Pinopode Formation Research Articles

Generation of epithelial-stromal assembloids as an advanced in vitro model of impaired adenomyosis-related endometrial receptivity

ObjectiveTo create a novel, more advanced in vitro model of human endometrium, using so-called assembloids, looking to explore endometrial receptivity in adenomyosis. DesignEvaluation of assembloid responsiveness to hormonal stimulation by immunohistochemistry, ELISA and scanning electron microscopy (SEM). SettingUniversity-based research unit in gynecology. SubjectsTwelve women, 6 of whom were affected by adenomyosis. InterventionOrganoids (in the form of glandular fragments) and stromal fibroblasts were collected from endometrial biopsies. The two populations were combined inside an extracellular matrix to create 3D assembloids, which were then exposed to hormonal stimulation (β-estradiol for 48 hours, followed by β-estradiol/progesterone/cyclic adenosine monophosphate for 72 hours) to mimic the window of implantation. Main outcome measuresGlycodelin, leukemia inhibitory factor (LIF) and homeobox A10 (HOXA10) expression, prolactin secretion and pinopode development. ResultsEndometrial organoids and stromal cells were successfully isolated from women with and without adenomyosis and combined to generate the assembloid model. Upon stimulation, assembloids from both groups acquired a more secretory phase-like phenotype, as demonstrated by histology, and were shown to be positive for glycodelin, LIF and HOXA10 by immunohistochemistry. Adenomyotic assembloids expressed significantly lower levels of LIF and HOXA10 within the stromal compartment after stimulation than did healthy assembloids in the same condition. ELISA revealed prolactin secretion in vitro, showing an upward trend in hormonally treated assembloids from both healthy and affected women. By SEM, fully formed pinopodes were discerned on the epithelial surface of healthy assembloids after stimulation, but they were absent in case of adenomyosis. ConclusionPrimary assembloids can be generated from endometrial biopsies from both healthy subjects and women affected by adenomyosis. These assembloids are amenable to hormonal stimulation and mimic secretory phase-specific characteristics of endometrial tissue in vivo, including glycodelin, LIF and HOXA10 expression, and pinopode formation. Assembloids from adenomyosis appear to be less sensitive to hormonal treatment, showing reduced expression of LIF and HOXA10 in the stromal compartment and failing to form pinopodes. All in all, endometrial assembloids may serve as an advanced preclinical model of adenomyosis-related impaired endometrial receptivity, opening up new horizons in understanding and treating the condition.

GLUT4 in Mouse Endometrial Epithelium: Roles in Embryonic Development and Implantation.

GLUT4 is involved in rapid glucose uptake among various kinds of cells to contribute to glucose homeostasis. Prior data have reported that aberrant glucose metabolism by GLUT4 dysfunction in the uterus could be responsible for infertility and increased miscarriage. However, the expression and precise functions of GLUT4 in the endometrium under physiological conditions remain unknown or controversial. In this study, we observed that GLUT4 exhibits a spatiotemporal expression in mouse uterus on pregnant days 1–4; its expression especially increased on pregnant day 4 during the window of implantation. We also determined that estrogen, in conjunction with progesterone, promotes the expression of GLUT4 in the endometrial epithelium in vivo or in vitro. GLUT4 is an important transporter that mediates glucose transport in endometrial epithelial cells (EECs) in vitro or in vivo. In vitro, glucose uptake decreased in mouse EECs when the cells were treated with GLUT4 small interfering RNA (siRNA). In vivo, the injection of GLUT4-siRNA into one side of the mouse uterine horns resulted in an increased glucose concentration in the uterine fluid on pregnant day 4, although it was still lower than in blood, and impaired endometrial receptivity by inhibiting pinopode formation and the expressions of leukemia inhibitory factor (LIF) and integrin ανβ3, finally affecting embryonic development and implantation. Overall, the obtained results indicate that GLUT4 in the endometrial epithelium affects embryo development by altering glucose concentration in the uterine fluid. It can also affect implantation by impairing endometrial receptivity due to dysfunction of GLUT4.

Electroacupuncture Improves Pregnancy Outcomes in Rats with Thin Endometrium by Promoting the Expression of Pinopode-Related Molecules.

A thin endometrium affects the success of assisted reproduction due to low endometrial receptivity. Acupuncture improves endometrial receptivity and promotes the formation of pinopodes, the ultrastructure marker implantation window. However, the specific underlying mechanisms remain unclear. In this study, the efficacy of acupuncture treatment and its underlying mechanism were investigated by analyzing pregnancy rate, pinopode formation, and related molecular markers in thin endometrium model rats. Absolute ethanol (95%) was injected into the uteruses of female Sprague-Dawley rats to construct a thin endometrium model. In this model, acupuncture stimulation at EX-CA1, SP6, and CV4 ameliorated the pregnancy rate. Significantly increased embryo implantation, endometrial thickness, numbers of glands, and blood vessels were observed in the electroacupuncture (EA) group compared to the model group. The number of pinopodes in the EA group was abundant, with a shape similar to that of the control group. Additionally, significantly higher expression levels of pinopode-related markers, including integrin αvβ3, homeobox A10 (HOXA10), heparin-binding EGF-like growth factor (HBEGF), estrogen receptor alpha (ERα), and progesterone receptor (PR), were observed in the EA group than those in the model group. In conclusion, EA had a positive effect on the endometrial receptivity of thin endometrium model rats by improving pinopode formation through multiple molecular targets.

Effects of soy isoflavonoids (genistein and daidzein) on endometrial receptivity.

Objective(s):This study aimed to examine the effects of genistein and daidzein on endometrial receptivity by histopathological, immunohistochemical, and biochemical techniques.Materials and Methods:In this study, 72 female Sprague-Dawley rats were randomly divided into 8 groups. The endometrial receptivity model was applied to identified groups. Experimental animals were given periorally 10 mg/kg and high 40 mg/kg doses of genistein and daidzein for 5 days by gavage. At the end of the experiment, uterine tissues were evaluated histopathologically, immunohistochemically, and biochemically. Results:When histopathological findings were examined, significant decreases in pinopod formation were observed in high dose genistein and daidzein groups. When compared with the endometrial receptivity group, immunohistochemical staining findings showed a significant decrease in the expression of integrin β3, integrin αvβ3, LIF, and HOXA10 and an increase in MUC 1 expression in the high dose of genistein and daidzein groups. In biochemical evaluations, it was determined that genistein and daidzein increased estrogen levels and decreased progesterone levels in a dose-dependent manner. Conclusion:Genistein and daidzein have a negative effect on endometrial receptivity. Therefore, individuals with a risk of infertility should pay attention to the consumption of genistein and daidzein.

3D Bioprinting a human iPSC-derived MSC-loaded scaffold for repair of the uterine endometrium

Common events in the clinic, such as uterine curettage or inflammation, may lead to irreversible endometrial damage, often resulting in infertility in women of childbearing age. Currently, tissue engineering has the potential to achieve tissue manipulation, regeneration, and growth, but personalization and precision remain challenges. The application of “3D cell printing” is more in line with the clinical requirements of tissue repair. In this study, a porous grid-type human induced pluripotent stem cell-derived mesenchymal stem cell (hiMSC)-loaded hydrogel scaffold was constructed using a 3D bioprinting device. The 3D-printed hydrogel scaffold provided a permissive in vitro living environment for hiMSCs and significantly increased the survival duration of transplanted hiMSCs when compared with hiMSCs administered locally in vivo. Using an endometrial injury model, we found that hiMSC transplantation can cause early host immune responses (the serological immune response continued for more than 1 month, and the local immune response continued for approximately 1 week). Compared with the sham group, although the regenerative endometrium failed to show full restoration of the normal structure and function of the lining, implantation of the 3D-printed hiMSC-loaded scaffold not only promoted the recovery of the endometrial histomorphology (endometrial tissue and gland regeneration) and the regeneration of endometrial cells (stromal cells and epithelial cells) and endothelial cells but also improved endometrial receptivity functional indicators, namely, pinopode formation and leukemia inhibitory factor and αvβ3 expression, which partly restored the embryo implantation and pregnancy maintenance functions of the injured endometrium. These indicators were significantly better in the 3D-printed hiMSC-loaded scaffold group than in the unrepaired (empty) group, the hiMSCs alone group and the 3D scaffold group, and the empty group showed the worst repair results. Our study confirm that the 3D-printed hiMSC-loaded hydrogel scaffold may be a promising material for endometrial repair.

DYSREGULATED EMBRYONIC PLACENTATION IS MORE SIGNIFICANTLY ASSOCIATED WITH RISK OF EARLY SPONTANEOUS PREGNANCY LOSS THAN MATERNAL UTERINE SIGNALING

Biochemical pregnancy loss (BPL) rates have remained persistent at 12-15% despite significant developments in in vitro fertilization including improved embryo culture, aneuploidy screening, and endometrial receptivity testing. In order to elucidate factors important for early pregnancy maintenance that could lead to BPL, we utilized a mouse model to investigate sites of implantation corresponding to viable embryos or spontaneous embryo resorption (miscarriage). Uterine and placental gene expression of implantation sites corresponding to either viable or spontaneously resorbing embryos from early murine pregnancy. Female wild-type, outbred CF-1 pregnant mice were identified on day 10.5 of embryonic development for collection of uterine implantation sites containing either viable embryos (controls) or embryos that were in the early stage of resorption and not yet hemorrhagic. Total RNA was isolated from embryonic placentas (n=6 per group) and maternal uterine tissue (n=5 per group). Quantitative real-time PCR was performed on the QuantStudio 5 (Applied Biosystems) for key implantation and early pregnancy genes including: Cd81, Glrx, Bdnf, Cox2, Ptgs2, Vegfa, and Icos. Gene expression data were analyzed by the REST software (Qiagen) and Student’s t-test, with significance at P<0.05. Embryonic placental expression of Cd81 was significantly increased in embryo resorption sites (P<0.001). Elevated Cd81 inhibits cytotrophoblast invasion, indicating that insufficient placentation may have contributed to early pregnancy loss. Expression of embryonic placental Glrx was decreased in resorption sites (P<0.01), suggesting that heightened oxidative stress hindered placentation and/or maternal-fetal communication resulting in fetal loss. On the maternal side, expression of the paracrine growth factor Bdnf was decreased in uterine tissue from embryo resorption sites (P<0.01). Previous studies have reported increased Bdnf in the pregnant uterus and reduced Bdnf in uterine flushings from repeat implantation failure patients. In contrast, genes involved in tissue remodeling, angiogenesis, and decidualization (Cox2, Ptgs2, Vegfa), immune balance (Icos), trophoblast invasion (Cd81), or pinopode formation (Glrx) were not differentially expressed in maternal uterine tissue between viable and resorbing implantation sites. The etiology of BPL is likely diverse and complex. Our preliminary data suggest that dysregulated embryonic placentation is more significantly associated with risk of early pregnancy loss than maternal uterine signaling. As lipid composition and amino acid metabolism are important for implantation, ongoing studies will utilize liquid chromatography mass spectrometry to identify metabolite changes that correlate with these observed significant embryonic placentation changes.

PECULIARITIES OF PINOPODS FORMATION IN ENDOMETRIUM DEPENDING ON LUTEAL PHASE SUPPORT IN CYCLES OF IN VITRO FERTILIZATION

The formation of pinopods occurring in the middle of the luteal phase is a major indicator of the quality of the endometrium. The study is aimed at studying the peculiarities of pinopod formation in the endometrium, depending on the method of maintaining the luteal phase in the treatment of infertility by means of in vitro fertilization. To this end, 50 women being the oocyte donors were examined. They were subdivided into five subgroups depending on the luteal phase support scheme. On the 5th day after receiving oocytes, women underwent endometrial tube biopsy to scan electron microscopy and determine foam events in the endometrium. It has been proven that in patients receiving highly purified progesterone for subcutaneous administration of 25 mg (1 ml) once a day and 90 mg of intra−vaginal progesterone as a gel, normal development of pinopods was observed in 60.0 % of cases. This scheme proved to be the most effective. Therefore, for the success of extracorporeal fertilization, the process of forming pinopods, which depends on the scheme of maintaining the luteal phase of the cycle, is important. It is noted that the common negative features in the formation of pinopods in women during the period of "implantation windows" is a decrease in the number of pinopod events, an increase in the number of foam events, the presence of areas of pinopod absence, mosaicism of their sizes, forms and stages of development. Key words: pinopods, luteal phase support, endometrium, in vitro fertilization.

Adrenomedullin improves fertility and promotes pinopodes and cell junctions in the peri-implantation endometrium.

Implantation is a complex event demanding contributions from both embryo and endometrium. Despite advances in assisted reproduction, endometrial receptivity defects persist as a barrier to successful implantation in women with infertility. We previously demonstrated that maternal haploinsufficiency for the endocrine peptide adrenomedullin (AM) in mice confers a subfertility phenotype characterized by defective uterine receptivity and sparse epithelial pinopode coverage. The strong link between AM and implantation suggested the compelling hypothesis that administration of AM prior to implantation may improve fertility, protect against pregnancy complications, and ultimately lead to better maternal and fetal outcomes. Here, we demonstrate that intrauterine delivery of AM prior to blastocyst transfer improves the embryo implantation rate and spacing within the uterus. We then use genetic decrease-of-function and pharmacologic gain-of-function mouse models to identify potential mechanisms by which AM confers enhanced implantation success. In epithelium, we find that AM accelerates the kinetics of pinopode formation and water transport and that, in stroma, AM promotes connexin 43 expression, gap junction communication, and barrier integrity of the primary decidual zone. Ultimately, our findings advance our understanding of the contributions of AM to uterine receptivity and suggest potential broad use for AM as therapy to encourage healthy embryo implantation, for example, in combination with in vitro fertilization.

Altered expression of hormone receptor, integrin β3 and pinopode in the endometrium of luteal phase defect women

Objective: To invistigate estrogen receptor (ER), progesterone receptor (PR), integrin β3, and pinopode expression in luteal phase deficiency (LPD) women.Methods: There were 52 nulligravidas consecutive infertile patients undergoing a routine assistant reproduction consultation included in this study. An endometrial biopsy sample was randomly obtained between days 4 and 10 of the luteal phase. Endometrial morphology was examined with scanning electron microscopy. Expressions of ER, PR, integrin β3 were determined in the endometrium of LPD patients with immunohistochemistry.Results: The incidence of LPD was 15.3% (8/52) in this study. On day luteinizing hormone (LH) surge + 9∼LH + 10, noted regressing pinopodes resembling a day LH + 7∼LH + 8 in the endometrium of the control group. The expressions of ER and PR in glandular epithelium were significantly increased in endometrium of LPD than that in the control group (p < 0.05). In contrast, there was a statistically significant decrease expression of the integrin β3 in women from the group of LPD (p < 0.05).Conclusion: The altered expression of ER and PR may be associated with the expression variation of integrin and pinopode formation in endometrium of LPD women. This alteration may imply the association of low rates of cycle fecundity and high rates of embryonic loss in LPD women.

The effect of sperm activation on pinopod formation in endometrial epithelium

IntroductionEndometrial receptivity is crucial in implantation of the developing embryo in the endometrium and formation of the pregnancy. In this study, possible effect of sperm and uterine endometrial contact on formation of pinopod, an important element in morphological differentiation necessary for implantation, was investigated. Materials and methodsIn this experimental study, 42 female Spraque–Dawley albino rats and 14 male Spraque–Dawley albino rats (total 56 rats) were used. Vasectomy was performed in half of the male rats. For each group, two distinct branches were formed with 21 females and 7 males: Group 1 (non-vasectomized) and Group 2 (vasectomized). Cases were sacrificed and evaluated every day from Day 1 to Day 3. Scanning electron microscopic (SEM) images were analyzed according to different stages of pinopod development on different days. Pinopods were classified as developing, developed and regressing pinopod. The average number of pinopods were calculated by counting the pinopods at four endometrial regions examined for each rat and total number was divided by 4. Same procedure was done for all rats in every group. Results were compared among the groups. For statistical analysis among the groups, *Independent Samples Test (Mean±Std) and **Mann Whitney U Test (Median (25–75%)) were used. ResultsThe most important finding in SEM examination of uterus removed on the first day following mating from female rats that were copulated with non-vasectomy male rats which comprised the first group of the study was that heads of the sperms in the uterus were embedded in endometrial epithelium. Similarly, examination of the endometrium of uterus that were removed on postcoital second day revealed small number of developed pinopods (average 0.39) (P=0.902). Examination was done by taking the developing pinopods within image area into account and number of developing pinopods in endometrium epithelium of the rats in first group (average 20.61) was higher than that of second group (average 12.86) (P<0.001). Examination of endometrium of the uterus that were removed on third postcoital day revealed less number of developing pinopods in both groups. Examination of first group revealed an average of 1.21 developing pinopod, whereas the average number of developing pinopod in second group was 2.25 (P=0.011). Examination based on the count of developed pinopods revealed that number of pinopods in first group (average 13.79) was higher than second group (average 8.96) (P<0.001). Regressing pinopod images were observed in only endometrium that were taken on postcoital third day in the second group. DiscussionIn this study, it was clearly shown that sperms were entered into endometrial epithelium with their heads. It can be suggested that they might have a facilitating effect for pinopod formation by reacting with endometrial epithelium as a result of this invasion. It would be beneficial to demolish the other factors triggering pinopod formation to investigate whether presence of sperm alone in the uterus has an effect on pinopod formation.

Endometrial receptivity markers in infertile women stimulated with letrozole compared with clomiphene citrate and natural cycles

Though various advantages associated with the use of letrozole as an ovulation inducing drug are documented, there is inadequate information regarding its effect on endometrial receptivity. Expression of endometrial receptivity markers including αvβ3 integrin, L-selectin, LIF, and pinopods during the implantation window in infertile women stimulated with letrozole or clomiphene citrate (CC), and spontaneous cycles is investigated. A total of 36 women were included in the study out of which 16 women were diagnosed with primary unexplained infertility and the other 20 women with severe oligoasthenozoospermic/azoospermic male partners. Both groups were sub-divided into three groups; women stimulated with letrozole, or with CC, and the third group was allowed to ovulate spontaneously (natural cycles). Women having natural cycles and not given any drug were considered as controls. Once ovulation was confirmed by ultrasonography, endometrial samples were collected on the seventh day post-ovulation and analyzed. In women with unexplained infertility treated with letrozole and CC, epithelial and stromal expression of αvβ3 integrin, L-selectin, leukemia inhibitory factor (LIF), and pinopod formation was found to be significantly higher as compared to controls. Expression of these receptivity markers was found to be comparable amongst the letrozole, CC, and control groups in women with severe oligoasthenozoospermic/azoospermic male partners. Cell cycle analysis showed similar cell cycle phase fractions on comparing the CC and the letrozole groups. Stimulation with letrozole and CC appears to enhance endometrial receptivity in women with unexplained infertility. However, letrozole and CC did not have any significant effect on the endometrial receptivity markers of women with severe oligoasthenozoospermic/azoospermic male partners.

Evaluation of Pinopodes Expression on the Mouse Endometrium Immediately before Implantation by Treatment with HMG/HCG and Sildenafil Citrate Administration.

Sildenafil citrate is a new drug and has special properties that bring about nitric oxide effects on vascular smooth muscle. The aim of this study was to assess the effects of ovarian stimulation and sildenafil citrate injection on pinopode expression in mice. THIRTY ADULT FEMALE MICE WERE RANDOMLY DIVIDED INTO THREE GROUPS: control, hyperstimulated and hyperstimulated +sildenafil citrate injection. In experimental groups mice received 7.5 IU human menopausal gonadotropin (HMG) and then after 48 hr received 7.5 IU human chorionic gonadotropic (HCG) hormones. After that every two females were put with one male in one cage for mating. Hyperstimulated +sildenafil citrate group were injected with sildenafil citrate (3 mg/mouse) every 24 hr for three days, after HMG injection. The animals were sacrificed by cervical dislocation 96 hr after HMG injection, and their uterine specimens (the middle one-third) were prepared for transmission electron microscope studies. Electron microscopy observations showed that in the control group there were long and short microvilli while no developed pinopodes were observed, however, in the two other groups, well developed pinopodes were expressed 4 days after HMG injection. The results showed that hyperstimulation of mice with sildenafil citrate may be more helpful in formation of pinopodes and implantation.

No correlation between pinopode formation and LIF and MMP2 expression in endometrium during implantation window

Implantation depends on two factors - embryo and endometrium. The period of maximal endometrial receptivity is a poorly understood phenomenon. We decided to look at three possible markers of implantation: pinopodes, leukemia inhibitory factor, and matrix metalloproteinase 2 and their correlations. We included in the study 23 idiopathic infertility patients and 21 patients with recurrent spontaneous abortions of unknown etiology. Twenty one fertile patients were also recruited. A biopsy was used for endometrial dating according to the Noyes and Hertig criteria, and assessed for the presence of pinopodes via a scanning electron microscope. Endometria were examined in Real Time-Polymerase Chain Reaction cycles for the mRNA expression of leukemia inhibitory factor (LIF) and matrix metalloproteinase 2 (MMP2). No difference was found in the stage of pinopodes development, nor in the coverage of endometrial surface between the studied groups. The expression level for LIF mRNA was lower in control patients compared to idiopathic infertility and recurrent miscarriage patients. No difference was detected in the expression of MMP2 between all studied groups. No correlation was found between pinopodes development stage and LIF and MMP2 expressions in endometrium. Of the studied factors, LIF and pinopodes show the most promise as potential markers of endometrial receptivity. However, the results achieved suggest that these markers are independent of each other.

No correlation between pinopode formation and LIF and MMP2 expression in endometrium during implantation window

No correlation between pinopode formation and LIF and MMP2 expression in endometrium during implantation window

Optimization of cryocycles by using pinopode detection in patients with multiple implantation failure: preliminary report

The aim of this study was to investigate pinopode formation patterns in patients with a history of multiple IVF failures and to evaluate if their detection with subsequent modification of protocols using frozen–thawed embryos could help to increase the pregnancy and live-birth rates in these patients. The study included 55 women with at least three implantation failures. On-time pinopodes were present in only 12.7% of cases, the rest showed acceleration, delay, arrest or asynchronization of pinopode formation. Patients with anomalies of pinopode development were divided into two subgroups, one of which had the standard protocol and the other a modified protocol, in which the duration of progesterone administration before embryo transfer was changed according to pinopode formation pattern. In nine patients with the modified protocol, with asynchronized and arrested pinopodes, simultaneous transfer of embryos of different ages was fulfilled. The implantation, pregnancy and take-home-baby rates for the modified and standard protocols were 28.0% versus 6.0%, 52.4% versus 12.0% and 47.6% versus 12.0%, respectively. Detection of inappropriately timed pinopode formation with subsequent synchronization of embryonic development and endometrial maturation allowed improvements in the effectiveness of programmes using frozen–thawed embryos in women with multiple implantation failure.

The expression of receptivity markers in the fallopian tube epithelium

Pinopodes represent the morphological and integrins, the biomolecular markers of endometrial receptivity. We studied using scanning electron microscopy, the expression of pinopodes on tubal samples and their corresponding endometria, from 21 women of reproductive age (7 from proliferative phase, 7 from day LH +5 and 7 from day LH +7). In addition, we examined the immunohistochemical staining of integrins alpha v beta 3, alpha v beta 5 and their ligands, fibronectin (FN) and osteopontin (OPN) in the same tubal epithelium samples. Pinopodes were detected on the tubal epithelium exclusively during day LH +7, coincident with their formation in the endometrium and synchronous to alpha v beta 3 sharp increase in the oviduct epithelium, suggesting a regulation similar to the endometrium. In contrast, alpha v beta 5, FN and OPN remained unchanged during the cycle. These results show for the first time the formation of pinopodes in the tubal epithelium at the time of endometrial receptivity and correlate it with the upregulation of the intact dimmer alpha v beta 3 in the tubes.

Haploinsufficiency for Adrenomedullin Reduces Pinopodes and Diminishes Uterine Receptivity in Mice1

Adrenomedullin (AM) is a multifunctional peptide vasodilator that signals through a G-protein-coupled receptor when the receptor, called calcitonin receptor-like receptor (CL), is associated with a receptor activity-modifying protein 2 (RAMP2). We demonstrated previously that haploinsufficieny for each of these genes led to reduced maternal fertility, and that even a modest genetic reduction of AM peptide caused maternal defects in implantation, placentation, and fetal growth. Here, we further demonstrate that Adm(+/-) female mice displayed reduced pregnancy success rates that were not caused by defects in folliculogenesis, ovulation, or fertilization. The poor fertility of Adm(+/-) female mice could not be rescued by transfer of wild-type blastocysts, which suggested an underlying defect in uterine receptivity. In fact, we found that Adm, Calcrl, and Ramp2 gene expressions are tightly and spatiotemporally regulated in the luminal epithelial cells of the uterus during the estrus cycle and the peri-implantation period. RAMP3, which also generates an AM receptor when associated with CL, had a diametrically opposite expression pattern than that of Adm, Calcrl, and Ramp2 and was most robustly induced in the stroma of the uterus. Finally, we discovered that Adm(+/-) female mice have a substantially reduced number of pinopodes on the uterine luminal epithelial surface, which is indicative and possibly causative of the poor uterine receptivity. Taken together, our studies identify a new class of pharmacologically tractable proteins that are involved in establishing uterine receptivity through the regulation of pinopode formation.

Disturbances in the LIF pathway in the endometrium among women with unexplained infertility

To study the expression of leukemia inhibitory factor (LIF), its receptors LIFR and gp130, and its inhibitor SOCS1 in endometria from fertile women and infertile women with unexplained infertility. Signaling through the LIF pathway is involved in maintenance of a receptive state of human endometrium. Impaired endometrial receptivity may be a cause of female infertility. Prospective clinical study. Hospital-based IVF unit and university-affiliated reproductive research laboratories. Twenty-six healthy fertile women and 14 women with unexplained infertility. Endometrial biopsy. Pinopode formation, expression of LIF, LIFR, gp130, and SOCS1 protein and mRNA in endometrial biopsies. The expression of LIFR in the endometrium was negatively correlated to the expression of SOCS1 and positively correlated to the formation of pinopodes. In control fertile women, simultaneous intense apical staining of LIFR and gp130 together with faint SOCS1 staining was observed in epithelial cells, whereas the opposite was seen in most women with unexplained infertility. Unexplained infertility in some women might be explained by disturbances in the LIF pathway in midsecretory-phase endometrium.

Endometrial Mucin-1 and Pinopode in Peri-implantation Phase in Ovarian High Responders during Controlled Ovarian Hyperstimulation Cycles

Objective To investigate effects of ovarian high response on endometrial mucin-1 (MUC1) and pinopode in peri-implantation phase in controlled ovarian hyperstimulation (COH) cycles. Methods Ovarian high response was defined as serum E 2 > 15 000 pmol/L on the day of hCG administration in COH cycle using GnRH agonist and recombinant FSH (n=8). Healthy and fertile women were used as the natural control (n=10). Endometrial biopsies were performed on the day of LH+7/hCG+7. Pinopode formation was observed by scanning electron microscope. Expression of MUC1 was detected with quantitative Real-time PCR and immunohistochemistry. Results In high response group, the lumen surface was covered with variant pinopodes and microvillous. The expression of MUC1 mRNA in high response group was lower than that in the natural control (P Conclusion Asynchronized pinopode appearance and lower expression of MUC1 during peri-implantation period were the characteristics of endometrium in high response group, which may provide a clue of decreased endometrial receptivity in the supraphysiological hormone milieu.

Alteration of maternal MEIS1 expression by in vivo gene transfection affects implantation

HOXA10 is a well-known transcription factor necessary for embryonic uterine development and for adult endometrial receptivity. The TALE family of cofactors, including Meis and Pbx, provide HOX target gene specificity in development and myeloid differentiation. We have previously identified the expression of meis1 dramatically increased during the midsecretory phase of the menstrual cycle, corresponding to the time of implantation. The aim of this study was to observe the function of meis1 in embryonic implantation. The present study was designed to alter the level of the meis 1 in endometrium by the liposome-mediated gene transfection techniques, then observe the effect on endometrial receptivity. We transfected mice on day 2 post coitus with pcDNA4/TO plus meis1, pTER plus MEIS1 siRNA or respective controls consisting of empty pcDNA4/TO and pTER by intrauterine lipofection. The expression of endometrial pinopodes on day 4.5 pregnant mice was determinced by scanning electron microscopy. The expression of intergrin-β3 was measured by PT-PCR and immunochemistry on day 4.5. The pregnant rate and implantation rate was observed on day 9 between each group. 1. Blocking meis1 expression by transfection of meis1 siRNA into the cycling mouse uterus before implantation dramatically reduced average implantation rate. Constitutively expressing meis1 in the uterus optimized the number of implantation sites. 2. Increased meis 1 expression significantly increased intergrin-β3 expression. Decreased meis 1 expression significantly reduced intergrin-β3 expression. 3. Blocking meis 1 expression before implantation dramatically decreased pinopod number. Constitutively expressing meis 1 in the uterus just before the normal time of pinopod formation resulted in increased pinopod number. Maternal meis1 expression is essential for implantation.