Syndecan-1 knock-down in decidualized human endometrial stromal cells leads to significant changes in cytokine and angiogenic factor expression patterns

<p>Abstract</p> <p>Background</p> <p>Successful embryonic implantation depends on a synchronized embryo-maternal dialogue. Chemokines, such as chemokine ligand 1 (CXCL1), play essential roles in the maternal reproductive tract leading to morphological changes during decidualization, mediating maternal acceptance towards the semi-allograft embryo and induction of angiogenesis. Chemokine binding to their classical G-protein coupled receptors is essentially supported by the syndecan (Sdc) family of heparan sulfate proteoglycans. The aim of this study was to identify the involvement of Sdc-1 at the embryo-maternal interface regarding changes of the chemokine and angiogenic profile of the decidua during the process of decidualization and implantation in human endometrium.</p> <p>Methods</p> <p>A stable Sdc-1 knock-down was generated in the immortalized human endometrial stromal cell line St-T1 and was named KdS1. The ability of KdS1 to decidualize was proven by Insulin-like growth factor binding 1 (IGFBP1) and prolactin (PRL) confirmation on mRNA level before further experiments were carried out. Dot blot protein analyses of decidualized knock-down cells vs non-transfected controls were performed. In order to imitate embryonic implantation, decidualized KdS1 were then incubated with IL-1beta, an embryo secretion product, vs controls. Statistical analyses were performed applying the Student's t-test with p < 0.05, p < 0.02 and p < 0.01 and one way post-hoc ANOVA test with p < 0.05 as cut-offs for statistical significance.</p> <p>Results</p> <p>The induction of the Sdc-1 knock-down revealed significant changes in cytokine and angiogenic factor expression profiles of dKdS1 vs decidualized controls. Incubation with embryonic IL-1beta altered the expression patterns of KdS1 chemokines and angiogenic factors towards inflammatory-associated molecules and factors involved in matrix regulation.</p> <p>Conclusions</p> <p>Sdc-1 knock-down in human endometrial stroma cells led to fulminant changes regarding cytokine and angiogenic factor expression profiles upon decidualization and imitation of embryonic contact. Sdc-1 appears to play an important role as a co-receptor and storage factor for many cytokines and angiogenic factors during decidualization and implantation period, supporting proper implantation and angiogenesis by regulation of chemokine and angiogenic factor secretion in favour of the implanting embryo.</p

Installing oncofertility programs for common cancers in optimum resource settings (Repro-Can-OPEN Study Part II): a committee opinion

The main objective of Repro-Can-OPEN Study Part 2 is to learn more about oncofertility practices in optimum resource settings to provide a roadmap to establish oncofertility best practice models. As an extrapolation for oncofertility best practice models in optimum resource settings, we surveyed 25 leading and well-resourced oncofertility centers and institutions from the USA, Europe, Australia, and Japan. The survey included questions on the availability and degree of utilization of fertility preservation options in case of childhood cancer, breast cancer, and blood cancer. All surveyed centers responded to all questions. Responses and their calculated oncofertility scores showed three major characteristics of oncofertility practice in optimum resource settings: (1) strong utilization of sperm freezing, egg freezing, embryo freezing, ovarian tissue freezing, gonadal shielding, and fractionation of chemo- and radiotherapy; (2) promising utilization of GnRH analogs, oophoropexy, testicular tissue freezing, and oocyte in vitro maturation (IVM); and (3) rare utilization of neoadjuvant cytoprotective pharmacotherapy, artificial ovary, in vitro spermatogenesis, and stem cell reproductive technology as they are still in preclinical or early clinical research settings. Proper technical and ethical concerns should be considered when offering advanced and experimental oncofertility options to patients. Our Repro-Can-OPEN Study Part 2 proposed installing specific oncofertility programs for common cancers in optimum resource settings as an extrapolation for best practice models. This will provide efficient oncofertility edification and modeling to oncofertility teams and related healthcare providers around the globe and help them offer the best care possible to their patients

hCG stimulates angiogenic signals in lymphatic endothelial and circulating angiogenic cells

Human chorionic gonadotropin (hCG) has long been associated with the initiation and maintenance of pregnancy, where angiogenesis plays an important role. However, the function of hCG in angiogenesis and the recruitment of vascular active cells are not fully understood. In this study, the role of hCG and its receptor in circulating angiogenic and human endothelial cells, including lymphatic, uterine microvascular, and umbilical vein endothelial cells, was examined. Immunohistochemistry and immunoblot analysis were used to detect LH/hCG receptor expression and the expression of hCG-induced angiogenic molecules. HIF-1α was determined via ELISA and downstream molecules, such as CXCL12 and CXCR4, via real-time PCR. Chemotaxis was analyzed using Boyden chambers. Our results show that the LH/hCG receptor was present in all tested cells. Furthermore, hCG was able to stimulate LH/hCG-receptor-specific migration in a dose-dependent fashion and induce key angiogenic molecules, including HIF-1α, CXCL12, and CXCR4. In conclusion, our findings underscore the importance of hCG as one of the first angiogenic molecules produced by the conceptus. hCG itself alters endothelial motility, recruitment, and expression of pro-angiogenic molecules and may therefore play an important role in vascular adaption during implantation and early placental formation

hCG stimulates angiogenic signals in lymphatic endothelial and circulating angiogenic cells

Human chorionic gonadotropin (hCG) has long been associated with the initiation and maintenance of pregnancy, where angiogenesis plays an important role. However, the function of hCG in angiogenesis and the recruitment of vascular active cells are not fully understood. In this study, the role of hCG and its receptor in circulating angiogenic and human endothelial cells, including lymphatic, uterine microvascular, and umbilical vein endothelial cells, was examined. Immunohistochemistry and immunoblot analysis were used to detect LH/hCG receptor expression and the expression of hCG-induced angiogenic molecules. HIF-1α was determined via ELISA and downstream molecules, such as CXCL12 and CXCR4, via real-time PCR. Chemotaxis was analyzed using Boyden chambers. Our results show that the LH/hCG receptor was present in all tested cells. Furthermore, hCG was able to stimulate LH/hCG-receptor-specific migration in a dose-dependent fashion and induce key angiogenic molecules, including HIF-1α, CXCL12, and CXCR4. In conclusion, our findings underscore the importance of hCG as one of the first angiogenic molecules produced by the conceptus. hCG itself alters endothelial motility, recruitment, and expression of pro-angiogenic molecules and may therefore play an important role in vascular adaption during implantation and early placental formation

Interferon stimulated gene 15 expression at the human embryo−maternal interface

Purpose In early pregnancy the dialogue between maternal endometrium and embryo is a key process in establishing a receptive decidua and placental network. Decidual ISG15 induction is thought to promote pregnancy maintenance and development. ISG15 is involved in RNA splicing, cytoskeletal organization, stress response and further intracellular processes. Methods ISG15 expression was examined immunohistologically in paraffin-embedded human placental and decidual tissue samples of all pregnancy trimesters on adjacent sections (first trimester n = 5, second n = 5, third n = 3). Samples were processed using a protocol applying a rabbit polyclonal ISG15 antibody. A mouse monoclonal cytokeratin seven antibody was utilized to identify the different placental departments and decidual glands. Staining results and anatomical features were evaluated blindly with strict rating criteria. Results ISG15 expression was identified in first and second trimester tissue samples. ISG15 localized especially to the extravillous cytotrophoblasts in the maternal wall and in maternal blood vessel. Expression was detected in cytotrophoblast progenitor cells in the placental villi and the cell column with a maximum in the first trimester. The syncytial layer stained positive in first and second trimester samples. Third trimester samples showed no expression of ISG15 at all. Conclusions ISG15 abundance in the human placenta is an interesting finding, with implications for placental development, fetal growth and potential defense mechanism against infections. The maximal expression of ISG15 in the first and second trimester of pregnancy suggests that ISG function is needed when placental and embryo development is enormous and embryo susceptibility to external influences is high.</p

Interferon stimulated gene 15 expression at the human embryo−maternal interface

Purpose In early pregnancy the dialogue between maternal endometrium and embryo is a key process in establishing a receptive decidua and placental network. Decidual ISG15 induction is thought to promote pregnancy maintenance and development. ISG15 is involved in RNA splicing, cytoskeletal organization, stress response and further intracellular processes. Methods ISG15 expression was examined immunohistologically in paraffin-embedded human placental and decidual tissue samples of all pregnancy trimesters on adjacent sections (first trimester n = 5, second n = 5, third n = 3). Samples were processed using a protocol applying a rabbit polyclonal ISG15 antibody. A mouse monoclonal cytokeratin seven antibody was utilized to identify the different placental departments and decidual glands. Staining results and anatomical features were evaluated blindly with strict rating criteria. Results ISG15 expression was identified in first and second trimester tissue samples. ISG15 localized especially to the extravillous cytotrophoblasts in the maternal wall and in maternal blood vessel. Expression was detected in cytotrophoblast progenitor cells in the placental villi and the cell column with a maximum in the first trimester. The syncytial layer stained positive in first and second trimester samples. Third trimester samples showed no expression of ISG15 at all. Conclusions ISG15 abundance in the human placenta is an interesting finding, with implications for placental development, fetal growth and potential defense mechanism against infections. The maximal expression of ISG15 in the first and second trimester of pregnancy suggests that ISG function is needed when placental and embryo development is enormous and embryo susceptibility to external influences is high.</p

Physiological and anatomical aspects of the reproduction of mice with reduced Syndecan-1 expression

Abstract Background Syndecan-1 is a heparan sulfate proteoglycan acting as a co-receptor for cytokines and growth factors mediating developmental, immunological and angiogenic processes. In human, the uteroplacental localization of Syndecan-1 and its reduced expression in pregnancy-associated pathologies, such as the intrauterine growth restriction, suggests an influence of Syndecan-1 in embryo-maternal interactions. The aim of the present study was to identify the effect of a reduced expression of Syndecan-1 on the reproductive phenotype of mice and their progenies. Methods Reproductive characteristics have been investigated using animals with reduced Syndecan-1 and their wildtype controls after normal mating and after vice versa embryo transfers. Female mice were used to measure the estrus cycle length and the weight gain during pregnancy, as well as for histological examination of ovaries. Male mice were examined for the concentration, motility, viability and morphology of spermatozoa. Organs like heart, lung, liver, kidney, spleen, brain and ovaries or testes and epididymis of 6-month-old animals were isolated and weighed. Statistical analyses were performed using two-tailed students t-test with P < .05 and P < .02, chi square test (P < .05) and Fisher’s Exact Test (P < .05). A linear and a non-linear mixed-effects model were generated to analyze the weight gain of pregnant females and of the progenies. Results Focusing on the pregnancy outcome, the Syndecan-1 reduced females gave birth to larger litters. However, regarding the survival of the offspring, a higher percentage of pups with less Syndecan-1 died during the first postnatal days. Even though the ovaries and the testes of Syndecan-1 reduced mice showed no histological differences and the ovaries showed a similar number of primary and secondary follicles and corpora lutea, the spermatozoa of Syndecan-1 reduced males showed more tail and midpiece deficiencies. Concerning the postnatal and juvenile development the pups with reduced Syndecan-1 expression remained lighter and smaller regardless whether carried by mothers with reduced Syndecan-1 or wildtype foster mothers. With respect to anatomical differences kidneys of both genders as well as testes and epididymis of male mice with reduced syndecan-1 expression weighed less compared to controls. Conclusions These data reveal that the effects of Syndecan-1 reduction are rather genotype- than parental-dependent

Metabolic and behavioral parameters of mice with reduced expression of Syndecan-1.

Energy balance is essential for all species. Ligand-receptor interactions mediate processes that regulate body activities like reproduction and metabolism based on the energy status. Such receptors are the heparan sulfate proteoglycans and specifically the family of syndecans. Therefore we investigated the differences of metabolic parameters of heterozygous Syndecan 1 mice (Sdc1+/-) with reduced expression of Sdc1 and the corresponding wild type mice. Sdc1+/- mice have a reduced body weight although they show increased leptin and decreased corticosterone levels. Furthermore, their food and water intake is increased. This is accompanied with less adipose tissue, smaller adipocytes and thus an increased density of adipocytes. For the detailed analysis of the metabolism the automated PhenoMaster system has been used, which allowed continuous and undisturbed recording of food and water intake, energy expenditure and movement. The reason for the lower body weight was the higher energy expenditure of these animals compared to controls. Additionally, female Sdc1+/- mice showed an increased locomotor activity. Referring to organs, the intestine in Sdc1+/- mice was heavier and longer, but no differences at the cellular level could be observed. These findings were independent of normal mating or vice versa embryo transfers of Sdc1+/- and wild type embryos in recipient females of the other genotype. Herein we showed that the reduced expression of Sdc1 led to an altered metabolism on fetal as well as on maternal side, which may play a role in the growth restriction observed in human pregnancy pathologies and in mice lacking Sdc1

Ninety-five orthotopic transplantations in 74 women of ovarian tissue after cytotoxic treatment in a fertility preservation network: tissue activity, pregnancy and delivery rates

Abstract STUDY QUESTION What is the success rate in terms of ovarian activity (menstrual cycles) as well as pregnancy and delivery rates 1 year after orthotopic ovarian transplantations conducted in a three-country network? SUMMARY ANSWER In 49 women with a follow-up >1 year after transplantation, the ovaries were active in 67% of cases and the pregnancy and delivery rates were 33 and 25%, respectively. WHAT IS KNOWN ALREADY Cryopreservation of ovarian tissue in advance of cytotoxic therapies and later transplantation of the tissue is being performed increasingly often, and the total success rates in terms of pregnancy and delivery have been described in case series. However, published case series have not allowed either a more detailed analysis of patients with premature ovarian insufficiency (POI) or calculation of success rates based on the parameter ‘tissue activity'. STUDY DESIGN, SIZE, DURATION Retrospective analysis of 95 orthotopic transplantations in 74 patients who had been treated for cancer, performed in the FertiPROTEKT network from 2008 to June 2015. Of those 95 transplantations, a first subgroup (Subgroup 1) was defined for further analysis, including 49 women with a follow-up period >1 year after transplantation. Of those 49 women, a second subgroup (Subgroup 5) was further analysed, including 40 women who were transplanted for the first time and who were diagnosed with POI before transplantation. PARTICIPANTS/MATERIALS, SETTING, METHODS Transplantation was performed in 16 centres and data were transferred to the FertiPROTEKT registry. The transplantations were carried out after oncological treatment had been completed and after a remission period of at least 2 years. Tissue was transplanted orthotopically, either into or onto the residual ovaries or into a pelvic peritoneal pocket. The success rates were defined as tissue activity (menstrual cycles) after 1 year (primary outcome) and as pregnancies and deliveries achieved. MAIN RESULTS AND THE ROLE OF CHANCE The average age of all transplanted 74 women was 31 ± 5.9 years at the time of cryopreservation and 35 ± 5.2 at the time of transplantation. Twenty-one pregnancies and 17 deliveries were recorded. In Subgroup 1, tissue was cryopreserved at the age of 30 ± 5.6 and transplanted at 34 ± 4.9 years. Ovaries remained active 1 year after transplantation in 67% of cases (n = 33/49), the pregnancy rate was 33% (n = 16/49) and the delivery rate was 25% (n = 12/49). In Subgroup 5, tissue was cryopreserved at the age 30 ± 5.9 years and transplanted at 34 ± 5.2 years. Ovaries remained active 1 year after transplantation in 63% of cases (n = 25/40), the pregnancy rate was 28% (n = 11/40) and the delivery rate was 23% (n = 9/40). The success rates were age dependant with higher success in women who cryopreserved at a younger age. In Subgroup 5, tissue was exclusively transplanted into the ovary in 10% (n = 4/40) of women and into a peritoneal pocket in 75% (n = 30/40), resulting in spontaneous conceptions in 91% of patients (n = 10/11). LIMITATIONS, REASONS FOR CAUTION The data were drawn from a retrospective analysis. The cryopreservation and transplantation techniques used have changed during the study period. The tissue was stored in many tissue banks and many surgeons were involved, leading to heterogeneity of the procedures. However, this does reflect the realistic situation in many countries. Although patients with POI were evaluated before transplantation to allow specific analysis of the transplanted tissue itself, the possibility cannot be excluded that residual ovarian tissue was also reactivated. WIDER IMPLICATIONS OF THE FINDINGS This is the largest case series worldwide to date and it confirms that cryopreservation and transplantation of ovarian tissue can be a successful option for preserving fertility. Persistent tissue activity 12 months after transplantation suggests that the pregnancy and delivery rates may increase further in the future. As transplantation into the peritoneum results in a high success rate, this approach may be an alternative to transplantation into the ovary. However, in order to establish the best transplantation site, a randomized study is required. STUDY FUNDING/COMPETING INTEREST This study was in part funded from the Deutsche Forschungsgemeinschaft (# DI 1525) and the Wilhelm Sander Foundation (2012.127.1) and did not receive any funding from a commercial company. No competing interests. TRIAL REGISTRATION NUMBER None