Delineating the prime mover action of progesterone for endometrial receptivity in primates.

Progesterone is essential for endometrial receptivity in primates. It is now evident that embryo-derived signal influences implantation stage endometrium under progesterone dominance, and collectively results in endometrial receptivity to implanting blastocyst. Previously, a few studies were performed using global gene profiling based on microarray technology to identify changes in gene expression between early luteal phase and mid luteal phase endometrium, however, the issue of combinatorial regulation by progesterone-dependent regulation and by embryo-derived signal on transcripts profiles during endometrial differentiation toward receptivity for blastocyst implantation in primates has not been addressed. the present review summarizes a few issues, specifically that of transforming growth factor β-tumour necrosis factor α (TGFβ-TNFα) pathways and signal transducer and activator of transcription (STAT) signalling system related to luteal phase progesterone action on endometrial receptivity in terms of its transcriptomic expression using a potent antiprogestin (mifepristone) in conception cycles of the rhesus monkey as a non-human primate model.

Expressional regulation of genes linked to immunity & programmed development in human early placental villi.

Background & objectives: During 6 to 8 wk of gestation, human placental villi show a complex pattern of morphogenesis. There is however, no large scale gene expression study exploring the temporal pattern of the developmental molecular networks in placental villi during the early weeks of gestation. We evaluated the transcriptome profiling of humn placental villus samples obtained from fertile women with voluntarily terminated normal pregnancies between 6-8 wk of gestation. Methods: Transcriptomic profiles of individual human placental villous samples from 25 women with normal pregnancies during 6 to 8 wk of gestation were examined using human whole genome expression arrays. Quantitative RT-PCR validation of copy numbers of transcripts for selected 15 genes and exploratory analysis of the microarray data revealed a high degree of quality assurance supportive of further clustering and differential analyses. Immunoblot and immunohistochemical analysis of selected five candidate proteins (CAGE1, CD9, SLC6A2, TANK and VEGFC) based on transcript profiles were done to assess the pattern of down stream informational flow. Results: A large number (~9K) of genes with known functions were expressed in the experimental samples. The clustering analysis identified three major expression clusters with gestational age, and four co-expressional clusters. Differential analysis identified a highly discrete regulatory process involving only about 160 genes. Immunochemical analysis of selected candidate proteins based on transcript profiles revealed generally synchronous expression in human early placental villi. Interpretation & conclusions: Several signaling pathways linked to immunity (COL1, JAK2, JAK3, IL12, IL13, IL15, IL27, STAT3 and STAT5) were downregulated, while genes of the enriched category of antiviral immunity (ATF/AP1, IL10R and OAS) were clearly over-expressed. Transcriptional integration supportive of programmed development was observed in first trimester placental villi and it included regulation of apoptosis and cell cycle progression (ARRB1, ATR, BLM, CHRNA7, CHRNB1, FYN, KPNA4, and MTOR/FRAP), autophagy (ATG4B, ATG14, BAD, and BCL2), cell adhesion (CD9 and FN1) and epithelial-to-mesenchymal transition (CALD1, FN1, HEY1, MMP2, and WNT3A).

Quantitative analysis of six gene products as candidate markers of early placental villi development in the human.

Early placental development is critical for successful pregnancy. Recently, we have reported that ~70 genes were differentially expressed in human placental villi between 6- and 8- weeks of gestation in cDNAbased expression arrays for ~400 PCR products, of which six specific gene products (COL4A4, CXCR4, ERBB2, HDAC1, HPRT1, and TNFRSF1A) appeared intriguing. In the present study we have examined expressions of these six candidate genes in placental villi obtained from 6-weeks, 7-weeks and 8-weeks (n = 6 for each group) human placental samples using quantitative real time RTPCR. We observed that there was considerable concordance (>95% confidence) in pair-wise analysis of transcript profiles between the two methods, however, absolute quantitative values as measured by quantitative RTPCR differed from those obtained from cDNA-based array analysis for 2 gene products (CXCR4 and ERBB2) out of 6 genes. No significant change was observed in the steady state expression of COL4A4 and HPRT1 during the time period examined. However, there was significant decrease in CXCR4 for 7-weeks (P< 0.01) and 8-weeks (P<0.05) samples, and significant (P<0.05) increase was seen for ERBB2 in 7-weeks and 8-weeks as compared to 6-weeks samples with no change between 7-weeks and 8-weeks samples. Moreover, significant (P< 0.05) increase for HDAC1 and decrease for TNFRSF1A was observed in 8-weeks samples as compared to 6-weeks samples with no change observed between 6-weeks and 7-weeks samples. We infer that it is essential that cDNA array-based data are verified in terms of quantitative estimates preferably by quantitative PCR before their use for any exploratory purpose. Taking together our previous array based data and the present study we conclude that a categorical balance exists between the expression of ERBB2 and HDAC1 genes affecting cell proliferation and differentiation in one hand, and CXCR4 and TNFRSF1A affecting chemotaxis, inflammatory response and apoptosis on the other hand. The expression of these genes appear important for the early development of human placental villi.

Small scale transcript expression profile of human first trimester placental villi analyzed by a custom-tailored cDNA array.

Human placental trophoblastic mass grows rapidly between 4 and 8 weeks of gestation making it highly vulnerable to external and internal challenges, however, there has been no reported study exploring the developmental molecular characteristics in human first trimester placental villi. In the present study, transcript expressions of human placental villi of normal pregnancies during 6 (n=6), 7 (n=6) and 8 (n=6) weeks of gestation using custom-tailored cDNA-based expression arrays for ~400 annotated human gene products were examined. Unsupervised and supervised analyses of expression data revealed that 386 (95%) genes were overtly involved in the first trimester placental villi, and these genes segregated into three clusters specifically corresponding to 6-, 7- and 8- weeks of gestation in principal component analysis. Bayesian prediction analysis based on relative expression levels of genes studied identified that expression patterns in 15 samples out of 18 samples showed concordance with high (0.8-1.0) confidence measures with the chronological age of the placenta, however, two samples collected during 7-weeks of gestation and one sample collected during 8-weeks of gestation were predicted to be 6- weeks sample with confidence measures between 0.6 and 0.5. Unsupervised hierarchical clustering analysis segregated the samples into two major branches; while one of them was composed of five 7-weeks samples only, the second major branch had three sub-branches: one of them was exclusively composed of three 8-week samples only, while other two subbranches were mainly composed of 6-weeks samples. K-means clustering analysis identified four optimal clusters of genes depending on the similarity of their relative expression for the set of genes studied across all the samples. Gene ontology (GO) based functional classifications of genes in K-means clusters revealed that the overall putative functions of co-regulated gene clusters were mutually comparable, however, specific genes related to ion homeostasis, metabolism, and VEGF activity specifically clustered in 8-weeks samples. Analysis of relative gene expression during in 6-8 weekplacental villi revealed that a large number of gene products were over represented by their either up-regulation (70 genes: ~18%) or down regulation (53 genes; ~14%) between 6 and 8 weeks villi samples and these genes are reportedly involved in biological processes like regulation of cell growth and proliferation, anti-apoptosis, angiogenesis, immune and inflammatory responses, extracellular matrix remodeling and multicellular organismal development involving almost all cellular components and molecular functions like signal transduction activity, transcription factor activity, nucleotide and protein binding, ion (especially calcium and zinc) binding and growth receptor activities. Interestingly, four genes (oxytocin receptor, tenascin C, TNF-R1 and retinol binding protein 1) showed differential regulation in human placental villi during 6-8 weeks of gestation, suggestive of an underlying network of regulation involving these factors in the developing placenta. To our knowledge, this is the first report indicating that these genes are involved in the early stage development of human placenta.

Morphological and functional characteristics of rabbit uterine epithelial cells grown on free floating collagen gel.

In the present study isolated uterine epithelial cells from normal rabbits were maintained in culture on free floating rat-tail collagen matrix, and the morphological characteristics of these cells were examined. Additionally, the pattern of protein synthesis and secretion by rabbit uterine epithelial cells grown on free floating collagen gels following estradiol and/or progesterone treatment in vitro was examined. Isolated epithelial cells cultured on collagen gels in complete medium containing serum attached to form monlayers, and eventually the gels became free floating and contracted giving rise to luminal arrangements. These cells were cytokeratin positive epithelial cells and were ultrastructurally polarized. These cells also exhibited differential upregulation and down regulation in the synthesis and secretion of proteins in response to estradiol, progesterone, and estradiol plus progesterone. Additionally, a permissive action between progesterone and estradiol in the synthesis of two species of secretory proteins was observed. It however remains to be examined whether different species of proteins produced in vitro in response to estradiol and progesterone bear any association with physiological states in reproductive cycle in this species.

Research in reproduction: the Indian scenario in the last decade [II].

Developing new, improved and totally safe, effective and acceptable contraceptives based on the recent advances in cellular and molecular biology of reproduction is a new challenge to biomedical scientists involved in research in reproductive biology. The present article reviews some of the major contributions made during the last decade by scientists working in India in developing new strategies and technologies for better human reproductive health and fertility regulation.

Research in reproduction: the Indian scenario in the last decade [I].

The physiology of reproduction attempts to decipher the biological basis of procreation. Fundamental advances in the biology of reproduction are essential to decipher this process. In this article, an attempt has been made to chronicle some recent advances in our understanding of the biology of reproduction during the last decade based on the contributions of scientists working in India.

Immunohistological localisation of vascular endothelial growth factor in human endometrium.

Several polypeptide growth factors regulate epithelial and stromal development in endometrium under the influence of estrogen and progesterone, and thereby regulate growth and differentiation of endometrium during menstrual cycle. However, little is known about the angiogenic growth factors that may affect endometrial vasculature throughout each menstrual cycle. Vascular endothelial growth factor (VEGF) is suggestively an important angiogenic growth factor in the female reproductive tract. The aim of the present study was to immunolocalize and assess semi-quantitatively VEGF immunostaining in cells of proliferative phase (n = 3), secretory phase (n = 6) and hyperplastic (n = 6) human endometrial samples. VEGF concentrations were significantly higher in glandular (P < 0.001) and stromal (P < 0.01) compartments of proliferative stage endometrium compared with those in secretory stage and hyperplastic endometrial samples, with no difference in the scores for glandular and stromal compartments between secretory stage and hyperplastic endometrial samples. Generally, glandular expression of VEGF was higher as compared to stromal compartment. Thus, it appears that endometrial VEGF expression and concentration are enhanced by estrogen, and may be correlated with neovascularization and increased vascular permeability during late proliferative period. Additionally, there was no enhancement in VEGF expression in hyperplastic glands, suggesting that regulation of glandular growth and that of angiogenesis in human endometrium operate through different mechanisms.

Hormonal requirement for blastocyst implantation and a new approach for anti-implantation strategy.

There is a growing awareness of a need for developing novel methods of contraceptive technology which should not only be effective in providing protection against conception but also take into consideration the reproductive health issues confronting men and women. This paper considers the process of embryo implantation as one such potential target. The hormonal basis of embryo implantation in primates has been discussed to indicate that progesterone, and not estrogen, from ovarian source is the primary determinant of embryo-endometrial maturation and their synchronization for implantation. Thus, low dose administration of the anti-progesterone, mifepristone, during early luteal phase has been shown to be an effective anti-implantation approach to for fertility control. Furthermore, the dissociation of endometrial-hormonal synchrony at the time of blastocyst implantation following the post-ovulatory mifepristone administration has been shown to be the physiological basis of its anti-implantation effect with undisturbed circulatory hormone profiles and ovarian functions. Further studies are required to appreciate the full potential and to mollify the limitations of this approach.

Breast lumps: a study of 10 years.

A retrospective study of lumps in the breast sent for histopathological examination in Calcutta National Medical College for a period of last 10 years was done. It was found that 2062 cases (6.82%) of histopathological examination of breast lump were studied out of a total 30208 specimens during that period. There were 1528 cases from benign breast lesions and 504 cases from the malignant breast lesions. The most common lesion found histologically was fibro-adenoma in 950 cases. Others included 150 cases of inflammatory breast diseases of which 36 cases were diagnosed as tuberculosis, 386 cases as fibrocystic disease and 42 cases as other benign neoplastic diseases. Out of 504 malignant cases 456 had carcinoma and 48 had sarcoma. Only 30 cases of male breast lesions were sent of which only a single case of infiltrating duct carcinoma was found, 28 cases had gynaecomastia and one case showed the evidence of fibrocystic disease.

Experiences with fine needle aspiration cytology as a diagnostic procedure for breast lesions in a Calcutta Hospital.

Nine hundred and forty cases of breast lesions were subjected to fine needle aspiration. Nine hundred twenty-four of them were females and sixteen were males. Smears from the 60 female cases were rejected as unsatisfactory. Of the remaining 864 female cases (aged 15 to 72 years) 704 (81.4%) were benign. Histopathological correlations were done in 448 cases with diagnostic accuracy of 97.3%. One hundred and sixty (18.5%) out of 864 females were malignant and cytohistological correlation was done in 142 cases with 98.6% corroboration. Ten of the 16 male cases were histologically examined with full corroboration. Overall accuracy considering both males and females was 98.6%. Though there was underdiagnosis involving 12 cases of infiltrating ductal carcinoma when the lesions were small and deeply situated there was no overdiagnosis in this study.

Incidence of malignancy in Bankura (a retrospective study).

A retrospective study on pattern of malignant lesions in a rural district of West Bengal from 1981-1990 was conducted in the department of pathology, BS Medical College, Bankura, West Bengal. A total number of 17,130 cases were received out of which 1976 were malignant lesions (11.53%). The peak incidence of cancer in both sexes was found between the 4th, 5th and 6th decades, ie, 31-60 years of age. Sex-wise distribution showed more incidence in female (1246-62.17%) than in male (730-37.83%). Cancer of the cervix (35.62%) showed the highest incidence in this series. Cancer of the larynx (11.94%) was the commonest cancer in males. Malignancy of the lymph node was observed in 11.43% cases. Malignancy of the lungs and vagina (< 0.5%) showed very low incidence.

Luteal phase contraception with mifepristone (RU 486) in the rhesus monkey.

Mifepristone (RU 486), an antiprogesterone, is a promising luteal phase contraceptive agent for human use. However, at present its use is limited by the practical constraint of determining the day of ovulation for an LH + 2 day administration of the drug as indicated from experimental and clinical studies. The aim of the present study was to identify the effective period of luteal phase (luteal phase window) when a single administration of mifepristone would induce antinidatory activity without disturbing menstrual cyclicity and ovulatory pattern in the rhesus monkey. RU 486 (2 mg/kg body weight in benzyl benzoate/olive oil, 1:3) was given to mated monkeys (n = 9) on cycle day 16 in the first treatment cycle (treatment group T1, n = 9), and in the following cycle on cycle day 20 (treatment group T2, n = 8). A single s.c. injection of this antiprogestin during early to midluteal phase (days 1-10 after ovulation, as determined from retrospective analysis of serum concentrations of estrogen and progesterone) provided a one hundred per cent protection against pregnancy, with no apparent side effects. There were no changes in cycle lengths (F = 3.5; P < 0.3), day of ovulation (F = 1.8; P < 0.7) and duration of menses (F = 3.5; P < 0.3) compared with the pre-treatment and post-treatment cycles. Pooled analyses of serum concentrations of estrogen and progesterone during luteal phases of T1 and T2 cycles also showed no variations with those in pre- and post-treatment cycles.(ABSTRACT TRUNCATED AT 250 WORDS)

Malignant lymphoma--a 15-year study report.

Analysis of incidence of different types of malignancies during 15 years showed occurrence of malignant lymphoma in 192 cases (4.1%). There were 82 cases (42.7%) of non-Hodgkin's lymphoma and 110 cases (57.3%) of Hodgkin's disease. Lymphocytic lymphoma, the major type observed among the non-Hodgkin's lymphoma with 58 cases (30.21%), showed a preponderance of well differentiated type (40 cases). Other cases in this group comprised mixed cellularity (2.08%), histiocytic (3.64%) and unclassified one (6.77%). Analysis of the Hodgkin's disease cases showed majority in it belonged to the mixed cellularity type (44.5%) followed by the lymphocytic predominant type (27.3%), lymphocytic depletion type (18.2%) and nodular sclerosing type (10%). The incidence of 14 cases of extranodal lymphomas was 7.3% among the lymphomas with maximum occurrence in the gastrointestinal tract (42.8%).

Contranidatory activity of early luteal phase administration of mifepristone in the rhesus monkey.

The contranidatory action of mifepristone (RU 486) given as a single application at different dosages to mated rhesus monkeys (Macaca mulatta) on second day after ovulation has been examined in the present study. In group 1, monkeys (n = 3) received only vehicle (benzyl benzoate: olive oil, 1:4, v/v) and were treated as controls. In group 2 monkeys (n = 4), RU 486 was given by gavage at 10 mg/kg in group 4 (n = 5). The patterns of cycles and profiles of serum estrogen and progesterone were monitored for assessing the occurrence of implantation and pregnancy. At a single dose of 10 mg/kg, RU 486 was found to be ineffective in preventing nidation, resulting pregnancy in three females out of four treated monkeys. Similarly, an s.c. administration of 1 mg/kg could provide pregnancy protection in two of the four treated monkeys. In these monkeys, however, the menstrual cycle characteristics were not affected as compared to pretreatment cycles. Interestingly, the administration of 2 mg/kg, s.c., RU 486 could provide a hundred percent pregnancy protection in mated monkeys, and there was no significant changes in the pattern of menstrual cycle characteristics. It appears that an early post-ovulatory administration of RU 486 may be successfully used in human as an effective once-a-month, early luteal phase contranidatory agent.

Is there any delta 5-3 beta hydroxysteroid dehydrogenase activity in preimplantation embryo of rhesus monkey?

This is the first report on the histochemical assessment of delta 5-3 beta hydroxysteroid dehydrogenase activity in all the preimplantation embryonic stages in the rhesus monkey (Macaca mulatta). An apparent stage dependent increase in enzyme activity was obtained, however, distinctively a high degree of non-specificity in enzyme reaction was noted primarily in morulae and blastocysts. Such marked non-specificity in the histochemical enzyme reaction for delta 5-3 beta hydroxysteroid dehydrogenase activity was not found in mouse blastocysts. High amounts of endogenous steroids present within rhesus embryos, or the participation of non-specific dehydrogenases could account for the observed non-specificity. Furthermore, the present report documents the pattern and degree of association (r = 0.9; P less than 0.01) between developmental stage and gestational age of preimplantation rhesus embryos, and thus provides a normal in situ cell cleavage rate of preimplantation embryo in the rhesus monkey.