藏本 吾郎

Infertility in women is associated with various uterine and ovarian disorders. Treatment strategies for infertility can range from medications to embryo implantation through assisted reproductive technology (ART). ART has enabled considerable progress; however, there is currently no treatment to replace the endometrium itself. Decidualization requires a complex interaction between endometrial tissue and estrogen and progesterone. We aimed to create a three-dimensional endometrial-like tissue model using in-vitro cell sheet engineering with rat endometrium, and culture cells at different progesterone concentrations to mimic local concentrations. Histological and morphological changes revealed that development of the endometrial-like tissue was not proportional to progesterone concentrations in terms of thickness, number of endometrial glands, or area fraction of intimal glands. These results suggest that decidualization may not be commensurate with the local endometrial progesterone concentration. Notably, the number of endometrial glands increased in the high concentration group and compaction occurred, indicating that the endometrial conditions in the high concentration group may be most conducive to increase pregnancy rates. These findings suggest that there may be an "optimal progesterone concentration" for decidualization, application of which may lead to new strategies for improving pregnancy rates in women with infertility.

RESEARCH QUESTION: This study aimed to retrospectively evaluate the efficacy of a hyaluronan-enriched transfer medium (HETM) for transfer failures and transfer of frozen embryos that have been graded as C at the time of transfer according to the Gardner classification of trophectoderm (TE). DESIGN: This study included 365 cycles of unsuccessful frozen-thawed embryo transfers in hormone replacement cycles graded C according to the Gardner classification of TE at the time of transfer. Clinical pregnancy rates were compared using the χ2 test, with the patients divided into two groups: one whose transfers did include HETM (HETM group) and one whose transfers did not include HETM (control group). As a subgroup analysis, patients with a TE grade of C at the time of transplantation were divided into two groups: those aged 39 years or younger and those aged 40 years or older at the time of transplantation. The clinical pregnancy rates of the groups with and without HETM were then compared. RESULTS: No difference in the clinical pregnancy rates between the HETM and control groups was observed. CONCLUSIONS: Hyaluronic acid is believed to favor implantation by promoting adhesion between the embryo and the endometrium, and there are reports of improved implantation and pregnancy rates as a result of HETM. However, the present results suggest limited effectiveness for HETM. Further case series should be conducted, and the suitability of its use as a treatment should be investigated.

OBJECTIVE: The study aimed to assess the feasibility of creating and transplanting human umbilical cord mesenchymal stem cell sheets applied to a rat model of hysterotomy, and additionally to determine benefits of human umbilical cord mesenchymal stem cell sheet transplantation in reducing uterine fibrosis and scarring. STUDY DESIGN: Human umbilical cord mesenchymal stem cell sheets are generated by culturing human umbilical cord mesenchymal stem cells on thermo-responsive cell culture plates. The temperature-sensitive property of these culture dishes facilitates normal cell culture in a thin contiguous layer and allows for reliable recovery of intact stem cell sheets without use of destructive proteolytic enzymes.We developed a rat hysterotomy model using nude rats. The rat uterus has two distinct horns: one horn provided a control/untreated scarring site, while the second horn was the cell sheet transplantation site.On day 14 following surgery, complete uteri were harvested and subjected to histologic evaluations of all hysterotomy sites. RESULTS: The stem cell sheet culture process yielded human umbilical cord mesenchymal stem cell sheets with surface area of approximately 1 cm2.Mean myometrial thickness in the cell sheet-transplanted group was 274 μm compared with 191 μm in the control group (p = 0.02). Mean fibrotic surface area in the human umbilical cord mesenchymal stem cell sheet-transplanted group was 95,861 μm2 compared with 129,185 μm2 in the control group. Compared with control horn sites, cell sheet-transplanted horns exhibited significantly smaller fibrotic-to-normal myometrium ratios (0.18 vs. 0.27, respectively, p = 0.029). Mean number of fibroblasts in cell sheet-transplanted horns was significantly smaller than the control horns (483 vs. 716/mm2, respectively, p = 0.001). CONCLUSION: Human umbilical cord mesenchymal stem cell sheet transplantation is feasible in a rat model of hysterotomy. Furthermore, use of stem cell sheets reduces fibroblast infiltration and uterine scar fibrotic tissue formation during hysterotomy healing, potentially mitigating risks of uterine scar formation. KEY POINTS: · Stem cell sheet transplanted to hysterotomy promotes myometrial regeneration and reduced fibrotic tissue formation.. · This study demonstrates the feasibility of using human umbilical cord mesenchymal stem cell sheets..

OBJECTIVE: To regenerate functional endometrium tissue using "cell sheet" techniques as a regenerative medicine approach to address endometrial disorders causing female factor infertility. DESIGN: In vivo experimental study. SETTING: Preclinical surgical and biomedical research laboratories. ANIMAL(S): Green fluorescent protein (GFP) transgenic rats [SD-Tg (CAG-EGFP) rats] and nude rats (F344/NJcl-rnu/rnu). INTERVENTION(S): GFP-positive rat uterine-derived cells as cell sheets were transplanted into resected rat uterine endometrial sites. Transplanted cell sheet areas were then analyzed using macroscopic observations and histological analysis including immunohistochemistry. Subsequently, crossbreeding was performed to establish fertility and confirm pregnancy in the rat-regenerated uterus. MAIN OUTCOME MEASURE(S): Morphologic and biochemical markers of regenerated endometrium and establishment of pregnancy in otherwise sterile animals. RESULT(S): After cell sheet transplantation, regenerated endometrium was confirmed as GFP-positive tissue engraftment both visually and under histological analysis. After crossbreeding, GFP-positive tissue areas and living fetuses were observed in the transplantation group. CONCLUSION(S): Cell sheet transplantation can regenerate endometrial tissue with histological structure and physiological function supporting pregnancy similar to normal endometrial tissue. Translation of this endometrial cell sheet transplantation method to human patients with endometrial disorders could yield a novel therapy for uterine infertility.

STUDY QUESTION: Can regenerative-medicine techniques using oral mucosal epithelial cell sheets (OMECS) provide a new treatment method for intrauterine adhesions (IUA) which cause female infertility? SUMMARY ANSWER: Transplantation of OMECS was confirmed to be effective in preventing IUA after endometrial damage in rats. WHAT IS KNOWN ALREADY: Uterine disorders such as IUA, commonly known as Asherman's syndrome, are one factor that can result in infertility. Clinical therapy for this kind of disease is targeted at the prevention of re-adhesion by surgical synechiotomy, administration of hormones after the operation, and the use of intrauterine devices. Recently, a new approach called 'cell-sheet engineering', which harvests confluent culture cells as a contiguous cell sheet having intact cell-cell junctions and an extracellular matrix, without having to use enzymatic treatment, has been developed for tissue regeneration. STUDY DESIGN, SIZE, DURATION: OMECS were prepared from rat oral mucosal tissues. An IUA model was made in rat uteri, and OMECS were transplanted into the model. Uteri transplanted with OMECS were compared with the non-transplanted control uteri by histological analysis at 1, 2 and 8 days after surgery (n = 3). PARTICIPANTS/MATERIALS, SETTING, METHODS: Oral mucosal tissues were resected from neonatal rats, and oral mucosal epithelial cells were collected with enzymatic treatment. An isolated cell suspension was seeded on a temperature-responsive cell culture-insert and incubated. After being detached from the insert, a cell sheet was transplanted onto the endometrium defect. At 1, 2 and 8 days after surgery, uteri were resected and examined. MAIN RESULTS AND THE ROLE OF CHANCE: Histological examination of the non-treated specimens at 1, 2 and 8 days after surgery did not show any uterine cavities typically caused by IUA. In contrast, the histology of uteri transplanted with OMECS immediately after endometrial damage showed the presence of uterine cavities, and furthermore, stratified squamous epithelial cells on the luminal surface (n = 3). LIMITATIONS, REASONS FOR CAUTION: The results of this study are difficult to apply directly to humans, because the structure and function of rat uteri are different from those of human. WIDER IMPLICATIONS OF THE FINDINGS: Transplantation of OMECS offers a reliable method not only to protect the woman's fertility from intrauterine re-adhesion after synechiotomy for IUA or uterine lumen adhesion but also to prevent adhesion after any intrauterine surgery in clinical cases.

Uterus is a female specific reproductive organ and plays critical roles in allowing embryo to grow. Therefore, the endometrial disorders lead to female infertility. Hence, the regeneration of endometrium allowing fertilized ovum to implant might be valuable in the field of fertility treatment. Recently, cell sheet engineering using a temperature-responsive culture dish has advanced in regenerative medicine. With this technology, endometrial cells were harvested as a contiguous cell sheet by reducing temperature. Firstly, mouse endometrial cell sheets were re-cultured for 3 days to evaluate the function. Histological analyses revealed that endometrial epithelial cell-specific cytokeratin 18 and female-specific hormone receptors, estrogen receptor β and progesterone receptor, were expressed. Furthermore, endometrial epithelial cells constructed epithelial layer at the apical side. Then, endometrial cell sheets from green-fluorescent-protein rat cells were transplanted onto the buttock muscle of nude rat for evaluating the function in vivo. Histological analyses showed that endometrial cell sheets reconstructed endometrium-like tissue, which was found to form uterus-specific endometrial glands having hormonal receptor to estrogen. In this study, endometrial cell sheets were speculated to contribute to the regeneration of functional endometrium as a new therapy.