Design of Materials for Bone Tissue Scaffolds

The strong impulse recently experienced by the manufacturing technologies as well as the development of innovative biocompatible materials has allowed the fabrication of high-performing scaffolds for bone tissue engineering. The design process of materials for bone tissue scaffolds represents, nowadays, an issue of crucial importance and the object of study of many researchers throughout the world. A number of studies have been conducted, aimed at identifying the optimal material, geometry, and surface that the scaffold must possess to stimulate the formation of the largest amounts of bone in the shortest time possible. This book presents a collection of 10 research articles and 2 review papers describing numerical and experimental design techniques definitively aimed at improving the scaffold performance, shortening the healing time, and increasing the success rate of the scaffold implantation process.

Role of adipokines in embryo implantation

Embryo implantation is a complex process in which multiple molecules acting together under strict regulation. Studies showed the production of various adipokines and their receptors in the embryo and uterus, where they can influence the maternal-fetal transmission of metabolites and embryo implantation. Therefore, these cytokines have opened a novel area of study in the field of embryo-maternal cross-talk during early pregnancy. In this respect, the involvement of adipokines has been widely reported in the regulation of both physiological and pathological aspects of the implantation process. However, the information about the role of some recently identified adipokines is limited. This review aims to highlight the role of various adipokines in embryo-maternal interactions, endometrial receptivity, and embryo implantation, as well as the underlying molecular mechanisms.

Uterine epithelial LIF receptors contribute to implantation chamber formation in blastocyst attachment

Recent studies have demonstrated that the formation of an implantation chamber composed of a uterine crypt, an implantation-competent blastocyst, and uterine glands is a critical step in blastocyst implantation in mice. Leukemia inhibitory factor (LIF) activates signal transducer and activator of transcription 3 (STAT3) precursors via uterine LIF receptors (LIFRs), allowing successful blastocyst implantation. Our recent study revealed that the role of epithelial STAT3 is different from that of stromal STAT3. However, both are essential for blastocyst attachment, suggesting the different roles of epithelial and stromal LIFR in blastocyst implantation. However, how epithelial and stromal LIFR regulate the blastocyst implantation process remains unclear. To investigate the roles of LIFR in the uterine epithelium and stroma, we generated Lifr-floxed/lactoferrin (Ltf)-iCre (Lifr eKO) and Lifr-floxed/anti-Mullerian hormone receptor type 2 (Amhr2)-Cre (Lifr sKO) mice with deleted epithelial and stromal LIFR, respectively. Surprisingly, fertility and blastocyst implantation in the Lifr sKO mice were normal despite stromal STAT3 inactivation. In contrast, blastocyst attachment failed, and no implantation chambers were formed in the Lifr eKO mice with epithelial inactivation of STAT3. In addition, normal responsiveness to ovarian hormones was observed in the peri-implantation uteri of the Lifr eKO mice. These results indicate that the epithelial LIFR-STAT3 pathway initiates the formation of implantation chambers, leading to complete blastocyst attachment, and that stromal STAT3 regulates blastocyst attachment without stromal LIFR control. Thus, uterine epithelial LIFR is critical to implantation chamber formation and blastocyst attachment.

P–403 Sodium tungstate increases embryo adhesion through a direct effect on endometrial cells

Study question Does sodium tungstate treatment induce a change in endometrial cells’ capacity to implant trophoblasts? Summary answer Administration of sodium tungstate to endometrial cells increases trophoblast adhesion. What is known already Sodium tungstate (ST) has shown its capacity to modulate the activity of cytokines, such as leptin, an activator of an obligatory signalling cascade in the embryo-implantation process. STAT3, a signal transducer molecule critical for the embryo implantation process, is also known to be activated by ST. Still, ST’s effect on implantation using biological systems has never been studied. Embryo implantation process and endometrium roles are complicated to study in vivo due to a lack of animal models and appropriate techniques. In vitro techniques using immortalised cell lines allows a first approach to study early implantation stages, such as embryo adhesion. Study design, size, duration An in vitro study was carried out using a human endometrial carcinoma cell line (HEC–1-A) treated with sodium tungstate for 24 and 48h, and choriocarcinoma cell spheroids (JAr). Different times of treatment and concentrations were studied. Each experiment was performed in triplicate. Participants/materials, setting, methods Confluent endometrial HEC–1-A cultures were treated with ST at concentrations (0–150mM) and withaferin A (1mM), negative control for embryo adhesion. After the treatment period, HEC–1-A cultures were washed with ST-free culture medium to eliminate ST. Immediately, 15 JAr trophoblast spheroids were added to cultures and coincubated with gentle agitation for 30, 60 and 90 minutes. An inverted light microscope was used to count adhered and floating spheroids, and determine the trophoblast adherence ratio. Main results and the role of chance HEC–1-A cells treated with ST showed normal morphology and growth at all doses except 150mM. At the highest dose tested, the cells’ culture was still viable (negative blue trypan staining) and maintained morphology, but the adhesion to the plate surface was affected. Doses from 0.15 to 15mM were used to perform adhesion assays. HEC–1-A cells treated with ST for 24h showed an increased capacity to adhere JAr trophoblast spheroids. Adhesion rates reached significant differences at doses of 1.5 and 15mM after 60 and 90 minutes of coincubation. After 90 minutes, untreated cells reached 32.8% adhesion rate, while 1.5 and 15mM ST-treated cells reached 54.6% and 53.4% respectively (p < 0.05 ST vs untreated). Thus, the increment of trophoblast adhesion rate induced by ST reached 66%. Lower adhesion rates were observed after 60 minutes of coincubation but were also significant with a relative increase of 49.1% at 1.5mM and 50.5% at 1.5mM when compared with untreated cells (p < 0.05) Longer treatments (48h) showed similar trends to 24h-treatments, but with a lower extent of ST effect on HEC–1-A receptivity. Maximum adhesion rates were also observed at 90 minutes of coincubation and 1.5 and 15mM doses. The Mean adhesion rate increase was >40% with both doses. Limitations, reasons for caution: The current study is the first approach to evaluate sodium tungstate effect on endometrium using an in vitro model. Future research using in vivo models should be performed to assess sodium tungstate effect on endometrium receptivity and its potential as a fertility treatment. Wider implications of the findings: We conclude that the direct effect of sodium tungstate on endometrial cells increases embryo adhesion rate. These results open a new research line to a potential treatment in human reproduction management with sodium tungstate to solve the unmet need of inducing embryo implantation. Trial registration number Not applicable

P–356 Oral administration of sodium tungstate to a swine model improves embryo implantation rate

Study question Does sodium tungstate treatment improve embryo implantation and therefore, fertility in large mammals? Summary answer Oral administration of sodium tungstate increases embryo implantation and reproductive efficiency in large mammals. What is known already Sodium tungstate (ST) has shown its capacity to modulate critical molecules in the embryo implantation process. ST showed a positive effect on PCOS-like model to restore ovulation and fertility. Moreover, ST proved to act directly on the endometrium to increase embryo adhesion in in vitro assays. There is an inherent difficulty in studying implantation using in vivo models due to the close communication between ovary, embryo and endometrium. For the current study, the Large-White swine breed has been selected because of its high efficiency in ovulatory and fertilisation processes, minimising low embryo quality interferences in the implantation process. Study design, size, duration A randomised, blinded, prospective, placebo-controlled study was performed to evaluate ST effect on fertility, ovulation, and embryo implantation rates in swine, which is characterised by a high fertilisation rate but a limiting implantation rate. Forty-four primiparous Large-White sows (8 months old) were orally-treated with ST or placebo for 44–46 days, from 10 days prior to starting a progestin-based treatment for ovulation induction to gestational days 11th–13th (i.e., the window of implantation in swine). Participants/materials, setting, methods: Animals were randomised in treatment groups based on body weight ranges and housed individually in temperature-controlled conditions. 2.5g ST (diluted in 5ml of distilled water) or vehicle were once-daily orally administered with a syringe. Sows responding to ovulation-induction protocols were inseminated with high-quality sperm from untreated pigs and euthanised at gestational days 28–30 (1st pregnancy trimester) to recover genital tracts. Pregnancy, number of ovulations, number of viable/non-viable implanted embryos and fetal measurements were immediately recorded. Main results and the role of chance All 44 sows involved in the study responded to ovulation induction and were inseminated, but 4 females were excluded from the study because of uterine anatomical abnormalities (unicornuate uterus) or abnormalities during pregnancy. Hence, 19 ST-treated and 21 placebo sows were eligible.There were no differences in pregnancy rate (pregnancy was observed in 17 ST-treated sows 19 placebo-treated sows; 89.47% and 90.48%, respectively) or number of ovulations (21.5±4.1 vs 21.8±2.9 in placebo and treated animals, respectively; p = 0.300). However, implantation rate was significantly improved in ST-treated animals, since the number of implanted embryo was found to be increased by 15% per sow in the ST-treated group; which means two additional good-quality embryos per sow (16.5±3.2 in the ST group vs 14.4±3.9 in the placebo group, p < 0.05). The percentage of viable implantations, calculated as the number of viable embryos divided by the total number of viable and non-viable implanted embryos was also increased by the ST treatment (91.6±7.9 vs 96.2±4.7 in treated vs placebo groups, p < 0,05). Finally, there were no effects of the treatment on the foetal phenotype, body mass and size. Limitations, reasons for caution The current study is the first attempt to evaluate ST effect on reproductive outcomes, in healthy large mammals. Having in mind that the selected model is high reproductive efficient, further studies assessing ST effects in infertile and sub-fertile mammals should be performed to elucidate ST activity in suboptimal fertility conditions. Wider implications of the findings: Sodium tungstate treatment proves, for the first time, the improvement of fertility in healthy large mammals. Sodium tungstate treatment improves endometrial implantation and therefore, fertility efficiency. Thus, after subsequent further research, sodium tungstate may become a potential treatment for improving embryo implantation, an unmet medical need. Trial registration number Not applicable

Characteristics of Endometrial Cells and the Factors that Influence the Implantation Process

Infertility is a global health problem faced by 8 - 10% of couples in the world. It means 50 - 80 million couples have infertility problem. Knowledge about comprehensive and profound reproductive system is main asset for managing infertility. When look at various factors those contribute to women infertility, so the role of endometrium is very important. The optimal endometrial environment for receiving blastocyst is a major factor in the process of implantation and pregnancy. This study attempts to review and compare sources from research article, case report, and reviews from reputable international journals. Research by Germeyer et all (2010) shows that optimal growth and development of endometrium are factors those greatly contributes for successful implantation which is continued to pregnancy. The same thing is revealed by Neykova review (2020) which states the success implantation process is determined by the quality of the embryo, the readiness of the endometrium to accept the result of conception and interaction, and communication of molecular cells. Currently, various studies are being developed for evaluating endometrium condition through genetic improvement, the use of new biomarkers, non-invasive method even sophisticated method such as proteomic technique Endometrial Receptivity Assay (ERA) in order to solve women infertility problem.

Embryo Genetics

Advances in embryo and reproductive genetics have influenced clinical approaches to overcome infertility. Since the 1990s, many attempts have been made to decipher the genetic causes of infertility and to understand the role of chromosome aneuploidies in embryo potential. At the embryo stage, preimplantation genetic testing for chromosomal abnormalities and genetic disorders has offered many couples the opportunity to have healthy offspring. Recently, the application of new technologies has resulted in more comprehensive and accurate diagnoses of chromosomal abnormalities and genetic conditions to improve clinical outcome. In this Special Issue, we include a collection of reviews and original articles covering many aspects of embryo diagnosis, genome editing, and maternal–embryo cross-communication during the implantation process.