Digital multiplexed mRNA analysis of functionally important genes in single human oocytes and correlation of changes in transcript levels with oocyte protein expression.

OBJECTIVE: To investigate functionally important transcripts in single human oocytes with the use of NanoString technology and determine whether observed differences are biologically meaningful. DESIGN: Analysis of human oocytes with the use of NanoString and immunoblotting. SETTING: University-affiliated reproductive medicine unit. PATIENTS: Women undergoing in vitro fertilization. INTERVENTION: Human oocytes were analyzed with the use of NanoString or immunoblotting. MAIN OUTCOME MEASURES: The abundance of transcripts for ten functionally important genes-AURKA, AURKC, BUB1, BUB1B (encoding BubR1), CDK1, CHEK1, FYN, MOS, MAP2K1, and WEE2-and six functionally dispensable genes were analyzed with the use of NanoString. BubR1 protein levels in oocytes from younger and older women were compared with the use of immunoblotting. RESULT(S): All ten functional genes but none of the six dispensable genes were detectable with the use of NanoString in single oocytes. There was 3- to 5-fold variation in BUB1, BUB1B, and CDK1 transcript abundance among individual oocytes from a single patient. Transcripts for these three genes-all players within the spindle assembly checkpoint surveillance mechanism for preventing aneuploidy-were reduced in the same oocyte from an older patient. Mean BUB1B transcripts were reduced by 1.5-fold with aging and associated with marked reductions in BubR1 protein levels. CONCLUSION(S): The abundance of functionally important transcripts exhibit marked oocyte-to-oocyte heterogeneity to a degree that is sufficient to affect protein expression. Observed variations in transcript abundance are therefore likely to be biologically meaningful, especially if multiple genes within the same pathway are simultaneously affected.

Immunofluorescence staining of spindles, chromosomes, and kinetochores in human oocytes.

Understanding how human oocytes execute chromosome segregation is of paramount importance as errors in this process account for the overwhelming majority of human aneuploidies and increase exponentially with advancing female age. The spindle is the cellular apparatus responsible for separating chromosomes at anaphase. For accurate chromosome segregation, spindle microtubules must establish appropriately configured attachments to chromosomes via kinetochores. With regard to understanding the mechanistic basis for human aneuploidies therefore, it will be important to explore the molecular underpinnings of spindle structure and the interaction of its microtubules with chromosomes in human oocytes. Here we describe a technique for simultaneously immunolabelling chromosomes, spindle microtubules and kinetochores in human oocytes.

Adenomyosis reduces pregnancy rates in infertile women undergoing IVF.

High-resolution transvaginal ultrasound has facilitated the diagnosis of adenomyosis. This study determined the prevalence of this finding in infertile women and its effect on the outcome of IVF/intracytoplasmic sperm injection (ICSI). This prospective study evaluated 275 consecutive women, commencing IVF/ICSI for the first time. Inclusion criteria were adequate ovarian reserve. Women with fibroids or a previous myomectomy were excluded. All women were screened for adenomyosis by transvaginal ultrasound on three separate occasions. The control group included 256 women and the adenomyosis group included 19 women. There was no significant difference in the ages of women, FSH, cause of infertility, body mass index, total dose of gonadotrophin used and number of oocytes collected between the two groups. However, women with adenomyosis had a higher mean antral follicle count (P=0.006). The clinical pregnancy rate (22.2% versus 47.2%) and ongoing pregnancy rate (11.1% versus 45.9%) were significantly lower in women with adenomyosis and the miscarriage rate (50.0% versus 2.8%) was significantly higher in women with adenomyosis (all P<0.001). Ultrasound evidence of adenomyosis is found in a significant number of women presenting with infertility and has a negative impact on the outcome of IVF/ICSI. This paper suggests that a common condition known as adenomyosis is associated with a reduced success following fertility treatment such as IVF. The diagnosis of adenomyosis has been greatly facilitated by the advent of high-resolution transvaginal ultrasound. This was a study including 275 consecutive women who were commencing IVF for the first time. Comparing women who did not have adenomyosis and those that did, the clinical and ongoing pregnancy rates were both lower in women with adenomyosis (22.2% versus 47.2% and 11.1% versus 45.9%, respectively). So, fewer women with adenomyosis became pregnant and had an ongoing pregnancy. The miscarriage rate was higher in women with adenomyosis compared with those without (50.0% versus 2.8%). We conclude that ultrasound evidence of adenomyosis is found in a significant number of women presenting with infertility and has a negative impact on the outcome of IVF.

The distribution and pathological staging of colorectal cancers in ethnic groups.

BACKGROUND: There is little data on the distribution and histological staging of colorectal cancer in ethnic groups in the United Kingdom. A study to investigate this was undertaken at a hospital serving a multi-ethnic population. PATIENTS AND METHODS: Case analysis from a prospective database of all colorectal cancers between 2000-2004 was performed. Data was recorded on the distribution of cancer, operative procedures and Dukes' staging. RESULTS: A total of 256 patients (118 females) were diagnosed with colorectal cancer, of whom 214 (83.6%) underwent resection. There were 39 Afro-Caribbeans 66.2 years +/- 12.6 years; mean [age +/- s.d.], 14 Asians 64 years +/- 12.6 years, 34 Mediterraneans 67.7 years +/- 9.9 years and 176 Caucasian British 74.3 years +/- 11.3 years. Right-sided colonic lesions were more frequent in Afro-Caribbeans, whilst left-sided lesions were more frequent in Mediterraneans. The incidence of Dukes'A cancer was high in Mediterraneans whilst Dukes' C cancer was commoner in Caucasians and Afro-Caribbeans. CONCLUSION: Ethnic patients present with colorectal cancer at significantly younger ages. Afro-Caribbeans have significantly more right-sided and Mediterraneans more left-sided cancer. Afro-Caribbean and Caucasian patients present with more advanced cancer compared to Mediterraneans. This has implications on the investigation, as well as screening in ethnic minority patients.