Prenatal to peripubertal exposure to Di(2-ethylhexyl) phthalate induced endometrial atrophy and fibrosis in female mice.

Di(2-ethylhexy) phthalate (DEHP) is a widely used plasticizer that is ubiquitously found in the environment. Using a mouse model, we investigated the impact of early life DEHP exposure ranging from the prenatal to peripubertal developmental period of the female reproductive system. Pregnant female mice were allocated to three groups as follows: control, 100 mg/kg/day, and 500 mg/kg/day DEHP treatment. DEHP exposure was introduced through feeding during pregnancy (3 weeks) and lactation (3 weeks). After weaning, the offspring were also exposed to DEHP through feeding for another 2 weeks. Observations were conducted on female offspring at 10 and 24 weeks. The number of live offspring per dam was significantly lower in the high-DEHP-exposed group (500 mg/kg/day) compared to the control group (7.67 ± 1.24 vs. 14.17 ± 0.31; p < 0.05) despite no difference in pregnancy rates across the groups. Low-DEHP exposure (100 mg/kg/day) resulted to a decreased body weight (36.07 ± 3.78 vs. 50.11 ± 2.11 g; p < 0.05) and decreased left uterine length (10.60 ± 1.34 vs. 14.77 ± 0.82 mm; p < 0.05) in 24-week- old female mice. As early as 10 weeks, endometrial atrophy and fibrosis were observed, and endometrial cystic hyperplasia was noted in female mice at 24 weeks. Our study is the first to demonstrate that female mice exposed to DEHP in the early life developed endometrial fibrosis in the female offspring. Further studies on the consequences of these observations in fecundity and other reproductive functions are warranted.

4,4'-(9-Fluorenylidene)dianiline (BAFL) is antiestrogenic and has adverse effects on female development in CD-1 mice.

Many phenolic compounds have been found to have endocrine disrupting activities, but their arylamine analogs, the phenolic hydroxyl groups substituted by aniline amino groups, have rarely been reported. 4,4'-(9-Fluorenylidene)dianiline (BAFL) is an arylamine analog of fluorene-9-bisphenol (BHPF) and BHPF has been reported to be a strong antiestrogen which could cause endometrial atrophy, ovarian damage and adverse pregnancy outcomes in animals. BAFL has been widely used as material to synthetize polymers, such as polyimides, polyamide, and polyamine, for various uses since the 1970s. Here, we assessed the antiestrogenicity of BAFL using a variety of methods and looked into its impacts on the development of females in CD-1 mice. With the aid of a yeast estrogen screen assay, we found BAFL possessed obviously antiestrogenic activity (IC50 = 8.15 × 10-6 M), which close to that of tamoxifen and BHPF. Using a 10-d mouse uterotrophic assay, we found that BAFL obviously decreased uterine weight in a dose-dependent way. Histological analyses of mouse uteri revealed that BAFL induced marked endometrial atrophy and inhibited the uterine development. Immunohistochemical analyses showed that Sprr2d, an estrogen-responsive gene encoding protein, was mainly expressed in endometrial epithelial cells and BAFL decreased the areas and levels of Sprr2d staining in mouse uteri. It was clear from uterine transcriptome investigations that BAFL significantly downregulated the expressions of multiple genes responding to estrogen. Molecular docking showed that BAFL could effectively occupy the antagonist-binding pocket of hERα, and one of the amino groups of BAFL formed hydrogen bonds with the side chains of Arg394 and Glu353 in the receptor. These results indicated that BAFL exhibited clearly antiestrogenic characteristics and could interfere with normal female development in mice, which should be avoided using in commodities that come into direct contact with humans. Moreover, this study indicated that the arylamine analogs of phenolic endocrine disrupting chemicals might also have endocrine disrupting activities.

Antiestrogenic property of 9,9-bis[4-(2-hydroxyethoxy)phenyl]fluorene (BPEF) and its effects on female development in CD-1 mice.

Identifying chemicals with endocrine disrupting properties linked to disease outcomes is a key concern, as stated in the WHO-UNEP 2012 report on endocrine-disrupting chemicals. The chemical 9,9-bis[4-(2-hydroxyethoxy)phenyl]fluorene (BPEF) is widely and increasingly applied in synthesizing fluorene-based cardo polymers with superior optical, thermal and mechanical properties for various uses. However, little toxicological information is available regarding its safety. Here, we studied the endocrine disrupting property of BPEF by multiple toxicological tools and investigated its effects on female development in adolescent mice. Using the yeast two-hybrid bioassay, BPEF showed strong antiestrogenicity which was similar to that of tamoxifen, an effective antiestrogenic drug. In adolescent CD-1 mice, BPEF significantly decreased the uterine weight at relatively low doses and induced marked endometrial atrophy. Immunohistochemical staining and transcriptome analyses of the mice uteri revealed that BPEF could repressed the expressions of estrogen-responsive genes. Molecular simulation indicated that BPEF could be docked into the antagonist pocket of human estrogen receptor α, and the formation of hydrogen bonds and hydrophobic interactions between BPEF and the active site of receptor maintained their strong binding. All of the data demonstrated that BPEF possessed strong antiestrogenic property and might disrupt female development, suggesting it should be avoided in making products that might directly expose to people, particularly immature women.

Stem cell paracrine actions in tissue regeneration and potential therapeutic effect in human endometrium: a retrospective study.

OBJECTIVE: Determining genetic and paracrine mechanisms behind endometrial regeneration in Asherman's syndrome and endometrial atrophy (AS/EA) patients after autologous CD133+ bone marrow-derived stem cell (CD133+ BMDSC) transplantation. DESIGN: Retrospective study using human endometrial biopsies and mouse models. SETTING: Fundación-IVI, IIS-La Fe, Valencia, Spain. SAMPLES: Endometrial biopsies collected before and after CD133+ BMDSC therapy, from eight women with AS/EA (NCT02144987) from the uterus of five mice with only left horns receiving CD133+ BMDSC therapy. METHODS: In human samples, haematoxylin and eosin (H&E) staining, RNA arrays, PCR validation, and neutrophil elastase (NE) immunohistochemistry (IHQ). In mouse samples, PCR validation and protein immunoarrays. MAIN OUTCOME MEASURES: H&E microscopic evaluation, RNA expression levels, PCR, and growth/angiogenic factors quantification, NE IHQ signal. RESULTS: Treatment improved endometrial morphology and thickness for all patients. In human samples, Jun, Serpine1, and Il4 were up-regulated whereas Ccnd1 and Cxcl8 were down-regulated after treatment. The significant decrease of NE signal corroborated Cxcl8 expression. Animal model analysis confirmed human results and revealed a higher expression of pro-angiogenic cytokines (IL18, HGF, MCP-1, MIP2) in treated uterine horns. CONCLUSIONS: CD133+ BMDSC seems to activate several factors through a paracrine mechanism to help tissue regeneration, modifying endometrial behaviour through an immunomodulatory milieu that precedes proliferation and angiogenic processes. Insight into these processes could bring us one step closer to a non-invasive treatment for AS/EA patients. TWEETABLE ABSTRACT: CD133+ BMDSC therapy regenerates endometrium, modifying the immunological milieu that precedes proliferation and angiogenesis.

Hormonal Biophysiology of the Uterus.

Intrauterine development of the uterus is promoted by the hormonal influence of the maternal steroid sex hormones on the female fetus. The cyclic activity of the endometrium starts at puberty, at menarche, and is controlled by the pituitary hormones (FSH and LH) and steroid ovarian hormones, the latter acting on the target tissue-the endometrium. The proliferative and secretory cyclic changes of the endometrium prepare the uterus for implantation of a fertilized ovum. Ovarian failure to secrete steroid hormones results in the menopausal gradual atrophy of the endometrium.

[Improved management of patients in the detection of fluid in the uterine cavity during ultrasound examination of small pelvis in postmenopausal women].

In carrying out preventive ultrasound in postmenopausal often reveals the presence of fluid in the uterine cavity and exhibiting as serozometra, whereby the patient is sent to the gynecological hospital to perform hysteroscopy. The analysis of 55 case histories of patients with serozometra treated in gynecological departments of Volgograd 2013-2015 years. The findings suggest that serozometra the majority (82,4 %) of the cases is not accompanied by endometrial pathology , thus intrauterine intervention was performed unfounded. In identifying serozometra advisable dynamic observation, followed by the need to address the issue of hysteroscopy.

Autologous cell therapy with CD133+ bone marrow-derived stem cells for refractory Asherman's syndrome and endometrial atrophy: a pilot cohort study.

STUDY QUESTION: Could cell therapy using autologous peripheral blood CD133+ bone marrow-derived stem cells (BMDSCs) offer a safe and efficient therapeutic approach for patients with refractory Asherman's syndrome (AS) and/or endometrial atrophy (EA) and a wish to conceive? SUMMARY ANSWER: In the first 3 months, autologous cell therapy, using CD133+ BMDSCs in conjunction with hormonal replacement therapy, increased the volume and duration of menses as well as the thickness and angiogenesis processes of the endometrium while decreasing intrauterine adhesion scores. WHAT IS KNOWN ALREADY: AS is characterized by the presence of intrauterine adhesions and EA prevents the endometrium from growing thicker than 5 mm, resulting in menstruation disorders and infertility. Many therapies have been attempted for these conditions, but none have proved effective. STUDY DESIGN, SIZE, DURATION: This was a prospective, experimental, non-controlled study. There were 18 patients aged 30-45 years with refractory AS or EA were recruited, and 16 of these completed the study. Medical history, physical examination, endometrial thickness, intrauterine adhesion score and neoangiogenesis were assessed before and 3 and 6 months after cell therapy. PARTICIPANTS/MATERIALS, SETTING, METHODS: After the initial hysteroscopic diagnosis, BMDSC mobilization was performed by granulocyte-CSF injection, then CD133+ cells were isolated through peripheral blood aphaeresis to obtain a mean of 124.39 million cells (range 42-236), which were immediately delivered into the spiral arterioles by catheterization. Subsequently, endometrial treatment after stem cell therapy was assessed in terms of restoration of menses, endometrial thickness (by vaginal ultrasound), adhesion score (by hysteroscopy), neoangiogenesis and ongoing pregnancy rate. The study was conducted at Hospital Clínico Universitario of Valencia and IVI Valencia (Spain). MAIN RESULTS AND THE ROLE OF CHANCE: All 11 AS patients exhibited an improved uterine cavity 2 months after stem cell therapy. Endometrial thickness increased from an average of 4.3 mm (range 2.7-5) to 6.7 mm (range 3.1-12) ( ITALIC! P = 0.004). Similarly, four of the five EA patients experienced an improved endometrial cavity, and endometrial thickness increased from 4.2 mm (range 2.7-5) to 5.7 mm (range 5-12) ( ITALIC! P = 0.03). The beneficial effects of the cell therapy increased the mature vessel density and the duration and intensity of menses in the first 3 months, with a return to the initial levels 6 months after the treatment. Three patients became pregnant spontaneously, resulting in one baby boy born, one ongoing pregnancy and a miscarriage. Furthermore, seven pregnancies were obtained after fourteen embryo transfers, resulting in three biochemical pregnancies, one miscarriage, one ectopic pregnancy, one baby born and one ongoing pregnancy. LIMITATIONS, REASONS FOR CAUTION: Limitations of this pilot study include the small sample size and the lack of control group. WIDER IMPLICATIONS OF THE FINDINGS: This novel autologous cell therapy is a promising therapeutic option for patients with these incurable pathologies and a wish to conceive. STUDY FUNDING/COMPETING INTERESTS: This study was funded by the Spanish Ministry of Science and Innovation (SAF 2012-31017, Principal Investigator C.S.), Spanish Ministry of Health (EC11-299, Principal Investigator C.S.) and Regional Valencian Ministry of Education (PROMETEOII/2013/018, Principal Investigator C.S.). Four authors (X.S., I.C., A.P. and C.S.) are co-inventors of the patent resulting from this work (Application number: 62/013,121). S.C., C.A., F.R., J.F., J.P. and J.R. have no conflict of interest in relation to this work. TRIAL REGISTRATION NUMBER: This study was registered with ClinicalTrials.gov (NCT02144987).

Evolution of biotechnological advances and regenerative therapies for endometrial disorders: a systematic review.

BACKGROUND: The establishment and maintenance of pregnancy depend on endometrial competence. Asherman syndrome (AS) and intrauterine adhesions (IUA), or endometrial atrophy (EA) and thin endometrium (TE), can either originate autonomously or arise as a result from conditions (i.e. endometritis or congenital hypoplasia), or medical interventions (e.g. surgeries, hormonal therapies, uterine curettage or radiotherapy). Affected patients may present an altered or inadequate endometrial lining that hinders embryo implantation and increases the risk of poor pregnancy outcomes and miscarriage. In humans, AS/IUA and EA/TE are mainly treated with surgeries or pharmacotherapy, however the reported efficacy of these therapeutic approaches remains unclear. Thus, novel regenerative techniques utilizing stem cells, growth factors, or tissue engineering have emerged to improve reproductive outcomes. OBJECTIVE AND RATIONALE: This review comprehensively summarizes the methodologies and outcomes of emerging biotechnologies (cellular, acellular, and bioengineering approaches) to treat human endometrial pathologies. Regenerative therapies derived from human tissues or blood which were studied in preclinical models (in vitro and in vivo) and clinical trials are discussed. SEARCH METHODS: A systematic search of full-text articles available in PubMed and Embase was conducted to identify original peer-reviewed studies published in English between January 2000 and September 2023. The search terms included: human, uterus, endometrium, Asherman syndrome, intrauterine adhesions, endometrial atrophy, thin endometrium, endometritis, congenital hypoplasia, curettage, radiotherapy, regenerative therapy, bioengineering, stem cells, vesicles, platelet-rich plasma, biomaterials, microfluidic, bioprinting, organoids, hydrogel, scaffold, sheet, miRNA, sildenafil, nitroglycerine, aspirin, growth hormone, progesterone, and estrogen. Preclinical and clinical studies on cellular, acellular, and bioengineering strategies to repair or regenerate the human endometrium were included. Additional studies were identified through manual searches. OUTCOMES: From a total of 4366 records identified, 164 studies (3.8%) were included for systematic review. Due to heterogeneity in the study design and measured outcome parameters in both preclinical and clinical studies, the findings were evaluated qualitatively and quantitatively without meta-analysis. Groups using stem cell-based treatments for endometrial pathologies commonly employed mesenchymal stem cells (MSCs) derived from the human bone marrow or umbilical cord. Alternatively, acellular therapies based on platelet-rich plasma (PRP) or extracellular vesicles are gaining popularity. These are accompanied by the emergence of bioengineering strategies based on extracellular matrix (ECM)-derived hydrogels or synthetic biosimilars that sustain local delivery of cells and growth factors, reporting promising results. Combined therapies that target multiple aspects of tissue repair and regeneration remain in preclinical testing but have shown translational value. This review highlights the myriad of therapeutic material sources, administration methods, and carriers that have been tested. WIDER IMPLICATIONS: Therapies that promote endometrial proliferation, vascular development, and tissue repair may help restore endometrial function and, ultimately, fertility. Based on the existing evidence, cost, accessibility, and availability of the therapies, we propose the development of triple-hit regenerative strategies, potentially combining high-yield MSCs (e.g. from bone marrow or umbilical cord) with acellular treatments (PRP), possibly integrated in ECM hydrogels. Advances in biotechnologies together with insights from preclinical models will pave the way for developing personalized treatment regimens for patients with infertility-causing endometrial disorders such as AS/IUA, EA/TE, and endometritis. REGISTRATION NUMBER: https://osf.io/th8yf/.

Endometrial regeneration in Asherman's syndrome and endometrial atrophy using Wharton's jelly-derived mesenchymal stem cells.

OBJECTIVES: Reconstruction of the endometrium in patients with endometrial atrophy and Asherman's syndrome using Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs). MATERIAL AND METHODS: Prospective pilot study, with the inclusion of two patients. RESULTS: After administration of WJ-MSCs into the uterine cavity, endometrial reconstruction was achieved in both patients. Pregnancy was achieved in one of them, after transfer of a frozen embryo, completed by delivery around the due date. CONCLUSIONS: Endometrial atrophy and Asherman's syndrome, is one of the most frustrating clinical situations we face in assisted reproductive procedures. The use of Wharton's jelly-derived mesenchymal stem cells in restoring the normal function of the endometrium, could become an easy and accessible therapeutic medal, for this endometrial dysfunction, which is so difficult to treat.

Comparison of different sources of platelet-rich plasma as treatment option for infertility-causing endometrial pathologies.

OBJECTIVE: To study the effect of human plasma from different sources, namely, umbilical cord blood and adult blood platelet-rich plasma (PRP), on the regeneration of endometrial damage. DESIGN: Composition analysis, in vitro approaches, and a preclinical murine model using plasma to promote endometrial regeneration. SETTING: Hospital and university laboratories. PATIENT(S)/ANIMAL(S): Adult plasma from four Asherman syndrome/endometrial atrophy patients and one fertile woman, commercial umbilical cord plasma, and uterine-damaged NOD/SCID mice model were used. INTERVENTION(S): Endometrial stromal cells from primary culture and an endometrial stem cell line were cultured in vitro, and uterine-damaged NOD/SCID mice were treated with plasma samples from several origins. MAIN OUTCOME MEASURE(S): To investigate the possible beneficial effects of PRP from Asherman syndrome/endometrial atrophy patients. To test if plasma from human umbilical cord blood had a stronger effect than adult PRP in endometrial regeneration. To demonstrate if PRP from Asherman syndrome/endometrial atrophy patients was as effective as PRP from a healthy woman and could therefore be used for autologous treatment. RESULT(S): All plasma samples contained molecules with a high potential for regeneration (stem cell factor, platelet-derived growth factor BB, thrombospondin-1, von Willebrand factor). Furthermore, the highest increase in in vitro proliferation and migration rate was found when endometrial stromal cells were treated with umbilical cord plasma; adult PRP also revealed a significant increment. In the mouse model, a higher expression of Ki67 and Hoxa10 in the endometrium was detected after applying adult PRP, and the proteomic analysis revealed a specific protein expression profile depending on the treatment. The damaged uterine tissue showed more proregenerative markers after applying umbilical cord plasma (Stat5a, Uba3, Thy1) compared with the other treatments (nonactivated umbilical cord plasma, activated adult PRP, and no treatment). CONCLUSION(S): Human PRP possesses regeneration properties usable for endometrial pathologies. Besides that, these regenerative effects seem to be more apparent when the source of obtaining is umbilical cord blood.

Birth of a healthy infant after bone marrow-derived cell therapy.

Bone marrow-derived cell (BMDC) therapy has numerous applications as potential biological cells for use in regenerative medicine. Here, we present an original case of endometrial atrophy associated with genital tuberculosis in a woman who achieved a live birth with BMDC. This 27-year-old woman came to our center with endometrial atrophy and primary infertility. She had a past history of genital tuberculosis and amenorrhea. Her husband's semen quality was normal. The patient was counseled for hysteroscopy due to thin endometrium and advised in vitro fertilization (IVF) with donor eggs in lieu of poor ovarian reserve. Several attempts of IVF with hormone replacement therapy (HRT) were made, but the desired thickness of the endometrium was not achieved. Uterine artery injection of BMDC through interventional radiology was given, followed by HRT for three months, which resulted in improved endometrium. This was subsequently followed by IVF with donor egg. The treatment resulted in the conception and delivery of a 3.1-kg baby boy through lower segment caesarean section with no antenatal, intranatal or postnatal complications. Recently, there has been massive interest in stem cells as a novel treatment method for regenerative medicine, and more specifically for the regeneration of human endometrium disorders like Asherman syndrome and thin endometrium, which was the reason behind using this strategy for treatment.

Autologous Bone Marrow-Derived Stem Cell Therapy for Asherman's Syndrome and Endometrial Atrophy: A 5-Year Follow-up Study.

BACKGROUND: Based on the role of bone marrow (BM) stem cells in regeneration of endometrium, refractory cases of Asherman's syndrome (AS) and endometrial atrophy (EA) may benefit with BM-derived intrauterine stem cell instillation. Aims and Objectives: To evaluate the role of BM-derived autologous stem cell therapy in endometrial regeneration and restoration of menstruation and fertility in refractory cases of AS and EA. SETTING: This study was conducted at a tertiary care center. DESIGN: This was a prospective, single-arm longitudinal study. MATERIALS AND METHODS: Twenty-five cases with refractory AS or EA were included. BM-derived mononuclear stem cells were instilled into the subendometrial zone followed by oral estrogen therapy for 3 months. Menstrual flow and endometrial thickness (ET) were assessed at 3, 6, and 9 months and 5 years. RESULTS: Statistical analysis was carried out using statistical software STATA version 12.0. Mean prestem cell transfer ET (mm) was 3.3 ± 1.0. At the end of 3 months, there was a significant increase in ET (mm) to 5.1 ± 1.9 (P = 0.001), but there was no significant change at 6 months (5.6 ± 1.5; P = 0.164), at 9 months (6.1 ± 1.7; P = 0.135), or at the end of 5 years. Six of the seven amenorrheic patients resumed menses. Three patients had a successful pregnancy outcome. CONCLUSION: Intrauterine stem cell treatment is a promising novel approach for refractory cases of AS and EA.