Clinical utility of chlormadinone acetate (Lutoral™) in frozen-thawed embryo transfer with hormone replacement.

PURPOSE: The clinical utility of chlormadinone acetate tablets (Lutoral™), an orally active progestin which has been available since June 2007, was compared to an in-house vaginal suppository formulation of progesterone used between 2006 and 2007 for assisted reproductive technology (ART). METHODS: We retrospectively evaluated the efficacy and safety of chlormadinone acetate by comparing the pregnancy rates and the incidences of birth defects and hypospadias in frozen-thawed embryo transfer cycles using the in-house vaginal progesterone and those using chlormadinone acetate for luteal phase support. RESULTS: The pregnancy rates in the frozen-thawed embryo transfer cycles were 31.2% (259/831) with vaginal progesterone for luteal phase support and 31.6% (4228/13 381) with chlormadinone acetate (no significant difference). In the cycles resulting in live birth following administration of chlormadinone acetate between July 2007 and December 2015, the incidence of birth defects was 2.8% (80/2893), and the incidence of hypospadias was 0.03% (1/2893). CONCLUSIONS: These results indicate that the pregnancy rate following frozen-thawed embryo transfer using chlormadinone acetate for luteal phase support was comparable with that using vaginal progesterone, with no increased risk of birth defects, including hypospadias, which has been a concern following the use of progestins.

Long-term outcomes of freeze-all strategy: A retrospective analysis from a single ART center in Japan.

PURPOSE: To demonstrate the benefits of the freeze-all strategy for in vitro fertilization treatment based on retrospective analyses. METHODS: Post-thaw embryo survival rates of slow-frozen embryos in 294 cycles and vitrified embryos in 12 195 cycles were assessed. Progesterone (P4) and estradiol (E2) levels per mature oocyte by age category were assessed in 9081 cycles and pregnancy rates with fresh embryo transfer and frozen-thawed embryo transfer by P4 level were assessed in 1535 cycles. RESULTS: The survival rates of frozen-thawed embryos were 92.5% with slow freezing and 99.1% with vitrification. P4 levels on the day of human chorionic gonadotropin (hCG) injection showed a trend toward an increase with age. The pregnancy rate per mature oocyte with fresh embryo transfer decreased dependently upon P4 level, while that with frozen-thawed embryo transfer was not affected by P4 level. The pregnancy rates with frozen-thawed embryo transfer were higher than those with fresh embryo transfer in patients aged 42 years or younger. CONCLUSIONS: The freeze-all strategy is a valuable treatment option which allows the separation of an embryo transfer cycle from an oocyte retrieval cycle, especially for patients with high P4 levels at oocyte retrieval and patients of advanced maternal age.

Performance of anti-Müllerian hormone (AMH) levels measured by Beckman Coulter Access AMH assay to predict oocyte yield following controlled ovarian stimulation for in vitro fertilization.

PURPOSE: We evaluated the performance of anti-Müllerian hormone (AMH) measured by the Beckman Coulter fully automated Access assay to predict oocyte yield following controlled ovarian stimulation (COS) for in vitro fertilization (IVF). METHODS: The correlation between the Access assay and the pre-mixing method with Generation II ELISA assay (Gen II pre-mix assay) was assessed using 230 blood samples. The relationship of AMH level measured by the Access assay and the actual number of oocytes retrieved following COS was assessed using 3296 IVF cycles. The performances of AMH, follicle stimulating hormone (FSH), and estradiol (E2) in predicting the responses to COS were also evaluated by constructing receiver operating characteristic (ROC) curves. RESULTS: The AMH levels measured just before oocyte retrieval by the Access assay and the number of oocytes retrieved following COS showed a good correlation with R = 0.655. The ROC analysis revealed that the sensitivity of AMH was comparable with or lower than that of E2 but higher than that of FSH. CONCLUSIONS: With the improved Access AMH assays, AMH was as sensitive as E2 and could become an accurate marker of ovarian response to COS in more than 3000 Japanese IVF patients.

Anti-N-methyl-d-aspartate receptor limbic encephalitis associated with mature cystic teratoma of the fallopian tube.

Anti-N-methyl-d-aspartate receptor (NMDAR) limbic encephalitis is the most common form of paraneoplastic encephalitis that is associated with teratomas. Because tumor removal leads to better clinical outcomes, it is essential to reveal the location of the teratomas. This is the first reported case of anti-NMDAR encephalitis associated with teratoma of the fallopian tube. Salpingo-oophorectomy improved neurological symptoms and immunohistochemical examinations indicated the expression of NMDAR on neuroglial cells within the fallopian tube teratoma. Teratomas of the fallopian tube cause anti-NMDAR encephalitis; the imaging analysis and exploratory laparoscopies of the fallopian tube as well as of the ovary should be considered. Surgical removal of both fallopian tubes and ovaries with a normal appearance should be considered for patients in whom immunotherapy is not effective.

A new Krüppel-like factor 1 mutation (c.947G > A or p.C316Y) in humans causes ß-thalassemia minor.

Here we describe a Japanese patient with mild ß-thalassemia (ß-thal) with an intact ß-globin gene but a new missense mutation of c.947G > A or p.C316Y in the erythroid Krüppel-Like Factor (KLF1) gene which is strongly associated with the expression of the ß-globin gene. The association of the KLF1 mutation with ß-thal, is here described. The p.C316Y mutation occurred at one of the cysteines that constitute the second zinc finger motif of KLF1, and would have changed the zinc finger conformation to impair the DNA binding properties or the promoter function of the ß-globin gene. Our expression study found that the mutant KLF1 gene had a markedly negative effect on the ß-globin gene expression, or 7.0% of that of its normal counterpart. A presumed heterozygous state, or equimolar presence of the mutant and normal KLF1s reduced the expression rate to 70.0% of the normal alone. This degree of the decrease may explain the very mild phenotype of the patient's ß-thal. Furthermore, the patient's whole-exome analysis using next-generation sequencing revealed that the ß-thal defect is caused by only this KLF1 gene mutation. The Hb A2 and Hb F levels that are frequently elevated in KLF1 mutations were elevated by 4.1 and 1.3%, respectively, in this case. The contribution to their elevation by KLF1: p.C316Y is uncertain.

[Hemoglobinopathies in Japan: characteristics and comparison with those of other ethnic groups].

Abnormalities of hemoglobin (Hb), or hemoglobinopathies, are classified into Hb abnormalities that arise from altered quality induced mainly by amino acid substitution. Furthermore, thalassemia is a quantitative abnormality of normal Hb. Most hemoglobinopathies are inherited disorders. The abnormal Hb known to date comprise more than 210 types in the Japanese population. The rate of thalassemia in Japan is less than that in endemic regions, but the frequencies of ß- and α- thalassemias are 1/1,000 and 1/3,500 in the general population, respectively, so not particularly rare. The mutation spectrum is different from that of endemic regions, probably because Japanese have been historically isolated islanders. Japanese hemoglobinopathy generally has minor symptoms, which are different from those in endemic areas where thalassemia exhibits major clinical manifestations. This might be why useful knowledge is obtained in our laboratory which concentrates on detailed observation of clinical data in addition to genetic analysis. We have, in fact, discovered new clinical characteristics and the significance of hemoglobinopathy, especially of minor or intermediate thalassemia. This approach is quite different from that in other countries coping with only the major type. By focusing on this novel approach, we aim to contribute to improving diagnostic technology for patients.

Oxidation status of ß-thalassemia minor and Hb H disease, and its association with glycerol lysis time (GLT50).

ß-Thalassemia (ß-thal), especially ß-thalassemia major (ß-TM), is reported to be related to reactive oxygen species (ROS) and enhanced oxidation status. It is reflected by increased malondialdehyde (MDA), by membrane lipid peroxidation and decreased by the newly developed total antioxidant capacity (TAC). However, there is less evidence for ß-thal minor and Hb H (ß4) disease on its association with oxidation status. On the other hand, hemolysis by glycerol lysis time (GLT50) is invariably prolonged in thalassemia. The reason for the prolongation of GLT50 is not well understood. The aim of this study was to investigate the oxidation state in ß-thal minor and Hb H disease and to find out the association of the oxidation with the prolongation of GLT50. Blood samples from 39 subjects (33 with ß-thal minor, six with Hb H disease) were collected from individuals living in Japan. The clinical screening tests and molecular identification of the thalassemias were performed. Malondialdehyde and TAC were measured using spectrophotometric analyses. In ß-thal minor and Hb H disease, the plasma MDA level was significantly elevated and the TAC reduced. A highly reversed correlation between MDA and TAC was noted. Their GLT50 levels were evidently prolonged, and the GLT50 has significant correlations with MDA and TAC. ß-Thalassemia minor and mild Hb H disease are evidently in a milieu of reduced redox state, and GLT50 prolongation in ß-thal minor and Hb H disease has a close correlation with the oxidation state, possibly by oxidative impairment of the membrane protein of the red cell.

Detection of beta-thalassemia/hemoglobin E disease in samples which initially were diagnosed as homozygous hemoglobin E.

BACKGROUND: Differentiation of beta-thalassemia/HbE disease from homozygous HbE in samples containing HbA2/E > 75% and HbF < 15% is difficult. The aim of this study is to observe the possibility of using Hb typing and hematological parameters to identify both disorders. METHODS: Multiplex amplification refractory mutation system (MARMS)-PCR for beta-thalassemia codons 17 (A > T), 41/42 (-TCTT), 71/72 (+A), and IVSI-ntl (G > T) mutations and ARMS-PCR for HbE were performed in 67 samples that contained HbA2/E > 75% and HbF < 15%. RESULTS: Beta-thalassemia/HbE disease was identified in 10 of 67 (14.93%) samples. Levels of hemoglobin, hematocrit, and mean corpuscular volume (MCV) of beta-thalassemia/HbE disease were significantly lower than those of homozygous HbE whereas, levels of HbF were significantly higher. CONCLUSIONS: In places where the molecular analysis is not available, HbF > 5% in combination with MCV < 55 fL, hemoglobin < 100 g/L, and hematocrit < 0.30 L/L could be used for screening of beta-thalassemia/HbE disease.

A new ß(0)-thalassemia mutation (codon 102, AAC>ATCAC) in coexistence with a heterozygous P4.2 Nippon gene.

A new ß-thalassemia (ß-thal) frameshift mutation was found at codon 102 (AAC>ATCAC) in a 17-year-old Japanese male and his 14-year-old sister. Both demonstrated a more severe phenotype than the usual ß-thal minor with mild hemolytic involvement. No mRNA derived from the thalassemic allele, or ß(T)mRNA, was detected in the sequencing analysis of the whole mRNA (cDNA). However, the ß(T)mRNA from the whole ßmRNA was specifically amplified by amplification refractory mutation system (ARMS), and was actually found to be present. Furthermore, quantitative polymerase chain reaction (q-PCR) demonstrated a negligible amount of ß(T)mRNA. Thus, their more severe phenotype was not caused by the "dominant type" ß-thal in which a considerable amount of the ß(T)mRNA would be expected. In fact, our proband had a total ßmRNA level that was mostly normal. Thus, the cause of a ß-thal phenotype by the frameshift mutation was ascribed to the reduced amount of mRNA. We further searched for the cause of their severe phenotype. However, factors that exacerbated the phenotype of ß-thal, such as α-globin gene triplication, coexisting iron deficiency and infection were not found. Finally, we noticed that the red cell morphology revealed ovalocytosis and small numbers of stomatocytes that were seen in the hereditary spherocytosis (HS), especially by P4.2 mutations. The sequence of the P4.2 gene disclosed heterozygous P4.2 Nippon, or missense mutation at codon 142 (GCT>ACT) on exon 3, the most common mutation of Japanese HS. Frequent mutations of other membrane proteins, Band 3 and ankyrin that are common cause of HS in the Japanese population, other than P4.2, were not detected. When HS by P4.2 Nippon develops it is homozygous, and no P4.2 protein is observed in sodium dodecilsulphate-polyacrylamide gel electrophoresis (SDS-PAGE), while in our case the amount of the P4.2 was almost normal in the SDS-PAGE. However, there are several reports that revealed more severe phenotypes of ß-thal by the coexisting abnormality of membrane protein. It is uncertain, but the presence of heterozygous P4.2 Nippon may be associated with the exacerbation of the phenotype of ß-thal minor.

Identification of a novel mutation in the ß-globin gene 3' untranslated region [+1,506 (A>C)] in a Japanese male with a heterozygous ß-thalassemia phenotype.

ß-Thalassemia (ß-thal) is characterized by the absent or reduced production of ß-globin chains. The precise molecular lesion that causes decreased ß-globin synthesis in ß(+)-thal is difficult to predict when mutations occur in the locus control region (LCR), the promoter, the introns or 3' untranslated regions (3'UTRs). Among them, the role of the 3'UTR of ß-globin gene in mRNA stability is poorly understood, mainly due to very few cases that have mutations in this region. So far, only three mutations have been reported in the 3'UTR of ß-globin gene. Although, it is speculated that some of these reported mutations could be associated with mRNA stability, the precise molecular basis still remains unclear. We report here a novel mutation in the ß-globin gene 3'UTR [+1,506 (A>C)] in a 31-year-old Japanese male with hematological parameters suggestive of heterozygous ß-thal. Further functional studies on this novel mutation reported here, may help in understanding of the regulation and expression of the ß-globin gene and its products.

A 25-year observation of a Japanese female patient with Hb Nottingham who has two children with the same disorder.

We undertook a 25-year observation of a female patient with an unstable variant, Hb Nottingham or ß98(FG5)Val→Gly, GTG>GGG. The proband was diagnosed with Hb Nottingham at the age of 9 years. Splenectomy was performed in order to successfully aid her height growth due to chronic anemia at the age of 11, although anemia improvement was transient. She experienced pregnancy/delivery twice, at age 23 and 26, respectively. During both pregnancies, a large number of nucleated red blood cells (NRBCs) appeared in her peripheral blood. No developmental delay of the fetus was noted in either pregnancy, and she gave birth without any maternal complications or perinatal problems. Both babies were diagnosed with Hb Nottingham. To the best of our knowledge, this is the first report of a long-term observation of a proband with Hb Nottingham, including her pregnancy/delivery and the neonatal course of her children with the same disorder.

The +1,506 (A>C) mutation in the 3' untranslated region affects ß-globin expression.

The 3' untranslated region (3'UTR) is known to be important to mRNA stability but the stabilization mechanism on the ß-globin gene is not fully elucidated. We speculated in our previous report that +1,506 (A>C) mutation (HGVS nomenclature: *32A>C) on the ß-globin 3'UTR causes ß-thalassemia (ß-thal) in order to destabilize the mRNA. To investigate further, we studied the expression efficiency for the mutation with a luciferase assay. We made recombinant pGL4.74 vectors in which the luciferase 3'UTR was replaced with the wild-type and mutant 3'UTR of the ß-globin gene. For a comparison experiment, recombinant vectors were made not only for this mutation but also six other mutations in the ß-globin 3'UTR which bring about ß-thal or affect mRNA stability. The +1,506 mutation led to a 30.0% lower protein expression than normal in this assay. We concluded that this mutation destabilizes mRNA and consequently decreases the ß-globin amount to finally cause ß-thal. Our study highlights the crucial area of ß-globin 3'UTR for protein expression.

[Establishment of multiplex hybri-probe method--analysis using the polymorphism of UGT1A1 gene].

There are a number of methods for gene analysis of a point mutation and deletion/insertion of several nucleotides. In 2011, we reported an improved hybridization probe methods (Hybri-Probe method) that are highly sensitive and accurate, and excellent in cost and time effectiveness. Here, we have developed the Multiplex Hybri Probe method for several types of mutations or polymorphisms including the microsatellite polymorphisms, especially of palindromic sequence such as (TA)n and (GC)n. In addition, several mutations are analyzed at a time. In this research we focused on the three types of polymorphism on the Uridine diphosphate glucuronyltransferase (UGT) gene. Design of the probes for the detection of UGT1A1*6(211 G --> A G71R) and UGT1A1*27 (686 C --> A P229Q) was not difficult because the mutations were a single base substitution. However, UGT1A1*28 (A (TA)6TAA --> A (TA)7TAA) has tandem and palindromic sequence. Since the probes to detect the mutation in such sequence resulted in failure, we made several mismatches, i.e., TATATATATATA --> TATGTGTATATA. As a result, the probes designed for the three polymorphisms above did not overlap in the Tm and were separated by approximately 10 degree intervals between 63.2 degrees C and 37.5 degrees C. In this Multiplex Hybri-Probe method, the three kinds of probe are added tube into the PCR product in the same tube and followed by the measurement of the Tm using the LightCycler. Thus, it is very simple, and performed by one step for any mutation including the microsatellite polymorphisms, and it also has good cost performance and favorable time efficiency. It takes two hours including the running time of PCR for completion of the analysis. It may also be available to the detection of other gene abnormalities.

Internet-based approach to population screening for common hemoglobinopathies in United Arab Emirates.

This article reports on efforts to overcome common hurdles that were faced during population-based screening for common hemoglobinopathies in the United Arab Emirates. An Internet-based approach was designed and implemented to increase the acceptance of the screening program. The process involved: an awareness campaign, a simple bilingual (Arabic/English) online consent form and registration process, the use of a barcode for sample labeling, an equipment upgrade, electronic communication of a successful registration process, test results, and a counseling process. Before the implementation of the Internet-based system, great concern was noted among the clients in terms of the availability of accurate and timely test results, the need for pretest and post-test counseling, and the way that their personal health information was handled. Lapses in information exchange between the clients who participated in the screening program for the carrier state of inherited disorders and the screening laboratory posed significant challenges. The emphasis on confidentiality and the ease of access to services was instrumental in increasing the level of acceptance of these services in our community. Based on an analysis of > 10,000 samples, we conclude that Internet-based reporting holds much promise for improving the quality of care that clients receive.

[Improved hybridization probe method for genetic analysis of beta-thalassemia].

There are a number of methods for genetic diagnosis of a point mutation and/or deletion/insertion of several nucleotides. However, none of these methods are satisfactory with respect to sensitivity, accuracy and cost effectiveness. In 2003, the Hybridization Probe method was first reported, but it was unsatisfactory. We attempted to improve this method by separating the step of using real-time PCR with melting curve analysis. In our improved method, or Hybri Probe method, the sensitivity and accuracy of Tm were remarkably improved by employing H2O and Hi-Di Formamide. In order to detect several beta-thalassemia mutations, relevant probes of basically wild sequence were prepared, and optimal conditions for detecting mutations were studied. This method yielded highly sensitive and accurate results. This Hybri Probe method is available for SNPs and microsatellite polymorphisms, and it may also be useful for the detection of gene abnormalities other than those in beta-thalassemia.

Molecular identification of the novel Gγ-ß hybrid hemoglobin: Hb Gγ-ß Ulsan (Gγ through 13; ß from 19).

Gene fusion is a very rare mechanism that produces hemoglobin variants. Less than ten types of ß-like hybrid globins have been reported to date. Herein we identified the first hybrid hemoglobin between Gγ- and ß-globins in a five-year-old Korean male who had thalassemia minor feature and triplication of the HBA2 gene (αα/αααα). The novel globin originated from a 27,707-base pair deletion spanning from the HBG2 to HBB gene (NG_000007.3:g.42947_70653del). Its protein sequence included 13 N-terminal amino acids from Gγ-globin, five common amino acids from Gγ- and ß-globins, and 128 amino acids from ß-globin (Gγ through 13; ß from 19). Molecular genetic analyses characterized the hybrid DNA and RNA. Mass spectrometry and de novo protein sequencing successfully identified the fusion peptide in the hybrid hemoglobin. We named this novel hybrid Hb Gγ-ß Ulsan. The novel hemoglobin constituted 37.0% of the total hemoglobin and showed reduced oxygen affinity.

Glycogen storage disease type Ia (GSD Ia) during pregnancy: report of a case complicated by fetal growth restriction and preeclampsia.

Glycogen storage disease type Ia (GSD Ia) leads to disturbed glycogenolysis and gluconeogenesis due to a deficiency in the enzyme glucose-6-phosphatase. A patient with GSD Ia showed hypoglycemia and proteinuria without dietary management since early pregnancy. The patient's condition was complicated by hypertension with increase in proteinuria at 22 weeks of gestation. In spite of administration of antihypertensive drugs and dietary management, the disease became more severe with deterioration in the fetal status and inhibition of fetal growth. Thus, a cesarean section was performed at 26 weeks of gestation. The delivered male infant weighing 412 g died at 2 days after birth. The patient's blood pressure had normalized within 3 months after delivery, while proteinuria persisted.

Successful pregnancy after artificial insemination in a case of human seminal plasma allergy.

Human seminal plasma allergy in women is uncommon, but causes a variety of serious reactions, including urticaria, dyspnea and vomiting, in those that are affected. Semen barriers, such as condoms, are the most widely advocated method for avoiding these reactions. However, this is not acceptable to couples who wish to have children. We present a case of a woman with human seminal plasma allergy who became pregnant after the eighth cycle of artificial insemination using washed sperm from her spouse. (Reprod Med Biol 2008; 7: 119-122).