Hiromitsu Shirasawa

Novel method for immunofluorescence staining of mammalian eggs using non-contact alternating-current electric-field mixing of microdroplets.

Recently, a new technique was developed for non-catalytically mixing microdroplets. In this method, an alternating-current (AC) electric field is used to promote the antigen-antibody reaction within the microdroplet. Previously, this technique has only been applied to histological examinations of flat structures, such as surgical specimens. In this study, we applied this technique for the first time to immunofluorescence staining of three-dimensional structures, specifically, mammalian eggs. We diluted an antibody against microtubules from 1:1,000 to 1:16,000, and compared the chromatic degree and extent of fading across dilutions. In addition, we varied the frequency of AC electric-field mixing from 5 Hz to 46 Hz and evaluated the effect on microtubule staining. Microtubules were more strongly stained after AC electric-field mixing for only 5 minutes, even when the concentration of primary antibody was 10 times lower than in conventional methods. AC electric-field mixing also alleviated microtubule fading. At all frequencies tested, AC electric-field mixing resulted in stronger microtubule staining than in controls. There was no clear difference in a microtubule staining between frequencies. These results suggest that the novel method could reduce antibody consumption and shorten immunofluorescence staining time.Recently, a new technique was developed for non-catalytically mixing microdroplets. In this method, an alternating-current (AC) electric field is used to promote the antigen–antibody reaction within the microdroplet. Previously, this technique has only been applied to histological examinations of flat structures, such as surgical specimens. In this study, we applied this technique for the first time to immunofluorescence staining of three-dimensional structures, specifically, mammalian eggs. We diluted an antibody against microtubules from 1:1,000 to 1:16,000, and compared the chromatic degree and extent of fading across dilutions. In addition, we varied the frequency of AC electric-field mixing from 5 Hz to 46 Hz and evaluated the effect on microtubule staining. Microtubules were more strongly stained after AC electric-field mixing for only 5 minutes, even when the concentration of primary antibody was 10 times lower than in conventional methods. AC electric-field mixing also alleviated microtubule fading. At all frequencies tested, AC electric-field mixing resulted in stronger microtubule staining than in controls. There was no clear difference in a microtubule staining between frequencies. These results suggest that the novel method could reduce antibody consumption and shorten immunofluorescence staining time.

Preventive effect of human papillomavirus vaccination on the development of uterine cervical lesions in young Japanese women

To examine the effect that human papillomavirus (HPV) vaccination has had in Akita Prefecture, by comparing cervical cytology results from women aged 20–24 years with or without HPV vaccination.

In vitro maturation and cryopreservation of oocytes retrieved from intra-operative aspiration during second enucleation for ovarian tumor: A case report

Highlights • We reported oocyte collection from an ovarian tumor with a single ovary.• Intra-operative retrieval of oocytes may be useful for preserving fertility.• We have done in vitro maturation for immature oocytes with ovarian enucleation.

Time-lapse monitoring reveals that vitrification increases the frequency of contraction during the pre-hatching stage in mouse embryos

Contraction during the blastocyst stage is observed during embryonic development of various mammals, including humans, but the physiological role of this process is not well understood. Using time-lapse monitoring (TLM), we studied the influence of vitrification and contractions on embryonic development in mice. Mouse embryos were cultured at the 2-cell stage. At the 8-cell stage, embryos were randomly divided into a fresh group (FG) and vitrified group (VG) and observed for up to 144 h. Strong contractions (i.e., contractions causing a decrease in volume of more than 20% and expansion of the perivitelline space) occurred significantly more often in unhatched embryos than hatching embryos in both groups. Regarding hatching embryos, contractions in the pre-hatching stage were significantly more frequent in the VG than the FG. Furthermore, mRNA expression levels of genes related to contractions were determined at three time points, the 8-cell stage, early blastocyst stage, and 20 h after blastocoel formation, with quantitative reverse transcription-polymerase chain reaction. There was no significant difference in Hspa1a expression between the FG and VG, but Hspa1a overexpression was observed just after thawing and tended to decrease gradually thereafter in some blastocysts. Furthermore, in the VG, Atp1a1 tended to show higher expression in the strong contraction group than in the weak contraction group. Overall, vitrification is an excellent method for cryopreservation but could increase contractions in the pre-hatching stage and may increase energy demands of the embryo. Observation of contraction by TLM may improve the evaluation of embryo quality.

In vitro maturation of human immature oocytes for fertility preservation and research material

In recent years, the importance of fertility preservation (FP) has increased. In vitro maturation (IVM), an important technique in FP, has started to be used in the clinic, but controversies persist regarding this technique. Here, a survey of IVM for FP is provided.

Perinatal Management of Pregnancy Complicated by Autosomal Dominant Emery–Dreifuss Muscular Dystrophy

Introduction Autosomal dominant Emery–Dreifuss muscular dystrophy (AD-EDMD) is rare compared with other forms of muscular dystrophy and is characterized by cardiac conduction defects. Here, we present the case of a patient diagnosed with AD-EDMD during the first trimester of pregnancy who developed acute preeclampsia and subsequently, congestive heart failure (CHF) following cesarean section. Case A 36-year-old, gravida 0 para 0 woman was diagnosed with AD-EDMD by genetic testing during the first trimester of pregnancy, and she suddenly developed preeclampsia and partial HELLP (hemolytic anemia, elevated liver enzymes, and low platelets) syndrome at 33 weeks of gestation. The patient subsequently developed CHF following cesarean section. Conclusion CHF can occur as a direct result of the cardiac defects arising due to EDMD, and therefore, careful prenatal and postpartum management is recommended for such cases.

A case of acute aortic dissection in a woman with Marfan syndrome at 29 weeks’ gestation

If severe, e.g. ectopic vesicae, a separate corrective procedure is required. During the 7th week of embryo development, the cloacal membranes separate, creating the anal opening for the hindgut and ventral opening for the urogenital sinus with the perineal body forming between the two. Th e posterior aspect closes with the ectoderm and recanalises 2 weeks later. Development of imperforate anus is caused by a lack of recanalisation during the 9th week (Herman and Teitelbaum 2012). Concurrent ectopic positioning of the anal opening results in a fi stula. Imperforate anus is commonly categorised into ‘ high ’ and ‘ low ’ lesions, depending on whether the rectum passed through the levator muscle (Waters 1982). Low lesions are less severe and may be repaired in a single stage procedure without the need for a colostomy. Our patient had a high lesion without fi stula. Th e absence of a fi stula predisposes to bowel obstruction and therefore a diverting colostomy was performed early in life. PSARP was fi rst described by DeVries and Pe ñ a in 1982 for the repair of high anorectal malformations. A median sagittal incision from the sacrum to the anal dimple allows generous exposure of the anorectal region. Th e superfi cial and deep layers of the external sphincter are identifi ed by electrostimulation and split in the midline. Th e dorsal and ventral portions of the levators are divided to allow the terminal bowel through. Th e bowel is frequently ectatic and needs to be tailored prior to reconstruction of the sphincter. Th e rectum and vagina may also be closely joined and share a common wall. Th is requires careful dissection and mobilisation of the terminal bowel. An anal canal is constructed and the bowel mucosa is sutured to the skin to create the anal opening. Th ere is a paucity of evidence regarding the best mode of delivery for such patients. Breech (2010) suggested that patients who have had a simple repair of low imperforate anus are candidates for vaginal delivery, provided the perineal body is adequate (at least 3 cm; www.bardmedical.com/POPQ). Patients with repair of cloacal anomaly (confl uence of urinary tract, vagina and rectum), extensive surgical repair, a neovagina or interposition graft should be delivered abdominally. A consultant should be present, as there is a risk of operative diffi culty due to adhesions, distorted anatomy and potential risk of injury to bowel and bladder. Th e patient should be counselled appropriately. An increasing number of women who have undergone such corrective surgery will present to the obstetrician in pregnancy. Early consultant obstetrician involvement is crucial to make a defi nitive care plan for such patients. A full history and clinical assessment of continent status, strength of pelvic fl oor and the size and extent of scarring of the perineal body will assist in deciding the best mode of delivery. Details of the type of lesion (high or low) and corrective surgery(ies) performed should be obtained from the patient ’ s GP or paediatric hospital. A colorectal opinion may be sought, as in this case.

Associations of environmental exposures to methylmercury and selenium with female infertility: a case–control study

Background: Methylmercury exposure is a common health risk resulting from daily fish intake. However, studies addressing the link between methylmercury and infertility are limited and also inconsistent. In addition, no previous epidemiological studies have accounted for the interaction between methylmercury and selenium. We aimed to investigate the association between environmental exposures to metals and female fertility. Methods: This case‐control study included 98 infertile women receiving fertility treatment (infertile group) and 43 female workers in their thirties (control group) who provided blood samples and returned a questionnaire on lifestyles and dietary characteristics. Blood levels of mercury, lead, cadmium, arsenic, manganese, zinc, and selenium were compared between the groups. Spearman correlation analyses between anti‐Müllerian hormone and the metals were conducted. Results: The mean selenium level in blood (± SD) and the selenium/mercury molar ratio were significantly lower in the infertile group (189 ± 25 &mgr;g/L and 94.6 ± 44.3, respectively) than in the control group (200 ± 25 &mgr;g/L and 118.4 ± 70.5). By contrast, blood mercury levels after adjusting for blood selenium and age were significantly higher in the infertile group than in the control group. Multiple logistic regression analyses with the adjustment for the other metals and potential confounders confirmed significant associations of infertility with elevated mercury and reduced selenium levels. No significant correlations were observed between anti‐Müllerian hormone and metals. Conclusions: Methylmercury and selenium exposures appear to have adverse and protective effects on female fertility, respectively. This is the first report to suggest the antagonistic interaction between methylmercury and selenium in relation to human female fertility.

The location of “8”-shaped hatching influences inner cell mass formation in mouse blastocysts

The hatching of a blastocyst where the blastocyst portions on the inside and the outside of the zona pellucida feature a figure-of-eight shape is termed “8”-shaped hatching; this type of hatching has been reported to affect the proper presentation of the inner cell mass (ICM) in both human and mouse embryos. Here, our aim was to investigate the factors that affect ICM presentation during “8”-shaped hatching. We performed IVF by using B6D2F1 female mice and ICR male mice, and used the 104 captured blastocysts. Embryos were maintained in KSOM at 37°C in a 5% CO2, 5% O2, and 90% N2 environment, and their growth behavior was monitored individually and continuously using time-lapse cinematography. At 120 h after insemination, embryos were immunostained and examined under a confocal microscope. We used the hatching form to identify “8”-shaped hatching, and we classified the “8”-shaped-hatching blastocysts into two groups, one in which the hatching site was near the ICM center, and the other in which the hatching site was far from the ICM center. We measured each group for ICM size and the number of Oct3/4-positive cells. Of the 95 hatching or hatched embryos, 74 were “8”-shaped-hatching blastocysts, and in these embryos, the ICM was significantly wider when the hatching site was near the ICM than when the hatching site was far from the ICM (P = 0.0091). Moreover, in the “8”-shaped-hatching blastocysts in which the ICM was included in the blastocyst portion outside the zona pellucida―the portion defined as the “outside blastocyst”―after the collapse of this outside blastocyst, the ICM adhered to the trophectoderm of the outside blastocyst, opposite the hatching site. Our results indicate that in “8”-shaped-hatching blastocysts, the hatching site and the collapse of outside blastocyst affect ICM formation. Thus, the assessment of “8”-shaped hatching behaviors could yield indices for accurately evaluating embryo quality.

A case of placental site trophoblastic tumor complicating nephrotic syndrome in which hysteroscopic biopsy did not yield a definitive diagnosis

Placental site trophoblastic tumor (PSTT) is the rarest subtype of gestational trophoblastic neoplasm. We present a case of PSTT complicating nephrotic syndrome. A 32-year-old woman experienced irregular menstrual bleeding and lower extremity edema 18 months after delivery. She was diagnosed with nephrotic syndrome and exaggerated placental site based on the hysteroscopic biopsy results. During follow-up, transvaginal color Doppler ultrasound showed an enlarged uterus filled with a hypervascular mass. Positron emission tomography–computed tomography showed diffuse accumulation in the entire uterus. The patient was diagnosed with PSTT only after total hysterectomy. Postoperatively, serum β-human chorionic gonadotropin decreased to within the normal range and her nephrotic syndrome resolved. She has remained without evidence of recurrence for 15 months. It is difficult to diagnose PSTT definitively. Most patients with PSTT are of reproductive age, therefore, to maintain fecundity, therapy development is expected.