 3D-PD and endometrial biopsy on precisely day LH + 7. Immunohistochemistry using vWF was employed to identify micro vessels in endometrial biopsy specimens followed by the use of morphometric technique to measure the mean vessel diameter and volume fractions. The vascularization index (VI), flow index (FI) and vascularization flow index (VFI) assessed by 3D-PD were calculated for both the endometrial and sub-endometrial regions. There were no significant correlations between any of the ultrasonographic measurements (endometrial thickness, endometrial volume, endometrial VI/FI/VFI, sub-endometrial volume, sub-endometrial VI/FI/VFI) and morphometric features (number of micro vessel, mean diameter of micro vessel and volume fraction measurement of vessel). This study indicates that endometrial vascularity assessed by 3D-PD could not be used to reflect changes in micro vessels of the endometrium at the time of embryo implantation in women with unexplained recurrent miscarriage.