title: Diagnosis of abnormal human fertilization status based on pronuclear origin and/or centrosome number.
abstract: OBJECTIVE
  Normally fertilized zygotes generally show two pronuclei (2PN) and the extrusion of the second polar body. Conventional in vitro fertilization (c-IVF) and intracytoplasmic sperm injection (ICSI) often result in abnormal monopronuclear (1PN), tripronuclear (3PN), or other polypronuclear zygotes. In this study, we performed combined analyses of the methylation status of pronuclei (PN) and the number of centrosomes, to reveal the abnormal fertilization status in human zygotes.
  
  
  METHODS
  We used differences in DNA methylation status (5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC)) to discriminate between male and female PN in human zygotes. These results were also used to analyze the centrosome number to indicate how many sperm entered into the oocyte.
  
  
  RESULTS
  Immunofluorescent analysis shows that all of the normal 2PN zygotes had one 5mC/5hmC double-positive PN and one 5mC-positive PN, whereas a parthenogenetically activated oocyte had only 5mC staining of the PN. All of the zygotes derived from ICSI (1PN, 3PN) had two centrosomes as did all of the 2PN zygotes derived from c-IVF. Of the 1PN zygotes derived from c-IVF, more than 50 % had staining for both 5mC and 5hmC in a single PN, and one or two centrosomes, indicating fertilization by a single sperm. Meanwhile, most of 3PN zygotes derived from c-IVF had a 5mC-positive PN and two 5mC/5hmC double-positive PNs, and had four or five centrosomes, suggesting polyspermy.
  
  
  CONCLUSIONS
  We have established a reliable method to identify the PN origin based on the epigenetic status of the genome and have complemented these results by counting the centrosomes of zygotes.
authors.affiliation: Fertility Research Centre, Mio Fertility Clinic, 2-1-1, Kuzumo-Minami, Yonago, Tottori, 683-0008, Japan.
authors.forename: Yoshiteru
authors.initials: Y
authors.lastname: Kai
authors.affiliation: Reproductive Centre, Mio Fertility Clinic, 2-1-1, Kuzumo-Minami, Yonago, Tottori, 683-0008, Japan.
authors.forename: Kyoko
authors.initials: K
authors.lastname: Iwata
authors.affiliation: Reproductive Centre, Mio Fertility Clinic, 2-1-1, Kuzumo-Minami, Yonago, Tottori, 683-0008, Japan.
authors.forename: Yumiko
authors.initials: Y
authors.lastname: Iba
authors.affiliation: Fertility Research Centre, Mio Fertility Clinic, 2-1-1, Kuzumo-Minami, Yonago, Tottori, 683-0008, Japan. yasmio@mfc.or.jp.|Reproductive Centre, Mio Fertility Clinic, 2-1-1, Kuzumo-Minami, Yonago, Tottori, 683-0008, Japan. yasmio@mfc.or.jp.
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Mio
mesh_terms: D044503:5-Methylcytosine / Q000032:analysis / Q000378:metabolism / Q000031:analogs & derivatives; D000782:Aneuploidy; D016022:Case-Control Studies; D002467:Cell Nucleus / Q000235:genetics*; D018385:Centrosome; D003596:Cytosine / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D005260:Female; D005307:Fertilization in Vitro; D005455:Fluorescent Antibody Technique; D005821:Genetic Techniques; D006801:Humans; D008297:Male; D020554:Sperm Injections, Intracytoplasmic; D013094:Spermatozoa / Q000502:physiology; D015053:Zygote / Q000502:physiology*
chemical_list: C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D003596:Cytosine
keywords: 5-Hydroxymethylcytosine (5hmC); Abnormal fertilization; Centrosome; Monopronuclear (1PN) zygote; Tripronuclear (3PN) zygote