Yoko Izumi

Fertilization and pregnancy using cryopreserved testicular sperm for intracytoplasmic sperm injection with azoospermia

OBJECTIVES To report fertility for men with obstructive (OA) and nonobstructive (NOA) azoospermia after frozen and thawed spermatozoa recovered from the seminiferous tubules and intracytoplasmic sperm injection (ICSI) and to evaluate the factors other than spermatozoa. DESIGN Retrospective clinical analysis. SETTING Male infertility clinic for testicular sperm extraction and freezing/thawing (cryoTESE) and assisted reproductive technologies clinic for ICSI. PATIENT(S) Seventy-four men with OA and 140 men with NOA undergoing attempted cryoTESE-ICSI. INTERVENTION(S) Seventy-three couples with OA underwent a total of 184 cryoTESE-ICSI cycles, and 46 couples with NOA underwent a total of 75 cryoTESE-ICSI cycles. MAIN OUTCOME MEASURE(S) The numbers of eggs at metaphase II injected, two-pronuclei oocytes, normal cleaved embryos, embryos transferred, transfer cycles, biochemical pregnancies, and clinical pregnancies, as well as the implantation and delivery rates, were examined. RESULT(S) Fertilization rate in NOA was significantly lower than in OA. Neither the pathology, the source, nor the quantity of spermatozoa had any effect on fertilization or pregnancy rates. Maternal age had no effect on fertilization or embryo cleavage, but did dramatically affect the implantation, pregnancy, and delivery rates in NOA and OA. CONCLUSION(S) Good pregnancy rates were achieved without significant differences among the sperm sources. The pregnancy and the delivery rate were dependent strictly on the age of the female partner but not on her ovarian reserve.

Molecular changes induced by bisphenol-A in rat Sertoli cell culture.

Bisphenol-A (BPA) is an industrial chemical and is known to act as an endocrine disrupter. This study was designed to evaluate how BPA regulates Sertoli cell (SC) signal molecules. Purified rat SCs were cultured and treated with BPA (200 µmol/l) at various time points. Western blot analysis was used to determine the activation of extracellular signal-related kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), p38 mitogen activated protein kinase (MAPK), nuclear factor kappa B (NF-κB), cyclooxygenase-1,2 (COX-1, 2), estrogen receptor-α (ER-α), and androgen receptor (AR). The levels of transferrin (TF), prostaglandin E2 (PGE2), and prostaglandin F2α (PGF2α) in culture medium were quantified by ELISA. Interleukin (IL)-1β and IL-6 mRNAs were measured by quantitative real-time PCR (QRT-PCR). Compared with the control, BPA activated the phosphorylation of ERK1/2 (p-ERK1/2) through 30 min to 6 h. TF was down-regulated at 6 and 24 h. Furthermore, IL-1β was up-regulated at 30 min and IL-6 was up-regulated at 1 and 24 h. ERK activity inhibitor (PD98059, 10 µmol/l) inhibited these molecular changes. These results reveal the possibility that BPA may have adverse effects on spermatogenesis via ERK1/2.

Genome-wide copy number analysis and systematic mutation screening in 58 patients with hypogonadotropic hypogonadism

OBJECTIVE To clarify the molecular basis of hypogonadotropic hypogonadism (HH). DESIGN Genome-wide copy number analysis by array-based comparative genomic hybridization and systematic mutation screening of 29 known causative genes by next-generation sequencing, followed by in silico functional assessment and messenger RNA/DNA analyses of the mutants/variants. SETTING Research institute. PATIENT(S) Fifty-eight patients with isolated HH (IHH), combined pituitary hormone deficiency (CPHD), and syndromic HH. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Frequency and character of molecular abnormalities. RESULT(S) Pathogenic defects were identified in 14 patients with various types of HH, although oligogenicity was not evident in this patient group. As rare abnormalities, we identified a submicroscopic deletion involving FGFR1 and an SOX3 polyalanine deletion in patients with IHH, and a WDR11 splice site mutation in a patient with CPHD. No disease-associated polymorphism was detected in the 58 patients. CONCLUSION(S) The present study provides further evidence that mutations and deletions in the known causative genes play a relatively minor role in the etiology of HH and that submicroscopic rearrangements encompassing FGFR1 can lead to IHH as a sole recognizable clinical feature. Furthermore, the results indicate for the first time that polyalanine deletions in SOX3 and mutations in WDR11 constitute rare genetic causes of IHH and CPHD, respectively.

Loss-of-Function SOX10 Mutation in a Patient with Kallmann Syndrome, Hearing Loss, and Iris Hypopigmentation

Background: Kallmann syndrome (KS) is a clinically and genetically heterogeneous disorder consisting of hypogonadotropic hypogonadism and anosmia. KS is occasionally associated with deafness. Recently, mutations in SOX10, a well-known causative gene of Waardenburg syndrome (WS) characterized by deafness, skin/hair/iris hypopigmentation, Hirschsprung disease, and neurological defects, have been identified in a few patients with KS and deafness. However, the current understanding of the clinical consequences of SOX10 mutations remains fragmentary. Case Report: A Japanese male patient presented with sensory deafness, blue irises, and anosmia, but no hair/skin hypopigmentation, Hirschsprung disease, or neurological abnormalities. He showed no pubertal sex development at 15.1 years of age. Blood examinations revealed low levels of FSH and testosterone. Results: Molecular analysis detected a de novo p.Leu145Pro mutation in SOX10, which has previously been reported in a patient with WS and Hirschsprung disease. The mutation was predicted to be probably damaging. The mutant protein barely exerted in vitro transactivating activity. Conclusions: These results highlight the significance of SOX10 haploinsufficiency as a genetic cause of KS with deafness. Importantly, our data imply that the same SOX10 mutations can underlie both typical WS and KS with deafness without skin/hair hypopigmentation, Hirschsprung disease, or neurological defects.

Paradoxical gain-of-function mutant of the G-protein-coupled receptor PROKR2 promotes early puberty

The human genome encodes ~750 G‐protein‐coupled receptors (GPCRs), including prokineticin receptor 2 (PROKR2) involved in the regulation of sexual maturation. Previously reported pathogenic gain‐of‐function mutations of GPCR genes invariably encoded aberrant receptors with excessive signal transduction activity. Although in vitro assays demonstrated that an artificially created inactive mutant of PROKR2 exerted paradoxical gain‐of‐function effects when co‐transfected with wild‐type proteins, such a phenomenon has not been observed in vivo. Here, we report a heterozygous frameshift mutation of PROKR2 identified in a 3.5‐year‐old girl with central precocious puberty. The mutant mRNA escaped nonsense‐mediated decay and generated a GPCR lacking two transmembrane domains and the carboxyl‐terminal tail. The mutant protein had no in vitro signal transduction activity; however, cells co‐expressing the mutant and wild‐type PROKR2 exhibited markedly exaggerated ligand‐induced Ca2+ responses. The results indicate that certain inactive PROKR2 mutants can cause early puberty by enhancing the functional property of coexisting wild‐type proteins. Considering the structural similarity among GPCRs, this paradoxical gain‐of‐function mechanism may underlie various human disorders.

Hypogonadotropic hypogonadism in a female patient previously diagnosed as having waardenburg syndrome due to a sox10 mutation

Hypogonadotropic hypogonadism (HH) is a clinically and genetically heterogeneous condition that can be associated with several additional clinical features such as anosmia, cleft palate, and hearing loss [1]. HH with anosmia is referred to as Kallmann syndrome (KS). More than 20 genes are known to underlie HH and/or KS, although mutations in these genes account for only a minor portion of the etiology of HH/KS [1–4]. In 2013, Pingault et al. identified SOX10 mutations in seven patients with KS [5]. Furthermore, Pingault et al. found that genetic knockout of Sox10 disrupted migration of GnRH cells in murine fetuses [5]. Subsequently, Vaaralahti et al. identified an additional KS patient with a SOX10mutation [6]. These results indicate that SOX10 mutations constitute rare genetic causes of KS. Currently, SOX10 is known as one of the causative genes of Waardenburg syndrome (WS), a rare genetic disorder characterized by hearing loss and hypopigmentation in the skin, hair, and eye [7]. Indeed, hearing impairment with or without gray/white hair was found in most of the KS cases reported by Pingault et al. and Vaaralahti et al. [5, 6]. However, detailed clinical assessment of the SOX10 mutation-positive patients and functional assays of the SOX10 mutants remain fragmentary. Thus, genetic links between HH/KS and WS have not been fully established. Here, we performed molecular and clinical analyses of a previously reported patient with WS due to a frameshift mutation in SOX10.

SOX3 Overdosage Permits Normal Sex Development in Females with Random X Inactivation

Submicroscopic duplications involving SOX3 and/or its flanking regions have been identified in 46,XX individuals both with and without disorders of sex development, raising the question whether SOX3 overdosage is sufficient to induce testicular development in genetically female individuals. Here, we report a mother-daughter pair with female phenotypes and random X inactivation. The individuals carry complex X chromosomal rearrangements leading to a copy number gain of genomic regions involving SOX3 and its upstream region. The amplified DNA fragments were detected at Xq27. These results provide evidence that SOX3 overdosage permits normal sex development in 46,XX individuals with random X inactivation.

Current status of preimplantation genetic diagnosis in Japan.

This is a retrospective study aimingto clarify the current status of preimplantation genetic diagnosis (PGD) in Japan. Our data were collected from 12 facilities between September 2004 and September 2012, and entered into a database. A majority of PGD in Japan was performed for balanced structural chromosomal abnormalities in couples with recurrent miscarriage. PGD for monogenic diseases was performed only in two facilities. The average maternal age was 38 years for monogenic diseases and 40 years for chromosomal abnormalities. Overall there have been671 cycles to oocyte retrieval reported. Of these cycles, 85% (572 cycles)were for chromosomal abnormalities, and 15% (99 cycles) for monogenic diseases. Diagnosis rates in the current study were 70.8% for monogenic diseases and 94.0% for chromosomal abnormalities. Rates of embryo transfer of PGD were 62.7% for monogenic diseases and 25.5% for chromosomal abnormalities. Clinical pregnancy rates per embryo transfer were 12.0% for monogenic diseases and 35.6% for chromosomal abnormalities. Our study is the first PGD report from all facilities which had the approval of the ethics committee of the Japanese Society of Obstetrics and Gynecology. We have built a basis for gathering continuous PGD data in Japan.

First report of detection of lysophosphatidic acids (LPAs) and analysis of LPA quantity in a human embryo-conditioned medium

Abstract Background. Lysophosphatidic acid (LPA) receptor 3 expressed in the uterus plays a critical role in uterine receptivity for the embryo. We investigated whether human embryos produce LPA by analysis of a human embryo-conditioned medium (HECM). Methods. LPAs in HECM were extracted by the Bligh-Dyer method. Extracted LPAs were converted to trimethylsilyl (TMS) derivatives. Gas chromatography (GC)/ selected ion monitoring (SIM) and GC / mass spectrometry (MS) were used to analyze LPAs derivatized with TMS. Monitoring and quantitative analysis of LPAs were performed using GC/SIM with [M-15]+ ion derived from LPAs. Identification of LPAs was confirmed by GC/MS. Results. LPA-C16:0, 16:1, 18:0, 18:1 and 18:2 were detected in HECM. The concentration of LPAs was calculated by comparing each detected peak area with a corresponding standard LPA. Conclusion. This study is the first study in which five molecular species of LPAs produced by human embryos were detected in HECM and their quantities analyzed.