Early Luteal Phase Research Articles

Comparison of antral follicle count across the menstrual cycle with Antimullerian hormone and ovarian response to controlled hyperstimulation related to ovarian reserve

To compare antral follicle count (AFC) at early follicular (EF), late follicular (LF) and luteal (L) phases with AFC at day 1 of the ovarian stimulation (S1), antimullerian hormone (AMH) and oocytes yield, in women with low, normal or high ovarian reserve.

Elevated serum interleukin-1β levels and interleukin-1β-to-interleukin-1 receptor antagonist ratio 1 week after embryo transfer are associated with ectopic pregnancy

To investigate whether interleukin-1β (IL-1β) and interleukin-1 receptor antagonist (IL-1RA) serum levels in the early luteal phase differ in IVF cycles that result in an ectopic pregnancy (EP) when compared with other outcomes. Retrospective cohort. Not applicable. A total of 307 women whose serum samples were available, with the following IVF outcomes: 103 live births, 80 negative pregnancy tests, 52 biochemical pregnancies, 47 EPs, and 25 miscarriages. Serum samples were obtained on cycle days 24 and 28 (cycle day 14 = day of egg retrieval). Levels of IL-1β and IL-1RA were determined by quantitative ELISA performed by blinded personnel. IL-1β and IL-1RA levels, IL-1β-to-IL-1RA ratio versus cycle outcome. The IL-1β levels were predictive of an EP. At cycle days 24 and 28 the mean IL-1β levels were higher in patients with an EP (127.1 pg/mL and 166.9 pg/mL, respectively) than in women with any other IVF outcome (15.8-55.3 pg/mL and 14.8-75.5 pg/mL, respectively). At cycle day 24 the IL-1β-to-IL-1RA ratio was 0.18 in the ectopic group versus 0.01-0.09 in the other groups. Elevated IL-1β levels and IL-1β-to-IL-1RA ratio as early as 4 days before the first pregnancy test are associated with an EP. If confirmed by prospective studies, clinical application of these findings could potentially improve EP detection.

Effects of 2,2′,4,4′-tetrabromodiphenyl ether (BDE47) on the enzymes of phase I (CYP2B1/2) and phase II (SULT1A and COMT) metabolism, and differences in the action of parent BDE-47 and its hydroxylated metabolites, 5-OH-BDE-47 and 6-OH-BDE47, on steroid secretion by luteal cells

In this study we determined the effects of BDE-47 on the expression and activity of phase I (CYP2B1/2) and phase II (SULT1A and COMT) enzymes, and assessed the actions of BDE-47 and its metabolites on luteal steroidogenesis. Luteal cells collected during early (ELP), middle (MLP) and late (LLP) luteal phase were exposed to BDE-47 (0.5, 25, and 50ng/ml) or metabolites (2.5, 5 and 25ng/ml). BDE-47 decreased CYP2B1/2 activity and expression but had no effect on SULT1A or COMT. BDE-47 exerted a stimulatory action on estrogen secretion in MLP and an inhibitory in LLP, but had no effect on progesterone secretion. 5-OH-BDE-47 and 6-OH-BDE-47 decreased progesterone, but had no effect on estrogen secretion. ConclusionsThe inhibitory effect of BDE-47 on CYP2B1/2 suggests the possibility of BDE-47 accumulation in the corpus luteum; by affecting steroid secretion and steroidogenesis enzymes, BDE-47 and its metabolites can be responsible for shortening luteal phase.

Daily low-dose hCG stimulation during the luteal phase combined with GnRHa triggered IVF cycles without exogenous progesterone: a proof of concept trial.

Can the luteal phase support be improved in terms of efficacy, hormonal profiles and convenience as compared with today's standard care? Daily low-dose rhCG supplementation in GnRHa triggered IVF cycles can replace the traditional used luteal phase support with exogenous progesterone. A bolus of hCG for final maturation of follicles in connection with COS may induce the risk of OHSS and the luteal phase progesterone levels rise very abruptly in the early luteal phase. This is a proof-of-concept study conducted as a three arm RCT with a total of 93 patients. First patient enrolled in January 2012 and the study finished in January 2014. Normal responder women undergoing IVF/ICSI treatment in a university hospital. One arm served as control, where women followed a standard antagonist protocol. Two study arms were included both having 125 IU hCG daily for luteal phase support without exogenous progesterone after using a GnRHa trigger for ovulation induction. In both study arms exogenous FSH was stopped on stimulation day 6 and replaced by exogenous hCG that was initiated on either stimulation day 2 or day 6. Blood samples were obtained on the day of ovulation induction, on the day of oocyte pickup (OPU) and day OPU + 7. The mean serum levels of hCG did not exceeded the normal physiological range of LH activity in any samples. Mid-luteal progesterone levels were significantly higher in the two study groups receiving daily low-dose hCG for luteal phase support as compared with the control group (control group: 177 ± 27 nmol/l; study group 1: 334 ± 42 nmol/l; study group 2: 277 ± 27 nmol/l; (mean ± SEM). No differences in reproductive outcome were seen between groups. The number of patients included is limited and conclusions need to be verified in a larger RCT. Endogenous production of progesterone may become more attractive as the luteal phase support with levels of LH-like activity only in the physiological range and may, from the patients' point of view, replace inconvenient exogenous progesterone preparation. Further hCG may reduce the cost of stimulation and may collectively be used for stimulation of the follicular phase, ovulation induction and for luteal phase support. An unrestricted grant from ARTS Biologics made this study possible. None of the authors has any competing interests to declare. ClinicalTrial.gov number: NCT01504139. 28 December 2011.

Stimulatory effect of luteinizing hormone, insulin-like growth factor-1, and epidermal growth factor on vascular endothelial growth factor production in cultured bubaline luteal cells

The purpose of this study was to evaluate the temporal (24, 48, and 72 hours) and dose-dependent (0, 5, 10, and 100 ng/mL of LH, insulin-like growth factor 1 [IGF-1], and EGF) in vitro expression and secretion patterns of vascular endothelial growth factor (VEGF) in luteal cell culture during different stages of estrous cycle in water buffaloes. Corpus luteum samples from ovaries of early luteal phase (ELP; Days 1–4), midluteal phase (Days 5–10), and late luteal phase (Days 11–16) were collected from a local slaughterhouse. The samples were then processed and cultured in (serum containing) appropriate cell culture medium and incubated separately with three factors (LH, IGF-1, or EGF) at the previously mentioned three dose–duration combinations. At the end of the respective incubation periods, VEGF was assayed in the spent culture medium by ELISA, whereas the cultured cells were used for VEGF mRNA expression by quantitative real-time polymerase chain reaction. The results of the present study disclosed dose- and time-dependent stimulatory effects of LH, IGF-1, and EGF on VEGF production in bubaline luteal cells. The VEGF expression and secretion from the cultured luteal cells were highest during the ELP, intermediate in the midluteal phase, and lowest in the late luteal phase of the estrous cycle for all the three tested factors. Comparison of the results of the three treatments depicted EGF as the most potent stimulating factor followed by IGF-1 and LH. Immunocytochemistry findings in luteal cell culture of ELP agreed with the VEGF expression and secretion. In conclusion, mRNA expression, protein secretion, and immunolocalization of VEGF data clearly indicated for the first time that LH, IGF-1, and EGF play an important role in stimulating luteal angiogenesis in buffalo CL. The highest expression and secretion of VEGF in the ELP might be associated with the development of blood vessels in early growth of CL, which in turn gets augmented by the aforementioned factors emphasizing their regulatory role in luteal angiogenesis. Further studies are however necessary to divulge more information on other factors which regulate VEGF secretion in bubaline CL and the synergistic effects existing among such growth factors.

Cholesterol Levels In Bligon Goats During Estrous Cycle

The study was aimed to assess the status of cholesterol levels during the estrous cycle in goats. The present study was conducted by using 4 female cycling bligon goats (Capra hircus), aged 2-3 years, weighed 27-45 kgs, and clinically healthy and Venous blood sampling was done on days 1 (estrus), 3-4 (early luteal), 10-16 (late luteal), 17-20 (proestrus) using a syringe and collected in tubes containing lithium heparin. Plasma wasseparated by centrifugation, put into 1.5 ml Eppendorf tube and stored at a temperature of -20° C until analysis performed. Cholesterol levels were measured by using spectrophotometric method. Cholesterol concentrations during the estrous cycle were analyzed by General Linear Model Repeated Measure. The average of cholesterollevels in bligon goats is 52.20 ± 11.11 – 64.50 ± 18.46 mg/dl. The results showed that the concentration of cholesterol was between phases during the estrous cycle was not significantly different (P>0.05). However, there was a decreasing tendency of cholesterol concentration at the early luteal phase (52.20 mg/dl) and at the late luteal (53.19 mg/dl) compared to the levels at the follicular phase (65.50 mg / dl at estrus and 59.22 mg / dl at proestrus ). It can be concluded that the plasma level of cholesterol does not change significantly during theestrous cycle.

Clear cell papillary renal cell carcinoma: a distinct low-grade renal tumour

To study the clinicopathologic features of clear cell papillary renal cell carcinoma (CCPRCC). The clinical, morphologic and immunohistochemical characteristics of 6 cases of CCPRCC were reviewed, with analysis of follow-up data. There were altogether 3 men and 3 women. The mean age of patients was 56 years. The size of tumors ranged from 1.0 to 4.5 cm in greatest dimension. They had solid or solid-cystic cut surface. Histologically, the tumors were encapsulated and showed several morphologic patterns, with tubules, papillae, acini, interconnecting ribbons and macro/microcysts lined by single layer of cells with clear or small amount of eosinophilic cytoplasm and low-grade nuclei (corresponding to Fuhrman grade 1 or 2). Mitotic figures were rarely seen. Characteristically, there was linear arrangement of the nuclei away from the basement membrane, conferring an appearance similar to that of endometrial glands in early secretory phase. Tubules and cysts contained serosanguineous fluid or colloid-like secretion were identified. No foamy histiocytes, psammomatous calcifications or hemosiderin was present in the papillary areas. Two of the tumors showed focal or extensive angioleiomyoma/leiomyoma-like components. No coagulative necrosis, sarcomatoid dedifferentiation, nor microscopic vascular invasion was observed. Immunohistochemically, all tumors showed strong co-expression of CK7 and CA9 (with characteristic "goblet" staining pattern). The staining for EMA, CK (AE1/AE3), vimentin, CK8, CK18, CK19 and PAX-8 were also positive in all cases. Ki-67 was expressed in less than or about 5% of the tumor cell nuclei. The staining for CD10, P504S, CD117, TFE3 and TFEB was negative. Follow-up data were available in all patients, with mean duration of 14 months (range = 7 to 27 months). All of the patients were disease-free after operation. CCPRCC is a special type of low-grade renal neoplasm with characteristic histopathologic and immunohistochemical features. It needs to be distinguished from clear cell renal cell carcinoma or papillary renal cell carcinoma.

Angiogenesis in The Ovary - The Most Important Regulatory Event for Follicle and Corpus Luteum Development and Function in Cow - An Overview.

In the ovary, the development of new capillaries from pre-existing ones (angiogenesis) is a complex event regulated by numerous local factors. The dominant regulators of angiogenesis in ovarian follicles and corpora lutea are the vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), angiopoietin (ANPT) and hypoxia-inducible factor (HIF) family members. Antral follicles in our study were classified according to the oestradiol-17-beta (E2) content in follicular fluid (FF) and were divided into five classes (E2 < 0.5, 0.5-5, 5-20, 20-180 and >180 ng/ml FF). The corresponding sizes of follicles were 5-7, 8-10, 10-13, 12-14 and >14 mm, respectively. Follicle tissue was separated in theca interna (TI) and granulosa cells (GC). The corpora lutea (CL) in our study were assigned to the following stages: days 1-2, 3-4, 5-7, 8-12 13-16 and >18 of the oestrous cycle and months 1-2, 3-4, 6-7 and >8 of pregnancy. The dominant regulators were measured at mRNA and protein expression levels; mRNA was quantified by RT-qPCR, hormone concentrations by RIA or EIA and their localization by immunohistochemistry. The highest expression for VEGF-A, FGF-2, IGF-1 and IGF-2, ANPT-2/ANPT-1 and HIF-1-alpha was found during final follicle maturation and in CL during the early luteal phase (days 1-4) followed by a lower plateau afterwards. The results suggest the importance of these factors for angiogenesis and maintenance of capillary structures for final follicle maturation, CL development and function.

Relationship between estradiol and progesterone concentrations and cognitive performance in normally cycling female cynomolgus monkeys

Preclinical research has demonstrated that cognitive function may be influenced by estradiol (E2) and progesterone (P4) concentrations, although few cognition studies involve normally cycling females. The present study examined cognitive performance in normally cycling female cynomolgus macaques (n=14), a species with similarities to humans in brain organization and a nearly identical menstrual cycle to women. Initial assessments compared cognitive measures to circulating concentrations of E2 and P4 (n=12). Once a relationship was characterized between hormones and cognitive performance, the menstrual cycle was divided into four distinct phases: early follicular (EF), late follicular (LF), early luteal (EL) and late luteal (LL), verified by the onset of menses and serum concentrations of E2 and P4. Concentrations of E2 were highest during the LF phase and P4 concentrations peaked during the EL phase. All monkeys were trained on two cognitive tasks: reversal learning, involving simple discrimination (SD) and reversal (SDR), which measured associative learning and behavioral flexibility, respectively (n=3–4 per phase) and a delayed match-to-sample (DMS) task which assessed working memory (n=11). P4 concentrations were positively correlated with number of trials and errors during acquisition of SD performance, but not during acquisition of the SDR task or maintenance of the reversal-learning task. Across the menstrual cycle, significantly fewer errors were made in the SDR task during the LF phase, when E2 concentrations were high and P4 concentrations low. Working memory, assessed with the DMS task, was not consistently altered based on previously characterized menstrual cycle phases. These findings demonstrate a relationship between P4, E2 and cognitive performance in normally cycling cynomolgus monkeys that is task dependent. Knowledge of these interactions may lead to a better understanding of sex-specific cognitive performance.

A specific association between facial disgust recognition and estradiol levels in naturally cycling women.

Subtle changes in social cognition are associated with naturalistic fluctuations in estrogens and progesterone over the course of the menstrual cycle. Using a dynamic emotion recognition task we aimed to provide a comprehensive description of the association between ovarian hormone levels and emotion recognition performance using a variety of performance metrics. Naturally cycling, psychiatrically healthy women attended a single experimental session during a follicular (days 7–13; n = 16), early luteal (days 15–19; n = 14) or late luteal phase (days 22–27; n = 14) of their menstrual cycle. Correct responses and reaction times to dynamic facial expressions were recorded and a two-high threshold analysis was used to assess discrimination and response bias. Salivary progesterone and estradiol were assayed and subjective measures of premenstrual symptoms, anxiety and positive and negative affect assessed. There was no interaction between cycle phase (follicular, early luteal, late luteal) and facial expression (sad, happy, fearful, angry, neutral and disgusted) on any of the recognition performance metrics. However, across the sample as a whole, progesterone levels were positively correlated with reaction times to a variety of facial expressions (anger, happiness, sadness and neutral expressions). In contrast, estradiol levels were specifically correlated with disgust processing on three performance indices (correct responses, response bias and discrimination). Premenstrual symptoms, anxiety and positive and negative affect were not associated with emotion recognition indices or hormone levels. The study highlights the role of naturalistic variations in ovarian hormone levels in modulating emotion recognition. In particular, progesterone seems to have a general slowing effect on facial expression processing. Our findings also provide the first behavioural evidence of a specific role for estrogens in the processing of disgust in humans.

Premenstrual syndrome: management and pathophysiology

Premenstrual syndrome (PMS) is 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. Women with PMS experience affective or somatic symptoms that cause severe dysfunction in social or occupational realms. Although evidence for a hormonal abnormality has not been established, the symptoms of the premenopausal disorders are related to the production of progesterone by the ovary. The progesterone metabolites may bind to a neurosteroid-binding site on the membrane of the neurotransmitters. Thus, ovulation suppression is an area of focus for diagnostic and treatment options. Many treatment studies have focused on suppression of ovulation with gonadotropin-releasing hormone analogs (GnRHa), high doses of transdermal estrogen, and bilateral oophorectomy all have positive evidence as treatment options for prevention of PMS. However, because of these limitations and their substantial intensive care, these do not appear to be appropriate methods for conventional treatment of PMS. Serotonergic antidepressants, selective serotonin reuptake inhibitors, are well-established, highly effective, and first-line pharmacologic therapy.

Functional expression of transient receptor potential channels in human endometrial stromal cells during the luteal phase of the menstrual cycle.

Are members of the transient receptor potential (TRP) channel superfamily functionally expressed in the human endometrial stroma? The Ca(2+)-permeable ion channels TRPV2, TRPV4, TRPC6 and TRPM7 are functionally expressed in primary endometrial stromal cells. Intercellular communication between epithelial and stromal endometrial cells is required to initiate decidualization, a prerequisite for successful implantation. TRP channels are possible candidates as signal transducers involved in cell-cell communication, but no fingerprint is available of the functional distribution of TRP channels in the human endometrium during the luteal phase of the menstrual cycle. Endometrial biopsy samples (previously frozen) from patients of reproductive age with regular menstrual cycles, who were undergoing diagnostic laparoscopic surgery for pain and/or infertility, were analysed. Samples were obtained from the menstrual (Days 1-5, n = 3), follicular (Days 6-14, n = 6), early luteal (Days 15-20, n = 5) and late luteal (Days 21-28, n = 5) phases. In addition, a total of 13 patient samples taken during the luteal phase were used to set up primary cell cultures for further experiments. Quantitative real-time PCR (qRT-PCR), immunocytochemistry, Fura2-based Ca(2+)-microfluorimetry and whole-cell patch clamp experiments were performed to study the functional expression pattern of TRP channels. Specific pharmacological agents, such as Δ(9)-tetrahydrocannabinol, GSK1016790A and 1-oleoyl-2-acetyl-glycerol, were used to functionally assess the expression of TRPV2, TRPV4 and TRPC6, respectively. Expression of TRPV2, TRPV4, TRPC1, TRPC4, TRPC6, TRPM4 and TRPM7 was detected at the mRNA level in endometrial biopsies (n = 19) and in primary endometrial stromal cell cultures obtained from patients during the luteal phase (n = 5) of the menstrual cycle. Messenger RNA levels of TRPV2, TRPC4 and TRPC6 were significantly increased (P < 0.01) in the late luteal phase compared with the early luteal phase. Immunocytochemistry experiments showed a positive staining for TRPV2, TRPV4, TRPC6 and TRPM7 in the plasma membrane and in the cytoplasm of primary endometrial stromal cells. Ca(2+)-microfluorimetry revealed significant increases (P < 0.001) in intracellular Ca(2+) levels when stromal cells were incubated with specific activators of TRPV2, TRPV4 and TRPC6. Further functional characterization was performed using whole-cell patch clamp experiments. Taken together, these data provide evidence for the functional activity of TRPV2, TRPV4, TRPC6 and TRPM7 channels in primary stromal cell cultures. Although mRNA levels are detected for TRPV6, TRPC1, TRPC4 and TRPM4, the limited supply of specific antibodies and lack of selective pharmacological agents restricted any additional analysis of these ion channels. Embryo implantation is a dynamic developmental process that integrates many signalling molecules into a precisely orchestrated programme. Our findings identified certain members of the TRP superfamily as candidate sensors in the epithelial-stromal crosstalk. These results are very helpful to unravel the signalling cascade required for successful embryo implantation. In addition, this knowledge could lead to new strategies to correct implantation failure and facilitate the development of novel non-hormonal contraceptives. This work was supported by grants from the Research Foundation-Flanders (G.0856.13N to J.V.), the Research Council of the KU Leuven (OT/13/113 to J.V. and T.D. and PF-TRPLe to T.V.) and by the Planckaert-De Waele fund (to J.V.). K.D.C. and K.H. are funded by the FWO Belgium. None of the authors have a conflict of interest.

Serum kisspeptin levels in normal and overweight Saudi females and its relation with anthropometric indices.

BACKGROUND AND OBJECTIVESAnimal studies suggest that undernutrition and low body weight are associated with low kisspeptin expression and vice versa. This led us to hypothesize that obese and overweight individuals might have significant differences in kisspeptin levels compared with normal-weight individuals.DESIGN AND SETTINGSA prospective cohort study of 1-year duration involving young Saudi female students in the University of Dammam.SUBJECTS AND METHODSYoung Saudi females studying in a Saudi university were divided into 2 groups: normal weight with a body mass index (BMI)=18.5–24.99 and overweight/obese with a BMI ≥25. Serum kisspeptin levels were measured by enzyme-linked immunosorbent assay in early follicular, pre-ovulatory, and luteal phases, and anthropometric parameters were recorded.RESULTSMean serum kisspeptin levels in the overweight/obese group (follicular phase: 266.88 [45.53], pre-ovulatory phase: 477.12 [21.18], and luteal phase: 735.13 [52.59]) did not differ significantly compared with the normal-weight group (follicular phase: 259.82 [20.50], pre-ovulatory phase: 448.36 [24.58], and luteal phase 708.18 [49.47]) in any of the menstrual phase. In any of the menstrual phase, kisspeptin levels were not found to be correlated with any of the anthropometric variable in any group.CONCLUSIONSerum kisspeptin levels in overweight and obese young females may not differ significantly compared with normal-weight females, and there may not be any correlation between serum kisspeptin and anthropometric indices.

Impact of serum estradiol levels on the implantation rate of cleavage stage cryopreserved-thawed embryos transferred in programmed cycles with exogenous hormonal replacement.

To investigate the impact of late follicular phase serum estradiol (E2) levels on implantation and pregnancy outcomes of cleavage stage cryopreserved/thawed embryos transferred in programmed cycles with exogenous hormonal replacement. Retrospective cohort analysis of IVF patients with transfer of cryopreserved-thawed day-3 embryos in E2 and progesterone (P4) supplemented cycles (n = 208cycles). implantation and pregnancy rates according to late follicular phase serum E2 levels and early secretory phase E2/P4 ratios. Logistic regression performed for embryo implantation and for pregnancy outcome in relation to E2 (day 15), P4 (day 15 and 16), before (crude analysis) and after adjustment (adjusted analysis) for baseline characteristics (including age, BMI, serum basal cycle day 3 FSH levels, embryo quality, endometrial lining thickness) showed no significant association. Similarly, ROC analysis showed no impact of cycle day 16 E2/P4 ratio. Neither late follicular phase serum E2 nor the early E2/P4 ratio were able to predict implantation or pregnancy outcome of day-3 cryopreserved-thawed embryos transferred in artificially programmed cycles.

Endocannabinoid regulation in human endometrium across the menstrual cycle.

Humans produce endogenous cannabinoids (endocannabinoids), a group of molecules that activate the same receptors as tetrahydrocannabinol. Endocannabinoids play important roles in reproduction in multiple species, but data in human endometrium are limited. Because endocannabinoids such as anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) often act within tissues as paracrine factors, their effects can be modulated by changes in expression of locally produced synthetic and degradative/oxidative enzymes. The objective of this study was to localize and quantify expression of these key synthetic and degradative/oxidative enzymes for AEA and 2-AG in human endometrium throughout the menstrual cycle. Key synthetic enzymes include N-arachidonyl-phosphatidylethanolamine phospholipase-D (NAPE-PLD), diacylglycerol-lipase a (DAGL-α, and DAGL-β. Key degradative enzymes include fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL); cyclooxygenase 2 (COX2) is an oxidative enzyme. Endometrial samples were collected in 49 regularly cycling, normal women. Protein localization and expression were achieved by immunohistochemistry and messenger RNA (mRNA) expression by real-time reverse transcriptase polymerase chain reaction. No significant cycle-dependent mRNA expression was observed except that of COX2 (P = .002), which demonstrated maximum expression in the proliferative phase. During the secretory phase, NAPE-PLD protein had increased expression in luminal (P = .001), stromal (P = .007), and glandular (P = .04) epithelia, while FAAH had increased glandular (P = .009) and luminal (P = .01) expression. Increased expression in glandular epithelia was identified for MAGL (P = .03). The COX2 had increased luminal expression during the early secretory phase (P < .0001). In conclusion, maximal expression of degradatory/oxidative enzymes in the secretory phase may foster decreased endocannabinoid tone during implantation.

193 PROGESTERONE CONCENTRATIONS AND MESSENGER RNA EXPRESSION OF PROGESTERONE RECEPTORS IN ENDOMETRIAL TISSUE OF BOVINE UTERINE HORN IPSILATERAL AND CONTRALATERAL TO CORPUS LUTEUM

Generally, conception is established in the uterine horn ipsilateral to the corpus luteum (CL) in cattle. When a bovine embryo is transferred into the uterine horn contralateral to CL, conception rate is low. Since progesterone (P4) is essential for the establishment of pregnancy in cattle, locational effects of P4 released from CL at the uterus may cause the differences in fertility. The aim of this study was to determine the endometrial tissue P4 concentrations (EndP4) and the mRNA expression of nuclear progesterone receptor (PGR), and progesterone receptor component-1 (PGRMC-1) and -2 (PGRMC-2) in the endometrial tissues from the ipsi- and contralateral horn. The uteruses of Holstein cows were obtained at a local abattoir. Endometrial tissues were collected from both horns. Based on ovarian morphology, the oestrus cycle of the cow was estimated as follows: early luteal phase (ELP, Day 5–6, Day 0 = oestrus), mid luteal phase (MLP, Day 8–12), late luteal phase (LLP, Day 15–17), and follicular phase (FP, Day 18–20). EndP4 was measured by enzyme immunoassay. Expressions of mRNA were analysed by real-time RT–PCR. Two-way factorial ANOVA and the Steel-Dwass test were applied for a multiple comparison of means. The interrelations between both parameters were expressed by Spearman correlation coefficient. At ELP and MLP, EndP4 in ipsi-horn were higher than that in contra-horn (P &lt; 0.05, see Table 1). Higher mRNA expression of PGRMC-1 in ipsi-horn was observed at ELP compared with contra-horn (P &lt; 0.05). Expressions of mRNA for PGR and PGRMC-2 were similar in both horns. In ipsi-horn at ELP, EndP4 was positively correlated with PGRMC-1 mRNA (r = 0.87, P &lt; 0.05), but was negatively correlated with PGR mRNA (r = –0.76, P &lt; 0.05). However, in contra-horn, EndP4 has no correlation to mRNA expression of P4 receptors. In conclusion, EndP4 was influenced by the location of CL and stage of oestrus cycle. Higher expression of PGRMC-1 mRNA in endometrial tissue of ipsi-horn at ELP might be up-regulated by higher EndP4. These locational effects of CL on uterus may provide an intrauterine environment suitable for embryo development. Table 1.End P4 and expression of mRNA for P4 receptors in bovine uterine horn1

125 EFFECT OF BOVINE OVIDUCTAL FLUID ON DEVELOPMENT AND QUALITY OF IN VITRO-PRODUCED BOVINE EMBRYOS

Although fertilization and early embryonic development take place in the oviduct, the consequences of tubal fluid supplementation during in vitro embryo culture have not been explored. The aim of this study was to evaluate the effect of bovine oviducal fluid (bOF) supplementation during in vitro embryo culture of bovine embryos on their development and quality. The bOF was aspirated from oviducts of slaughtered heifers in the early luteal phase. In vitro-produced zygotes were cultured in SOF (C–; n = 927) or SOF + 5% FCS (C+; n = 872) or in SOF + bOF (n ~900/group) at different concentrations (0.62, 1.25, 2.5, 5, 10, or 25%) in 10 replicates. Blastocysts on Days 7/8 were used for quality evaluation through (a) differential cell count, (b) survival after vitrification/warming, and (c) gene expression (qRT-PCR). One-way ANOVA (development and quality) and t-test (cell count) were used for statistical analysis. The bOF concentrations over 5% were detrimental for blastocysts development (&lt;7% at Day 7) and were discarded. Embryos cultured in absence of FCS exhibited a delay in the kinetics of blastocyst development; at Day 7, the groups cultured without FCS (bOF 0.62–2.5% and C–) had fewer blastocysts (range:12.0 ± 1.7 to 17.4 ± 1.5%) compared with C+ group (22.9 ± 1.2%). However, blastocyst yield at Day 9 was similar in 0.62 and 1.25 bOF groups (27.5 ± 1.7% and 27.5 ± 1.2%, respectively) compared with C+ (27.7 ± 1.0%) and significantly higher than 2.5 bOF (22.7 ± 1.5%) and C– (21.5 ± 1.4%; P &lt; 0.05). In terms of blastocyst quality, 48 h after vitrification/warming, embryos from bOF 1.25%, 0.62%, and C– groups survived significantly higher than C+ (61.3 ± 2.1; 61.6 ± 4.1; 59.3 ± 3.2; and 30.3 ± 2.5, respectively; P &lt; 0.05). This difference was even higher at 72 h (53.6 ± 1.7; 57.7 ± 3.8; 56.1 ± 2.9; and 25.9 ± 2.3%, respectively; P &lt; 0.05). Total cell number of the embryos cultured in bOF 1.25 and 0.62% groups were significantly higher than C+ and C– (165.1 ± 4.7 and 156.2 ± 4.2 v. 143.1 ± 4.9 and 127.7 ± 4.9, respectively), which was associated with an increased TE cell number in 1.25 and 0.62% bOF groups (119.9 ± 3.7 and 127.0 ± 4.5, respectively). Culture with 2.5% bOF had no effect on either blastocyst yield or quality. Gene expression analysis was performed in 1.25% bOF, C–, and C+ groups. The result suggested a higher glucose (SCL2A1) and lipid (CYP51 and FADS1) metabolism in those groups cultured without serum. Gene DNMT3A and the imprinted gene IGF2R were also significantly up-regulated in bOF and C– compared with C+ (P &lt; 0.05). Interestingly, AQP3, a gene positively correlated with survival after vitrification, was significantly up-regulated in bOF compared with C– and C+ (P &lt; 0.05). In conclusion, in vitro culture with low concentrations of bOF has a positive effect in development and quality of bovine embryos cultured in absence of FCS. Funded by the Spanish Ministry of Science and Innovation (AGL2012–37510).

12 EFFECT OF HUMAN CHORIONIC GONADOTROPIN (hCG) ADMINISTRATION ON DAY 2 OR DAY 5 AFTER OESTRUS ON PREGNANCY RATE IN HIGH-YIELDING DAIRY COWS

In cattle, ~40% of embryonic loss occurs in the period from Day 8 to Day 16 of pregnancy. A significant proportion of embryo loss may be due to inadequate circulating progesterone (P4) concentrations. Low P4 concentrations have also been implicated as a causative factor in the low pregnancy rates (PR) observed in high-yielding dairy cows. Administration of hCG during the early luteal phase stimulates hypertrophy of the original corpus luteum (CL) and, depending on the day of administration, induces ovulation of the first-wave dominant follicle and formation of a functional accessory CL, which increases circulating P4 concentrations. The aim of this study was to examine whether administration of hCG on Day 2 or Day 5 after oestrus after timed AI (TAI) would lead to an increase in pregnancy rates in dairy cattle. Lactating Holstein cows (n = 194) from 12 commercial dairy herds in Southern Spain (37.8833° N, 4.7667° W) with an average milk production at 37.8 L/cow per day and typically with a PR to first AI of ~30% were randomly assigned based on their body condition score (2.65 ± 0.05; mean ± SEM), parity (2.60 ± 0.09), and days in milk (75.06 ± 0.63) to 1 of 3 treatments and administered a single intramuscular injection of 3000 IU of hCG (4 mL of Veterin Corion) either (1) on Day 2 = 36 h after TAI (n = 65; hCG2 group), (2) Day 5 = 108 h after TAI (n = 64; hCG5 group), or (3) 4 mL of saline on Day 2 = 36 h after TAI (n = 65; control group). Cows were synchronized using a 7-day Ovsynch TAI protocol that included a P4-releasing intravaginal device (PRID DELTA 1.55 g). First, gonadotropin-releasing hormone (Cystorelin 100 mg) treatment was administered at PRID insertion (Day 0) followed by 25 mg Dinoprost (prostaglandin F2α: Enzaprost T) on Day 7 at PRID withdrawal. Then, 56 h later, the second gonadotropin-releasing hormone (100 mg) treatment was administered and all cows were inseminated 16 h later. Pregnancy was diagnosed by ultrasonography 28 to 32 days after TAI. Logistic regression model and chi-squared test were used to analyse data. Pregnancy rate to AI was significantly higher in the hCG2 and hCG5 groups than in the control group (43.1 and 45.3%, v. 27.7%; P &lt; 0.05). A treatment-by-parity interaction was observed; while pregnancy rate for primiparous cows was not affected by treatment, multiparous cows from the hCG2 group had greater pregnancies per AI than those in the control group (47.2% v. 21.1%, respectively; P &lt; 0.05). In conclusion, these preliminary results suggest that hCG administration on Day 2 and 5 after oestrus increases PR at first postpartum AI in Holstein cows. In addition, hCG on Day 2 increases the fertility in multiparous cows.This study was funded by the Spanish Ministry of Science and Innovation (AGL2012–37510) and partially supported by Ceva Salud Animal S.A., Spain – synchronization protocol and DFV, Spain – hCG treatments.

Alteration in endometrial proteins during early- and mid-secretory phases of the cycle in women with unexplained infertility.

BackgroundCompromised receptivity of the endometrium is a major cause of unexplained infertility, implantation failure and subclinical pregnancy loss. In order to investigate the changes in endometrial protein profile as a cause of unexplained infertility, the current study was undertaken to analyze the differentially expressed proteins of endometrium from early-secretory (LH+2) to mid-secretory phase (LH+7), in women with unexplained infertility.Methods2-D gel electrophoresis was performed to analyze the proteomic changes between early- (n = 8) and mid-secretory (n = 8) phase endometrium of women with unexplained infertility. The differentially expressed protein spots were identified by LC-MS analysis and validated by immunoblotting and immuno-histochemical analysis in early- (n = 4) and mid-secretory (n = 4) phase endometrium of infertile women. Validated proteins were also analyzed in early- (n = 4) and mid-secretory (n = 4) phase endometrium of fertile women.ResultsNine proteins were found to be differentially expressed between early- and mid- secretory phases of endometrium of infertile women. The expression of Ras-related protein Rap-1b, Protein disulfide isomerase A3, Apolipoprotein-A1 (Apo-A1), Cofilin-1 and RAN GTP-binding nuclear protein (Ran) were found to be significantly increased, whereas, Tubulin polymerization promoting protein family member 3, Superoxide dismutase [Cu-Zn], Sorcin, and Proteasome subunit alpha type-5 were significantly decreased in mid- secretory phase endometrium of infertile women as compared to early-secretory phase endometrium of infertile women. Validation of 4 proteins viz. Sorcin, Cofilin-1, Apo-A1 and Ran were performed in separate endometrial biopsy samples from infertile women. The up-regulated expression of Sorcin and down-regulated expression of Cofilin-1 and Apolipoprotein-A1, were observed in mid-secretory phase as compared to early-secretory phase in case of fertile women.ConclusionsDe-regulation of the expression of Sorcin, Cofilin-1, Apo-A1 and Ran, during early- to mid-secretory phase may have physiological significance and it may be one of the causes for altered differentiation and/or maturation of endometrium, in women with unexplained infertility.

Sex differences in acute hormonal and subjective response to naltrexone: The impact of menstrual cycle phase

Women often exhibit larger hormonal and subjective responses to opioid receptor antagonists than men, but the biological mechanisms mediating this effect remain unclear. Among women, fluctuations in estradiol (E2) and progesterone (P4) across the menstrual cycle (MC) affect the endogenous opioid system. Therefore, the goal of the current study was to compare acute naltrexone response between women in the early follicular phase of the MC (low E2 and P4), women in the luteal phase of the MC (high E2 and P4), and men. Seventy healthy controls (n=46 women) participated in two morning sessions in which they received 50mg naltrexone or placebo in a randomized, counterbalanced order. Women were randomized to complete both sessions in either the early follicular (n=23) or luteal phase of the MC. Serum cortisol, salivary cortisol, prolactin, luteinizing hormone (LH), and subjective response were assessed upon arrival to the laboratory and at regular intervals after pill administration. In luteal and early follicular women but not men, naltrexone (vs. placebo) increased serum cortisol and prolactin levels from baseline; however, the naltrexone-induced increases in these hormones were significantly greater in luteal women than early follicular women. Additionally, only luteal women demonstrated an increase from baseline in salivary cortisol levels and the severity of adverse drug effects in response to naltrexone. In sum, the results indicate that luteal phase women are more sensitive to acute hormonal and subjective effects of naltrexone than early follicular women and men. These findings may have important implications for the use of naltrexone in women.