Chromosome screening using culture medium of embryos fertilised in vitro: a pilot clinical study.

BACKGROUND: Previous studies from this as well as other research groups suggested that non-invasive chromosome screening (NICS) with embryo culture medium can be used to identify chromosomal ploidy and chromosomal abnormalities. We here report a series of clinical cases utilizing the technology. METHODS: A total of 45 couples underwent in vitro fertilisation during a period between February 2016 and February 2017. Karyotyping revealed normal chromosomes in both partners in 23 couples, and chromosomal rearrangements in at least one partner in 22 couples. Intracytoplasmic sperm injection (ICSI) was used for fertilization. NICS was carried out using embryo culture medium at the blastocyst stage via multiple annealing and looping-based amplification cycles, whole-genome amplification and next-generation sequencing. RESULTS: A total of 413 embryos were obtained; 170 blastocysts were subjected to NICS. The screening showed euploidy in 79 embryos, aneuploidy in 52 embryos, and mosaic ploidy for 33 embryos. The rate of euploidy was comparable in couples with normal karyotype (50.7%; 38/75) vs. chromosomal rearrangement (43.2%; 41/95). A total of 52 euploid embryos (50 oocyte retrieval cycles) were transferred in 43 women. Biochemical pregnancy rate was 72.0% (36/50). Clinical pregnancy rate was 58.0% (29/50). The rate of spontaneous miscarriage was 3/29 (none with chromosomal aneuploidy). A total of 27 healthy babies were delivered. CONCLUSIONS: NICS could identify embryo chromosomal abnormalities in couples either with or without chromosomal rearrangement, with satisfying clinical outcomes.

Noninvasive chromosome screening of human embryos by genome sequencing of embryo culture medium for in vitro fertilization.

Preimplantation genetic screening (PGS) is widely used to select in vitro-fertilized embryos free of chromosomal abnormalities and to improve the clinical outcome of in vitro fertilization (IVF). A disadvantage of PGS is that it requires biopsy of the preimplantation human embryo, which can limit the clinical applicability of PGS due to the invasiveness and complexity of the process. Here, we present and validate a noninvasive chromosome screening (NICS) method based on sequencing the genomic DNA secreted into the culture medium from the human blastocyst. By using multiple annealing and looping-based amplification cycles (MALBAC) for whole-genome amplification (WGA), we performed next-generation sequencing (NGS) on the spent culture medium used to culture human blastocysts (n = 42) and obtained the ploidy information of all 24 chromosomes. We validated these results by comparing each with their corresponding whole donated embryo and obtained a high correlation for identification of chromosomal abnormalities (sensitivity, 0.882, and specificity, 0.840). With this validated NICS method, we performed chromosome screening on IVF embryos from seven couples with balanced translocation, azoospermia, or recurrent pregnancy loss. Six of them achieved successful clinical pregnancies, and five have already achieved healthy live births thus far. The NICS method avoids the need for embryo biopsy and therefore substantially increases the safety of its use. The method has the potential of much wider chromosome screening applicability in clinical IVF, due to its high accuracy and noninvasiveness.

Characterization of a pituitary-tumor-derived cell line, TtT/GF, that expresses Hoechst efflux ABC transporter subfamily G2 and stem cell antigen 1.

The anterior lobe of the pituitary gland is composed of five types of endocrine cells and of non-endocrine folliculo-stellate cells that produce various local signaling molecules. The TtT/GF cell line is derived from pituitary tumors, produces no hormones and has folliculo-stellate cell-like characteristics. The biological function of TtT/GF cells remains elusive but several properties have been postulated (support of endocrine cells, control of cell proliferation, scavenger function). Recently, we observed that TtT/GF cells have high resistance to the antibiotic G418 and low influx for Hoechst 33342, indicating the presence of ATP-binding cassette (ABC) transporters that efflux multiple drugs, i.e., a property similar to that of stem/progenitor cells. Therefore, we examine TtT/GF cells for the presence of ABC transporters, for the efflux ability of Hoechst 33342 and for those genes characteristic of TtT/GF cells. Real-time polymerase chain reaction (PCR) for ABC transporters demonstrated that Abcb1a, Abcb1b and Abcg2, regarded as stem cell markers, were characteristically expressed in TtT/GF cells but not in Tpit/F1 and LßT2 cells. Furthermore, the remarkable low-efflux ability of Hoechst 33342 from TtT/GF cells was confirmed by using inhibitors and contrasted with the abilities of Tpit/F1 and LßT2 cells. The high and specific expression of stem cell antigen 1 (Sca1) in TtT/GF cells was confirmed by real-time PCR. We also demonstrated those genes that are expressed abundantly and characteristically in TtT/GF, suggesting that TtT/GF cells have unique characteristics similar to those of stem/progenitor cells of endothelial or mesenchymal origin. Thus, the present study has revealed an intriguing property of TtT/GF cells, providing a new clue for an understanding of the function of this cell line.

Alterations in the frequency of trinucleotide repeat dynamic mutations in offspring conceived through assisted reproductive technology.

STUDY QUESTION: How does the frequency of trinucleotide repeat dynamic mutations in offspring conceived through assisted reproductive technology (ART) compare with the frequency of these mutations in control offspring conceived from spontaneous pregnancies? SUMMARY ANSWER: There is a slight increase in dynamic mutation instability in offspring conceived through ART compared with the naturally conceived offspring. WHAT IS KNOWN ALREADY: There is evidence to suggest that ART can increase the risk of birth defects and karyotypic abnormalities. However, the accumulating evidence of an association between ART and de novo genetic aberrations is controversial. STUDY DESIGN, SIZE, DURATION: A prospective clinical observational study was performed on 246 families recruited from an in vitro fertilisation (IVF) centre at a tertiary-care, university-affiliated teaching hospital from 2008 to 2012. The study included 147 ART families [75 IVF and 72 intracytoplasmic sperm injection (ICSI)] in the study group and 99 natural-conception families in the control group. PARTICIPANTS, SETTING, METHODS: Parental, umbilical cord and infant peripheral blood samples were collected, and the trinucleotide repeats of the ATN1, AR, ATXN1, ATXN3, Huntington, DMPK and FMR-1 genes were investigated between the generations; these genes were chosen due to their ability to undergo dynamic mutation. The frequencies and sizes of the mutational repeats, as well as the intergenerational instability, were measured. MAIN RESULTS AND THE ROLE OF CHANCE: In 2466 transmissions identified in the ART offspring, 2.11% (n = 52/2466) of the alleles were unstable upon transmission, while in the control group offspring, the frequency of dynamic mutation was 0.77% (n = 10/1300); this difference was statistically significant (P < 0.01). The unstable transmission alleles were detected in 32 (2.48%) of the 1288 alleles from the IVF offspring and in 20 (1.70%) of the 1178 alleles from the ICSI offspring; both of these frequencies were significantly different from that of naturally conceived offspring (0.77%) (P < 0.01 and P < 0.05, respectively). However, there were no significant differences in the sizes of the mutational repeats or in the rates of expansion or contraction among the three groups (P > 0.05). The repeat copy numbers of the examined genes were found to be within the normal ranges in all parents and infants. LIMITATIONS, REASONS FOR CAUTION: One strength of our study is the relatively large sample size; we were able to detect mutations in seven common dynamic genes, and this large sample size allowed us to detect unstable alleles. Although we observed a clear alteration in the frequency of dynamic mutation in the ART offspring compared with controls, further studies are urgently needed to confirm this observation and determine the cause of this phenomenon. WIDER IMPLICATIONS OF THE FINDINGS: DNA microsatellite analysis provides an important tool to assess genomic instability. In this study, we report an association between ART and the frequency of dynamic mutation. The instability could be a reflection of the core infertility problem, the controlled ovarian hyperstimulation and/or the in vitro culture conditions.

Detection of human herpesviruses (HHVs) in semen of human male infertile patients.

Recently we demonstrated an ectopic expression of the human herpesvirus 1 thymidine kinase (HHV1-TK) gene by functioning of an intrinsic endogenous promoter in the transgenic rat (TG-rat), suggesting that HHV1 infection in humans induces expression of the TK gene with the ectopic promoter in the testis and results in accumulation of HHV1-TK protein, triggering male infertility similar to that in the TG-rat. Hence, in this study, we started to investigate a relationship between infection of herpesvirus and human male infertility. Semen was donated by Chinese male infertile patients (153 men, aged 21-49 years) with informed consent, followed by DNA preparation and analysis by PCR and DNA sequencing. Semen volume, sperm number and density, and sperm motility were examined. DNAs of HHV1, HHV4, HHV5 and HHV6 were confirmed by PCR, electrophoresis and DNA sequencing. Finally, virus DNA was identified in 59 patients (39%). The number of carriers was 39 (25%) for HHV1, 6 (4%) for HHV4, 33 (22%) for HHV5 and 3 (2%) for HHV6, respectively. Moreover, double-infection was found in 22 out of 59 specimens (37%), most of which were double-infection of HHV1 and HHV5 (15 out of 22 carriers). Though slight severity was present in some of the carriers, the relationship between virus infection and sperm impairment was not conclusive. Accordingly, it is essential to examine whether the viral HHV1-TK gene is expressed in the testis of the infertile human HHV carrier.

[A new puncture needle with multiple holes for testicular sperm extraction].

OBJECTIVE: To evaluate a new puncture needle with multiple holes (National Invention Patent of China: ZL 2010202466554) in testicular sperm extraction for infertile males. METHODS: This study included 215 azoospermia patients, who underwent testicular sperm extraction with a new puncture needle with multiple holes (group A, n = 133), by open biopsy (group B, n = 37), or with a fine needle (group C, n = 45). RESULTS: The first-time success rate was 100% in group A, 19% in B and 100% in C. The average operation time was obviously shorter in group A ([3 +/- 1] min) than in B ([15 +/- 3] min) and C ([7 +/- 2] min). The rate of postoperative complications was 3.0% in group A, significantly lower than in B (21.6%) and C (11.1%). CONCLUSION: The new puncture needle with multiple holes, with its advantages of accuracy, high first-time success rate, minimal invasiveness and low rate of complications, deserves to be generally applied in testicular sperm extraction.

Accumulated HSV1-TK proteins interfere with spermatogenesis through a disruption of the integrity of Sertoli-germ cell junctions.

Transgenic rats show spermatid-specific ectopic expression of the reporter gene, herpes simplex virus type1 thymidine kinase (HSV1-TK), in the testes and have demonstrated male infertility. However, the disruption of spermatogenesis and the underlying molecular mechanisms in these transgenic animals have not been well clarified. In this study, light and electron microscopic observations were performed to characterize the morphological changes in the testes. To explore the molecular mechanisms of male infertility in the HSV1-TK transgenic rat, cDNA microarray and quantitative real-time PCR analyses were performed. The seminiferous tubules of 3-month-old transgenic rats showed morphological alterations including seminiferous epithelial sloughing, vacuolization, and degeneration of spermatogenic cells, suggesting a failure of Sertoli-germ cell interaction. Components of the epididymal lumen from transgenic rats included abnormal spermatozoa, degenerating round spermatids and abnormal elongated spermatids indicating an appearance of direct impairment of spermiogenesis. cDNA microarray and real-time PCRanalyses revealed significant changes (P<0.05) in the gene expression level in six genes, testin, versican, mamdc1, fgf7, ostf1 and cnot7. Among them, testin drew most of our attention, since the testin gene is a sensitive marker for disruption of Sertoli-germ cell adhesion. Thus, our results suggest that the accumulation of HSV1-TK in the spermatids not only directly interferes with spermiogenesis but also disrupts spermatogenesis through a disruption of Sertoli-germ cell adhesions. It is important to explore the testicular actions of the HSV1-TK protein in transgenic experimental models and thereby gain clues to find an appropriate treatment for HSV-infected patients exhibiting human male infertility, as has been recently observed.

Failure to detect significant association between estrogen receptor-alpha gene polymorphisms and endometriosis in Japanese women.

OBJECTIVES: The aim of the study was to test whether estrogen receptor 1 (ESR1) gene polymorphisms are correlated with the risk of the development of endometriosis in Japanese women, as a preliminary study. METHODS: To compare allelic frequencies and genotype distributions, a case-control study of 100 affected women and 143 women with no evidence of disease was performed using 10 microsatellite repeat markers and 66 single-nucleotide polymorphisms (SNPs) in the ESR1 gene region. RESULTS: Although our results might be insufficient to detect genetic susceptibility, owing to the small sample size and low genetic power, statistical analysis of the differences in allelic frequency between the cases and controls at each microsatellite locus demonstrated that no microsatellite locus in the ESR1 gene displayed a significant association with the disease when multiple testing was taken into account. Also, there were no statistically significant differences in the SNP allele frequencies and genotypes between the cases and controls when multiple testing was taken into account. CONCLUSION: The findings in our pilot study suggest that ESR1 polymorphisms do not contribute to endometriosis susceptibility.

Lack of an association human dioxin detoxification gene polymorphisms with endometriosis in Japanese women: results of a pilot study.

OBJECTIVES: Endometriosis is a chronic disease caused by the presence of endometrial tissue in ectopic locations outside the uterus. Chronic exposure to the environmental pollutant dioxin has been correlated with an increased incidence in the development of endometriosis in non-human primates. We have therefore examined whether there is an association between the polymorphisms of ten dioxin detoxification genes and endometriosis in Japanese women. METHODS: This was a pilot study in which 100 patients with endometriosis and 143 controls were enrolled. The prevalence of five microsatellite and 28 single nucleotide polymorphism markers within ten dioxin detoxification genes (AhR, AHRR, ARNT, CYP1A1, CYP2E1, EPHX1, GSTM1, GSTP1, GSTT1, NAT2) was examined. RESULTS: Taking into account that this analysis was a preliminary study due to its small sample size and genetic power, the results did not show any statistically significant difference between the cases and controls for any of the allele and genotype frequency distributions examined. In addition, no significant associations between the allele/genotype of all polymorphisms and the stage (I-II or III-IV) of endometriosis were observed. CONCLUSION: Based on the findings of this pilot study, we conclude the polymorphisms of the ten dioxin detoxification genes analyzed did not contribute to the etiology of endometriosis among our patients.

Placental and maternal serum inhibin A in patients with preeclampsia and small-for-gestational-age.

AIM: The purpose of this study was to determine the levels of inhibin A simultaneously in the maternal serum and placental extract in preeclampsia (PE) with or without small-for-gestational-age (SGA) and normal controls at term, and to evaluate the relationship among changes in serum and placental inhibin A according to the severity of PE and PE with or without SGA. MATERIAL AND METHODS: This study involved 40 pregnant women; normal (n = 20), and PE (n = 20), the latter of who were classified into (i) mild (n = 10) and severe PE (n = 10); (ii) PE with SGA (n = 7) and without SGA (n = 13). Inhibin A concentrations were quantified by enzyme-linked immunosorbent assay (ELISA) in the maternal serum and placental extract. Inhibin-α subunit in the placenta was stained by immunohistochemistry (IHC), and its intensity was graded by a semiquantitative scoring method. RESULTS: There was a positive correlation in inhibin A concentrations between the serum and placental extract (r = 0.57, P < 0.001). Both maternal serum and placental inhibin A in PE groups were significantly higher than in controls, but there was no severity-dependent increase of inhibin A when compared with mild and severe PE. There was no difference in inhibin A levels between PE with and without SGA. Moreover, the inhibin-α subunit was predominantly abundant in the cytoplasm of the syncytiotrophoblasts, where the PE groups showed higher staining intensity than the controls (P < 0.000). CONCLUSION: Serum inhibin A level might be a useful biomarker for diagnosis and monitoring of PE.

An immunohistochemical study of metroplastic surgical specimens from patients with a septate uterus.

Purpose: To elucidate the etiology of recurrent pregnancy loss in patients with congenital uterine anomalies, an immunohistochemical technique was used to quantitatively evaluate the vascular arrangement of septate uteri with respect to vascular density and morphology. Methods: Nine specimens obtained from patients who had undergone metroplastic surgery for the treatment of a septate uterus and 10 control specimens from patients who had undergone a hysterectomy because of cervical carcinoma were used in this study. Formalin-fixed paraffin-embedded uterine specimens were then immunostained for CD34, which is specifically expressed in vascular endothelial cells. Results: The mean blood vessel count (mean ± SD) for the myometrium was 149.7 ± 22.7/field in the septate uteri and 162.2 ± 36.4/field in the control uteri; these values were not significantly different. However, the total vessel cross-sectional areas, as evaluated quantitatively using the KS400 image analysis system, were 10350.4 ± 1024.3 µm2/field for the septate uteri and 12002.9 ± 2232.3 µm2/field for the control uteri; these values were significantly different (p < 0.05). The vessel morphology expressed by vessel irregularity and deformity showed a characteristic change in the septate uterus. Conclusions: A significant difference in the distribution of the blood vessels existed between the septate and control uteri, presumably impairing blood flow in the myometrium and the adverse pregnancy outcome.

PROP1 coexists with SOX2 and induces PIT1-commitment cells.

Prophet of PIT1 (PROP1) is a pituitary-specific factor and responsive gene for the combined pituitary hormone deficiency in Ames dwarf mice and human patients. Our immunohistochemical studies demonstrated that PROP1 is consistently expressed in SOX2-expressing stem/progenitor cells in the rat pituitary from embryonic (E) to postnatal periods. At E13.5, all the cells in Rathke's pouch, the primordium of the pituitary, express PROP1. Afterward, PROP1-positive cells localize along the marginal cell layer, a putative stem cell niche in the pituitary, and stratify in the parenchyma of the anterior pituitary. In the embryonic period, PROP1 coexists transiently with PIT1, which is the anterior pituitary-specific factor and is a target of PROP1, but not any hormones. Thus, the present results imply a regulatory role of PROP1 not only in pituitary organogenesis but also in conversion of PIT1-lineage cells.

HSV type 1 thymidine kinase protein accumulation in round spermatids induces male infertility by spermatogenesis disruption and apoptotic loss of germ cells.

HSV type 1 thymidine kinase (HSV1-TK)-introduced transgenic rodents and HSV-infected humans were reported to suffer male infertility. The present study aimed to find novel clues to clarify the cause of HSV1-TK-induced male infertility using an HSV1-tk transgenic rat line. Two truncated HSV1-TK proteins, 37 and 39kDa, were produced and accumulated in the round spermatids, and their transcription initiation site was identified for the first time at the 65 base downstream of the translation start point of the full-length 43kDa HSV1-TK. Spermatozoa from those young transgenic rats showed malformed heads, looped tails, and missing cell membrane in heads and tails. Furthermore, age-dependent germ cell loss was observed. TUNEL assay suggested that this germ cell loss is caused by increased apoptotic germ cell death. These results suggest that the expression of HSV1-TK in testes brings about not only abnormal spermiogenesis but also a loss of germ cells due to apoptosis. These findings could provide a novel clue to elucidate the molecular mechanism underlying male infertility in transgenic animals and HSV-infected patients.

Presence of human herpes virus 1-thymidine kinase in testis of azoospermic infertile herpes-infected patients.

Infection with human herpes virus 1 (HHV1) is a suspected cause of human male infertility. However, the correlation between HHV1 infection and infertility is still unclear. We have previously generated transgenic rats that ectopically express the HHV1 thymidine kinase gene (HHV1-TK) in post-meiotic spermatids and found they had aberrant spermatogenesis and infertility. Therefore, we hypothesized that human infertility might be caused by HHV1 infection. Here, we examined whether HHV1-TK is expressed in human testis by analyzing the presence of its transcript and protein. Specimens were collected by biopsy from 30 azoospermic infertile male patients. RT-PCR and immunohistochemistry showed that 23 patients were positive for HHV1-TK expression, while seven patients were negative. Thus, we demonstrated HHV1-TK expression, indicating HHV1 infection, in the testis of human azoospermic infertile males for the first time; our findings represent a great advancement toward the verification of our hypothesis that HHV1-TK expression might cause human infertility.

Identification of PRTFDC1 silencing and aberrant promoter methylation of GPR150, ITGA8 and HOXD11 in ovarian cancers.

Methylated promoter CpG islands (CGIs) can be used to find novel tumor-suppressor genes and disease markers. In this study, to identify promoter CGIs aberrantly methylated in human ovarian cancers, we performed a genome-wide screening for differentially methylated DNA fragments using methylation-sensitive-representational difference analysis (MS-RDA). MS-RDA isolated 185 DNA fragments specifically methylated in an ovarian cancer cell line (ES-2), compared with a normal human ovarian surface epithelial cell line (HOSE6-3), and 33 of them were derived from putative promoter CGIs. Ten ovarian cancer cell lines were analyzed by methylation-specific PCR, and seven (GPR150, LOC222171, PRTFDC1, LOC339210, ITGA8, C9orf64 and HOXD11) of the 33 CGIs were methylated in one or more of the cell lines. Their downstream genes were barely expressed in cell lines without unmethylated DNA molecules by quantitative reverse-transcription-PCR. Demethylation of methylated cell lines with 5-aza-2'-deoxycytidine restored expression of two genes (PRTFDC1 and C9orf64). In primary ovarian cancers, CGIs of GPR150 (in 4 of 15 cancers), ITGA8 (2/15), PRTFDC1 (1/15), and HOXD11 (1/15) were methylated. Silencing of PRTFDC1 was revealed here for the first time, and aberrant methylation of GPR150, ITGA8 and HOXD11 could be candidate tumor markers.

Methylation and expression analysis of 15 genes and three normally-methylated genes in 13 Ovarian cancer cell lines.

Aberrant methylation of CpG islands (CGIs) in promoter regions of tumor-suppressor genes causes their silencing, and aberrant demethylation of normally methylated CGIs in promoter regions causes aberrant expression of cancer-testis antigens. Here, we comprehensively analyzed aberrant methylation of 15 genes and demethylation of three normally methylated genes in 13 ovarian cancer cell lines. RASSF1A was most frequently methylated (complete methylation in 7 and partial methylation in 4 cell lines), followed by ESR1 (5 and 2, respectively), FLNC (4 and 4), HAND1 (4 and 2), LOX (3 and 2), HRASLS (3 and 2), MGMT (3 and 0), CDKN2A (3 and 0), THBD (2 and 1), hMLH1 (2 and 0), CDH1 (1 and 1) and GSTP1 (1 and 0). hTERC and TIMP3 were only partially methylated in 7 and 2 cell lines, respectively. BRCA1 was not methylated at all. Aberrant demethylation of MAGE-A3, -B2 and -A1 was detected in 8, 4 and 3 cell lines, respectively. Gene expression was consistently absent in cell lines without unmethylated DNA molecules. Aberrant methylation was frequently observed in MCAS, RMUG-L (mucinous cell carcinomas), RTSG (poorly-differentiated carcinoma) and TYK-nu (undifferentiated carcinoma) while infrequent in HTOA, JHOS-2, and OV-90 (serous cell carcinomas). Aberrant demethylation was frequently observed in OV-90, OVK-18, and ES-2 cell lines. It was shown that aberrant methylation and demethylation were frequently observed in ovarian cancer cell lines, and these data will provide a basis for further epigenetic analysis in ovarian cancers.

Toll-like receptor 4 polymorphisms to determine acute pancreatitis susceptibility and severity: a meta-analysis.

AIM: To investigate the correlation of toll-like receptor 4 (TLR4) gene Asp299Gly and Thr399Ile polymorphisms and acute pancreatitis (AP) risk and severity. METHODS: To get a more precise estimation of the relationship, a comprehensive search was performed to examine all the eligible studies of TLR4 Asp299Gly and Thr399Ile polymorphisms and AP risk. The odds ratios with 95% confidence intervals were used to assess the strength of the association. Publication bias was analyzed by Begg's funnel plots. RESULTS: In total, six studies with 1255 cases and 998 controls were included in this meta-analysis. Totally, no significant associations were found between TLR4 Asp299Gly or Thr399Ile polymorphisms and AP risk using five models with high homogeneity (P > 0.05). Furthermore, stratification analysis by ethnicity or assay also found no significant association in these two polymorphisms (P > 0.05), and TLR4 Asp299Gly was not associated with AP severity (P > 0.05). In addition, no publication bias was found in these studies (P > 0.05). CONCLUSION: Our current meta-analysis suggests that TLR4 Asp299Gly and Thr399Ile polymorphisms may not be risk factors to AP susceptibility.

Does aberrant DNA methylation occur in human uterine leiomyomas? An attempt of genome-wide screening by MS-RDA.

OBJECTIVE: Uterine leiomyoma are very common benign tumors in women of reproductive age. However, the molecular mechanisms of cause and development of these tumors are poorly understood. This study attempts to examine whether or not aberrant DNA methylation occurred in these tumors. METHODS: We carried out a genome-wide screen for aberrant DNA methylation, adopting methylation-sensitive-representational difference analysis (MS-RDA) using normal adjacent myometria as tester and myoma tissue driver. CONCLUSION: A total of 192 clones identified by MS-RDA were sequenced, 27 DNA fragments derived from CpG islands (CGIs) were isolated, and seven of them were from CGI in the 5' regions of known genes, which include CHARC1, FAM44B, FLJ33655, HSUP, MLLT3, SLC16A1, and ZNF96. Then, methylation statuses of those CGIs were analyzed by methylation-specific polymerase chain reaction using 5 primary samples of human uterine leiomyoma. Aberrant DNA methylation did not observed in 7 genes in 5 human uterine leiomyoma eventually. This study is insufficient to identify aberrant DNA methylation occurring in the human uterine leiomyoma, a large population of primary samples and more attempts, such as the use of cell lines or primary monolayer cultures established from tissue samples, are warranted to clarify this issue.

Gene expression of lymphocyte prolactin receptor was suppressed in lactating mothers.

Prolactin (PRL) receptor (PRL-R) was proven to be ubiquitously expressed by cells in the immune system, while the physiological role of PRL was established in milk production in mammary glands. We analyzed the mRNA content of PRL-R in human lymphocytes in normo- and hyperprolactinemic conditions to document the presence of functioning PRL-R of human lymphocytes. Blood samples were obtained prior to treatment, and with written informed consent, from outpatients with ovarian dysfunction and hyperprolactinemia (n = 8; 19 ~ 41 y/o), from breast-feeding mothers after normal delivery (n = 12; 27 ~ 36 y/o), and from healthy volunteers: men (n = 9; 33 ~ 40 y/o) and women (n = 9; 26 ~ 36 y/o). Subsequently, total RNA was prepared from the lymphocytes separated. The quantity of PRL-R mRNA was examined by reverse transcription and polymerase chain reaction and normalized with a simultaneously measured amount of b actin. The resultant mRNA level of PRL-R was analyzed for its correlation with serum concentration of PRL measured by immunoassay. PRL-R mRNA levels of lymphocytes were significantly suppressed in lactating mothers, while there was a statistically significant negative correlation between PRL-R mRNA and serum PRL levels. However, there was no significant difference of PRL-R mRNA in the pathological condition of outpatients with ovarian dysfunction and/or hyperprolactinemia. While a few investigators reported the extra-mammary regulation on PRL-R by PRL, our data suggest that the PRL-R levels of circulating lymphocytes could be down-regulated by the elevated serum levels of PRL and that pituitary PRL may participate in regulating the expression of PRL-R genes on cells of the human immune system, especially in physiological circumstances such as in the postpartum period.

The highly related LIM factors, LMO1, LMO3 and LMO4, play different roles in the regulation of the pituitary glycoprotein hormone alpha-subunit (alpha GSU) gene.

LMO1, LMO3 and LMO4 were cloned from the adult porcine pituitary cDNA library. Amino acid sequences of porcine LMO1, LMO3 and LMO4 were highly conserved among mammalian species. Transfection assay of the pituitary-derived cell line L beta T2 was carried out using the pituitary alpha GSU (glycoprotein hormone alpha-subunit) promoter (-1059/+12 b) fused to pSEAP2-Basic vector as a reporter gene. The results demonstrated that, whereas LMO4 showed no apparent effect, alpha GSU promoter activity was markedly repressed by LMO1 but activated by LMO3, indicating the different roles of the three highly homologous proteins, LMO1, LMO3 and LMO4. Knockdown assay by LMO siRNAs (small interfering RNAs) confirmed the above results for LMO1 and LMO3, whereas that by LMO4 siRNA increased the expression, indicating different modes of action. RT-PCR (reverse transcription-PCR) for total RNAs of several cell lines showed that LMO1 and LMO4 mRNAs were present ubiquitously in all cell lines, except for LMO1 in L929 cells. In contrast, LMO3 mRNA was abundant only in L beta T4 and GH3 cells with only small amounts in L beta T2 and MtT/S cells, indicating the cell-type-specific function of this protein. Real-time analyses of porcine pituitary ontogeny revealed that the three LMO genes are expressed during the fetal period and decline immediately afterwards, followed by a remarkably low level of LMO3 and LMO4 after birth. RT-PCR of the porcine tissues examined showed ubiquitous expression of LMO4, whereas LMO1 and LMO3 are expressed tissue specifically. Thus the present study demonstrated that three highly related LIM cofactors, LMO1, LMO3 and LMO4, have different effects on alpha GSU gene expression in the pituitary glands.