Comparative transcriptomic characterization of a new mib mutant allele, mibnn2002, in zebrafish.

mibnn2002, identified from an allele screen, shows early segmentation defect and severe cell death phenotypes, which are different from those of other described mib mutant alleles. We have previously reported its defects in somitogenesis and identified its origin of mutation, a large deletion in LG2. The report here is a continuous study, where we applied the bioinformatics analysis to profile the genetic background of mibnn2002 mutants. By comparing the transcriptomic data of mibnn2002 mutants with those of AB wild-type, a total of 1945 differentially expressed genes were identified, including 685 up- and 1260 down-regulated genes. The Database for Annotation, Visualization and Integrated Discovery (DAVID) analysis and Ingenuity Pathway Analysis (IPA) identified the enriched pathways and their related biological functions. Our data further demonstrated that the defects in the somitogenesis were related to the down-regulated segmentation genes, such as foxc1a, smyhc1, myod1 and mylpfa.

Differences between NMRI and DBA/2J mice in the development of somites and susceptibility to methylnitrosourea-induced skeleton anomalies.

The development of DBA/2J mouse strain embryos is nearly 12 h - or 6 somite pairs - delayed as compared to the outbred NMRI mouse embryos of the same age on gestation days (GD) 8-12. To evaluate inter-strain differences in susceptibility to teratogens, dams were treated with methylnitrosourea (MNU, 5 mg/kg body weight i.p.) on defined gestation days (NMRI: GD 9, 91/2 or 10; DBA/2J: GD 10 or 101/2). Skeletal anomalies produced by MNU on both mouse strains varied with the GD of treatment. The pattern of anomalies produced by MNU on a given GD markedly differed between the two mouse strains, yet they were similar -with a few exceptions- when exposures at equivalent embryonic stages are compared. Findings from this study indicated that strain-dependent differences in the developmental stage of mouse embryos of the same gestational age occur, a possibility that has been often neglected when inter-strain differences in susceptibility to developmental toxicants are interpreted.

Differences between NMRI and DBA/2J mice in the development of somites and susceptibility to methylnitrosourea-induced skeleton anomalies

ABSTRACT The development of DBA/2J mouse strain embryos is nearly 12 h - or 6 somite pairs - delayed as compared to the outbred NMRI mouse embryos of the same age on gestation days (GD) 8-12. To evaluate inter-strain differences in susceptibility to teratogens, dams were treated with methylnitrosourea (MNU, 5 mg/kg body weight i.p.) on defined gestation days (NMRI: GD 9, 91/2 or 10; DBA/2J: GD 10 or 101/2). Skeletal anomalies produced by MNU on both mouse strains varied with the GD of treatment. The pattern of anomalies produced by MNU on a given GD markedly differed between the two mouse strains, yet they were similar -with a few exceptions- when exposures at equivalent embryonic stages are compared. Findings from this study indicated that strain-dependent differences in the developmental stage of mouse embryos of the same gestational age occur, a possibility that has been often neglected when inter-strain differences in susceptibility to developmental toxicants are interpreted.

Nuclear receptor corepressors Ncor1 and Ncor2 (Smrt) are required for retinoic acid-dependent repression of Fgf8 during somitogenesis.

Retinoic acid (RA) repression of Fgf8 is required for many different aspects of organogenesis, however relatively little is known about how endogenous RA controls gene repression as opposed to gene activation. Here, we show that nuclear receptor corepressors NCOR1 and NCOR2 (SMRT) redundantly mediate the ability of RA to repress Fgf8. Ncor1;Ncor2 double mutants generated by CRISPR/Cas9 gene editing exhibited a small somite and distended heart phenotype similar to that of RA-deficient Raldh2-/- embryos, associated with increased Fgf8 expression and FGF signaling in caudal progenitors and heart progenitors. Embryo chromatin immunoprecipitation studies revealed that NCOR1/2 but not coactivators are recruited to the Fgf8 RA response element (RARE) in an RA-dependent manner, whereas coactivators but not NCOR1/2 are recruited RA-dependently to a RARE near Rarb that is activated by RA. CRISPR/Cas9-mediated genomic deletion of the Fgf8 RARE in mouse embryos often resulted in a small somite defect with Fgf8 derepression caudally, but no defect was observed in heart development or heart Fgf8 expression. This suggests the existence of another DNA element whose function overlaps with the Fgf8 RARE to mediate Fgf8 repression by RA and NCOR1/2. Our studies support a model in which NCOR1/2 mediates direct RA-dependent repression of Fgf8 in caudal progenitors in order to control somitogenesis.

Embryotoxicity of nitrophenols to the early life stages of zebrafish (Danio rerio).

The nitrophenols (NPs) are water-soluble compounds. These compounds pose a significant health threat since they are priority environmental pollutants. In this study, 2-Nitrophenol (2NP) and 2,4-dinitrophenol (DNP) were examined for embryo and early life stage toxicity in zebrafish (Danio rerio). Acute toxicity and teratogenicity of 2NP and DNP were tested for 4 days using zebrafish embryos. The typical lesions observed were no somite formation, incomplete eye and head development, tail curvature, weak pigmentation (≤48 hours postfertilization (hpf)), kyphosis, scoliosis, yolk sac deformity, and nonpigmentation (72 hpf). Also, embryo and larval mortality increased and hatching success decreased. The severity of abnormalities and mortalities were concentration- and compound-dependent. Of the compounds tested, 2,4-DNP was found to be highly toxic to the fish embryos following exposure. The median lethal concentrations and median effective concentrations for 2NP are 18.7 mg/L and 7.9 mg/L, respectively; the corresponding values for DNP are 9.65 mg/L and 3.05 mg/L for 48 h. The chorda deformity was the most sensitive endpoint measured. It is suggested that the embryotoxicity may be mediated by an oxidative phosphorylation uncoupling mechanism. This article is the first to describe the teratogenicity and embryotoxicity of two NPs to the early life stages of zebrafish.

Retinoic Acid Activity in Undifferentiated Neural Progenitors Is Sufficient to Fulfill Its Role in Restricting Fgf8 Expression for Somitogenesis.

Bipotent axial stem cells residing in the caudal epiblast during late gastrulation generate neuroectodermal and presomitic mesodermal progeny that coordinate somitogenesis with neural tube formation, but the mechanism that controls these two fates is not fully understood. Retinoic acid (RA) restricts the anterior extent of caudal fibroblast growth factor 8 (Fgf8) expression in both mesoderm and neural plate to control somitogenesis and neurogenesis, however it remains unclear where RA acts to control the spatial expression of caudal Fgf8. Here, we found that mouse Raldh2-/- embryos, lacking RA synthesis and displaying a consistent small somite defect, exhibited abnormal expression of key markers of axial stem cell progeny, with decreased Sox2+ and Sox1+ neuroectodermal progeny and increased Tbx6+ presomitic mesodermal progeny. The Raldh2-/- small somite defect was rescued by treatment with an FGF receptor antagonist. Rdh10 mutants, with a less severe RA synthesis defect, were found to exhibit a small somite defect and anterior expansion of caudal Fgf8 expression only for somites 1-6, with normal somite size and Fgf8 expression thereafter. Rdh10 mutants were found to lack RA activity during the early phase when somites are small, but at the 6-somite stage RA activity was detected in neural plate although not in presomitic mesoderm. Expression of a dominant-negative RA receptor in mesoderm eliminated RA activity in presomitic mesoderm but did not affect somitogenesis. Thus, RA activity in the neural plate is sufficient to prevent anterior expansion of caudal Fgf8 expression associated with a small somite defect. Our studies provide evidence that RA restriction of Fgf8 expression in undifferentiated neural progenitors stimulates neurogenesis while also restricting the anterior extent of the mesodermal Fgf8 mRNA gradient that controls somite size, providing new insight into the mechanism that coordinates somitogenesis with neurogenesis.

The MURCS Association: Mullerian Duct Aplasia, Renal Hypoplasia and Cervicothoracic Somite Dysplasia - A Case Report.

We report on a 22 years old lady with aplasia of uterus and most of the vagina with normal secondary sexual characteristics, unilateral renal hypoplasia and anomalies of cervico throacic somites (MURCS Association), growth retardation, cardiac defect and congenital urethrovaginal fistula. Although there is a broad spectrum of anomalies described with MURCS association genitourinary fistula is not yet reported and reviewed in published articles. The relevance of this paper is to show the importance of further investigation in cases of primary amenorrhoea with mullerian agenesis to establish that the patient has MURCS association and not simply MRKH (Mayer Rokitansky-Kusterhauser Syndrome) syndrome. Consequently we should provide guidance to the patients and their families about the best way to conduct the case including genetic counseling and family screening.

Diagnostic pitfalls in the evaluation and management of amenorrhea in adolescents.

Amenorrhea is a common menstrual problem seen in adolescents. Amenorrhea has been shown to have a negative impact on adolescents' quality of life. In this paper we discuss the various causes and investigations of amenorrhea in adolescents and address management dilemmas for specific conditions. Specific approaches in dealing with adolescents using the HEADSS (Home, Education, Activity, Drugs, Sexual activity, Suicidal) approach are discussed.

Ultrasonography and MRI features of the Mayer-Rokitansky-Küster-Hauser syndrome.

Although Mayer-Rokitansky-Küster-Hauser syndrome is a rare condition with a reported incidence of 1/4500 female live births, it represents the second most common cause of primary amenorrhea and has psychologically devastating consequences. The radiologist plays a pivotal role in both making the accurate initial diagnosis of this condition and assessing findings that may contribute to treatment planning. The purpose of this article is to provide an overview of the capabilities of ultrasound and magnetic resonance imaging (MRI) for the diagnosis and management of this syndrome with emphasis on the relevant clinical and surgical findings and to describe potential associated abnormalities and differential diagnosis.

Long-term outcomes after percutaneous coronary intervention for chronic total occlusion (from the CREDO-Kyoto registry cohort-2).

Despite improving success rate of percutaneous coronary intervention (PCI) for chronic total occlusion (CTO) lesions, the clinical benefit of recanalization of CTO is still a matter of debate. Of 13,087 patients who underwent PCI in the CREDO-Kyoto registry cohort-2, 1,524 patients received PCI for CTO (CTO-PCI). Clinical outcomes were compared between 1,192 patients with successful CTO-PCI and 332 patients with failed CTO-PCI. In-hospital death tended to occur less frequently in the successful CTO-PCI group than in the failed CTO-PCI group (1.4% vs 3.0%, p = 0.053). Through 3-year follow-up, the cumulative incidence of all-cause death was not significantly different between the successful and failed CTO-PCI groups (9.0% vs 13.1%, p = 0.18), whereas the cumulative incidence of cardiac death was significantly less in the successful CTO-PCI group than in the failed CTO-PCI group (4.5% vs 8.4%, p = 0.03). However, after adjusting confounders, successful CTO-PCI was associated with lesser risk for neither all-cause death (hazard ratio 0.93, 95% confidence interval 0.64 to 1.37, p = 0.69) nor cardiac death (hazard ratio 0.71, 95% confidence interval 0.44 to 1.16, p = 0.16). The cumulative incidence of coronary artery bypass grafting (CABG) was remarkably less in patients with successful PCI compared with those with failed PCI (1.8% vs 19.6%, p <0.0001). In conclusion, successful CTO-PCI compared with failed PCI was not associated with lesser risk for 3-year mortality. However, successful CTO-PCI was associated with significantly less subsequent CABG.

Laparoscopic Davydov correction of a failed gracilis flap neovagina in a patient with Mayer-Rokitansky-Kuster-Hauser syndrome with a pelvic kidney.

The laparoscopic Davydov procedure is a surgical technique for creation of a neovagina in patients with Mayer-Rokitansky-Kuster-Hauser syndrome (MRKH) or vaginal agenesis. Herein we report its use in creating a neovagina after failure of a previous surgical attempt in a patient with a pelvic kidney, which has not been described to date. The patient, a 28-year-old woman with MRKH in whom creation of a neovagina using bilateral gracilis flaps had been attempted, was unable to have intercourse because of a shortened and scarred vagina. We successfully used the laparoscopic Davydov procedure to create a functional neovagina despite the previous surgery and the presence of a pelvic kidney. The Davydov procedure is an option for use in patients with MRKH with history of unsuccessful neovaginal surgery and can be performed in the presence of a pelvic kidney.

Magnetic resonance evaluation of Müllerian remnants in Mayer-Rokitansky-Küster-Hauser syndrome.

OBJECTIVE: To analyze magnetic resonance imaging (MRI) findings of Müllerian remnants in young females clinically suspected of Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome in a primary amenorrhea workup. MATERIALS AND METHODS: Fifteen young females underwent multiplanar T2- and transverse T1-weighted MRI at either a 1.5T or 3.0T MR imager. Two gynecologic radiologists reached consensus decisions for the evaluation of Müllerian remnants, vagina, ovaries, and associated findings. RESULTS: All cases had bilateral uterine buds in the pelvic cavity, with unilateral cavitation in two cases. The buds had an average long-axis diameter of 2.64 ± 0.65 cm. In all cases, bilateral buds were connected with fibrous band-like structures. In 13 cases, the band-like structures converged at the midline or a paramedian triangular soft tissue lying above the bladder dome. The lower one-third of the vagina was identified in 14 cases. Fourteen cases showed bilateral normal ovaries near the uterine buds. One unilateral pelvic kidney, one unilateral renal agenesis, one mild scoliosis, and three lumbar sacralization cases were found as associated findings. CONCLUSION: Typical Müllerian remnants in MRKH syndrome consist of bilateral uterine buds connected by the fibrous band-like structures, which converge at the midline triangular soft tissue lying above the bladder dome.

HOXA10 and HOXA13 sequence variations in human female genital malformations including congenital absence of the uterus and vagina.

Congenital genital malformations occurring in the female population are estimated to be 5 per 1000 and associate with infertility, abortion, stillbirth, preterm delivery and other organ abnormalities. Complete aplasia of the uterus, cervix and upper vagina (Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome) has an incidence of 1 per 4000 female live births. The molecular etiology of congenital genital malformations including MRKH is unknown up to date. The homeobox (HOX) genes HOXA10 and HOXA13 are involved in the development of human genitalia. In this investigation, HOXA10 and HOXA13 genes of 20 patients with the MRKH syndrome, 7 non-MRKH patients with genital malformations and 53 control women were sequenced to assess for DNA variations. A total of 14 DNA sequence variations (10 novel and 4 known) within exonic and untranslated regions were detected in HOXA10 and HOXA13 among our cohorts. Four HOXA10 and two HOXA13 DNA sequence variations were found solely in patients with genital malformations. In addition to mutations resulting in synonymous amino acid substitutions, in the HOXA10 gene a missense mutation was identified and predicted by computer analysis as probably damaging to protein function in two non-MRKH patients, one with a bicornate and the other patient with a septated uterus. A novel exonic HOXA10 cytosine deletion was also identified in a non-MRKH patient with a septate uterus and renal malformations resulting in a premature stop codon and loss of the homeodomain helix 3/4. This cytosine deletion and the missense mutation in HOXA10 were analysed by real time PCR and sequencing, respectively, in two additional larger cohorts of 103 patients with MRKH and 109 non-MRKH patients with genital malformations. No other patients were found with the cytosine deletion however one additional patient was identified regarding the missense mutation. Rare DNA sequence variations in the HOXA10 gene could contribute to the misdevelopment of female internal genitalia.

Chiari malformation type I: a case-control association study of 58 developmental genes.

Chiari malformation type I (CMI) is a disorder characterized by hindbrain overcrowding into an underdeveloped posterior cranial fossa (PCF), often causing progressive neurological symptoms. The etiology of CMI remains unclear and is most likely multifactorial. A putative genetic contribution to CMI is suggested by familial aggregation and twin studies. Experimental models and human morphometric studies have suggested an underlying paraxial mesoderm insufficiency. We performed a case-control association study of 303 tag single nucleotide polymorphisms (SNP) across 58 candidate genes involved in early paraxial mesoderm development in a sample of 415 CMI patients and 524 sex-matched controls. A subgroup of patients diagnosed with classical, small-PCF CMI by means of MRI-based PCF morphometry (n = 186), underwent additional analysis. The genes selected are involved in signalling gradients occurring during segmental patterning of the occipital somites (FGF8, Wnt, and retinoic acid pathways and from bone morphogenetic proteins or BMP, Notch, Cdx and Hox pathways) or in placental angiogenesis, sclerotome development or CMI-associated syndromes. Single-marker analysis identified nominal associations with 18 SNPs in 14 genes (CDX1, FLT1, RARG, NKD2, MSGN1, RBPJ1, FGFR1, RDH10, NOG, RARA, LFNG, KDR, ALDH1A2, BMPR1A) considering the whole CMI sample. None of these overcame corrections for multiple comparisons, in contrast with four SNPs in CDX1, FLT1 and ALDH1A2 in the classical CMI group. Multiple marker analysis identified a risk haplotype for classical CMI in ALDH1A2 and CDX1. Furthermore, we analyzed the possible contributions of the most significantly associated SNPs to different PCF morphometric traits. These findings suggest that common variants in genes involved in somitogenesis and fetal vascular development may confer susceptibility to CMI.

Uterine rudiments in patients with Mayer-Rokitansky-Küster-Hauser syndrome consist of typical uterine tissue types with predominantly basalis-like endometrium.

OBJECTIVE: To analyze the histologic and immunohistochemical structure of uterine rudiments focusing on the endometrium in a representative group of patients with Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome compared with a control group. DESIGN: Prospective comparative study. SETTING: University hospital. PATIENT(S): Forty-two patients with MRKH syndrome and 13 control subjects. INTERVENTION(S): Representative biopsies or whole uterine rudiments were removed during surgery and processed by a standardized procedure including immunohistochemical staining and analysis. MAIN OUTCOME MEASURE(S): Histologic structure, tissue types, hormone receptor expression, endometrial proliferative capacity, and type in correlation with cycle phase. RESULT(S): Twenty-two of the uterine rudiments showed a duct-like structure or small cavity, 17 of which contained endometrial epithelium and CD10-positive stroma. All rudiments contained an intact myometrial layer. Tubal epithelium and stroma were found in three rudiment samples. No significant differences were observed with regard to estrogen receptor (ER) or progesterone receptor (PR) expression in endometrium or myometrium. Interestingly, patients showed predominantly basalis-like endometrium with specific lack of CD90 expression and significantly lower proliferation compared with controls. CONCLUSION(S): All typical uterine tissues can be found in uterine rudiments of patients with MRKH syndrome. Expression of hormonal receptors in the latter and controls did not differ significantly. Endometrium shows predominantly basalis-like features in uterine rudiments.

Torsion of a hydrosalpinx in a virgin patient with Mayer-Rokitansky-Küster-Hauser syndrome: report of a rare condition and its possible etiology.

BACKGROUND: To describe a rare case of hydrosalpinx torsion in a virgin patient with Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome. CASE: A 25-year-old woman with previously diagnosed MRKH syndrome in whom lower abdominal pain led to discovery and resection of a hydosalpinx of unusual origin in a university hospital department of obstetrics and gynecology, Japan. RESULTS AND CONCLUSIONS: Laparoscopy revealed a twisted left-sided hydrosalpinx, and the mass was resected laparoscopically. Results of the blood test for Chlamydia trachomatis were positive, but results of the PCR test were negative. Our case was unusual in that hydrosalpinx is rare in virgin patients with MRKH. The cause of the hydrosalpinx was unclear, but one possibility is excess tubal secretions from the fallopian tube.

Follow-up in women with vaginal agenesis after laparoscopic creation of neovagina.

STUDY OBJECTIVE: To compare 2 different methods to create a neovagina in patients with Rokitansky syndrome and to describe their functional results during the follow-up. DESIGN: Descriptive study on functional outcome in 32 patients with Rokitansky syndrome (Canadian Task Classification Force II-2). SETTING: University tertiary Hospital. DESIGN AND PATIENTS: 18 patients (group 1) underwent our original technique. 14 patients (group 2) were operated on with the modified technique. INTERVENTION: Comparison between 2 different techniques of laparoscopic approach. RESULTS: Both groups had excellent surgical outcomes. The modified technique (group 2) obtained an optimal vaginal depth in fewer days than in the first group (p < .001). Consequently, the hospital stay was shorter in the second group (p < .001). Follow-up was 85 months for group 1 and 17.2 months for group 2. Functional outcome was excellent during the follow-up in both groups. CONCLUSION: Laparoscopic creation of a neovagina is a safe, minimally invasive treatment with good functional and sexual results.

Vault prolapse of sigmoid neovagina 26 years after vaginoplasty in Mayer-Rokitansky-Küster-Hauser syndrome: a case report.

Various operative methods have been devised to create a neovagina for patients with Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome. Sigmoid vaginoplasty, a common modality for vaginal reconstruction, is believed to have satisfactory long-term anatomical and functional results. We herein report a patient with MRKH syndrome and vault prolapse of a sigmoid neovagina 26 years after vaginoplasty. Biopsies from the neovagina revealed colonic mucosa. Bilateral iliococcygeus fascia fixation of the neovaginal vault was performed vaginally. The patient had a low Pelvic Organ Prolapse/Urinary Incontinence Sexual Questionnaire-12 (PISQ-12) score preoperatively, which further decreased postoperatively. Therefore, the surgery failed to achieve a good functional result. No recurrence of the prolapse was observed 2 years postoperatively.

Androgen insensitivity syndrome.

Androgen insensitivity syndrome (AIS) is a disorder caused by a mutation of the gene encoding the androgen receptor (AR; Xq11-q12). The prevalence of AIS has been estimated to be one case in every 20,000 to 64,000 newborn males for the complete syndrome (CAIS), and the prevalence is unknown for the partial syndrome (PAIS). The symptoms range from phenotypically normal males with impaired spermatogenesis to phenotypically normal women with primary amenorrhea. Various forms of ambiguous genitalia have been observed at birth. The diagnosis is confirmed by determining the exact mutation in the AR gene. PAIS individuals require precise diagnosis as early as possible so that the sex can be assigned, treatment can be recommended, and they can receive proper genetic counseling. After birth, differential diagnosis should be performed using other forms of abnormal sexual differentiation of primary amenorrhea. The treatment of AIS is based on reinforcement sexual identity, gonadectomy planning, and hormone replacement therapy. The prognosis for CAIS is good if the testicular tissue is removed at the appropriate time. For PAIS, the prognosis depends on the ambiguity of the genitalia and physical and psychosocial adjustment to the assigned sex.

Renal and urological abnormalities occurring with Mullerian anomalies.

OBJECTIVE: To describe the spectrum and frequency of renal and urological abnormalities in a cohort of patients with uterine anomalies. MATERIAL AND METHODS: We conducted an ethics committee approved review of 164 women referred to the imaging department from a specialist gynaecological anomaly clinic. Imaging acquired over 8 years was assessed and this was sufficient for assessment of the morphology of the gynaecological and renal tracts in 113 patients. RESULTS: Absent unilateral kidney was the most common abnormality (31.8%), most frequent in Type 3 uterine anomalies. Absent kidneys occurred in other anomaly types at lower frequency. Pelvic ureteric remnants were found in 9 of 36 patients with absent kidneys; these inserted ectopically, most commonly into the vagina. Urological abnormalities were found in 11 patients with two kidneys, including ectopic ureters, scarred kidneys and dysplastic kidney. Obstructed hemivaginas were associated commonly but not invariably with an absent kidney. CONCLUSION: Previously unreported renal and urological abnormalities have been described in patients with congenital uterine anomalies. These have significant clinical impact. Ectopic ureters can cause incontinence, and potentially cause pain and become infected. Knowledge of the ureteric course, including ureteric remnants, is essential before complex laparoscopic and vaginal surgery.