Late Proliferative Phase Research Articles

Injectable human recombinant collagen matrices limit adverse remodeling and improve cardiac function after myocardial infarction

Despite the success of current therapies for acute myocardial infarction (MI), many patients still develop adverse cardiac remodeling and heart failure. With the growing prevalence of heart failure, a new therapy is needed that can prevent remodeling and support tissue repair. Herein, we report on injectable recombinant human collagen type I (rHCI) and type III (rHCIII) matrices for treating MI. Injecting rHCI or rHCIII matrices in mice during the late proliferative phase post-MI restores the myocardium’s mechanical properties and reduces scar size, but only the rHCI matrix maintains remote wall thickness and prevents heart enlargement. rHCI treatment increases cardiomyocyte and capillary numbers in the border zone and the presence of pro-wound healing macrophages in the ischemic area, while reducing the overall recruitment of bone marrow monocytes. Our findings show functional recovery post-MI using rHCI by promoting a healing environment, cardiomyocyte survival, and less pathological remodeling of the myocardium.

Effect of hysteroscopic lysis of intrauterine adhesions in menstruating women on subsequent late proliferative phase endometrial thickness

Purpose: To determine if adhesiolysis of intrauterine adhesions improves or is detrimental to post-operative endometrial thickness in women with normal menstruation. Materials and Methods: Endometrial thickness by sonography was determined at the time of the mature dominant follicle, both in a cycle prior and then following hysteroscopic adhesiolysis. Results: The pre- and post-average endometrial thicknesses were exactly the same. Considering three endometrial thickness categories (4-5 mm poor prognosis, 5-6 mm fair prognosis,and ≥ 8 good prognosis) one woman went from fair to good, but three went from good to fair, and one went from good to poor. Conclusions: Through there were no significant differences, but there was a trend for diminished endometrial thickness. Thin endometria has been associated with diminished fecundity. This preliminary study should encourage a large prospective controlled study evaluating the effect of adhesiolysis on endometrial thickness and pregnancy rates in women with normal menses despite intrauterine adhesions.

AQP2 is regulated by estradiol in human endometrium and is associated with spheroid attachment invitro.

17β‑estradiol (E2) and aquaporin 2 (AQP2) are associated with endometrial receptivity, and E2 directly regulates AQP2 expression in endometrial cancer cells. The present study aimed to investigate the role of AQP2 in embryo implantation. Normal endometrial samples were collected at the Women's Hospital (Hangzhou, China) from women seeking invitro fertilization and embryo transfer; women with endometrial abnormalities were excluded from the study. Samples were categorized into early‑mid proliferative, late proliferative, early secretory, mid‑secretory and late secretory phase groups, according to the menstrual cycle. The mRNA and protein expression levels of AQP2 were assessed in normal human endometrium in response to E2 via reverse transcription‑quantitative polymerase chain reaction and western blotting, respectively. The effects of AQP2 on spheroid attachment were assessed using an invitro co‑culture assay with small interfering (si)RNA against AQP2. The highest expression levels of AQP2 were observed in the late proliferative and mid‑secretory phases, with the lowest levels detected in the early proliferative and late secretory phases. In addition, treatment with 10‑9 or 10‑7 M E2 for 24h upregulated AQP2 in the cultured endometrium. Knockdown of AQP2 by siRNA significantly decreased JAr spheroid attachment; however, this effect was significantly reversed when AQP2 siRNA‑transfected cells were treated with 10‑7 M E2. The results of the present study suggested that AQP2 expression levels in human endometrium may be mediated by estrogen, and low AQP2 expression levels may be a potential cause of impaired uterine receptivity.

Increased AIF-1-mediated TNF-α expression during implantation phase in IVF cycles with GnRH antagonist protocol.

Is allograft inflammatory factor-1 (AIF-1), a cytokine associated with inflammation and allograft rejection, aberrantly elevated in in vitro fertilization (IVF) cycles with gonadotropin-releasing hormone (GnRH) antagonist protocol with potential effects on endometrial receptivity? Our findings indicated AIF-1 is increased in IVF cycles with GnRH antagonist protocol and mediates greater TNF-α expression during implantation phase, which may be unfavorable for embryo implantation. Studies have shown that GnRH antagonist protocol cycles have lower implantation and clinical pregnancy rates than GnRH agonist long protocol cycles. Endometrial receptivity but not embryo quality is a key factor contributing to this phenomenon; however, the mechanism is still unknown. Implantation and pregnancy rates were studied in 238 patients undergoing their first cycle of IVF/ICSI between 2012 and 2014. Forty of these patients opted to have no fresh embryo replacement and were divided into two equal groups: (i) GnRH antagonist protocol and (ii) GnRH agonist long protocol, group 3 included 20 infertile women with a tubal factor in untreated cycles. During the same interval, endometrial tissues were taken from 18 infertile women with a tubal factor in the early proliferative phase, late proliferative phase, and mid-secretory phase of the menstrual cycle (n = 6/group). Microarray analysis, RT-qPCR, Western blot analysis, immunohistochemistry were used to investigate the expression levels of AIF-1 and the related cytokines (TNF-α, IL1β, IL1RA, IL6, IL12, IL15 and IL18). The effect of AIF-1 on uterine receptivity was modeled using in vitro adhesion experiments (coculture of JAR cells and Ishikawa cells). The expression of AIF-1 was the highest in early proliferative phase, decreasing thereafter in the late proliferative phase, and almost disappearing in the mid-secretory phase, indicating that low AIF-1 expression might be important for embryo implantation during implantation phase. Microarray results revealed that AIF-1 was upregulated in the antagonist group compared with the control group (fold change [FC] = 3.75) and the agonist (FC = 2.20) group. The raw microarray data and complete gene expression table were uploaded to GEO under the accession number of GSE107914. Both the mRNA and protein expression levels of AIF-1 and TNF-α were the higher in the antagonist group than in the other two groups (P < 0.05) which did not differ significantly (P > 0.05). The protein levels of TNF-α in both Ishikawa cells and primary endometrial cells were significantly increased (P < 0.05) at 96 h after transfection with the AIF-1 expression vector, indicating that TNF-α was mediated by AIF-1 in endometrial cells. Overexpression of AIF-1 in Ishikawa cells inhibited adhesion of JAR cells to them. Thus, increased AIF-1 might inhibit adhesion during implantation via raised TNF-α. The sample size of the microarray was small, which might weaken the accuracy of our results; however, the sample size of RT-qPCR and the Western blotting assays were sufficient to compensate for this deficiency in our study. In addition, the aberrant AIF-1 and thus TNF-α expression is one of many factors that may contribute to limiting implantation success. Therefore, further extensive in vitro mechanistic and in vivo animal studies are needed to assess the actual functional impact of this pathway. Anti-TNF-α therapy might mitigate the adverse effects of GnRH antagonist on endometrial receptivity and improve the implantation rate in GnRH antagonist protocols in IVF. This work was supported by grants from the National Natural Science Foundation of China, Grant numbers 81771656 and 81370763; Clinical research special fund of Chinese Medical Association, Grant number 16020480664; Shanghai Jiao Tong University Medicine-Engineering Fund, Grant number YG2017ZD11 and YG2017MS57; and the Merck-Serono China Research Fund for Fertility Agreement. P.C.K.L. is supported by a Canadian Institutes of Health Research Foundation Scheme Grant 143317. None of the authors has any competing interests.

P38 Mitogen-Activated Protein Kinase is Involved in the Pathogenesis of Endometriosis by Modulating Inflammation, but not Cell Survival.

Local pro-inflammatory environment and enhanced cell survival contribute to the endometriosis development. A serine/threonine kinase p38 mitogen-activated protein kinase (MAPK) mediates intracellular signaling of cytokine production, cell proliferation, and apoptosis in different cell types. The current study compares p38 MAPK activity in normal endometrium and endometriosis, and assesses role(s) of p38 MAPK on cytokine production and cell survival in endometriosis. Immunohistochemical levels of total and phosphorylated (active) p38 MAPK as well as its correlation with interleukin 8 (IL-8) expression, and cell proliferation and apoptosis were compared in normal human endometrium and endometriosis. The action of p38 MAPK on pro-inflammatory cytokine-induced IL-8 and monocyte chemotactic protein (MCP)-1 expression in endometriotic cells were assessed by enzyme-linked immunosorbent assay. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell survival, 5-bromo-2'-deoxyuridine incorporation, and Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assays were used to determine the function of p38 MAPK in cultured human endometriotic stromal cell proliferation and apoptosis. p38 MAPK activity was significantly higher in both eutopic and ectopic endometria compared to normal endometria during late proliferative and early secretory phases ( P < .05). Increased p38 MAPK activity in endometriotic cells correlated with IL-8 expression (Pearson correlation coefficient r = 0.83, P < .01), but not with apoptosis in vivo. The pro-inflammatory cytokines IL-1β and tumor necrosis factor (TNF)-α induced activation of p38 MAPK. Inhibition of p38 MAPK activity blocked IL-1β and TNF-α-induced IL-8 and MCP-1 secretion in cultured endometriotic stromal cells ( P < .05), but did not impact on endometriotic cell survival. These results suggest that rather than modulating cell survival, increased p38 MAPK activity in endometriotic cells contributes to the pathogenesis of endometriosis by promoting the local inflammatory milieu.

Decreased PECAM1-mediated TGF-β1 expression in the mid-secretory endometrium in women with recurrent implantation failure.

Is recurrent implantation failure (RIF) associated with decreased expression of platelet and endothelial cell adhesion molecule 1 (PECAM1) and transforming growth factor β1 (TGF-β1) in the endometrium during the implantation window? The present study demonstrates that the expression of PECAM1 and TGF-β1 is significantly decreased in the mid-secretory endometrium in women with RIF, which may account for embryo implantation failure. RIF has become a bottleneck issue that hampers the improvement of pregnancy rates in IVF-embryo transfer (IVF-ET). The causes of RIF are complex and may involve the dysregulation of various growth factors, metabolites, and inflammatory cytokines. At present, the precise pathogenesis of RIF has not been elucidated. This was a prospective case-control study. Endometrial tissue samples were obtained from January 2014 to December 2016 from two groups of women who had undergone IVF (RIF group, 22 women who underwent ≥3 ETs including a total of ≥4 good-quality embryos without pregnancy, control group, 18 women who conceived in their first treatment cycle). At the same time, samples were obtained from 18 women with infertility secondary to tubal factor in the early proliferative, late proliferative and mid-secretory phases of the menstrual cycle (n = 6 per group). Samples used for isolation of primary human endometrial epithelial cells and stromal cells (HEECs and HESCs) were collected in December 2017 from six women with infertility secondary to tubal factor. We investigated gene expression using integrative whole genome expression microarray analysis, including differentially expressed gene screening, principal component analysis, and functional enrichment analysis. RT-qPCR, western blotting, immunohistochemistry, immunofluorescence co-localization analysis and short hairpin RNA (shRNA) plasmid transfection in Ishikawa cell line, HEECs and HESCs were used to investigate the expression of PECAM1 and TGF-β1. Integrative data mining of whole-genome expression profiles identified cell adhesion as a key regulator in RIF. Database retrieval and literature review screened several novel cell adhesion-related genes that might participate in embryo implantation, which include PECAM1, intercellular adhesion molecule 2 (ICAM2), integrin subunit β2 (ITGB2), selectin P (SELP) and TEK receptor tyrosine kinase (TEK). Among these targets, the mRNA and protein levels of PECAM1 were significantly lower in the RIF group than those in the control group. During the menstrual cycles of women with secondary infertility, the protein expression level of PECAM1 was the lowest in early proliferative phase, slightly increased in late proliferative phase and was the highest in mid-secretory phase. While the expression level of HOXA10, an endometrial receptivity marker, kept at a low level in early proliferative phase and increased in late proliferative phase, then maintained at a high level in the mid-secretory phase. Furthermore, TGF-β1, mediated by PECAM1, was also decreased significantly in the RIF group. Using shRNA-based approach, we demonstrated that the depletion of PECAM1 significantly decreased the expression of TGF-β1 in Ishikawa cells, as well as in primary HEECs and HESCs. These results indicated that PECAM1 and TGF-β1 might play a pivotal role in modulating endometrial receptivity. Although we have shown that PECAM1 and TGF-β1 were down-regulated in the women with RIF, the molecular mechanism of the effect of the factors on the endometrial receptivity remain unclear. Our findings provide insight into the contribution of PECAM1 and TGF-β1 in regulating implantation, which could be used to develop potential therapeutic methods for RIF. This work was supported by grants from the National Natural Science Foundation of China (Nos. 81771656 and 81370763), Special fund for clinical research of the Chinese Medical Association (No. 16020480664), and the Merck Serono China Research Fund for Fertility Agreement. The authors have no competing interests.

Insufficient Angiogenesis: Cause of Abnormally Thin Endometrium in Subfertile Patients?

This study investigated subfertile patients with abnormally thin endometrium after infertility treatment. As they had adequate serum concentrations of hormones, an endometrial factor for subfertility was suspected. To elucidate the cause of subfertility, endometrial biopsies were taken in each patient in the late proliferative and mid-secretory phases of one menstrual cycle. Endometrial biopsies from women with normal menstrual cycles and confirmed fertility who were undergoing hysterectomy for benign uterine disease were used as positive controls. The tissue samples were investigated for steroid hormone receptor expression and for the proliferation marker Ki-67. Immunohistochemistry was performed with antibodies against the marker molecules for endometrial receptivity - β 3 integrin, VEGF, LIF, and CD56 (large granular lymphocytes, LGLs). The steroid hormone receptors for estrogen (E2) and progesterone (P) were expressed normally (at the first biopsy) and were down-regulated (at the second biopsy) within the cycle. Strikingly, all of the marker molecules investigated showed negative or weak and inadequate expression in the mid-secretory phase. Numbers of LGLs remained as low as in the proliferative phase. In contrast, fertile patients were found to express these marker molecules distinctly in the mid-secretory phase. It may be hypothesized that a severe deficiency of these angiogenesis-related marker molecules leads to defective development of the endometrium, which remains thin. Deficient angiogenetic development may thus provide an explanation for the endometrial factor that causes infertility. Further investigations will need to focus on identifying the regulating factors that act between steroid receptor activation and the expression of these marker molecules.

P27kip1 Is Required for Functionally Relevant Adult Hippocampal Neurogenesis in Mice.

We asked whether cell-cycle associated protein p27kip1 might be involved in the transition of precursor cells to postmitotic maturation in adult hippocampal neurogenesis. p27kip1 was expressed throughout the dentate gyrus with a strong nuclear expression in early postmitotic, calretinin-positive neurons and neuronally determined progenitor cells (type-3 and some type-2b), lower or absent expression in radial glia-like precursor cells (type-1) and type-2a cells and essentially no expression in granule cells. This suggested a transitory role in late proliferative and early postmitotic phases of neurogenesis. Inconsistent with a role limited to cell cycle arrest the acute stimuli, voluntary wheel running (RUN), environmental enrichment (ENR) and kainate-induced seizures increased p27kip1 expressing cells. Sequential short-term combination of RUN and ENR yielded more p27kip1 cells than either stimulus alone, indicating an additive effect. In vitro, p27kip1 was lowly expressed by proliferating precursor cells but increased upon differentiation. In p27kip1-/- mice neurogenesis was reduced in vivo, whereas the number of proliferating cells was increased. Accordingly, the microdissected dentate gyrus of p27kip1-/- mice generated more colonies in the neurosphere assay and an increased number of larger spheres with the differentiation potential unchanged. In p27kip1-/- monolayer cultures, proliferation was increased and cell cycle genes were upregulated. In the Morris water maze p27kip1-/- mice learned the task but were specifically impaired in the reversal phase explainable by the decrease in adult neurogenesis. We conclude that p27kip1 is involved in the decisive step around cell-cycle exit and plays an important role in activity-regulated and functionally relevant adult hippocampal neurogenesis. Stem Cells 2017;35:787-799.

Hormonal regulation of oviductal glycoprotein 1 (OVGP1; MUC9) in the macaque cervix: a novel indicator of progestogen action

Progestogen-only contraception is reported to act by suppressing ovulation and/or altering cervical mucus secretion. Unfortunately, the effect of contraceptives on mucus quality is difficult to assess accurately by traditional Insler score or post-coital tests, and the lack of a molecular test for mucus quality has created a roadblock to the development of new contraceptives that target the cervix. Oviductal glycoprotein 1 (OVGP1; MUC9) is a hormonally regulated, carbohydrate-rich glycoprotein secreted by oviductal epithelial cells [1]. In addition to the oviduct, the OVGP1 expression has been reported for the cervix of rabbits, [2] mice and women, but hormonal regulation of OVGP1 in the cervix is poorly understood. The objective of this study was to characterize hormonal regulation and localization of OVGP1 in the macaque cervix as a novel marker of progestogen action. Real-time PCR and immunohistochemical analysis of the macaque cervix. Adult ovariectomized rhesus macaques (Macaca mulatta) were treated sequentially with estradiol (E2) and then E2 plus progesterone (P) to create artificial menstrual cycles. Reproductive tracts were collected in the artificial proliferative phase (E2 alone; n=13) and in the artificial secretory phase after 1, 3, 7, and 14 days of P (n=4, 5, 5, and 10, respectively). Total RNA was isolated from cervical samples and analyzed for expression of OVGP1 by TaqMan® real-time PCR and presented relative to expression of ribosomal (S10) RNA. Representative samples were analyzed by immunohistochemistry on cryosections with polyclonal antibodies specific to OVGP1. Representative sections were also stained for estrogen receptor 1 (ESR1) and progesterone receptor (PGR) as independent assays for E2 and (P) action. OVGP1 transcript was upregulated in the proliferative phase, maximal in the late proliferative phase, and maintained through day 1 of the secretory phase. There was a significant decrease (P<0.01) in OVGP1 expression between secretory phase day 1 and day 3 and a further significant (P<0.05) decrease by secretory phase day 7 and 14. IHC revealed specific OVGP1 staining in columnar cells of the mid cervix. Staining was strongest in the apical portion of the epithelial cells consistent with secretion into the cervix mucus. As expected, ESR-1 and PGR was present in the cervix throughout the cycle. ESR1 and PGR staining was stronger in the cervix epithelium during the proliferative phase of the cycle. These results indicate that OVGP1 is expressed in the primate cervix as well as the primate oviduct, and strongly upregulated by estrogen in the proliferative phase and down-regulated by P during the secretory phase. The cervical epithelium would be easily accessible by biopsy or cervical brush, and OVGP1 could be assayed either by ELISA or by real-time PCR to provide a rapid and very sensitive method for identifying the effect of progestogen-based contraceptives on the cervix in clinical trials.

Failure to increase the thickness of thin endometria with intrauterine infusion of granulocyte colony stimulating factor (G-CSF)

Purpose: To corroborate or refute a previous study suggesting that intrauterine infusion of granulocyte colony stimulating factor (GCSF) could significantly improve endometrial thickness into more fertile levels when the endometrial thickness was ≤ five mm. Materials and Methods: Three women whose endometrial thickness never exceeded five mm on the in vitro fertilization-embryo transfer(IVF-ET) cycle or subsequent attempted frozen ETs using graduated estradiol had intrauterine infusion of G-CSF to the estradiol regimen. Results: Not one of the three women improved the endometrial thickness beyond five mm and none conceived on the G-CSF cycle. One woman had a subsequent pregnancy following a frozen ET with only a four-mm thickness with no infusion of G-CSF. Conclusions: These data do not confirm the efficacy of intrauterine infusion of G-CSF for poor endometrial thickness. Perhaps only certain cases will respond. A larger series could take time to accumulate since other studies have shown that only 0.3% of women with ≤ five mm endometrial thickness in the late proliferative phase during an IVF-ET cycle will not improve the endometrial thickness > five mm using graduated estradiol protocols.

The Doppler profile of ovarian endometrioma

Aims. To characterize the vascularity of endometriotic cysts using color Doppler ultrasound and to differentiate more accurately between endometriotic cysts and other pelvic cyst masses. Methods. Two prospective studies were initiated: the first included 50 patients diagnosed with ovarian endometriotic cysts and 50 patients without gynecological pathology, in whom the resistance index (RI) of the uterine artery, ovarian artery and endometrial arcuate arteries in the early secretory phase was determined by endovaginal Doppler ultrasound; the second study included 50 patients diagnosed with ovarian endometriotic cysts, in whom the RI of the uterine artery, ovarian artery and cyst wall arteries in the late proliferative phase and in the late secretory phase was determined. Results. In the early secretory phase, the vascular flow through the uterine artery was significantly higher in patients with ovarian endometrioma. These patients had increased endometrial vascularity in the early secretory phase compared to the control group. In the late secretory phase, the vascular flow through the ovarian artery was higher compared to the late proliferative phase in patients with ovarian endometrioma. The vascularization of the ovarian endometrioma wall was significantly increased in the late secretory phase compared to the late proliferative phase. Conclusions. Vascularization, described through the arrangement of vessels, vascular density and vascular resistance, is an important factor in the evaluation of ovarian endometrioma.

Therapeutic options for management of endometrial hyperplasia.

Endometrial hyperplasia (EH) comprises a spectrum of changes in the endometrium ranging from a slightly disordered pattern that exaggerates the alterations seen in the late proliferative phase of the menstrual cycle to irregular, hyperchromatic lesions that are similar to endometrioid adenocarcinoma. Generally, EH is caused by continuous exposure of estrogen unopposed by progesterone, polycystic ovary syndrome, tamoxifen, or hormone replacement therapy. Since it can progress, or often occur coincidentally with endometrial carcinoma, EH is of clinical importance, and the reversion of hyperplasia to normal endometrium represents the key conservative treatment for prevention of the development of adenocarcinoma. Presently, cyclic progestin or hysterectomy constitutes the major treatment option for EH without or with atypia, respectively. However, clinical trials of hormonal therapies and definitive standard treatments remain to be established for the management of EH. Moreover, therapeutic options for EH patients who wish to preserve fertility are challenging and require nonsurgical management. Therefore, future studies should focus on evaluation of new treatment strategies and novel compounds that could simultaneously target pathways involved in the pathogenesis of estradiol-induced EH. Novel therapeutic agents precisely targeting the inhibition of estrogen receptor, growth factor receptors, and signal transduction pathways are likely to constitute an optimal approach for treatment of EH.

Comparison of Uterine Receptivity between Fertile and Unexplained Infertile Women by Assessment of Endometrial and Subendometrial Perfusion Using Contrast-Enhanced Ultrasound: Which Index is Better—Peak Intensity or Area under the Curve?

The goal of this study was to compare uterine receptivity between women with normal fertility and those with unexplained infertility during natural cycles by assessment of endometrial and subendometrial perfusion using contrast-enhanced ultrasound (CEUS). We wanted to determine the better index: peak intensity (PI) or area under the curve (AUC). Thirty women with unexplained infertility were recruited into the study group, and 30 women with normal fertility were recruited into the control group. All women underwent CEUS during the late proliferative phase, ovulation phase, and implantation window of a menstrual cycle. Endometrial PI, endometrial AUC, subendometrial PI and subendometrial AUC were analyzed. In the late proliferative phase, the control group had a significantly higher endometrial PI (p < 0.001) as well as subendometrial PI (p < 0.001) and AUC (p = 0.004) than the study group. In the ovulation phase, the control group had a significantly higher endometrial PI (p < 0.001) and AUC (p = 0.021), as well as subendometrial PI (p < 0.001) and AUC (p = 0.003). During the implantation window, there were no significant differences between the two groups. Only subendometrial PI underwent a significant periodic change during the menstrual cycle in both groups. This finding was further confirmed by evaluation of the microvessel density of endometria. In conclusion, CEUS can be used to assess endometrial and subendometrial perfusion to evaluate uterine receptivity. Subendometrial PI was the most sensitive index compared with endometrial PI, endometrial AUC and subendometrial AUC.

Systemic EP4 Inhibition Increases Adhesion Formation in a Murine Model of Flexor Tendon Repair.

Flexor tendon injuries are a common clinical problem, and repairs are frequently complicated by post-operative adhesions forming between the tendon and surrounding soft tissue. Prostaglandin E2 and the EP4 receptor have been implicated in this process following tendon injury; thus, we hypothesized that inhibiting EP4 after tendon injury would attenuate adhesion formation. A model of flexor tendon laceration and repair was utilized in C57BL/6J female mice to evaluate the effects of EP4 inhibition on adhesion formation and matrix deposition during flexor tendon repair. Systemic EP4 antagonist or vehicle control was given by intraperitoneal injection during the late proliferative phase of healing, and outcomes were analyzed for range of motion, biomechanics, histology, and genetic changes. Repairs treated with an EP4 antagonist demonstrated significant decreases in range of motion with increased resistance to gliding within the first three weeks after injury, suggesting greater adhesion formation. Histologic analysis of the repair site revealed a more robust granulation zone in the EP4 antagonist treated repairs, with early polarization for type III collagen by picrosirius red staining, findings consistent with functional outcomes. RT-PCR analysis demonstrated accelerated peaks in F4/80 and type III collagen (Col3a1) expression in the antagonist group, along with decreases in type I collagen (Col1a1). Mmp9 expression was significantly increased after discontinuing the antagonist, consistent with its role in mediating adhesion formation. Mmp2, which contributes to repair site remodeling, increases steadily between 10 and 28 days post-repair in the EP4 antagonist group, consistent with the increased matrix and granulation zones requiring remodeling in these repairs. These findings suggest that systemic EP4 antagonism leads to increased adhesion formation and matrix deposition during flexor tendon healing. Counter to our hypothesis that EP4 antagonism would improve the healing phenotype, these results highlight the complex role of EP4 signaling during tendon repair.

Effect of Hysteroscopic Lysis of Intrauterine Adhesion in Menstruating Women on Subsequent Late Proliferative Phase Endometrial Thickness

Though there is little question that hysteroscopic lysis of intrauterine adhesions in women with Asherman's syndrome may correct an infertility problem, it is not clear if the presence of intrauterine adhesions not severe enough to inhibit menses contributes to infertility. More importantly, it is not clear whether hysteroscopic lysis improves or inhibits fecundity. Thin endometria at the time of peak follicular maturation has been associated with infertility.

Morphological and Ultrastructural Changes in Human Endometrium Following Low-Dose Levonorgestrel Contraceptive Intrauterine Systems (LNG-IUS-12) 13.5mg.

To study the effect of low-dose levonorgestrel contraceptive intrauterine systems (LNG-IUS-12) on endometrial morphology and ultrastructure. Twenty seven women who desired contraception were inserted LNG-IUS-12. Endometrial biopsies were collected during the late proliferative phase of the cycle before (control) and after the use of the LNG-IUS for 36months. The changes of morphology and ultrastructure of endometrium tissue were observed by light microscope and transmission electron microscope. After 36months of use, the endometrial gland reduced in number and some were atrophic. A decidual change of stroma was present. Electronic microscopy demonstrated the presence of low columnar, karyopyknosis, and chromatin margination in the glandular epithelial cell. Some vacuolar degeneration in partial mitochondria and rough endoplasmic reticulum presented light extension. Changes could be concluded as cell inhibition of proliferation. The low-dose levonorgestrel-releasing intrauterine systems has a suppressive effect on endometrium that is similar to LNG-IUS.

P26.19: Thin endometrium as a possible reason of missed pregnancy

We would like to present a case report of 5 patients who had missed pregnancy in the first trimester. All these patients had uterine curettage in our clinic and normal embryo karyotype was proved. In all this cases uterine curettage over time was followed by hysteroscopy and repeated curettage because of remains of chorionic tissue. One woman had hysteroscopy and repeated curettage for this reason twice: one time - after abortion and the second time - after delivery. In all these cases the removal of the chorionic debris was not performed easily, because of intense attachment to uterine wall. We retrospectively revised ultrasound examinations of these patients in the absence of pregnancy (some time before the missed pregnancy), before and after hysteroscopy and repeated curettage. It was found out that the endometrium of all these women in the late proliferative phase of menstrual cycle was quite thin - 4-6,4 mm maximum and normal three layer structure of endometrium was seen only in 2 patients. 4 of 5 patients had implantation in uterine corners or in the isthmus. On the echograms before and after hysteroscopy and repeated curettage Doppler signs of deep location of chorionic vessels were seen, in one patient the uterine wall abnormality extended to serous layer. We suggest the connection between thin endometrium and impaired process of chorion invasion and decidual formation (deep trophoblast invasion) especially in cases of low implantation and implantation in uterine corners where endometrium is known to be thinner and decidual formation is often impaired. It seems that some mechanisms of self-protection (uterine protection) are settled down in these cases and, as a result, termination of pregnancy occurs. Undoubtedly, this suggestion needs further investigation.

Failure to improve a thin endometrium in the late proliferative phase with uterine infusion of granulocyte-colony stimulating factor

Purpose: To determine if the treatment with uterine infusion of granulocyte colony-stimulating factor (G-CSF) can improve endometrial thickness in an infertile woman with a double uterus, who consistently showed a thin endometrium in the late proliferative phase either in controlled ovarian hyperstimulation (COH) IVF-ET cycles or with graduated estrogen/sildenafil protocols for frozen embryo transfer (ET). Materials and Methods: A single uterine infusion of G-CSF was performed in the late proliferative phase in a woman who only attained a five-mm thickness despite a high dose vaginal and oral estradiol regimen plus sildenafil. Results: No increase was found within a couple days. Conclusions: A previous four-case study in another center found 100% improvement in the endometrial thickness in women with consistently thin endometria. Perhaps the uterine anomaly in the present case prevented the response of the endometrium.

Asynchronous glands in the endometrium of women with recurrent reproductive failure

Of 969 non-consecutive endometrial biopsies performed for investigation of recurrent reproductive failure, 20 cases (2.1%) showed the striking presence of retarded or asynchronous endometrial glands in otherwise unremarkable mid or late secretory endometrium. These glands were characterised by tall columnar cells with crowded nuclei showing increased reactivity for the proliferative marker MIB-1, occasional mitoses, greatly reduced or absent secretion, and persistent expression of oestrogen receptors and usually progesterone receptors and their isoforms typical of late proliferative phase endometrium. The nearby endometrial stromal cells exhibited no discernibly reduced reactivity for calretinin. These changes were seen in single glands (even portions of glands), or clusters of glands, adjacent to normal late secretory type endometrial glands and set in pseudodecidualised stroma characteristic of late luteal phase. Some examples also displayed adjacent glands with intermediate features and it is speculated that firstly, this is a relatively common phenomenon in women with recurrent miscarriage or implantation failure and with an unknown potential to affect implantation. Secondly, it is an intrinsic defect of the endometrium and can occur in sequential endometrial biopsies in the same patient. Thirdly, it differs from previously described patterns of so-called luteal phase defect or deficient secretory phase in that it occurs in the demonstrated presence of adequate progesterone effect on the endometrium and is associated with persistence rather than exaggerated down-regulation of receptors. Nevertheless, supplementary progesterone therapy (vaginal pessaries) for the first trimester appeared to have a beneficial therapeutic effect on reproductive outcome in these patients.

Very Thin Endometria in the Late Proliferative Phase Is More Associated with Poor Pregnancy Rates Following Controlled Ovarian Hyperstimulation Than Graduated Estradiol Regimens Used for Frozen Embryo Transfer

From the early days of IVF-ET thin endometria at the time of peak follicular maturation was negatively associated with pregnancy rates (PRs). Though improvement in IVF technology has produced “heartier” embryos there is still a negative association with thin endometria and pregnancy outcome but not as severe as before. One option for thin endometria is to freeze all embryos and transfer in a subsequent cycle using a graduated estrogen protocol which usually but not always improves the endometrial thickness.