Early Luteal Phase Research Articles

Effect of cytokines and ovarian steroids on equine endometrial function: an in vitro study

Regulation of immune-endocrine interactions in the equine endometrium is not fully understood. The aims of the present study were to: (1) investigate the presence of tumour necrosis factor alpha (TNF), interferon gamma (IFNG), Fas ligand (FASLG) and their receptors in the mare endometrium throughout the oestrous cycle; and (2) assess endometrial secretory function (prostaglandins), angiogenic activity and cell viability in response to TNF, oestradiol (E2), progesterone (P4) and oxytocin (OXT). Transcription of TNF and FASLG mRNA increased during the early and late luteal phase (LP), whereas IFNG mRNA increased in late LP. Transcription of the mRNA of both TNF receptors was highest in the mid-LP. All cytokines and receptors were expressed in surface and glandular epithelium, as well as in the stroma. Expression of TNF and its receptor TNFRSF1A increased during the follicular phase (FP) and mid-LP. IFNG was expressed in the mid-LP, whereas its receptor IFNR1 was expressed in the in mid- and late LP. The highest expression of FASLG and FAS occurred during the late LP. OXT increased the secretion of prostaglandin (PG) E2 and PGF2α in the FP and mid-LP. In the mid-LP, E2 and P4+E2 stimulated PGF2α secretion, whereas TNF and P4 increased cell viability. All treatments, with the exception of P4, increased nitric oxide and angiogenic activity in both phases. The coordinated action of cytokines and ovarian hormones may regulate secretory, angiogenic and proliferative functions in the equine endometrium.

Effects of alcohol on the performance of the Tower of London task in relation to the menstrual cycle

It is well known that the metabolism of alcohol and cognitive functions can vary during the menstrual cycle. Also, both alcohol ingestion and hormonal variations during menstruation have been associated with characteristic changes in electroencephalographic (EEG) activity. The aim of the present study was to determine whether EEG activity during the performance of the Tower of London (TOL) task is affected by previous ingestion of alcohol and whether these EEG patterns vary in relation to different phases of the menstrual cycle. For this purpose, female participants consumed a moderate dose of alcohol or placebo during the follicular and early luteal phases of the menstrual cycle and then, 35 min after liquid ingestion, EEG activity was recorded during the performance of TOL. A deleterious effect of alcohol on TOL performance was potentiated in the follicular phase, related to a higher α1 relative power, probably as a result of the low progesterone levels characteristic of this menstrual phase. These data show the feasibility of examining the interaction of alcohol and menstrual cycle phases on cognitive performance by means of EEG recording, and contribute toward a better understanding of the brain mechanisms that underlie the cognitive changes that occur during the menstrual cycle under the effects of alcohol.

Alterations of Circulating Endothelial Cell and Endothelial Progenitor Cell Counts around the Ovulation

Circulating endothelial cells (CECs) and progenitor cells (CEPs) have been intensively studied as a promising tool for treating ischemic diseases and monitoring cancer treatments, but how the menstrual cycle affects the variation in their counts remains unclear. The aims of the study were to determine the influence of the menstrual cycle on the number of CECs and CEPs and to investigate the association of their counts with circulating hormones and angiogenesis-associated factors. CEP and CEC counts by flow cytometry and the CellSearch system and circulating factor levels were measured eight times during the menstrual cycle in 18 volunteers. The menstrual cycle was divided into six phases based on hormone concentrations. CEP counts peaked in the periovulatory and middle luteal phases with a drop in the early luteal phase. CEC counts showed no significant variation. There were significant correlations between the CEP counts and the serum concentrations of estradiol (E2), LH, and granulocyte colony-stimulating factor (G-CSF) (P < 0.0001, P < 0.0001, and P = 0.01, respectively). The difference in CEP counts between two adjacent phases was significantly correlated with that in E2, LH, G-CSF, and serum vascular endothelial growth factor (P < 0.0001, P < 0.0001, P = 0.02, and P = 0.006, respectively). CEP counts peaked in the periovulatory and middle luteal phases, with a drop in the early luteal phase, and were correlated with serum E2, LH, and G-CSF concentrations. Consideration of the variation in CEP counts would be important for the clinical application of CEPs.

Uterine artery blood flow in the periimplantation period in embryo transfer cycles

To assess the role of the uterine artery blood flow in the prediction of implantation in women undergoing embryo transfer during the periimplantation period. A total of 233 couples were included in this prospective study. All patients had embryo transfer, 125 were performed in in-vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) and 108 in cryo cycles. Ultrasound measurements were performed immediately before transfer. The pulsatility index (PI), Resistance index (RI) and the peak systolic velocity (PSV) were measured in both uterine arteries using endovaginal ultrasound. In IVF/ICSI cycles the doppler parameters PI (2.48 vs. 2.15), RI (0.78 vs. 1.30) and PSV (60 vs. 63) did not differ significantly between the pregnant and non-pregnant group. The pregnancy rate per transfer was similar in women showing an unilateral (24%), bilateral (33%) or no (27%) notch in the uterine blood flow. In cryo cycles the uterine artery blood flow parameters PI (3.2 vs. 3.0), RI (0.9 vs. 0.9) and PSV (53.2 vs. 51.2) did not differ either between pregnant and not pregnant patients. Previous studies were aiming at the measurement of arterial doppler parameters during the follicular phase which may not be adequate for the prediction of implantation. However, our results show that doppler studies during the early luteal phase of assisted reproductive technology cycles are not indicative for the likelihood of pregnancy, too.

Endometrial gene expression in the early luteal phase is impacted by mode of triggering final oocyte maturation in recFSH stimulated and GnRH antagonist co-treated IVF cycles

Do differences in endometrial gene expression exist after ovarian stimulation with four different regimens of triggering final oocyte maturation and luteal phase support in the same patient? Significant differences in the expression of genes involved in receptivity and early implantation were seen between the four protocols. GnRH agonist triggering is an alternative to hCG triggering in GnRH antagonist co-treated cycles, resulting in an elimination of early ovarian hyperstimulation syndrome. Whereas previous studies have revealed a low ongoing clinical pregnancy rate after GnRH agonist trigger due to a high early pregnancy loss rate, despite supplementation with the standard luteal phase support, more recent studies, employing a 'modified' luteal phase support including a bolus of 1500 IU hCG on the day of oocyte aspiration, have reported ongoing pregnancy rates similar to those seen after hCG triggering. A prospective randomized study was performed in four oocyte donors recruited from an oocyte donation program during the period 2010-2011. Four oocyte donors in a university IVF center each prospectively underwent four consecutive stimulation protocols, with different modes of triggering final oocyte maturation and a different luteal phase support, followed by endometrial biopsy on Day 5 after oocyte retrieval. The following protocols were used: (A) 10 000 IU hCG and standard luteal phase support, (B) GnRH agonist (triptorelin 0.2 mg), followed by 1500 IU hCG 35 h after triggering final oocyte maturation, and standard luteal phase support, (C) GnRH agonist (triptorelin 0.2 mg) and standard luteal phase support and (D) GnRH agonist (triptorelin 0.2 mg) without luteal phase support. Microarray data analysis was performed with GeneSpring GX 11.5 (RMA algorithm). Pathway and network analysis was performed with the gene ontology software Ingenuity Pathways Analysis (Ingenuity® Systems, www.ingenuity.com, Redwood City, CA, USA). Samples were grouped and background intensity values were removed (cutoff at the lowest 20th percentile). A one-way ANOVA test (P< 0.05) was performed with Benjamini-Hochberg multiple testing correction. Significant differences were seen in endometrial gene expression, related to the type of ovulation trigger and luteal phase support. However, the endometrial gene expression after the GnRH agonist trigger and a modified luteal phase support (B) was similar to the pattern seen after the hCG trigger (A). The study was performed in four oocyte donors only; however, it is a strength of the study that the same donor underwent four consecutive stimulation protocols within 1 year to avoid inter-individual variations. These endometrial gene-expression findings support the clinical reports of a non-significant difference in live birth rates between the GnRH agonist trigger and the hCG trigger, when the GnRH agonist trigger is followed by a bolus of 1500 IU hCG at 35 h post trigger in addition to the standard luteal phase support. This study was supported by an un-restricted research grant by MSD Belgium. EudraCT number 2009-009429-26, protocol number 997 (P06034).

Quantification of blood volume and vascular perfusion in the corpus luteum of rhesus macaques during the menstrual cycle

OBJECTIVE: To determine changes to luteal vascular flow and volume in rhesus monkeys using dynamic contrast enhanced ultrasound (d-CEU). DESIGN: In vivo, repeated measures. MATERIALS AND METHODS: Hormonal status of 8 adult cycling female macaques was monitored to detect the day of ovulation. D-CEU with Definity (Lantheus) microbubble perfusion was performed on the corpus luteum (CL) at the early, mid, mid-late and late luteal phase (LP; Mol Reprod Dev, 76: 367-378, 2009), as recently described for the uterus (Fertil Steril 95:1316, 2011). Vascular parameters were quantified in d-CEU video captures using a region of interest (ROI) focused on the CL. Video intensity (VI) was normalized to brachial artery flow to provide an estimate of blood volume (BV); change in VI (DVI/sec) was used to determine vascular perfusion rate (VP). RESULTS: Significant changes in BVand VP (P<001 and P1⁄40.07) were observed between stages of the LP. BV was negatively correlated with luteal day (P<0.02). In contrast, serum progesterone (P) levels correlated positively with BV (P1⁄40.04) and VP (P1⁄40.001). P area under curve (AUC) also positively correlated with AUC for BV (P1⁄40.03). In typical LPs, BV was highest during the early LP, before decreasing 3-fold in late LP CL. In contrast, VP peaked at mid LP, with values 5 and 11-fold greater than early and late LP, respectively. Two females were identified with luteal insufficiency based on peak P levels during the mid and mid-late LP (2.3 0.2 ng/ml), whereas 5 had normal LPs (6.2 1.2 ng/ml). Another female was identified with a luteal cyst on the contralateral ovary, and a functional CL that regressed before P levels declined. Insufficient LPs trended toward lower peak BVand VP, compared to typical LPs. CONCLUSION: D-CEU provides a non-invasive measurement of the dynamics of CL blood volume and flow during the luteal lifespan in the menstrual cycle. Reduced vascular perfusion is evident in animals with reduced P secretion and in animals with aberrant luteal function. Supported by: P51OD011092, R01HD020869 (RLS).

Expression of leptin and its receptor in corpus luteum during estrous cycle in buffalo (Bubalus bubalis)

Leptin is supposed to play a crucial role in ovarian luteal dynamics. The present study was aimed to investigate the importance of leptin and its receptors in buffalo corpus luteum (CL) obtained from different stages of the estrous cycle. Real-time RT-PCR (qPCR), western blot and immunohistochemistry techniques were applied to investigate mRNA expression, protein expression and localization of examined factors. Additionally to assess the contribution of leptin in progesterone production the expression profiles of StAR, P450scc and HSD were also investigated. In general, we demonstrated presence of leptin and its receptors in buffalo CL during the estrous cycle. The mRNA levels of leptin and its receptors were significantly up regulated in (P<0.05) in all the stages and highest levels were observed in mid and late luteal stages consistent with in vivo luteinization of buffalo CL and declined coincidental to luteal regression. The expression of StAR, P450scc and HSD factors maintained low in early luteal phase, after that level of expression increased steadily to show a significant rise (P<0.05) in mid luteal phase followed by gradual decline in late luteal phase and regressed CL and this correlates well with the Ob and ObR receptor activity, verifying their key role in progesterone and other steroids production in functional CL. As revealed by immunohistochemistry, leptin protein was localized predominantly in large luteal cells however leptin receptor (Ob-R) was localized in large luteal cells as well as in endothelial cells. It can be concluded from our study that leptin via its autocrine/paracrine effects play a significant role in promoting angiogenesis, steroidogenesis and also acts as key survival factor in bubaline CL.

Cyclic regulation of LPA3 in human endometrium

Lysophosphatidic acid (LPA) belongs to the group of lipid messengers, which plays a pivotal role in the establishment of implantation via its cellular receptor, LPA3. The aims of the present study were to characterize LPA3 mRNA and protein in human endometrium during the normal human menstrual cycle. Forty-three normally cycling volunteers without reproductive disorders were randomized to undergo endometrial sampling on a specific cycle day. Samples were assessed for relative LPA3 mRNA expression using real-time PCR and for LPA3 protein using immunohistochemistry and western blot. The expression of LPA3 mRNA significantly increased during the early and late secretory phase compared with other menstrual phases. LPA3 protein was localized to the epithelial and stromal cells and expression levels followed the same pattern as for LPA3 mRNA. In the normal human menstrual cycle, LPA3 mRNA and protein expression also change, indicating that this gene may be related to the function of the endometrium.

Endometrial thickness measurement throughout a menstrual cycle in non-obese infertile patients with polycystic ovary syndrome

To analyze the changes in the endometrial thickness in infertile polycystic ovary syndrome (PCOS) patients throughout an entire menstrual cycle and compare the changes to those seen in infertile patients without PCOS. This prospective, cross-sectional study was conducted in a total of 58 non-obese, infertile women with PCOS. The endometrial thickness was measured at three different times throughout the menstrual cycle by ultrasound. Age- and body mass index (BMI)-matched control group consisted of 62 non-obese infertile patients without PCOS. Demographic, hormonal and the ultrasonographic measurements of the two groups were compared. Day 3 levels of LH were significantly different between the groups (p=0.013). Ovarian volume measurement was significant between the groups (p=0001). All the endometrial thickness measurements in the early, mid-cycle and late luteal phases were also significantly different; p=0.001 for all. The study demonstrated an increased endometrial stripe measurements throughout a menstrual cycle in infertile patients with PCOS, when compared to infertile patients without PCOS.

Quantification of Immune Cells in the Primate Corpus Luteum During the Menstrual Cycle; Increases During the Late Luteal Phase Indicate a Potential Role in Luteolysis.

The potential role of the immune system in regulation of the mammalian ovary has primarily been investigated in domestic animal and rodent models; however the presence of immune cells in the human ovary has also been noted by immunohistochemistry. Recently, microarray-identified luteal mRNAs for immune regulatory components such as chemokines and cytokines, immune cell-associated genes, as well as gene signaling pathways associated with immune function were found to fluctuate during the lifespan of rhesus macaque corpora lutea (CL). To characterize immune cell populations in primate CL throughout the menstrual cycle, luteal tissue was collected from rhesus females at discrete intervals of the luteal phase, including early days 3-5 (n=3), mid days 6-8 (n=4), mid-late day 10 (n=4) and day 12 (n=3), as well as after onset of luteal regression (2 days n=2, and 3-4 days n=4, following a drop in serum progesterone levels to < 0.3 ng/ml). Luteal cells were enzymatically dispersed, counted and viability assessed. Wet weight (mg) of CL were significantly different across the luteal phase (P<0.03), but total numbers of viable cells did not differ between groups (P=0.8). Dispersed cells were then incubated with fluorescently-labeled antibodies specific for immune-cell surface proteins: CD11b (ITGAM; neutrophils including activated lymphocytes, granulocytes and monocytes), CD14 (monocytes/macrophages), CD16 (FCGR3; primarily natural killer cells as well as neutrophils and macrophages), and CD3 (CD3-epsilon/T cell receptor complex; T lymphocytes). Number of labeled cells for each CL (positive events in 10,000 cells counted) was assessed using a BD LSR II flow cytometry instrument for each antibody (FCS Express version 3, De Novo Software). A non-immune, isotype antibody matched for each cell surface specific marker was used to gate the positive cells within the CL. The number of positive events recorded for immune cells at each stage of the luteal phase was then analyzed by one-way ANOVA (SAS version 2.0). Both CD11b- and CD14-positive cells (neutrophils and macrophages) were constant until increasing significantly 3-4 days after onset of luteal regression (P<0.0001). Numbers of CD16-positive (natural killer cells) increased initially between early and mid luteal phase to plateau through the mid-late stages, until a further 3-fold increase by 3-4 days after onset of regression (P<0.005). Additionally, CD16-positive cells were the most abundant immune cell type in CL during the mid and mid-late luteal phases, and increased to levels comparable with CD11b-positive cells during luteal regression. Presence of CD3-positive lymphocytes tended to increase 3-4 days after regression (P=0.07), but were less abundant than other cell types. These data demonstrate a remarkable increase in cells of the innate immune system, (i.e. macrophages and neutrophils) in the primate CL associated with the latter stages of luteal regression. Natural killer cells may have a dual role in the CL to promote luteal structure and function during the mid-luteal phase, whereas macrophages, neutrophils and natural killer cells may participate in structural luetolysis at the end of the luteal lifespan. Supported by NIH grant R01HD020869 (RLS) and NCRR grant P51RR000163 (Support For National Primate Research Center).

Effect of transient high-energy diets just after ovulation on ovarian performance and metabolic status in cyclic ewes

The aim of this study was to investigate the influence of short-term treatment of high-energy diet after ovulation on ovarian function and metabolic status in ewe. Cycling ewes Ossimi were divided into treatment group (TG; n=6) and control group (CG; n=6). After ultrasonographic detection of ovulation (day 0), a high-energy diet (12.55MJ DE/kg diet; 125% of maintenance) was fed to TG from day 1 to day 4 after ovulation (4days). The high-energy diet consisted of 850g concentrate mixture and 150g alfalfa hay, plus ad libitum access to wheat straw. The CG was offered as maintenance diet (10MJ DE/kg diet) throughout the experiment. Follicular development was observed ultrasonographically every other day while blood samples were collected daily throughout the experiment for the analysis of glucose, total cholesterol, urea, triglycerides, total proteins, aspartate aminotransferase (AST), alanine transaminase (ALT) and glutamate oxaloacetate transaminase (GOT). Transient feeding of high-energy ration during early luteal phase of estrous cycle significantly influenced the concentration of glucose and some metabolic profiles. Total proteins were greater at first, third and fourth day after ovulation (ovulation is day 0) in TG when compared to CG ewes. Blood glucose concentration was greater in TG than that of CG at days 1, 2, 3 and 4 postovulation. The average number of small follicles (2–2.9mm) of the first follicular wave after ovulation was greater in TG ewes (23.3 follicles) than that of CG (2.2). There was no difference in the number of medium (3–5mm) and large sized follicles (>5mm) between CG and TG ewes. Moreover, the maximum size of large follicles did not differ between CG (5.6mm) and TG ewes (5.9mm). In conclusion, high-energy diet may improve number of small follicles and alter energy metabolite during early luteal phase in cycling ewes.

Impact of norgestomet supplementation during early luteal phase on subsequent luteal profiles and conception rate in buffalo: a preliminary study

The current study was aimed to establish the impact of progesterone supplementation (norgestomet progestagen) between days 4 to 10 post-ovulation on subsequent luteal profile and conception rate in buffaloes. The 28 Murrah buffaloes of second to fourth parity, having normal reproductive organs, were estrus synchronized by double PGF(2α) protocol at 11 days apart. The buffaloes were inseminated during mid- to late estrus and thereafter repeated at 24 h interval. The buffaloes were randomly assigned into two groups: (1) control (no treatment, n = 14) and (2) treatment group (CRESTAR ear implant, n = 14). The CRESTAR ear implant (3 mg, norgestomet progestagen) was inserted subcutaneous between days 4 to 10 post-ovulation. The ovaries were scanned at estrus and thereafter on days 4, 10, 16, 21, and 40 post-ovulation to examine the preovulatory follicle (POF) and corpus luteum (CL) diameter. Each ultasonography was followed by blood sample collection for analysis of plasma progesterone concentrations following ovulation. The conception rate was similar (p > 0.05) between treated and control buffaloes. The pregnant buffalo of the control group had larger (p < 0.05) POF diameter than nonpregnant counterparts. The CL diameter was similar (p > 0.05) in both treated and untreated control as well as in their pregnant and nonpregnant buffaloes of the respective groups. The plasma progesterone concentrations were higher (p < 0.05) in the treatment group on the day 10 post-ovulation as compared to the control buffaloes. It is concluded that norgestomet supplementation had no impact on conception rate and CL diameter but enhances the plasma progesterone concentrations following treatment in buffaloes.

Pathophysiology of premenstrual syndrome and premenstrual dysphoric disorder

Premenstrual syndrome (PMS) and premenstrual dysphoric disorder are triggered by hormonal events ensuing after ovulation. The symptoms can begin in the early, mid or late luteal phase and are not associated with defined concentrations of any specific gonadal or non-gonadal hormone. Although evidence for a hormonal abnormality has not been established, the symptoms of the premenstrual disorders are related to the production of progesterone by the ovary. The two best-studied and relevant neurotransmitter systems implicated in the genesis of the symptoms are the GABArgic and the serotonergic systems. Metabolites of progesterone formed by the corpus luteum of the ovary and in the brain bind to a neurosteroid-binding site on the membrane of the gamma-aminobutyric acid (GABA) receptor, changing its configuration, rendering it resistant to further activation and finally decreasing central GABA-mediated inhibition. By a similar mechanism, the progestogens in some hormonal contraceptives are also thought to adversely affect the GABAergic system. The lowering of serotonin can give rise to PMS-like symptoms and serotonergic functioning seems to be deficient by some methods of estimating serotonergic activity in the brain; agents that augment serotonin are efficacious and are as effective even if administered only in the luteal phase. However, similar to the affective disorders, PMS is ultimately not likely to be related to the dysregulation of individual neurotransmitters. Brain imaging studies have begun to shed light on the complex brain circuitry underlying affect and behaviour and may help to explicate the intricate neurophysiological foundation of the syndrome.

MRNA transcription of prostaglandin synthases and their products in the equine endometrium in the course of fibrosis

Accurate regulation of the reproductive cycle and successful implantation depend on proper functioning of the endometrium. The aim of this study was to determine whether mRNA transcription of specific enzymes responsible for prostaglandin (PG) synthesis (prostaglandin-endoperoxide synthase, PTGS-2; prostaglandin F2α synthase, PGFS; and prostaglandin E2 synthases, PGES) and PG concentrations in endometrial extracts would change in moderate (Kenney's Category II) and severe phases of fibrosis (Kenney's Category III; endometrosis), compared with healthy endometrium (Kenney's Category I), during the estrous cycle. Endometrial tissues samples were obtained from mares at the early (n = 12), mid (n = 12) and late (n = 12) luteal phases and the follicular phase (n = 12) of the estrous cycle. Additionally, all endometria were classified microscopically as belonging to Categories I and II or III according to the Kenney classification, resulting in allocation of 4 samples for each subcategory, e.g., mid luteal I, II and III. Relative mRNA transcription was quantified using Real-time PCR. Concentrations of PGE2 and PGF2α in the endometrial extracts were determined using enzyme-linked immunosorbent assay (EIA). In Category I, PTGS-2 mRNA transcription was upregulated at the mid (P < 0.05) and late luteal phases (P < 0.001) and at the follicular phase (P < 0.05) compared to the early luteal phase. PGFS mRNA transcription as well as PGF2α concentrations increased at the mid (P < 0.01) and late (P < 0.05) luteal phases compared to the early luteal phase in Category I. PGES mRNA transcription was higher at the mid (P < 0.01) and late luteal phases (P < 0.05) compared to the early luteal and follicular phases in Category I. Prostaglandin E2 concentration in Category I was higher at the mid luteal phase (P < 0.01) compared to all other phases of the estrous cycle. During incipient endometrosis (Category II) and under full endometrosis (Category III), PTGS-2, PGFS and PGES mRNA transcription and PG concentration were altered compared to the respective estrous phases in healthy endometria (P < 0.05). It may be concluded that serious changes in mRNA transcription of PG synthases and PG production that occur in the equine endometrium during the course of fibrosis in the estrous cycle could be responsible for disturbances leading to disorders of the estrous cycle and early embryo losses.

Combined mRNA microarray and proteomic analysis of eutopic endometrium of women with and without endometriosis

An early semi-invasive diagnosis of endometriosis has the potential to allow early treatment and minimize disease progression but no such test is available at present. Our aim was to perform a combined mRNA microarray and proteomic analysis on the same eutopic endometrium sample obtained from patients with and without endometriosis. mRNA and protein fractions were extracted from 49 endometrial biopsies obtained from women with laparoscopically proven presence (n= 31) or absence (n= 18) of endometriosis during the early luteal (n= 27) or menstrual phase (n= 22) and analyzed using microarray and proteomic surface enhanced laser desorption ionization-time of flight mass spectrometry, respectively. Proteomic data were analyzed using a least squares-support vector machines (LS-SVM) model built on 70% (training set) and 30% of the samples (test set). mRNA analysis of eutopic endometrium did not show any differentially expressed genes in women with endometriosis when compared with controls, regardless of endometriosis stage or cycle phase. mRNA was differentially expressed (P< 0.05) in women with (925 genes) and without endometriosis (1087 genes) during the menstrual phase when compared with the early luteal phase. Proteomic analysis based on five peptide peaks [2072 mass/charge (m/z); 2973 m/z; 3623 m/z; 3680 m/z and 21133 m/z] using an LS-SVM model applied on the luteal phase endometrium training set allowed the diagnosis of endometriosis (sensitivity, 91; 95% confidence interval (CI): 74-98; specificity, 80; 95% CI: 66-97 and positive predictive value, 87.9%; negative predictive value, 84.8%) in the test set. mRNA expression of eutopic endometrium was comparable in women with and without endometriosis but different in menstrual endometrium when compared with luteal endometrium in women with endometriosis. Proteomic analysis of luteal phase endometrium allowed the diagnosis of endometriosis with high sensitivity and specificity in training and test sets. A potential limitation of our study is the fact that our control group included women with a normal pelvis as well as women with concurrent pelvic disease (e.g. fibroids, benign ovarian cysts, hydrosalpinges), which may have contributed to the comparable mRNA expression profile in the eutopic endometrium of women with endometriosis and controls.

FOXO1 Expression and Regulation in Endometrial Tissue during the Menstrual Cycle and in Early Pregnancy Decidua

Aims: To determine FOXO1 (forkhead box O1) expression in the human endometrium during the menstrual cycle and decidua of early pregnancy, as well as FOXO1 regulation in endometrial stromal cells (ESC). Methods: Immunohistochemistry, RT-qPCR, and Western blot analyses evaluated cellular localization and altered FOXO1 expression in the endometrium during the menstrual cycle and decidua of early pregnancy (proliferative phase, n = 12; early-secretory phase, n = 7; mid-secretory phase, n = 10; late-secretory phase, n = 10; early pregnancy, n = 12). Using RT-qPCR and Western blot, we studied the regulation of FOXO1 by 8-bromo-cAMP, 4-pregnene-3,20-dione, 17β-estradiol, and human chorionic gonadotrophin in ESC (n = 5). Results: The expression level of FOXO1 in human endometrial tissue fluctuated with the menstrual cycle. If pregnancy occurred, the expression of FOXO1 was further increased (p < 0.05) and cAMP regulated FOXO1 expression in ESC. In addition, 4-pregnene-3,20-dione cooperatively stimulated FOXO1 expression with cAMP. We also observed FOXO1 expression during in vitro cAMP-induced decidualization. Conclusions: The pattern of FOXO1 expression suggests a potential role for FOXO1 in implantation and decidualization.

Relationship between pregnancy per artificial insemination and early luteal concentrations of progesterone and establishment of repeatability estimates for these traits in Holstein-Friesian heifers

Pregnancy per insemination is a major determinant of reproductive efficiency in cattle and is affected by concentrations of progesterone (P4) during early pregnancy. The relationship between pregnancy per artificial insemination (P/AI) and early luteal concentrations of P4, and repeatability of concentrations of P4 was examined on d 4, 5, 6, and 7 (day of standing estrus=d 0) in 118 Holstein Friesian heifers following 2 rounds of AI to 1 high-fertility sire. Repeatability estimates (Re) for P/AI were established following 4 rounds of AI. We found a linear and quadratic relationship between P/AI and concentrations of P4 on d 4 to 7 after estrus, as well as a linear and quadratic relationship between P/AI and the change in concentration of P4 from d 4 to 7 and from d 5 to 7. Optimum concentrations of P4 to maximize probability of P/AI were 2.5, 4.0, 5.0, 5.2, and 3.5ng/mL for d 4, 5, 6, and 7, and the change from d 4 to 7, respectively. Repeatability of P/AI following 4 rounds of AI was low (Re=0.07). Repeatability estimates for concentrations of P4 from cycle to cycle indicated low repeatability between d 4 (Re=0.05) and 7 (Re=0.20). These data indicated the importance of P4 in the early luteal phase for pregnancy survival, but also demonstrated that high concentrations of P4 on these days have a deleterious effect on embryo viability. Early luteal (d 4 to 5) concentrations of P4 were a reasonable predictor of concentrations on d 7 and could be used as a diagnostic tool to identify animals at risk of subsequent embryo loss.

Bovine large luteal cells show increasing de novo DNA methylation of the main ovarian CYP19A1 promoter P2

Transformation of the estrogen producing large dominant follicle into a functional progesterone producing corpus luteum involves profound and well-orchestrated changes in cell type-specific gene expression profiles, possibly involving epigenetic mechanisms of gene silencing. As an experimental paradigm to examine the involvement of de novo DNA methylation in the process of luteinization, the transcript abundance and promoter-specific DNA methylation levels of CYP19A1, which encodes the key enzyme for estrogen biosynthesis, were analyzed in enzymatically dispersed and purified large granulosa luteal cells of early- to mid-cycle bovine corpora lutea. To characterize the morphology and physiology of isolated corpora lutea, their weights and the respective plasma progesterone levels were analyzed. Transcript abundance of CYP19A1, HSD3B1, GHR, and of LHGCR was quantified by real-time PCR. Methylation levels were analyzed by bisulfite direct sequencing. The data indicated that corpora lutea weights and plasma progesterone levels significantly increased during the early luteal phase (days 3–6 of the cycle). The growth of small and large luteal cells was particularly pronounced between days 3 and 4. Large luteal cells are characterized by high HSD3B1 and GHR, but low LHCGR transcript abundance, whereas CYP19A1 expression was very low or undetectable. The DNA methylation levels of the main ovarian CYP19A1 promoter P2 significantly increased from day 5. In conclusion, the data indicated de novo DNA methylation approximately five days after the luteinizing hormone-induced down-regulation of CYP19A1 expression, suggesting that DNA methylation during the early luteal phase might play a role for permanent silencing of previously down-regulated genes.

Endometrial Phospholipase A2 Activity During the Oestrous Cycle and Early Pregnancy in Mares

The aim of this study was to determine phospholipase A2 (PLA2) kinetics and activity in the mare's endometrium during the oestrous cycle and early pregnancy. Phospholipase A2 is responsible for the liberation of arachidonic acid from phospholipids, which is the first limiting step in prostaglandins synthesis. Phospholipase A2 activity was measured using an assay based on the liberation of oleic acid from 1-palmitoyl-2-[(14) C] oleoyl phosphatidylcholine. The enzyme was shown to be calcium dependent, to have an optimum pH of 8 and an apparent Michaelis constant of 127 μM. Enzyme activity was low in the endometrium of early luteal phase tissue but increased significantly (p < 0.001) during the late luteal phase (5.39 ± 0.16; 3.48 ± 0.33, 6.85 ± 0.59, and 9.96 ± 1.23 nmol oleic acid released/mg protein at oestrus, and Days 3, 8 and 14 after ovulation, respectively). The mean PLA2 activity in endometrial tissue from pregnant mares (4.23 ± 0.74) was significantly lower (p < 0.01) than from cyclic animals during late dioestrus (9.96 ± 1.23). The results indicate that PLA2 activity in equine endometrium changes with the stage of the oestrous cycle and thus may be influenced by systemic hormone concentrations. The inhibitory effects of conceptus products on secretion of prostaglandin during early pregnancy were associated with a competitive inhibitor that decreased endometrial PLA2 activity.

Localization and hormonal regulation of endometrial matrix metalloproteinase-26 in the rhesus macaque.

The current understanding of hormonal regulation of matrix metalloproteinase-26 (MMP-26) in the primate endometrium is incomplete. The goal of this work was to clarify estrogen and progesterone regulation of MMP-26 in the endometrium of ovariectomized, hormone-treated rhesus macaques. Ovariectomized rhesus macaques (n= 66) were treated with estradiol (E(2)), E(2) plus progesterone, E(2) followed by progesterone alone or no hormone. Endometrium was collected from the hormone-treated animals during the early, mid- and late proliferative and secretory phases of the artificial menstrual cycle. MMP-26 expression was quantified by real-time PCR, and MMP-26 transcript and protein were localized by in situ hybridization and immunohistochemistry and correlated with estrogen receptor 1 and progesterone receptor (PGR). MMP-26 was localized to glandular epithelium and was undetectable in the endometrial stroma and vasculature. MMP-26 transcript levels were minimal in the hormone-deprived macaques and treatment with E(2) alone did not affect MMP-26 levels. Treatment with progesterone both in the presence and absence of E(2) stimulated MMP-26 expression in the early and mid-secretory phases (P < 0.001). MMP-26 expression preceded decidualization of endometrial stroma. MMP-26 levels then declined to baseline in the late secretory phase (P < 0.01) despite continued E(2) plus progesterone treatment. Loss of detectable MMP-26 expression in the late secretory phase was correlated with late secretory phase loss of glandular epithelial PGR. Endometrial MMP-26 expression is dependent on the presence of progesterone in the early secretory phase and then gradually becomes refractory to progesterone stimulation in the late secretory phase. In the macaque, MMP-26 is a marker of the pre-decidual, secretory endometrium. During the second half of the late secretory phase, and during decidualization, MMP-26 loses its response to progesterone concurrent with the loss of epithelial PGR. The decline in MMP-26 levels between the mid- and late secretory phases may play a role in the receptive window for embryo implantation.