Impact of abnormal karyotype on reproductive outcome in premature ovarian insufficiency.

Purpose: This study evaluated the reproductive potential of premature ovarian insufficiency (POI) patients with abnormal karyotypes undergoing infertility treatments. Methods: A retrospective analysis of infertility treatments in POI patients with an abnormal karyotype treatment. Clinical and laboratory data were analyzed. Results: The study group was forty-nine POI patients. Follicular growth was achieved in 29% (89/307) controlled ovarian stimulation (COS) cycles in 57% (28/49) of patients. Oocyte retrieval was attempted in 47% (23/49) of patients with a proportion of successful oocyte retrieval per oocyte pick-up (OPU) of 59.4% (41/69). The average number of retrieved oocytes was 2.4 ± 2.7 per patient and fertilization rate was 70.7% (29/41). Embryo transfer (ET) performed in eight patients with a total of nine ET attempts, resulting in 33.3% (3/9) of live birth rate per ET. Three patients delivered a healthy baby (6.1% (3/49) of live birth rate per patient). Mosaic Turner syndrome patients had a longer duration of amenorrhea and lower chances of successful follicular growth with OPU in 35.7% (5/14) of patients, whereas 47XXX had shorter duration of amenorrhea and COS with follicle growth with OPU in 83.3% (5/6). Conclusion: COS might provide an opportunity for POI women with abnormal karyotypes to conceive a biological offspring.

ZEB1 expression is a potential indicator of invasive endometriosis.

INTRODUCTION: Although endometriosis is a benign disease, it shares some features with cancers, such as invasiveness and the potential to metastasize. This study sought to investigate the epithelial-mesenchymal transition status in human endometriotic lesions. MATERIAL AND METHODS: Thirteen endometriosis patients and 10 control women without endometriosis undergoing surgery for benign indications were recruited. We examined the expression of E-cadherin, vimentin, and epithelial-mesenchymal transition-induced transcriptional factors, such as Snail and ZEB1, by immunohistochemistry. We evaluated the expression of each marker in epithelial cells of both endometriotic lesions (ovarian endometrioma, deep infiltrating endometriosis, adenomyosis) and normal endometria. The correlation between ZEB1 expression and serum level of CA125 was also investigated. RESULTS: Immunohistochemical analysis revealed that although E-cadherin, vimentin, and Snail were expressed in epithelia of normal endometria and endometriotic lesions, ZEB1 expression was only expressed in epithelia of endometriotic lesions. Additionally, ZEB1 was most frequently observed in epithelial cells of invasive endometriosis. The endometriosis patients with high serum CA125 level were more likely to have ZEB1-positive lesions. CONCLUSIONS: This is the first observation of ZEB1 expression in epithelial cells of benign disease. The preferential expression of ZEB1 in epithelial cells of endometriotic lesions suggests that these cells may have, at least in part, a higher level of mesenchymal features possibly via ZEB1-driven epithelial-mesenchymal transition than normal endometria and that ZEB1 can be a potential indicator of invasiveness or severity of endometriosis.

Live Birth Rate in Patients With Premature Ovarian Insufficiency During Long-Term Follow-Up Under Hormone Replacement With or Without Ovarian Stimulation.

We analyzed data from 466 patients with premature ovarian insufficiency (POI) who wished to have a biological child and were followed up while undergoing hormone replacement (HR) therapy with or without ovarian stimulation (OS) between April 2014 and December 2020. OS was conducted in 6891 cycles in 429 patients (Group OS), whereas only HR (Group HR) was conducted in 1117 cycles in 37 patients. The follicle growth rate was 48.3% (207/429) per patient in Group OS and 5.4% (2/37) in Group HR (p<0.01). There were 51 live births (LBs) in 50 patients during follow-up. In Group OS, the LB rate was 5.8% (47/807) in cycles where in vitro fertilization (IVF) and embryo transfer were attempted (Group IVF), and 1.3% (3/236) in cycles where intrauterine insemination/timed intercourse was attempted (p<0.01). No pregnancies occurred in Group HR. Among the patients in Group IVF, the LB rate was significantly higher in patients aged <35 years at the initiation of follow-up than in patients who started at later ages (p<0.01). Among the cases who achieved an LB, 39 were patients with idiopathic POI (Group IVF-1, n=297) and seven were patients who had undergone surgical treatment for benign ovarian tumors (Group IVF-2, n=50); however, no LBs occurred in patients who had undergone treatment for malignancy (n=17), and only one in patients with chromosomal abnormalities (n=22). The LB rate per case in the patients in Group IVF-1 and those aged <35 years at the start of follow-up (Group IVF-1-a) was 24.1% (26/108), which was higher than those of the other age groups. The LB rate per case in the patients in Group IVF-1-a with <4 years of amenorrhea was 37.3% (19/51), and that in the patients in Group IVF-2 with <4 years of amenorrhea was 21.2% (7/33). These results suggest that infertility treatment is possible in some patients with POI, especially those that can be classified in Group IVF-1-a and Group IVF-2 with <4 years of amenorrhea. Therefore, OS combined with HR therapy should be considered for such patients before attempts at oocyte donation.

The periphery of the human fetal adrenal gland is a site of angiogenesis: zonal differential expression and regulation of angiogenic factors.

CONTEXT: Although the inner fetal zone (FZ) of the mid-gestation human fetal adrenal (HFA) produces dehydroepiandrosterone sulfate, the function of the outer definitive zone (DZ) remains less clear. We have proposed that the DZ phenotype is that of a pool of progenitor cells, many of which are mitotically active. Recently, we studied HFA expression of a family of vascular endothelial cell-specific angiogenic factors, the angiopoietins (Angs), and demonstrated that Ang2 was localized predominantly in the periphery of the gland. Ang1 stabilizes, whereas Ang2 destabilizes, vessels, increasing responsiveness to angiogenic stimuli such as vascular endothelial growth factor (VEGF)-A and fibroblast growth factor (FGF)-2. OBJECTIVE: Our objective was to test the hypothesis that the periphery of the HFA is a site of angiogenesis. DESIGN: Studies were conducted involving RNA, frozen sections, and primary cell cultures from midgestation HFAs. MAIN OUTCOME MEASURES: Immunofluorescence, laser capture microdissection, and real-time quantitative RT-PCR were used. RESULTS: Double immunostaining demonstrated that proliferating endothelial cells were limited to the DZ and DZ/FZ border. Ang2 mRNA was primarily expressed in the DZ, whereas Ang1 mRNA was primarily in the FZ. VEGF-A and FGF-2 mRNA levels were higher in the DZ. FGF-2 (10 ng/ml) induced Ang2 mRNA by 4-fold in both zones of cells (P < 0.01, at 24 h), but not Ang1 or VEGF-A mRNA. CONCLUSION: Data suggest that angiogenesis occurs at the periphery of the HFA. The DZ-predominant expression of Ang2 may be explained, in part, by the parallel pattern of FGF-2 expression.

Genetically manipulated human embryonic stem cell-derived dendritic cells with immune regulatory function.

Genetically manipulated dendritic cells (DC) are considered to be a promising means for antigen-specific immune therapy. This study reports the generation, characterization, and genetic modification of DC derived from human embryonic stem (ES) cells. The human ES cell-derived DC (ES-DC) expressed surface molecules typically expressed by DC and had the capacities to stimulate allogeneic T lymphocytes and to process and present protein antigen in the context of histocompatibility leukocyte antigen (HLA) class II molecule. Genetic modification of human ES-DC can be accomplished without the use of viral vectors, by the introduction of expression vector plasmids into undifferentiated ES cells by electroporation and subsequent induction of differentiation of the transfectant ES cell clones to ES-DC. ES-DC introduced with invariant chain-based antigen-presenting vectors by this procedure stimulated HLA-DR-restricted antigen-specific T cells in the absence of exogenous antigen. Forced expression of programmed death-1-ligand-1 in ES-DC resulted in the reduction of the proliferative response of allogeneic T cells cocultured with the ES-DC. Generation and genetic modification of ES-DC from nonhuman primate (cynomolgus monkey) ES cells was also achieved by the currently established method. ES-DC technology is therefore considered to be a novel means for immune therapy.

Differential zonal expression and adrenocorticotropin regulation of secreted protein acidic and rich in cysteine (SPARC), a matricellular protein, in the midgestation human fetal adrenal gland: implications for adrenal development.

CONTEXT: Matricellular proteins are a group of secreted, multifunctional extracellular matrix glycoproteins that includes thrombospondins (TSPs), tenascin-C, and secreted protein acidic and rich in cysteine (SPARC). They may be implicated in the dynamic developmental processes of the human fetal adrenal (HFA) in which the outer, definitive zone (DZ) cells are postulated to proliferate, migrate centripetally, differentiate, and populate the inner, steroidogenic fetal zone (FZ). OBJECTIVE: The objective of the study was to identify a matricellular molecule that likely plays a major role in HFA development. DESIGN: Studies involved RNA, cryosections, and cell cultures from 14- to 23-wk HFAs and human adult adrenal RNA. MAIN OUTCOME MEASURES: Measures included transcripts encoding matricellular proteins, using real-time quantitative RT-PCR; SPARC localization by immunostaining; and ACTH regulation of SPARC expression and secretion by quantitative RT-PCR and Western blot. RESULTS: SPARC HFA mRNA was 100-, 700-, and 300-fold higher than TSP-1, TSP-2, and tenascin-C mRNA, respectively. HFA SPARC mRNA was 3-fold higher than adult adrenals (P < 0.005), comparable with levels in adult brain (positive control), whereas mRNAs encoding TSP-1 and TSP-2 were lower in fetal than adult adrenals. SPARC immunoreactivity was detected exclusively in the FZ, not DZ. ACTH, a key regulator of HFA growth and function, increased SPARC mRNA (by 1.7-fold at 1 nm, 48 h, P < 0.05) in isolated FZ cells but not DZ cells. ACTH up-regulation of SPARC protein was also detected in FZ cell lysates and culture medium. CONCLUSIONS: Results suggest a possible role for SPARC in development of functional and/or structural zonation of the HFA.

Peritoneal pregnancy with massive hemoperitoneum in early gestation: two case reports.

Peritoneal pregnancy may cause severe abdominal bleeding without genital bleeding as early as the fifth week of gestation. Awareness that pregnancy can exist in unusual locations is imperative.

Reduction in the vascular bed volume of uterine fibroids after hormonal treatment: evaluation with dynamic double-echo R2* imaging.

PURPOSE: To demonstrate the reduction in vascular bed volume (VBV) of uterine fibroids after administration of gonadotropin-releasing hormone analogue (GnRHa) using magnetic resonance (MR) imaging including dynamic double-echo R2* imaging (DDE-R2*I) and to assess the value of DDE-R2*I as a predictor of such reduction. METHODS: Twenty-one women with uterine intramural fibroids underwent MR imaging including DDE-R2*I before GnRHa treatment. DDE-R2*I was acquired using a single-section, double-echo, fast spoiled gradient recalled acquisition in the steady state (SPGR) sequence. We calculated the area under the curve (AUC) of the signal intensity on R2*I within a 3×3-cm²region of interest that served to represent the VBV. We repeated MR imaging after 2 administrations of GnRHa and repeated image analyses. We statistically analyzed correlations between (A) pre-treatment AUC (AUC(pre)) and AUC reduction and (B) AUC(pre) and volume reduction. RESULTS: The interval between the 2 MR studies ranged from 56 to 119 days (mean: 80.4 days). The average volume of the fibroids before GnRHa treatment was 647.8 mL compared with 463.4 mL after the therapy (decreased by an average of 28.5%; P<0.0001). Meanwhile, measured AUC was reduced by 55.3% (483.4 vs. 206.5; P<0.0001). AUC(pre) correlated with volume reduction (r=0.68), but not AUC reduction. CONCLUSIONS: We confirmed reduction in the VBV of fibroids using DDE-R2*I. The measurement of AUC(pre) on DDE-R2*I aids prediction of fibroid volume reduction but correlates poorly with the percentage of AUC reduction.

Pseudoaneurysm of the uterine artery after laparoscopic myomectomy.

OBJECTIVE: To describe a case of uterine pseudoaneurysm after laparoscopic myomectomy in a 36-year-old woman. DESIGN: Case report. SETTING: University hospital. PATIENT(S): A 36-year-old woman, 3 months after laparoscopic myomectomy, presenting with an intrauterine hypoechoic lesion measuring 5 cm in diameter. INTERVENTION(S): Uterine pseudoaneurysm was diagnosed by color Doppler ultrasound. MAIN OUTCOME MEASURE(S): Complete resolution of the pseudoaneurysm. RESULT(S): Spontaneous thrombosis was observed in the pseudoaneurysm. At 6-month follow-up, the uterus appeared normal. CONCLUSION(S): Our case presents the possibility of delayed occurrence of uterine pseudoaneurysm after laparoscopic myomectomy.

Association between patient age at the time of surgical treatment for endometriosis and aryl hydrocarbon receptor repressor polymorphism.

A cross-sectional comparative study among women who underwent surgical treatment for endometriosis revealed that frequency of the Ala/Ala genotype at aryl hydrocarbon receptor repressor (AHRR) Pro185Ala polymorphism was three times higher (27.6% vs. 9.9%) in the younger group (30 years). AHHR genotyping may help to identify a subpopulation of women who are susceptible to the earlier onset of endometriosis.

Prenatal diagnosis of long QT syndrome by non-invasive fetal electrocardiography.

Long QT syndrome is a high-risk condition associated with arrhythmia due to its sudden cause of death. Prenatal diagnosis of long QT syndrome, however, is impossible using the fetal echocardiogram. Here we present the first reported case of long QT syndrome in which a prenatal diagnosis was made using non-invasive fetal electrocardiogram. We consider that the non-invasive fetal electrocardiogram may be a good method for diagnosing fetal QT prolongation.

Expression of ovary-specific acidic protein in steroidogenic tissues: a possible role in steroidogenesis.

Ovary-specific acidic protein (OSAP) is a novel molecule discovered from a genomic project designed to identify ovary-selective genes in mice. Whereas public databases suggest extraovarian expression of OSAP, its tissue distribution has not yet been well documented. Thus, the expression profile of mouse and human OSAP was determined by quantitative real-time RT-PCR using RNAs isolated from various tissues. The results demonstrate that the human and mouse OSAP expression profiles are similar; OSAP is prominently expressed in steroidogenic tissues with the highest level of expression observed in the adrenal gland. Placenta served as an exception and possessed minimal level of OSAP mRNA. Immunohistochemical studies show that mouse OSAP localizes almost exclusively to the steroid-producing cells of the ovary, adrenal gland, and testis. Consistent with predictions made by several subcellular localization algorithms, dual labeling studies in Y-1 mouse adrenocortical cells indicate OSAP resides in the mitochondria. Because of its abundant expression in steroidogenic cells and mitochondrial localization, a role for OSAP in steroidogenesis was determined. OSAP silencing by specific small interfering RNAs significantly inhibits 8-bromoadenosine-cAMP-induced progesterone production in Y-1 cells. Reduction in OSAP levels results in mitochondrial fragmentation and a decrease in the cellular content of mitochondrial DNA, indicative of decreased mitochondrial abundance. Lastly, 8-bromoadenosine-cAMP does not regulate OSAP protein expression in Y-1 cells as is the case for other steroidogenic components known to be induced by cAMP. Collectively these results suggest that OSAP is involved in steroidogenesis, potentially through its ability to maintain mitochondrial abundance and morphology.

Histone acetylation and subcellular localization of chromosomal protein BRD4 during mouse oocyte meiosis and mitosis.

Most specific and general transcription factors (TFs) become dissociated from hypoacetylated mitotic chromosomes, which may contribute to transcriptional silencing during mitosis. Only some chromosomal proteins, such as bromodomain containing protein 4 (BRD4), have a potential to associate with mitotic chromosomes in a histone acetylation-dependent manner. It remains to be fully demonstrated whether similar displacement of nuclear factors takes place in meiotic oocytes whose chromosomes become globally deacetylated. To address this, we here examined the subcellular localization of BRD4 in conjunction with the acetylation status of histones in mouse oocytes. Immunofluorescence studies revealed that BRD4 preferentially localized to mitotic chromosomes in early embryos. In contrast, not only endogenous BRD4 but also exogenous BRD4 overexpressed by mRNA microinjection were displaced from meiotic chromosomes whose histones H3 and H4 were deacetylated. Treatment with trichostatin A (TSA), an inhibitor of histone deacetylases, induced histone hyperacetylation of meiotic chromosomes from which endogenous BRD4, however, remained dissociated. Finally, meiotic chromosomal localization of BRD4 could be achieved by BRD4 overexpression together with TSA-induced histone hyperacetylation. These results indicate that, unlike mitosis, histone acetylation is necessary but not sufficient for chromosomal localization of BRD4 during meiosis, suggesting that meiotic oocytes may have additional mechanism(s) for displacement of chromosomal proteins and TFs.

H1foo is indispensable for meiotic maturation of the mouse oocyte.

Oocyte-specific linker histone H1foo is localized in the oocyte nucleus, either diffusely or bound to chromatin, during the processes of meiotic maturation and fertilization. This expression pattern suggests that H1foo plays a key role in the control of gene expression and chromatin modification during oogenesis and early embryogenesis. To reveal the function of H1foo, we microinjected antisense morpholino oligonucleotides (MO) against H1foo into mouse germinal-vesicle stage oocytes. The rate of in vitro maturation of the antisense MO group was significantly lower than that of the control group. Eggs that failed to extrude a first polar body following injection of antisense MO arrested at metaphase I. Additionally, co-injection of in vitro synthesized H1foo mRNA along with antisense MO successfully rescued expression of H1foo and improved the in vitro maturation rate. There was no difference in the rate of parthenogenesis between the antisense MO and control groups. These results indicate that H1foo is essential for maturation of germinal vesicle-stage oocytes.

Pure uterine lipoma.

Pure fatty tumors of the uterus are extremely rare and usually develop in postmenopausal women. We present the first reported Japanese case of a pure uterine lipoma, in which a preoperative diagnosis was made by magnetic resonance imaging (MRI) and was pathologically confirmed postoperatively. As in our case, MRI is currently the best modality for determining the internal architecture of a tumor and the presence of fat. Because of the benign nature of a uterine lipoma, such an approach can avoid unnecessary surgery in asymptomatic patients.

Electroporation of cynomolgus monkey embryonic stem cells.

Efficient genetic modification of primate embryonic stem (ES) cells is essential for the application for both basic and preclinical research. The transfection efficiency of primate ES cells is reportedly lower than that of mouse ES cells. Cynomolgus monkey ES cells provide a powerful model for understanding human development and disease. We evaluated electroporation as a method to introduce foreign genes into cynomolgus monkey ES cells. Our examination has allowed us to establish a protocol producing about 100 stably transfected clones from 10(7) cynomolgus monkey ES cells. Differences in efficiency, however, were observed for other ES cell lines. We compared the transcriptional activities of the PGK-1, CMV, and SV40 promoters in cynomolgus monkey ES cells generating efficient G418 selection. Although the PGK-1 and SV40 promoters efficiently drove neo gene expression, the CMV promoter was significantly less transcriptionally active in cynomolgus monkey ES cells. Using this electroporation method, we established fluorescent cynomolgus monkey ES cell lines. These cells may be useful tools for tracing grafted cells in transplantation studies using a variety of functional cells derived from cynomolgus monkey ES cells.