Does tamoxifen therapy affect the hormone receptor expression and cell proliferation indices of endometrial polyps? An immunohistochemical comparison of endometrial polyps from postmenopausal women exposed and not exposed to tamoxifen.

OBJECTIVE: This study set out to test the null hypothesis that tamoxifen therapy would not affect the hormone receptor expression (oestrogen and progesterone receptors-ER and PR) or markers of cell proliferation/apoptosis (Ki67 and Bcl-2) of endometrial polyps from postmenopausal women exposed and not exposed to tamoxifen. METHODS: Endometrial polyps were prospectively obtained from women presenting with abnormal bleeding attending an out-patient hysteroscopy clinic who subsequently underwent endometrial polypectomy (16 from postmenopausal women not exposed to tamoxifen, 9 from women exposed to tamoxifen). Immunohistochemical staining for ER, PR, Ki67 and Bcl-2 was performed on polyps from both groups of women. Non-parametric statistical analysis was used (Mann-Whitney and Spearmans rank correlation). RESULTS: Endometrial polyps from tamoxifen users had significantly lower oestrogen receptor but increased progesterone receptor and Bcl-2 expression. There were no significant differences for proliferation markers (Ki67) between postmenopausal endometrial polyps exposed and not exposed to tamoxifen. CONCLUSIONS: Tamoxifen has a significant affect on hormone receptor expression and markers of apoptosis in endometrial polyps. The results support the hypothesis that tamoxifen promotes polyp growth by inhibiting apoptosis. The mechanism for this does not appear to be oestrogen receptor mediated.

Are endometrial polyps from pre-menopausal women similar to post-menopausal women? An immunohistochemical comparison of endometrial polyps from pre- and post-menopausal women.

OBJECTIVE: Do endometrial polyps from pre- and post-menopausal women have similar immunohistochemical expression of oestrogen and progesterone receptors (ER, PR) and markers of cellular proliferation/apoptosis (Ki67 and Bcl-2). DESIGN: Prospective cohort study. Non-parametric statistical analysis was used. SETTING: Polyps recruited from women attending an out-patient hysteroscopy clinic in a UK district general hospital. PATIENTS: Fourteen pre-menopausal and 16 post-menopausal women who presented with abnormal bleeding with endometrial polyps. INTERVENTIONS: Immunohistochemical staining was performed on endometrial polyps. MAIN OUTCOME MEASURES: Significant differences or correlations between hormone receptor expression (oestrogen and progesterone) and cell growth indices (Ki67 and Bcl-2). RESULTS: Endometrial polyps from pre- and post-menopausal women had significant differences in their expression of hormone receptors and Ki67. However, polyps from both groups of women had similarly increased levels of Bcl-2, an inhibitor of apoptosis. CONCLUSIONS: Pre- and post-menopausal polyps exhibit differing hormone receptor and proliferation markers, presumably a result of their hormonal milieu. However, both groups appear to have lost the usual control mechanisms for apoptotic regulation, this appears to be responsible for their growth.

An immunohistochemical comparison of endometrial polyps from postmenopausal women exposed and not exposed to HRT.

OBJECTIVE: Our study set out to test the null hypothesis that oestrogen containing continuous combined hormone replacement therapy (HRT) would not affect the hormone receptor expression (oestrogen and progesterone receptors-ER, PR) or markers of cell proliferation/apoptosis (Ki67 and Bcl-2) in endometrial polyps from postmenopausal women exposed and not exposed to HRT. DESIGN: Immunohistochemical staining for ER, PR, Ki67 and Bcl-2 was performed on polyps obtained from two groups of postmenopausal women. SETTING: Polyps were obtained from postmenopausal women attending an outpatient hysteroscopy clinic in a district general hospital (Bradford Royal Infirmary, UK). POPULATION: Twenty-five postmenopausal women presenting with abnormal bleeding subsequently diagnosed with endometrial polyps (16 from women not exposed to HRT, 9 from women exposed to HRT). METHODS: Semiquantitative immunohistochemistry was performed. MAIN OUTCOME MEASURES: Significant differences or correlations in either hormone receptor expression or markers of cell proliferation/apoptosis between the two groups of polyps. RESULTS: There were no significant differences for hormone receptor expression (ER and PR) between endometrial polyps exposed and not exposed to HRT. Bcl-2 expression was higher than Ki67 in both groups, but polyps from HRT users had increased levels reflecting decreased apoptosis in these polyps. CONCLUSIONS: HRT has no demonstrable effect on polyp ER and PR expression. However, HRT does appear to inhibit apoptosis and cell proliferation in endometrial polyps, which may affect polyp growth.

Lack of germline transmission of vector sequences following systemic administration of recombinant AAV-2 vector in males.

A potential consequence of systemic administration of viral vectors is the inadvertent introduction of foreign DNA into recipient germ cells. To evaluate the safety of in vivo recombinant adeno-associated virus (rAAV) mediated gene transfer approaches for hemophilia B, we explored the risk of germline transmission of vector sequences following intramuscular (IM) injection of rAAV in four species of male animals (mouse, rat, rabbit and dog). In vector biodistribution studies in mice and rats, there is a dose-dependent increase in the likelihood that vector sequences can be detected in gonadal DNA using a sensitive PCR technique. However, in dogs DNA extracted from semen is negative for vector sequences. To address this discrepancy, studies were done in rabbits, and both semen and testicular DNAs were analyzed for the presence of vector sequences. These studies showed that no AAV vector sequences were detected in DNA extracted from rabbit semen samples collected at time points ranging from 7 to 90 days following IM injection of 1 x 10(13) vector genomes rAAV (vg) per kg. In contrast, DNA extracted from gonadal tissue was positive for vector sequences, but the positive signals diminished in number and strength with time. By FISH analysis, AAV signals were localized to the testis basement membrane and the interstitial space; no intracellular signal was observed. We observed similar findings following hepatic artery administration of rAAV in rats and dogs, suggesting that our findings are independent of the route of administration of vector. Attempts to transduce isolated murine spermatogonia directly with AAV-lacZ were unsuccessful. In clinical studies human subjects injected IM with an AAV vector at doses up to 2 x 10(12) vg/kg have shown no evidence of vector sequences in semen. Together, these studies suggest that rAAV introduced into skeletal muscle or the hepatic artery does not transduce male germ cells efficiently. We conclude that the risk of inadvertent germline transmission of vector sequences following IM or hepatic artery injection of AAV-2 vectors is extremely low.

The end of the beginning for pluripotent stem cells.

Pluripotent stem cells can be expanded seemingly indefinitely in culture, maintain a normal karyotype and have the potential to generate any cell type in the body. As such they represent an incredible resource for the repair of diseased or damaged tissues in our bodies. These cells also promise to open a new window into the embryonic development of our species.

A national confidential survey of obstetrician's personal preferences regarding mode of delivery.

OBJECTIVE: To survey the personal preferences of obstetricians regarding mode of delivery, and relate these to hospital caesarean section rates. STUDY DESIGN: A confidential, questionnaire based survey to all obstetricians working in the Republic of Ireland (n=234). RESULTS: The response rate was 71% (n=165). Seven percent of Irish obstetricians would choose an elective caesarean section for themselves (or their partners) if they were primigravida with an uncomplicated, singleton cephalic presentation at term in the absence of any clinical indication. Caesarean section was the preferred mode of delivery for 38% of respondents if the estimated foetal weight was 4.5kg. There was a highly significant association between consultant obstetricians' personal preferences of towards caesarean section and their working in a hospital with a caesarean section rate greater than 16% (P<0.005). CONCLUSIONS: Irish obstetricians' personal preferences towards elective caesarean section for an uncomplicated, cephalic pregnancy at term are significantly lower than published data examining London based obstetricians' choices. There is a consistent trend against vaginal delivery if the obstetrician is female or younger. The association between a personal preference of the consultant for abdominal delivery and the caesarean section rate of the hospital that they work in may hamper efforts to decrease the rising numbers of caesarean sections.

Germ cell biology--from generation to generation.

Germ cells hold a unique place in the life cycle of animal species in that they are the cells that will carry the genome on to the next generation. In order to do this they must retain their DNA in a state in which it can be used to recapitulate embryonic development. In the normal life cycle, the germ cells are the only cells that retain this ability to recapitulate development, referred to as developmental totipotency. The molecular mechanisms regulating developmental potency are poorly understood. Recently its has been shown that germ cells can be turned into pluripotent stem cells when cultured in specific polypeptide growth factors that affect their survival and proliferation. The ability to manipulate developmental potency in germ cells with growth factors allows the underlying mechanisms to be dissected. Germ cells are also the only cells that undergo the unique reductive division of meiosis. This too is essential for the ability of germ cells to form the gametes that will carry the genome into the next generation. Arguably meiosis is the most important division in the life of a nascent organism. Defects in meiosis can result in embryonic or fetal loss or, if the animal survives, in the birth of an individual with chromosomal abnormalities. Recent advances in our understanding of meiosis have come from knockout mice and studies on genes identified through studies of human infertility. This review will focus on these two key aspects of germ cell biology, developmental potency and meiosis.

Hamster and rat fetal cells have low spontaneous mutation frequencies and rates.

Somatic cells of whole Syrian hamster fetuses (gestation day 13) were isolated and tested by an in vivo/in vitro mutation assay for spontaneous mutation frequencies using independent 6-thioguanine (6-TG), diphtheria toxin (DT), and ouabain mutation selection systems. Optimum conditions were ascertained. For 6-TG mutants, a total of 21 mutants were found in cells from 24 litters on 1993 plates, for an overall mutant frequency of 1.8 x 10(-7) per viable cell with 12 positive litters. In all, 26 litters were tested using DT; 77 mutants were found in 840 plates, yielding an overall mutant frequency of 2.6 x 10(-7), with 20 positive litters. No correlations or familial effects were found among 23 litters tested for both DT and 6-TG. Of 14 litters which were tested for ouabain mutants, 4 were positive, with a total of 5 mutants found on 988 plates, for an overall mutant frequency of 7.6 x 10(-8). For 14 F344 rat fetuses, the overall 6-TG spontaneous mutation frequency was determined to be 1.6 x 10(-7). From the data, estimates of mutation rates were calculated. For mutation to 6-TG resistance the rate was 8.3 x 10(-8), for mutation to DT resistance the rate was 8.1 x 10(-8) and for ouabain, the spontaneous mutation rate was 5.7 x 10(-8). For F344 rat, the spontaneous mutation rate was 1.1 x 10(-7). Induced mutant frequencies after in utero exposure to 1 mmol/kg N-ethyl-N-nitrosourea (ENU) were 311, 135 and 200 times the spontaneous value for 6-TG, DT and ouabain, respectively, for Syrian hamster fetal cells and 125 times the spontaneous 6-TG value for fetal F344 rat cells. Both spontaneous mutation frequencies and underlying spontaneous mutation rates are low, consistent with the view that fetal cells exercise extremely tight control over DNA fidelity.

Outcome of outpatient micro-hysteroscopy performed for abnormal bleeding while on hormone replacement therapy.

This retrospective observational study was carried out in a large district general hospital to review the outcome of outpatient micro-hysteroscopy performed on women with abnormal bleeding while on hormone replacement therapy. All women referred to the outpatient hysteroscopy unit with abnormal bleeding while on hormone replacement therapy between November 1994 and August 1998 had hysteroscopy performed using a 1.2 mm semi-rigid hysteroscope with a 2.5 mm sheath. Hysteroscopy was performed on 190 women. Ninety-two women (48.4%) had a normal uterine cavity, 38 (20%) had an atrophic endometrium, 52 (27.4%) were found to have endometrial polyps, seven (3.7%) had suspicious endometrium (histology showed two adenocarcinomas and three hyperplasias) and one patient (0.5%) had a submucous fibroid. Histological evaluation showed 145 (76.32%) specimens were benign, 37 (19.47%) specimens either contained no tissue or insufficient tissue for diagnosis, five (2.63%) showed hyperplasia and three (1.58%) were adenocarcinoma. Two hyperplasias and one focal adenocarcinoma were diagnosed in endometrial polyps. Nearly half of the women who had a hysteroscopy for abnormal bleeding while on hormone replacement therapy had a normal endometrial cavity. Almost one-third had endometrial pathology, of which the majority were endometrial polyps. The incidence of endometrial carcinoma was low. No abnormality was missed on hysteroscopy, but histology was normal in two patients with hysteroscopically suspicious endometrium.

Microlaparoscopy.

Microlaparoscopy is defined as using instruments with an outer sheath of less than 2 mm; as such, it represents the leading edge of fiberoptic and instrument design technology. Although still in its infancy, it has been proposed as the new standard instrument for abdominal entry and for the performance of some diagnostic and therapeutic procedures. It is already the instrument of choice for performing conscious laparoscopic procedures. The small size of the instrument makes it versatile, but it is important that they are used appropriately. In this way, microlaparoscopy offers significant advantages over conventional laparoscopy for surgeon and patient alike.

Cell sensitivity to transplacental mutagenesis by N-ethyl-N-nitrosourea is greatest during early gestation in the Syrian hamster.

The extremely high rate of cell division that occurs during early embryogenesis is hypothesized to predispose to high rates of mutation after chemical exposure. We tested this supposition experimentally. To probe the variation in susceptibility to mutation induction as a function of gestation stage, somatic cells of the developing Syrian hamster were isolated after transplacental treatment with N-ethyl-N-nitrosourea (ENU). Mutants were quantified using either 6-thioguanine (6-TG) or diphtheria toxin (DT) as selective agents. Several different approaches were used. In one, three litters were exposed on each gestation day and fetuses were removed on day 13. Maximum fetal sensitivity to ENU's genotoxic action was noted when treatment was at days 8 and 9, fewer mutants being obtained with earlier and later exposures. To compensate for the low numbers of target cells early in gestation, this experiment was repeated using larger numbers of litters exposed at the earlier time points, and the highest mutation frequency was now found to occur after treatment on gestation days 6 and 7. In the second approach, mutations were quantified in cells harvested 24 h after transplacental ENU exposure. Here again, embryos exposed at earlier times of gestation were more susceptible than those treated at later periods. Based on the total cell numbers in embryos and fetuses at each gestation day, we conclude that mutation frequency is maximal on day 6, corresponding to the primitive streak stage with extremely high rates of cell division.

Cell sensitivity to transplacental carcinogenesis by N-ethyl-N-nitrosourea is greatest in early post-implantation development.

In a clear demonstration of the changing sensitivity of the developing mammal to transplacental carcinogenesis, Ivankovic and Druckrey [S. Ivankovic, H. Druckrey, Transplacentare Erzeugung maligner Tumoren des Nervensystem: I. Athyl-nitroso-harnstoff (ANH) an BD IX-Ratten, Z. Krebsforsch. 71 (1968) 320-360] exposed pregnant BD IX rats to a pulse of N-ethyl-N-nitrosourea (ENU), a reactive carcinogen with a half-life of 20 min. No tumors were seen with ENU exposure before gestation day 12, but the multiplicity of neurogenic tumors increased steadily thereafter and was greatest with treatment on day 20, followed by a decline in sensitivity for the last three days of gestation. Similarly, a transplacental study of ENU in the Syrian hamster [B.A. Diwan, S. Rehm, J.M. Rice, Age- and dose-dependent transplacental carcinogenesis by N-nitrosoethylurea in Syrian golden hamsters, J. Cancer Res. Clin. Oncol. 122 (1996) 643-652] found that the numbers of tumors induced were greatest after exposure of late fetal stages. While these observations suggested that embryonic cells are refractory to carcinogenesis, an alternative explanation could be that a significant tumor yield was not observed because too few target cells were present in the embryo. I have resolved this issue by combining these published data with others on the numbers of neuroectodermal cells in the developing BD IX rat brain [R. Müller, M.F. Rajewsky, Elimination of O6-ethylguanine from the DNA of brain, liver, and other rat tissues exposed to ethylnitrosourea at different stages of prenatal development, Cancer Res. 43 (1983) 2897-2904] and total cell counts of successive developmental stages of the Syrian hamster fetus [P.J. Donovan, G.T. Smith, Cell sensitivity to transplacental mutagenesis by N-ethyl-N-nitrosourea is greatest during early gestation in the Syrian hamster, Mutation Res., 1999, this issue], allowing the risk per cell at different stages of gestation to be calculated. Sensitivity to carcinogenesis was found to be greatest early in gestation and to decrease as gestation proceeds. For the rat model, tumor frequency per cell changed from 1.3x10(-6) at day 12 exposure to 2.6x10(-8) at day 23 exposure, a 50-fold decrease. For the hamster model, the tumor-initiation rate decreased 1250-fold from 1.2x10(-5) at day 7 exposure to 9.6x10(-9) at day 13 exposure. Thus, two independent experiments with different rodent species demonstrate that sensitivity of individual cells to damage leading to transplacental carcinogenesis is greatest in the early fetus and lessens markedly as gestation proceeds, in parallel with changing sensitivity to mutation (Donovan et al., Mutat. Res., this issue).

The germ cell--the mother of all stem cells.

The germline, uniquely amongst the lineages of the embryo, carries the genome from generation to generation and is therefore the only lineage which retains true developmental totipotency. Paradoxically, when mouse primordial germ cells (PGCs) are introduced into a host blastocyst, they do not contribute to either the germline or the soma, suggesting that they are restricted in developmental potency. Conversely, in vivo PGCs give rise to embryonal carcinoma (EC) cells, the pluripotent stem cells of teratomas, benign tumors containing derivatives of the three primary germ layers. Similarly, PGCs can be converted in vitro into embryonic germ (EG) cells, pluripotent stem cells capable of giving rise to somatic and germline chimeras. The ability of PGCs to form EC cells in vivo and EG cells in vitro suggests that developmental potency of PGCs is regulateable. The molecular mechanisms controlling PGC growth and differentiation are gradually being elucidated through the characterization of sterile mutants and through the use of in vitro culture systems. Understanding how a PGC can give rise to a pluripotent stem cell could give significant insights into the regulation of developmental totipotency as well as having important implications for male fertility and the etiology of testicular cancer.

AIR-2: An Aurora/Ipl1-related protein kinase associated with chromosomes and midbody microtubules is required for polar body extrusion and cytokinesis in Caenorhabditis elegans embryos.

An emerging family of kinases related to the Drosophila Aurora and budding yeast Ipl1 proteins has been implicated in chromosome segregation and mitotic spindle formation in a number of organisms. Unlike other Aurora/Ipl1-related kinases, the Caenorhabditis elegans orthologue, AIR-2, is associated with meiotic and mitotic chromosomes. AIR-2 is initially localized to the chromosomes of the most mature prophase I-arrested oocyte residing next to the spermatheca. This localization is dependent on the presence of sperm in the spermatheca. After fertilization, AIR-2 remains associated with chromosomes during each meiotic division. However, during both meiotic anaphases, AIR-2 is present between the separating chromosomes. AIR-2 also remains associated with both extruded polar bodies. In the embryo, AIR-2 is found on metaphase chromosomes, moves to midbody microtubules at anaphase, and then persists at the cytokinesis remnant. Disruption of AIR-2 expression by RNA- mediated interference produces entire broods of one-cell embryos that have executed multiple cell cycles in the complete absence of cytokinesis. The embryos accumulate large amounts of DNA and microtubule asters. Polar bodies are not extruded, but remain in the embryo where they continue to replicate. The cytokinesis defect appears to be late in the cell cycle because transient cleavage furrows initiate at the proper location, but regress before the division is complete. Additionally, staining with a marker of midbody microtubules revealed that at least some of the components of the midbody are not well localized in the absence of AIR-2 activity. Our results suggest that during each meiotic and mitotic division, AIR-2 may coordinate the congression of metaphase chromosomes with the subsequent events of polar body extrusion and cytokinesis.

Derivation of pluripotent stem cells from cultured human primordial germ cells.

Human pluripotent stem cells would be invaluable for in vitro studies of aspects of human embryogenesis. With the goal of establishing pluripotent stem cell lines, gonadal ridges and mesenteries containing primordial germ cells (PGCs, 5-9 weeks postfertilization) were cultured on mouse STO fibroblast feeder layers in the presence of human recombinant leukemia inhibitory factor, human recombinant basic fibroblast growth factor, and forskolin. Initially, single PGCs in culture were visualized by alkaline phosphatase activity staining. Over a period of 7-21 days, PGCs gave rise to large multicellular colonies resembling those of mouse pluripotent stem cells termed embryonic stem and embryonic germ (EG) cells. Throughout the culture period most cells within the colonies continued to be alkaline phosphatase-positive and tested positive against a panel of five immunological markers (SSEA-1, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81) that have been used routinely to characterize embryonic stem and EG cells. The cultured cells have been continuously passaged and found to be karyotypically normal and stable. Both XX and XY cell cultures have been obtained. Immunohistochemical analysis of embryoid bodies collected from these cultures revealed a wide variety of differentiated cell types, including derivatives of all three embryonic germ layers. Based on their origin and demonstrated properties, these human PGC-derived cultures meet the criteria for pluripotent stem cells and most closely resemble EG cells.

A highly conserved centrosomal kinase, AIR-1, is required for accurate cell cycle progression and segregation of developmental factors in Caenorhabditis elegans embryos.

S. cerevisiae Ipl1, Drosophila Aurora, and the mammalian centrosomal protein IAK-1 define a new subfamily of serine/threonine kinases that regulate chromosome segregation and mitotic spindle dynamics. Mutations in ipl1 and aurora result in the generation of severely aneuploid cells and, in the case of aurora, monopolar spindles arising from a failure in centrosome separation. Here we show that a related, essential protein from C. elegans, AIR-1 (Aurora/Ipl1 related), is localized to mitotic centrosomes. Disruption of AIR-1 protein expression in C. elegans embryos results in severe aneuploidy and embryonic lethality. Unlike aurora mutants, this aneuploidy does not arise from a failure in centrosome separation. Bipolar spindles are formed in the absence of AIR-1, but they appear to be disorganized and are nucleated by abnormal-looking centrosomes. In addition to its requirement during mitosis, AIR-1 may regulate microtubule-based developmental processes as well. Our data suggests AIR-1 plays a role in P-granule segregation and the association of the germline factor PIE-1 with centrosomes.

Role of fibroblast growth factors and their receptors in mouse primordial germ cell growth.

Primordial germ cells (PGCs) are the embryonic progenitors of mature germ cells. During their proliferative stage, murine PGCs may be transiently cultured on mitotically inactive feeder layers. This culture system has permitted identification of several growth factors active toward PGCs. We and others have previously identified basic fibroblast growth factor (bFGF) as a powerful mitogen in this system. Here we characterize some of the functions of bFGF in PGC culture. Our data demonstrate that fibroblast growth factor (FGF) receptors I and II are present in the developing gonad and are consistent with expression of these receptors by PGCs. Moreover, PGCs can bind radiolabeled bFGF in vitro, demonstrating that the factor can act directly on these cells. While mitotic PGCs of either sex are shown to bind radiolabeled bFGF, oogonia that are undergoing meiotic arrest exhibit reduced bFGF binding, indicating potential developmental regulation of an FGF receptor.