Xiangbo Xu

A critical period of progesterone withdrawal precedes endometrial breakdown and shedding in mouse menstrual-like model

STUDY QUESTION Is there a critical period of progesterone (P4) withdrawal in a mouse menstrual-like model, and at what time after P4 withdrawal endometrial breakdown become irreversible? STUDY ANSWER Our results showed that a 12-16 h critical period of P4 withdrawal exists in the mouse menstrual-like model. WHAT IS KNOWN ALREADY P4 withdrawal is the trigger for endometrial breakdown and shedding during menstruation. To date, the molecular mechanisms responsible for endometrial breakdown have not been fully elucidated. In an ovariectomized macaque model, P4 replacement could reduce or block menses during a period of 36-48 h after P4 withdrawal, but after this, P4 supplementation did not reduce or block menses. Thus, in the macaque, a critical period of P4 withdrawal lasting 36-48 h exists before menses. STUDY DESIGN, SIZE, DURATION We created a mouse menstrual-like model and restored P4 at four time points. The total number of mice was 120 and the duration of treatment was 26 days. PARTICIPANTS, SETTING, METHODS A mouse menstrual model was characterized by endometrial morphology and plasma P4 levels. P4 was then replaced at 8, 12, 16 and 20 h after the removal of P4 implants. Vaginal smears, endometrial morphology, plasma P4 levels and expression patterns of matrix metalloproteinases (MMP-2, MMP-3, MMP-9, MMP-10, MMP-11 and MMP-13) were investigated. MAIN RESULTS AND THE ROLE OF CHANGE Replacement of P4 at 8 and 12 h blocked menstrual-like bleeding and endometrial shedding; however, replacement at 16 and 20 h did not suppress bleeding or shedding. Furthermore, P4 replacement at 12 h inhibited the expression of all latent or active MMPs; however, replacement at 16 h inhibited only pMMP-13. LIMITATIONS, REASONS FOR CAUTION Although determination of the critical period in vivo using a mouse model was successfully demonstrated, the mechanisms of P4 regulation need to be further explored. WIDER IMPLICATIONS OF THE FINDINGS The experimental opportunities provided by the mouse model will facilitate understanding the role of P4 in the regulation of menstruation and help to identify new targets for the clinical intervention of menstrual disorders.

Comprehensive Analysis of Leukocytes, Vascularization and Matrix Metalloproteinases in Human Menstrual Xenograft Model

In our previous study, menstrual-like changes in mouse were provoked through the pharmacologic withdrawal of progesterone with mifepristone following induction of decidualization. However, mouse is not a natural menstruation animal, and the menstruation model using external stimuli may not truly reflect the occurrence and development of the human menstrual process. Therefore, we established a model of menstruation based on human endometrial xenotransplantation. In this model, human endometrial tissues were transplanted subcutaneously into SCID mice that were ovarectomized and supplemented with estrogen and progestogen by silastic implants with a scheme imitating the endocrinological milieu of human menstrual cycle. Morphology, hormone levels, and expression of vimentin and cytokeratin markers were evaluated to confirm the menstrual-like changes in this model. With 28 days of hormone treatment, transplanted human endometrium survived and underwent proliferation, differentiation and disintegration, similar to human endometrium in vivo. Human CD45+ cells showed a peak of increase 28 days post-transplantation. Three days after progesterone withdrawal, mouse CD45+ cells increased rapidly in number and were significantly greater than human CD45+ cell counts. Mouse CD31+ blood vascular-like structures were detected in both transplanted and host tissues. After progesterone withdrawal, the expression levels of matrix metalloproteinases (MMP) 1, 2, and 9 were increased. In summary, we successfully established a human endometrial xenotransplantation model in SCID mice, based on the results of menstrual-like changes in which MMP-1, 2 and 9 are involved. We showed that leukocytes are originated from in situ proliferation in human xenografts and involved in the occurrence of menstruation. This model will help to further understand the occurrence, growth, and differentiation of the endometrium and the underlying mechanisms of menstruation.

Cyclooxygenase-2 Regulated by the Nuclear Factor-κB Pathway Plays an Important Role in Endometrial Breakdown in a Female Mouse Menstrual-like Model

The role of prostaglandins (PGs) in menstruation has long been proposed. Although evidence from studies on human and nonhuman primates supports the involvement of PGs in menstruation, whether PGs play an obligatory role in the process remains unclear. Although cyclooxygenase (COX) inhibitors have been used in the treatment of irregular uterine bleeding, the mechanism involved has not been elucidated. In this study, we used a recently established mouse menstrual-like model for investigating the role of COX in endometrial breakdown and its regulation. Administration of the nonspecific COX inhibitor indomethacin and the COX-2 selective inhibitor DuP-697 led to inhibition of the menstrual-like process. Furthermore, immunostaining analysis showed that the nuclear factor (NF)κB proteins P50, P65, and COX-2 colocalized in the outer decidual stroma at 12 to 16 hours after progesterone withdrawal. Chromatin immunoprecipitation analysis showed that NFκB binding to the Cox-2 promoter increased at 12 hours after progesterone withdrawal in vivo, and real-time PCR analysis showed that the NFκB inhibitors pyrrolidine dithiocarbamate and MG-132 inhibited Cox-2 mRNA expression in vivo and in vitro, respectively. Furthermore, COX-2 and NFκB inhibitors similarly reduced endometrial breakdown, suggesting that NFκB/COX-2-derived PGs play a critical role in this process. In addition, the CD45(+) leukocyte numbers were sharply reduced following indomethacin (COX-1 and COX-2 inhibitor), DuP-697 (COX-2 inhibitor), and pyrrolidine dithiocarbamate (NFκB inhibitor) treatment. Collectively, these data indicate that NFκB/COX-2-induced PGs regulate leukocyte influx, leading to endometrial breakdown.

ROS are critical for endometrial breakdown via NF-κB-COX-2 signaling in a female mouse menstrual-like model.

Progesterone withdrawal triggers endometrial breakdown and shedding during menstruation. Menstruation results from inflammatory responses; however, the role of reactive oxygen species (ROS) in menstruation remains unclear. In this study, we explored the role of ROS in endometrial breakdown and shedding. We found that ROS levels were significantly increased before endometrial breakdown in a mouse menstrual-like model. Vaginal smear inspection, morphology of uterine horns, and endometrial histology examination showed that a broad range of ROS scavengers significantly inhibited endometrial breakdown in this model. Furthermore, Western blot and immunohistochemical analysis showed that the intracellular translocation of p50 and p65 from the cytoplasm into the nucleus was blocked by ROS scavengers and real-time PCR showed that cyclooxygenase-2 (COX-2) mRNA expression was decreased by ROS scavengers. Similar changes also occurred in human stromal cells in vitro. Furthermore, Western blotting and real-time PCR showed that one ROS, hydrogen peroxide (H2O2), promoted translocation of p50 and p65 from the cytoplasm to the nucleus and increased COX-2 mRNA expression along with progesterone maintenance. The nuclear factor κB inhibitor MG132 reduced the occurrence of these changes in human stromal cells in vitro. Viewed as a whole, our results provide evidence that certain ROS are important for endometrial breakdown and shedding in a mouse menstrual-like model and function at least partially via nuclear factor-κB/COX-2 signaling. Similar changes observed in human stromal cells could also implicate ROS as important mediators of human menstruation.

Effect of daily milk supplementation on serum and umbilical cord blood folic acid concentrations in pregnant Han and Mongolian women and birth characteristics in China

Many studies have demonstrated the efficacy of folic acid (FA) supplementation in prevention of neural tube defects (NTDs), although the extent of NTDs varies among individuals of different races and ethnic origin. China is a multi-ethnic country with no standard practice for FA-fortified food. Milk is consumed by women, but little is known about the effects of milk on folate concentration in maternal blood and neonatal umbilical cord blood in Han and Mongolian women after stopping taking the supplement for a month and five month, respectively. The objective of this study was to determine whether only daily consumption of liquid milk can increase the blood folate concentration in pregnant women and whether there are differences in blood folate concentrations between Han and Mongolian women after cessation of FA supplementation. Of the 4052 women enrolled in the parallel group design study. Three thousand five hundred and twenty-six women had confirmed pregnancies and were randomized to receive liquid milk or not until delivery. Women who consumed the liquid milk had significantly increased serum folate concentrations at 16 and 32 weeks of gestation as well as cord blood at birth compared to control groups in both ethnic groups. Infants born to women drinking milk also had better the term birth weight and height, which may be related to the increased concentration of folate. In conclusion, daily consumption of milk can increase the serum folate concentration in pregnant Han and Mongolian women in China (differences in the efficacy of FA and milk supplementation) and may enhance birth outcomes.

Repeated Abortion Affects Subsequent Pregnancy Outcomes in BALB/c Mice

Aim In this study, we aimed to establish a mouse model of repeated medical termination of pregnancy in order to determine subsequent outcomes. Methods A model of mifepristone (RU 486)-induced medical abortion was established in BALB/c mice to facilitate the investigation of the impact of medical abortion on subsequent pregnancies, including litter sizes and newborn birth weights. Pregnant mice were sacrificed to examine midterm pregnancy status, investigate the frequency of fetal resorption, and measure placental function gene expression by real-time PCR and immunohistochemistry. Offspring liver mRNA was harvested for real-time PCR to determine gene expression and assess the effects of abortion on offspring development. Results Mice subjected to 2 previous medical abortions experienced spontaneous abortions in subsequent pregnancies. Medical abortion caused reduced reproductive capacity and affected placental dysfunction, with reduced expression of tissue factor (TF) and genes encoding proteins involved in metabolic functions relevant to pregnancy, such as 11β-hydroxysteroid dehydrogenase 1/2 (11β-HSD1/2) and glucocorticoid receptor (GR). Reduced expression was also observed for platelet endothelial cell adhesion molecule-1 (CD31) and vascular endothelial growth factor (VEGF). In offspring from subsequent pregnancies, genes involved in lipid metabolism, which may enhance key lipid transcription factors, such as PPARA and PPARG, as well as GR/11β-HSD1, were downregulated in the liver. In addition, the sperm motility of the F1 males reduced. Conclusion Repeated medical abortion impaired the reproductive function of female mice, significantly affecting the outcomes of subsequent pregnancies. The impact of repeated abortions on the offspring of subsequent pregnancies was also noteworthy and deserves further exploration. Thus, this model provides a useful means to study the mechanisms underlying the above phenomena, which will ultimately benefit the health of women and their children.

Vascular endothelial growth factor (VEGF) regulation by hypoxia inducible factor-1 alpha (HIF1A) starts and peaks during endometrial breakdown, not repair, in a mouse menstrual-like model

STUDY QUESTION How is vascular endothelial growth factor (VEGF) expression regulated by hypoxia inducible factor 1 alpha (HIF1A) during menstruation? SUMMARY ANSWER After progesterone (P4) withdrawal, HIF1A was activated and it directly up-regulated VEGF mRNA expression and this regulation was the highest during endometrium breakdown in the mouse menstrual-like model. WHAT IS KNOWN ALREADY VEGF, an important angiogenic factor, is known to be essential for endometrial repair, particularly in angiogenesis and re-epithelialization. However, its upstream regulation has not been fully clarified. HIF1 is the first transcription factor response to hypoxia and is closely associated with angiogenesis; it is also an upstream regulator of VEGF mRNA. STUDY DESIGN, SIZE, DURATION We investigated the changes in the expression of HIF1A and VEGF after P4 withdrawal and after HIF1A inhibition. The total number of mice used was 62. The treatment duration in the mouse menstrual-like model was 8 days. PARTICIPANTS/MATERIALS, SETTING, METHODS The mouse menstrual-like model and mouse and human decidual endometrial stromal cells were established to mimic menstruation. Protein and mRNA expressions of HIF1A and VEGF were investigated by immunohistochemistry, Western blot and quantitative PCR. The direct interaction between HIF1A and the Vegf promoter was also investigated by chromatin immunoprecipitation. HIF1A inhibition in vivo and in vitro was achieved by administration of an HIF1A inhibitor and by siRNA knockdown, respectively. MAIN RESULTS AND THE ROLE OF CHANCE HIF1A was translocated to the nucleus from 8 to 16 h after P4 withdrawal, while VEGF mRNA expression was the highest at 12 h. HIF1A directly bound to Vegf promoter during endometrial breakdown, which peaked at 12 h. HIF1A inhibition suppressed VEGF mRNA and protein expression in the mouse menstrual-like model and decidualized stromal cells. Inhibition of HIF1A also suppressed endometrial breakdown. LIMITATIONS, REASONS FOR CAUTION Although HIF1A regulation of VEGF mRNA was confirmed in the mouse menstrual-like model and decidual endometrium stromal cells, the functional regulation of VEGF protein was not further determined. WIDER IMPLICATIONS OF THE FINDINGS Here, we report that the functional regulation of VEGF was complicate in menstruation. We also report that HIF1A plays a key role in endometrial breakdown. STUDY FUNDING/COMPETING INTERESTS The National Nature Science Foundation of China (No. 30901608), the National Basic Research Program of China (2010CB530403) and the National Science and Technology Support Program (No. 2012BAI32B05). The authors have no conflicts of interest to disclose. TRIAL REGISTRATION NUMBER This study is not a clinical trial.

Endometrial breakdown with sustained progesterone release involves NF-κB-mediated functional progesterone withdrawal in a mouse implant model.

Irregular uterine bleeding is a major side effect of long‐acting progestogen‐only contraceptives in women, and is the primary reason women discontinue their use. In this study, a mouse model of endometrial breakdown was established using a subcutaneous progesterone implant to understand how irregular bleeding begins. Although progestogens sustained decidualization, endometrial breakdown was still observed in this model. We, therefore, hypothesized that endometrial breakdown might involve functional progesterone withdrawal. Using co‐immunoprecipitation assays, we observed the constitutive activation of nuclear factor kappa‐b (NF‐κB) p65 and its interaction with the progesterone receptor (PGR); moreover, transcriptional activity of the PGR was also repressed by NF‐κB activity in primary mouse and human decidual stromal cells that mimic progesterone maintenance. Yet the ratio of PGR‐B to PGR‐A was not increased in the mouse model. In vivo comparison of endometrial breakdown induced by progesterone withdrawal to that seen during sustained progesterone exposure, in the presence of NF‐κB inhibitors, revealed that NF‐κB‐mediated functional progesterone withdrawal is involved in endometrial breakdown in this implant model. These data prompt further studies to determine the homology of this functional progesterone withdrawal mechanism in human endometrium. Mol. Reprod. Dev. 83: 780–791, 2016.