Use of ultrasound imaging for the diagnosis of abnormal uterine bleeding in the bonnet macaque ( Macaca radiata).

Ultrasound is a powerful, low-cost, non-invasive medical tool used by laboratory animal veterinarians for diagnostic imaging. Sonohysterography and transvaginal ultrasound are frequently used to assess uterine anomalies in women presenting with abnormal uterine bleeding (AUB). In the present study, we have evaluated the abdominal ultrasound of bonnet monkeys ( n = 8) showing spontaneous ovulatory ( n = 5) and anovulatory ( n = 3) AUB. The ovulatory ( n = 5) macaques showed cyclic AUB for 7-8 days. The anovulatory ( n = 3) macaques had irregular AUB with menstrual cycles of 40-45 days. The B-mode abdominal, colour Doppler and 3D ultrasound scans were performed during the proliferative phase of the menstrual cycle. Ultrasound examination revealed endometrial polyps in five macaques and endometrial hyperplasia in three animals. The width and length of endometrial polyps was around 0.5-1 cm (average 0.51 ± 0.23 cm × 0.96 ± 0.16 cm) with significant increase in endometrial thickness ( P < 0.0002). 3D ultrasound also showed a homogeneous mass in the uterine cavity and colour Doppler ultrasound showed increased vascularity in the endometrial polyps. Endometrial hyperplasia characteristically appeared as a thickened echogenic endometrium ( P < 0.0002). This study demonstrates the use of non-invasive ultrasound techniques in the diagnosis of AUB in macaques.

Proliferation and decidualization of endometrial stromal cells during embryo-attachment stage in bonnet monkeys (Macaca radiata).

We report embryo-induced alterations occurring in endometrial stromal cells (ESCs) during the embryo-attachment stage in bonnet monkeys (Macaca radiata). Laser micro-dissected ESCs obtained from pregnant and non-pregnant animals were compared for levels of selected proliferation and decidualization-associated factors by analysis with quantitative real-time polymerase chain reaction or immunohistochemistry. Stromal cells exhibited extensive cellular proliferation, as indicated by cellular compaction and significantly higher (P < 0.05) levels of proliferating cell nuclear antigen and of estrogen receptor 1, c-Myc, and Cyclin D1 transcripts in pregnant animals as compared with non-pregnant animals. A significant decrease (P < 0.05) was observed in the transcript levels of stromal interleukin-6 (IL-6) in pregnant animals. Cell proliferation was accompanied by a significant increase (P < 0.001) in the levels of decidualization-associated molecules such as IL-1ß in the luminal and glandular epithelium and of stromal insulin-like growth-factor-binding protein-1 (IGFBP-1) and prostaglandin-endoperoxide synthase-2 (PTGS-2) proteins. In pregnant animals, proliferation was evident throughout the gestational stroma, whereas decidualization was more pronounced in the embryo-attachment zone than in the non-attachment zone. To our knowledge, this is the first report of alterations in the endometrial stroma during the embryo-attachment stage in a non-human primate model.

Stimulation of ovarian stem cells by follicle stimulating hormone and basic fibroblast growth factor during cortical tissue culture.

BACKGROUND: Cryopreserved ovarian cortical tissue acts as a source of primordial follicles (PF) which can either be auto-transplanted or cultured in vitro to obtain mature oocytes. This offers a good opportunity to attain biological parenthood to individuals with gonadal insufficiency including cancer survivors. However, role of various intra- and extra-ovarian factors during PF growth initiation still remain poorly understood. Ovarian biology has assumed a different dimension due to emerging data on presence of pluripotent very small embryonic-like stem cells (VSELs) and ovarian germ stem cells (OGSCs) in ovary surface epithelium (OSE) and the concept of postnatal oogenesis. The present study was undertaken to decipher effect of follicle stimulating hormone (FSH) and basic fibroblast growth factor (bFGF) on the growth initiation of PF during organ culture with a focus on ovarian stem cells. METHODS: Serum-free cultures of marmoset (n=3) and human (young and peri-menopausal) ovarian cortical tissue pieces were established. Cortical tissue pieces stimulated with FSH (0.5 IU/ml) or bFGF (100 ng/ml) were collected on Day 3 for histological and molecular studies. Gene transcripts specific for pluripotency (Oct-4A, Nanog), early germ cells (Oct-4, c-Kit, Vasa) and to reflect PF growth initiation (oocyte-specific Gdf-9 and Lhx8, and granulosa cells specific Amh) were studied by q-RTPCR. RESULTS: A prominent proliferation of OSE (which harbors stem cells) and transition of PF to primary follicles was observed after FSH and bFGF treatment. Ovarian stem cells were found to be released on the culture inserts and retained the potential to spontaneously differentiate into oocyte-like structures in extended cultures. q-RTPCR analysis revealed an increased expression of gene transcripts specific for VSELs, OGSCs and early germ cells suggestive of follicular transition. CONCLUSION: The present study shows that both FSH and bFGF stimulate stem cells present in OSE and also lead to PF growth initiation. Thus besides being a source of PF, cryopreserved ovarian cortical tissue could also be a source of stem cells which retain the ability to spontaneously differentiate into oocyte-like structures in vitro. Results provide a paradigm shift in the basic understanding of FSH action and also offer a new perspective to the field of oncofertility research.

Endometrial epithelial cell modifications in response to embryonic signals in bonnet monkeys (Macaca radiata).

The present investigation reports embryo-induced modifications in the epithelial cells of the endometrium in a primate species. In vivo, epithelial cell response to the embryonic signals was assessed at the embryo attachment stage in the gestational uterus of bonnet monkeys (Macaca radiata) and in vitro response was investigated by treating human endometrial epithelial cell line (Ishikawa) with human embryo conditioned media (CM). Endometrial epithelial (EE) cells at the embryo attachment stage in bonnet monkeys revealed higher proliferation accompanied by significant up regulation (p < 0.05) in the expression of estrogen receptor (ER)α and down regulation (p < 0.05) in ERß expression. Further gestational EE cells showed higher (p < 0.001) expression of mucin-1, except in the embryo attachment site. Also, observed were significantly higher expression (p < 0.05) and altered cytoplasmic distribution of α(v) and ß(3) integrins, when compared to non-pregnant animals. In pregnant animals, the embryo attachment zone showed differential expression of immunoreactive integrins as compared to the non-attachment zone. This suggested the role of embryo secreted factors in modulation of the epithelial cell profile. In vitro studies partially supported this assumption. Significantly higher proliferation (p < 0.05), as well as increased expression of ERα, integrin ß(3) and mucin-1 (p < 0.05) were observed in Ishikawa cells, on stimulation with CM. Taken together, these results indicated the proliferation and modulation in the expression of estrogen receptors and cell adhesion molecules in the EE cells; at the embryo attachment stage in bonnet monkeys. Further it is likely that embryo secreted factors contribute to some of these modifications in EE cells. This report is the first account of discrete cellular events, which occur in the uterine epithelium, at the embryo attachment stage in a primate species.

Expression of endometrial protein kinase a during early pregnancy in bonnet monkeys (Macaca radiata).

Embryo-induced signaling pathways are considered to be important for initiation and sustenance of pregnancy. However many of these pathways remain to be deciphered in primates. In the present study, differential display RT-PCR was used to identify genes or gene fragments that are differentially expressed in endometrium of bonnet monkeys (Macaca radiata) on Day 6 of pregnancy. Of several fragments found to be differentially expressed, a fragment of 567 base pair (named GG1) was characterized in detail. GG1 was highly represented in endometrium of pregnant animals compared with that of nonpregnant animals. Sequencing analysis revealed homology of this fragment to exons 7, 8, 9, and 10 and surprisingly to intron 6 of cAMP-dependent protein kinase A (PKA) regulatory type I alpha (tissue-specific extinguisher 1) (PRKAR1A). The increased expression of this fragment in gestational endometrium was confirmed by quantitative PCR studies. Two transcripts of 3.0 kilobase (kb) and 1.5 kb were detected in Northern blot probed with labeled GG1. Protein expressions of alpha regulatory (PRKAR1A) and alpha catalytic (PRKCA) subunits of PKA were also higher in gestational endometrium compared with that in nongestational endometrium. Further in vitro studies using human endometrial explants demonstrated regulation of PRKAR1A (or GG1) and prostaglandin-endoperoxide synthase 2 or cyclooxygenase 2 (PTGS2) by estradiol. This is the first study to date on the differential expression of PKA in primate endometrium during early pregnancy and its in vitro regulation by estradiol.

Endometrial modifications during early pregnancy in bonnet monkeys (Macaca radiata).

The present study was undertaken to investigate endometrial modifications that occur before embryo invasion in bonnet monkeys (Macaca radiata). These changes were analysed in luminal epithelium, glandular epithelium and stroma of endometrial functionalis on Day 6 post ovulation from pregnant and non-pregnant animals (n = 4 each) by transmission electron microscopy. Distinct features (i.e. loss of columnar shape by epithelial cells, changes in mitochondrial size and diffused apicolateral gap junctions) were observed in the luminal and glandular epithelium in pregnant animals. Stromal compaction was also observed in pregnant animals. Further, immunogold localisation studies demonstrated significantly higher expression (P < 0.05) of oestrogen receptor alpha, an oestrogen-regulated gene, in the glandular epithelium and stroma of the endometrium in pregnant animals compared with non-pregnant animals. Expression of two other genes known to be regulated by oestradiol, namely beta-actin and cyclo-oxygenase-1, were also significantly higher (P < 0.05) in the endometria of pregnant animals. These studies demonstrate marked changes in the endometrium before embryo invasion in bonnet monkeys. These studies also indicate altered oestrogenic activity in the uterine milieu before embryo invasion.

Enhanced expressions of endometrial tumour necrosis factor alpha and its receptors during early pregnancy in bonnet monkeys.

Tumour necrosis factor alpha (TNF-alpha), a pro-inflammatory cytokine may play an active role in stimulating inflammatory reactions during pregnancy. However, the expression of endometrial TNF-alpha has not been investigated especially during early pregnancy, a phenomenon invariably accompanied by inflammatory reaction. In the present study, the endometrial expressions of TNF-alpha and its receptors (TNFR1 and TNFR2) during early pregnancy, when the embryo lies free in the zona hatched state in the uterine lumen, were analyzed by immunohistochemistry. The endometrial expressions of TNF-alpha, TNFR1 and TNFR2 were found to be significantly up-regulated (p < 0.05) in the glandular epithelium on day 6 post-ovulation in pregnant animals. The alteration in the expression of these molecules may contribute to the induction of local inflammatory reactions during implantation.

Endometrial expression of immunomodulatory cytokines and their regulators during early pregnancy in bonnet monkeys (Macaca radiata).

BACKGROUND: It is well established that endometrium undergoes extensive histological changes during implantation and subsequent stages of pregnancy in rodents as well as primates. Our previous investigation using a non-human primate model has demonstrated that morphological alterations are initiated even before the embryo invades the endometrium. The present study was undertaken to determine whether the embryo-induced morphological changes are accompanied by any alteration in the protein levels of the immunomodulatory cytokines and their regulators in the preimplantation stage endometrium. METHODS: The endometrial expression of immunosuppressive factors such as transforming growth factor beta2 (TGFbeta2), glycodelin (PP14), leukaemia inhibitory factor (LIF) and interleukin-6 (IL-6) were analysed on day 6 post-ovulation in pregnant and non-pregnant bonnet monkeys (Macaca radiata) using immunohistochemical methods. RESULTS: The endometrial expression of TGFbeta2, TGFbeta2 receptor, PP14 and IL-6 were significantly up-regulated (p < 0.05) in pregnant animals as compared to non-pregnant animals, whereas the expression of LIF and its receptor remained unaltered in pregnant animals. CONCLUSIONS: Expression levels of some immunomodulatory cytokines in endometrium are significantly increased even before the embryo invades the endometrium. The altered cytokine expression profile in endometrium probably contributes towards generating a conducive environment for the embryo survival, growth and development in the uterus.

Morphological events in the primate endometrium in the presence of a preimplantation embryo, detected by the serum preimplantation factor bioassay.

BACKGROUND: Hormonal modulation of the endometrium towards receptivity is well established; however, the role of embryonic stimuli in modulation of the endometrium prior to implantation, especially in primates, is unknown. The aim of the present study was to evaluate the endometrial histology when the embryo was present in its vicinity prior to implantation. METHODS: Preimplantation factor (PIF) bioassay was used as a tool to detect the presence of an embryo in the uterine lumen of mated bonnet monkeys (Macaca radiata) (n=9). The control group comprised seven non-mated animals. The specificity of the PIF bioassay for the presence of an embryo was tested by studies in pregnant humans and monkeys. The effects of embryonic stimuli on the endometrial morphology were analysed by routine haematoxylin-eosin staining. The expressions of CD34, an endothelial cell marker, alpha-smooth muscle actin (alpha-SMA), a marker for blood vessel maturation, and prolactin, a marker of endometrial decidualization, were studied by immunohistochemistry. RESULTS: That PIF is embryo specific was established by its presence in sera of pregnant humans, monkeys and also in embryo culture media. Six mated bonnet monkeys were found to be PIF positive. Morphologically, the endometria from these PIF-positive animals showed the presence of the pre-epithelial plaque reaction, increased angiogenesis and stromal compaction. The significantly increased number of CD34- and alpha-SMA-positive blood vessels (P<0.05) in the endometria of PIF-positive animals indicated increased angiogenesis in response to embryonic stimuli. The endometrial expression of immunoreactive prolactin was also significantly increased (P<0.05) in the PIF-positive animals, indicating decidualization. CONCLUSIONS: Using PIF as a marker to detect early pregnancy in bonnet monkeys, we have shown that the embryo induces a pre-epithelial plaque type of reaction, increased angiogenesis and decidual reaction in the endometrium prior to implantation.

Preclinical evaluation of magainin-A as a contraceptive antimicrobial agent.

OBJECTIVE: To evaluate the safety and contraceptive efficacy of magainin-A in monkeys. DESIGN: Controlled laboratory study. SETTING: Department of Immunology, National Institute for Research in Reproductive Health, Parel, Mumbai, India. ANIMAL(S): Male and female bonnet monkeys (Macaca radiata). INTERVENTION(S): Animals were treated intravaginally with 1 mg of magainin-A before attempted conception, as well as daily for 14 days to assess local and systemic toxicity. MAIN OUTCOME MEASURE(S): Suitability of magainin-A for the control of pregnancy and sexually transmitted infections. RESULT(S): Complete sperm immobilization was observed within 20 seconds after the exposure to magainin-A (800-1,000 microg) in vitro. Intravaginal administration of 1 mg of magainin-A blocked conception in monkeys. When magainin-A was administered intravaginally for 14 consecutive days, no treatment-related abnormalities were observed in menstrual cycle length, vaginal epithelial cell morphology, and hematologic/serum biochemical profiles. The peptide inhibited the growth of sexually transmitted infection-causing pathogens but not HIV-1 and HIV-2. CONCLUSION(S): Magainin-A can be used as an effective and safe intravaginal contraceptive compound with additional protection against sexually transmitted infection-causing pathogens.