Early Luteal Phase Research Articles

Carboxypeptidase Inhibitor LXN Expression in Endometrial Tissue Is Menstrual Cycle Phase-Dependent and Is Upregulated in Endometriotic Lesions.

Endometriosis is a chronic hormone-dependent disease characterized by the spread of endometrial cells outside the uterus, which form endometriotic lesions and disrupt the functions of the affected organs. The etiopathogenesis of endometriosis is still unclear, and thus it is important to examine the genes that may contribute to the establishment of endometriotic lesions. The aim of this study was to investigate the expression of new potential candidate gene latexin (LXN), an inhibitor of carboxypeptidases, in endometrium and endometriotic lesions to elucidate its possible role in endometriosis development. LXN expression in tissues was assessed using quantitative reverse transcription PCR (qRT-PCR) analysis and immunohistochemical staining (IHC). The functions of LXN were examined using Transwell and MTT assays. qRT-PCR analysis revealed that LXN expression in endometrium was menstrual cycle-dependent, being lowest in the early-secretory phase and highest in the late-secretory phase and was significantly upregulated in endometriotic lesions. IHC confirmed LXN expression in endometrial stromal cells, and in vitro assays demonstrated that knockdown of LXN effectively reduced the migratory capacity of endometrial stromal cells while promoting cell viability. In conclusion, our results showed that LXN can be involved in the pathogenesis of endometriosis by regulating the proliferation and migration activity of endometriotic stromal cells.

Effect of early postmenopause and premenopause on resting-state electroencephalographic and their correlation with ovarian hormone levels.

This study aimed to compare the effect of the early postmenopausal period on resting-state electroencephalographic spectral power with that of the premenopausal period and to analyze the correlation between electroencephalographic spectral power values and endogenous ovarian hormone levels. This study involved 13 early postmenopausal women and 10 premenopausal women in the early follicular, 10 in the ovulatory phase, and 10 in the early luteal phase who underwent resting-state quantitative electroencephalographic spectral power with eyes closed and endogenous ovarian hormone measurements. The delta, theta, alpha1, alpha2, beta1, and beta2 absolute power were compared between the early postmenopausal and premenopausal groups. Correlations between electroencephalographic spectral power values and 17β estradiol, progesterone, follicle-stimulating hormone (FSH), and luteinizing hormone levels were analyzed in early postmenopausal women. Compared with the premenopausal group, the early postmenopausal group showed significantly higher resting-state theta power in the frontal region, alpha1 and alpha2 power in the frontal and central regions, beta1 power in the frontal, central, parietal, and occipital regions, and beta2 power in the centroparietal region. Beta2 power values were positively correlated with FSH levels. The current findings highlight that early postmenopausal women show greater resting-state alpha and beta power, which suggests cortical excitability of fast frequency bands involved in states of alertness, focus of attention, cognition, and emotion. Additionally, we emphasized the effect of FSH levels on fast cortical activation in early postmenopausal women.

O-076 Novel method to enhance preantral follicle growth by granulocyte colony-stimulating factor administration improves embryos and pregnancy rate in poor ovarian reserve: a randomized controlled trial

Abstract Study question Whether priming with granulocyte colony-stimulating factor (G-CSF) before ART improves embryo and pregnancy rate while increasing anti-Müllerian hormone (AMH) in patients with poor ovarian reserve. Summary answer G-CSF priming improved embryonic development and pregnancy rate in ART with poor ovarian reserve, simultaneously increasing serum AMH. Enhanced preantral follicle growth likely was responsible. What is known already Eleven years ago, we treated repeated implantation failure in 10 women with diminished ovarian reserve by administering G-CSF with embryo transfers. No implantation succeeded directly, but serendipitously 3 of the 10 spontaneously became pregnant 2 months later. One of these pregnancies involved twins in a 45-year-old woman without ovulation induction. We hypothesized that G-CSF administration might have stimulated preantral follicle growth, with improved ovulation after 2 months. In animal models with diminished ovarian reserve, G-CSF administration consistently increased ovarian preantral follicles and AMH. Therefore, we designed this clinical trial. Study design, size, duration Patients were prospectively randomized to priming or control groups. All patients initially underwent conventional infertility treatment for 2 consecutive cycles in which the priming group but not controls received a subcutaneous G-CSF priming injection during the early luteal phase. Each group then underwent 1 cycle of IVF/ICSI and fresh embryo transfer (IVF/ICSI-fresh ET), followed by cryopreserved ET if needed until live birth or embryo depletion. AMH was measured before and after priming. Participants/materials, setting, methods Hundred ART patients 20 to 42 years old with AMH below 2 ng/mL were randomized to priming or control groups (50 patients each). None had over 1 ART failure, day-3 follicle-stimulating hormone (FSH) above 30 IU/L, uterine anomalies, or a partner with azoospermia. Main results and the role of chance Fertilization rate, embryonic development, and implantation rate by fresh ET were significantly improved by G-CSF priming. Clinical and ongoing pregnancy rates by IVF/ICSI-fresh ET were significantly higher with priming (30% and 26% in 47 ART patients; 3 delivered with conventional treatment) than in controls (12% and 10% in 49 ART patients; 1 dropped out). With priming, significantly more patients achieved cryopreservation of redundant blastocysts (53% with priming vs. 24% in controls). The cumulative live birth rate was 32% in 50 patients with priming, significantly higher than 14% in 49 controls (relative risk, 2.8; 95% confidence interval, 1.04-7.7). Infants derived from priming had no congenital anomalies, while infant weights, birth weeks, and Apgar scores were similar between groups. Among 4 variables (age, day-3 FSH, AMH, and priming), logistic regression significantly associated age and priming with cumulative live birth. Priming significantly increased serum AMH. The efficacy of G-CSF priming was not associated with genotypes for killer-cell immunoglobulin-like receptors. No adverse effects of priming were observed. Limitations, reasons for caution Enhancement of preantral follicle growth by G-CSF administration needs to be confirmed histologically as this clinical trial could not allow ovarian biopsies for its purpose of infertility treatments. Wider implications of the findings This study proposes a novel, simple, and safe treatment for poor ovarian reserve. In addition, enhancement of preantral follicle growth by G-CSF priming may also improve ovulatory dysfunctions in polycystic ovary syndrome with elevated AMH, reflecting pooling of retarded/arrested growing preantral follicles. Trial registration number UMIN000013956

P-452 A decrease in serum progesterone-levels (P4) during early luteal phase in modified-natural frozen embryo transfer (mNC-FET) is significantly associated with lower pregnancy rates

Abstract Study question Does a serum progesterone-level (P4) increase (+ΔP4) or decrease (-ΔP4) through luteal phase (LP) compromise ongoing pregnancy rate (OPR) or miscarriage rate (MR) in mNC-FET? Summary answer A P4 decrease (-ΔP4) through luteal phase in mNC-FET is significantly associated with lower OPR and higher MR compared to P4 increase (+ΔP4) What is known already Increasing evidence has been recently reported in literature according to the relevance of P4 round FET but also on b-HCG day in artificial and natural FET (AC-FET; NC-FET). Otherwise, mNC-FET gives the chance for triggering and scheduling FET, with less days of monitoring for patients, but keeping the advantages described according to lower risk of preeclampsia through the relaxin production by the corpus luteum. Nevertheless, no studies have been reported according to reproductive outcomes and P4 increase or decrease through the luteal-phase in mNC-FET, as it has been done in fresh embryo transfer (IVF-transfer) Study design, size, duration This is a retrospective observational study led at a single university-affiliated fertility center. Overall, 504 women undergoing mNC-FET between October 2019-October 2023 were included for the analysis. Among them 373 (74%) were homologous-FET cycles (hom-FET), 123 (24%) heterologous-FET cycles (oocyte donation) (het-FET) and 8 (1.6%) embryo donation-FET cycles (don-FET) Participants/materials, setting, methods mNC-FETs were performed seven days after HCG-trigger (HCG+7) and on the sixth day of vaginal progesterone treatment. P4-levels were measured on the day before FET (D5-P4) and on β-HCG day (β-P4). Pregnancy outcomes were analysed according to + ΔP4 or -ΔP4 between those two time intervals through the LP in mNC-FET. Progesterone treatment continued until the 10th week of pregnancy.Chi-squared test was used to compare proportions and ROC-curve analysis to identify the best cutoffs for outcomes Main results and the role of chance Patients’ age at oocyte retrieval was 33.5 and 38.4 at FET. No differences were described according to epidemiological features. Mean β-P4 and D5-P4 (ng/mL) were 25.15 ± 12.8 and 24.88 ± 6.9 respectively. ΔP4 was reported as positive (+ΔP4) in 52.2% (n = 263), and negative (-ΔP4) in 47.8% patients (n = 241). Mean ΔP4 was +0.26 ± 13 ng/mL. Overall reproductive outcomes were: OPR 47.8% and miscarriage rate (MR) 12.3%. OPR was significantly lower (15.8% vs 77.2%, p = 0.00) and MR significantly higher (19.1% vs 11%, p = 0.099) in case of -ΔP4 compared to + ΔP4. Predicting factors for OPR were b-HCG (ROC-AUC (95% CI) 0.920 (0.896-0.945)), β-P4 (ROC-AUC (95% CI) 0.860 (0.827-0.893)) and ΔP4 (ROC-AUC (95% CI) 0.859 (0.826-0.893)). The optimal cut-off value for OPR were β-HCG > 45.15 UI, β-P4 >22.6 ng/mL and ΔP4 > 0.73 ng/mL, with a sensitivity and a specificity of 0.84-0.75, 0.97-0.78 and 0.83-0.78 respectively. OPR in patients with β-HCG > 45.15 UI and β-P4 >22.6 ng/mL was significantly higher (81.7%) compared to patients with β-HCG > 45.15 UI or β-P4 > 22.6 ng/mL (53%), and patients with β-HCG ≤ 45.15 UI and β-P4 ≤ 22.6 ng/mL (2.6%) (p < 0.001) Limitations, reasons for caution The main limitation of this study is its retrospective nature and that conclusions cannot be extrapolated to other methods for endometrial preparation for FET, although conflicting pregnancy outcomes according P4-levels have also been described in either natural or artificial FET. No live birth rate was available at the period study Wider implications of the findings ΔP4 through LP seems to be relevant for pregnancy outcomes in mNC-FET as -ΔP4 significantly reduces OPR and increases MR compared to + ΔP4. Evaluation of ΔP4 between the day before FET (D5-P4) and on β-HCG day (β-P4) may be a useful biomarker to predict ongoing pregnancy rates in mNC-FET cycles Trial registration number Not applicable

O-266 Multi-omics landscape of uterine fluid-derived extracellular vesicles across the menstrual cycle

Abstract Study question Do extracellular vesicles (EVs) mirror the cyclic physiological changes in endometrial tissue, reflecting the endometrial maturation along the entire menstrual cycle? Summary answer Multi-omics data highlights the importance of EVs during the mid-secretory phase and EVs cargo reflects the state of the tissue throughout the menstrual cycle. What is known already Extracellular vesicles isolated from uterine fluid (UF) during the mid-secretory phase have been characterized for their pivotal role in embryo-maternal communication, particularly in the context of microRNAs (miRNAs) and mRNA. EVs have been posited as potential biomarkers in a minimally invasive liquid biopsy approach for assessing endometrial receptivity, hopefully replacing the endometrial tissue biopsy-based testing. However, the dynamics of EVs and their cargo throughout the distinct phases of the menstrual cycle remain unknown. This is the first study to elucidate UF-EV content throughout the maturation of the endometrium. Study design, size, duration Whole-exome RNA and small-RNA sequencing was performed on 15 paired samples, comprising UF and endometrial biopsy (EB), obtained from healthy female subjects across the menstrual cycle. This initial dataset was expanded with 35 additional UF samples. Cumulatively, the analysis encompassed samples (EB and UF) across the menstrual cycle’s four distinct phases: proliferative (n = 10), early-secretory (ES) (n = 17), mid-secretory (MS) (n = 20), and late-secretory (LS) (n = 18). Additionally, 20 UF-EV samples were used for flow cytometry surface proteome analysis. Participants/materials, setting, methods Participants were excluded when infertility-associated conditions were found and included when they had at least one child born, with no hormonal medications used for three months before the sampling. EVs were extracted from UF with size-exclusion chromatography (SEC) and subsequently with glycosaminoglycan-based precipitation. The EVs were characterized with nanoparticle tracking analysis, western blot, transmission electron microscopy and flow-cytometry surface proteome analysis (MACSPlex) for 37 surface markers. Single-end RNA and small-RNA sequencing were performed on NextSeq1000. Main results and the role of chance The transcriptome, miRNome, and protein surface markers of UF-EVs were characterized throughout the menstrual cycle. Transcriptome profiling was performed in comparison to the preceding phase. During the ES phase, 5,507 differentially expressed genes (DEGs) were observed, most of them being downregulated and associated with G-coupled-protein signaling, while in the MS phase, 867 DEGs were identified associated with nucleosome assembly and cell differentiation. Interestingly in the LS phase, the histone genes associated with nucleosome assembly were upregulated compared to MS, highlighting that histone dynamics change corresponding to the menstrual cycle. The exploration of gene ontology revealed consistent enrichment of genes related to extracellular vesicles in UF-EV and EBs across the menstrual cycle. The miRNA analysis of UF-EVs and EB showed significant overlap in miRNA expression level especially in the MS phase. Furthermore, we found 16 unique miRNAs in the mid-secretory phase whose targets were associated with embryo morphogenesis and cell adhesion. MACSPlex analysis revealed that CD9 and CD63, canonical EV markers, were significantly abundant during the MS phase, indicative of an enriched secretion of EVs during the preparation of the endometrium for embryo implantation. These insights underscore that the cargo of UF-EVs reflects the dynamic changes in the endometrial tissue. Limitations, reasons for caution The research on UF-EVs faced notable challenges due to the irregular and non-standardized collection methods of UF in gynecological practice. This variability in UF collection introduces potential confounding factors. The study also operated with a limited sample size, underscoring the need for further validation. Wider implications of the findings Our study indicates that the UF-EVs reflect the menstrual cycle-related molecular changes and UF-EV analysis could be potentially used to follow the menstrual cycle-related molecular processes in the uterine environment. Trial registration number not applicable

O-302 Feasibility and efficacy of a flexible and simplified ovarian stimulation protocol for oocyte donors, combining random-start corifollitropin alpha and progestin primed ovarian stimulation protocol (PPOS)

Abstract Study question Is the total number of retrieved oocytes in donors equivalent, according to the onset phase of stimulation with corifollitropin alpha in a random-start PPOS protocol? Summary answer The total number of retrieved and mature oocytes were not different after starting stimulation in the early, mid or late follicular or luteal phases. What is known already Recent studies have shown the ability to obtain oocytes of equivalent quality after follicular or luteal phase ovarian stimulation and have also demonstrated the efficacy and safety of the PPOS protocol in oocytes donors. In order to provide a more friendly, simplified and flexible ovarian stimulation protocol reducing the burden for donors, we studied the impact on oocyte yield of combining a random start corifollitropin alpha stimulation with the concomitant prevention of premature LH surge by progestin administration. Study design, size, duration This multicenter open-labelled RCT was performed in 4 oocytes donation centers from July 2020 to September 2023 to assess the equivalence in terms of number of retrieved oocytes of a delayed onset of corifollitropin alpha stimulation with concomitant administration of desogestrel in Mid Follicular Phase (MFP/D4-D7 of the natural cycle), Late Follicular Phase (LFP/D8-D11), Ovulatory Phase (OP/D12-D15) or Luteal Phase (LP/D16-D30) compared to the standard start in Early Follicular Phase (EFP/D1-D3). Participants/materials, setting, methods From 110 randomized oocyte donors,102 reached triggering of ovulation with triptoreline acetate and OPU : EFP n = 22, MFP n = 20, LFP n = 19, OP n = 18 and LP n = 23. The sample size was calculated to show an equivalence between groups in total number of retrieved oocytes. Secondary endpoints were the number of mature oocytes, premature LH surge and Ovarian Hyperstimulation Syndrome rates. Differences were tested using a ANOVA test or a Pearson’s Chi2 test, as appropriate. Main results and the role of chance Donors were 33 [29.2-35.0] years old [95% Confidence interval] with a mean BMI of 23.4 [21.1-26.3] kg/m2. The mean number (SD) of retrieved oocytes was 14.2 (9.1). In intention to treat analysis, there was no significant difference between the 5 groups, regarding the total number of retrieved oocytes. The mean numbers of retrieved oocytes from the 5 groups were 12.5 (7.7), 13.2 (8.0), 15.7(9.8), 17.8 (11.4), and 12.8(8.3), respectively in the EPF group, the MPF group, the LPF group, the OP group and the FP group, with no significant differences between the groups (p = 0.30). The mean (SD) number of mature oocytes was 11.8 (7.6) with no significant differences between the 5 groups. Only one premature LH surge was reported in the MFP group (0.98%). Four minor to moderate OHSS were reported (3.9%). Limitations, reasons for caution The main limitation of our study is the small sample size responsible for a lack of power. But to our knowledge, this is the first RCT to compare the number of oocytes retrieved, according to the onset phase of ovarian stimulation in a random-start PPOS protocol, for oocyte donors. Wider implications of the findings The proportion of ovarian stimulation cycles with freeze-all is constantly increasing worldwide. Therefore simplified, ultra-flexible ovarian stimulation protocols could be proposed to maintain the spread of the workload along the working days without programming, with fewer injections and reduced burden for patients, with the same efficacy. Trial registration number NCT03895099

Physicochemical characteristics and protein profile of oviductal and uterine fluids from domestic sheep

Oviductal (OF) and uterine (UF) fluids are a complex mixture of ions and macromolecules dissolved in water, derived from the secretions of secretory cells and transudates of the circulatory system. Through proteomics, OF and UF have been analyzed in different domestic species throughout the estrous cycle or during the first days of pregnancy. Therefore, the aim of this study was to evaluate the volume, osmolarity, concentration and distribution pattern of proteins, as well as the identification of OVGP1, HSP70 and ezrin proteins for their importance in reproductive physiology, in OF and UF from adult criollo type domestic sheep during the early luteal phase of the estrous cycle. An average of 3.2±1.5 uL OF and 17±0.5 uL UF per reproductive system were obtained; osmolarity was 343±20.8 mOsm kg-1 and 280±96.2 mOsm kg-1 and protein concentration was 71.9±23.8 g L-1 and 21.8±1.1 g L-1, respectively. In the protein distribution pattern, 20 bands were observed in the OF and 14 bands in the UF. Of these, 14 and 8 were specific for OF and UF, respectively, and 6 were common for both. The spectra of the protein molecular weights were 24–324 and 29–353 kDa for OF and UF, respectively. The presence of OVPG1, HSP70 and ezrin proteins in both fluids was identified, being in greater quantity in the OF (P < 0.0005). The volume recovered from the UF was five times greater than that of the OF. Both osmolarity and protein concentration were higher in OF than in UF (1.2 and 3 times higher). The pattern of protein distribution between the OF and UF was different, being more complex in the OF. OVGP1, HSP70 and ezrin were identified in the OF and UF, and were found in greater quantities in the OF.

In vitro fertilization outcome based on the detailed early luteal phase trajectory of hormones: a prospective cohort study

BackgroundOvarian stimulation and the use of human chorionic gonadotropin (hCG) for triggering oocyte maturation in women undergoing in vitro fertilisation (IVF) introduces several differences in luteal phase hormone levels compared with natural cycles that may negatively impact on endometrial receptivity and pregnancy rates after fresh embryo transfer. Exogenous luteal phase support is given to overcome these issues. The suitability of a pragmatic approach to luteal phase support is not known due to a lack of data on early phase luteal hormone levels and their association with fertility outcomes during IVF with fresh embryo transfer. This study determined early luteal phase profiles of serum progesterone, 17-hydroxyprogesterone and hCG, and associations between hormone levels/hormone level profile after hCG trigger and the live birth rate in women undergoing IVF with fresh embryo transfer.MethodsThis prospective single center, cohort study was conducted in Vietnam from January 2021 to December 2022. Women aged 18–38 years with normal ovarian reserve and undergoing controlled ovarian stimulation using a gonadotropin-releasing hormone antagonist protocol were included. Serum hormone levels were determined before trigger, at 12, 24 and 36 h after hCG, and daily from 1 to 6 days after oocyte pick-up. Serum hormone level profiles were classified as lower or upper. The primary outcome was live birth rate based on early luteal phase hormone level profile.ResultsNinety-five women were enrolled. Live birth occurred in 19/69 women (27.5%) with a lower progesterone profile and 13/22 (59.1%) with an upper progesterone profile (risk ratio [RR] 2.15; 95% confidence interval [CI] 1.28–3.60), and in 6/31 (19.4%) versus 26/60 (43.3%) with a lower versus upper serum 17-hydroxyprogesterone profile (RR 2.24; 95% CI 1.03–4.86). Nearly 20% of women had peak progesterone concentration on or before day 3 after oocyte pick-up, and this was associated with significantly lower chances of having a life birth.ConclusionsThese data show the importance of proper corpus luteum function with sufficient progesterone/17-hydroxyprogesterone production for achievement of pregnancy and to maximize the chance of live birth during IVF.Trial RegistrationNCT04693624 (www.clinicaltrials.gov).

Endometrial Proliferative Phase-Centered View of Transcriptome Dynamics across the Menstrual Cycle.

The endometrium, the inner mucosal lining of the uterus, undergoes complex molecular and cellular changes across the menstrual cycle in preparation for embryo implantation. Transcriptome-wide analyses have mainly been utilized to study endometrial receptivity, the prerequisite for successful implantation, with most studies, so far, comparing the endometrial transcriptomes between (i) secretory and proliferative endometrium or (ii) mid-secretory and early secretory endometrium. In the current study, we provide a complete transcriptome description of the endometrium across the entire menstrual cycle and, for the first time, comprehensively characterize the proliferative phase of the endometrium. Our temporal transcriptome analysis includes five time points including the mid-proliferative, late proliferative (peri-ovulatory phase), early secretory, mid-secretory, and late secretory phases. Thus, we unveil exhaustively the transitions between the consecutive proliferative and secretory phases, highlighting their unique gene expression profiles and possible distinct biological functions. The transcriptome analysis reveals many differentially expressed genes (DEGs) across the menstrual cycle, most of which are phase-specific. As an example of coordinated gene activity, the expression profile of histone-encoding genes within the HIST cluster on chromosome 6 shows an increase in cluster activity during the late proliferative and a decline during the mid-secretory phase. Moreover, numerous DEGs are shared among all phases. In conclusion, in the current study, we delineate the endometrial proliferative phase-centered view of transcriptome dynamics across the menstrual cycle. Our data analysis highlights significant transcriptomic and functional changes occurring during the late proliferative phase-an essential transition point from the proliferative phase to the secretory phase. Future studies should explore how the biology of the late proliferative phase endometrium impacts the achievement of mid-secretory endometrial receptivity or contributes to molecular aberrations leading to embryo implantation failure.

Changes on corpus luteum structure and progesterone synthesis pathway after hCG or GnRH treatment during the early luteal phase in sheep

This study investigated the effect of hCG or GnRH on structural changes of the corpora lutea (CL) and the regulation of the expression of steroidogenic enzymes involved in P4 secretion in post-ovulatory (po-CL) and accessory CL (acc-CL). Sixty-four ewes were assigned to three groups receiving: 300 IU of hCG (hCG) or 4 µg Buserelin (GnRH) or 1 mL of saline solution (Control) on Day (d) 4 post artificial insemination (FTAI). Laparoscopic ovarian were performed on d 4, 14 and, 21 post-FTAI to determine the numbers of CL. Blood samples were collected for serum LH and P4 analysis. On d 14 post-FTAI, both CL were removed from the ovary to determine large luteal cell (LLC) number and to evaluate the expression of steroidogenic enzymes (HSD3B1, STAR, CYP11A1). Only hCG and GnRH treated ewes generated acc-CL. The LLC in both po- and acc-CL were significantly greater in the hCG group compared to GnRH and Control groups (P<0.05). Overall, hCG group showed the greatest immunodetection of HSD3B1and STAR in both po- and acc-CL (P<0.05). rnRNA expression of HSD3B1, STAR and CYP11A1 in the acc-CL tended to be greater in hCG group than in GnRH group (P<0.1). The LH concentration was increased in GnRH group (P<0.05) and P4 concentration was greater in hCG group compared to the other groups (P<0.05). In conclusion, administration of hCG has a notably impact on acc-CL development and the expression of steroidogenic enzymes compared to GnRH treatment in ewes. This leads to elevated P4 concentration and improved luteal function.

Changes in the mechanical properties of the thigh and lower leg muscle-tendon units during the early follicular and early luteal phases.

This study aimed to determine changes in the muscle and tendon stiffness of the thigh and lower leg muscle-tendon units during the early follicular and early luteal phases, and check for possible relations between muscle and tendon stiffness in each phase. The sample consisted of 15 female university students with regular menstrual cycles. The basal body temperature method, ovulation kit, and salivary estradiol concentration measurement were used to estimate the early follicular and early luteal phases. A portable digital palpation device measured muscle-tendon stiffness in the early follicular and early luteal phases. The measurement sites were the rectus femoris (RF), vastus medialis (VM), patellar tendon (PT), medial head of gastrocnemius muscle, soleus muscle, and Achilles tendon. No statistically significant differences in the thigh and lower leg muscle-tendon unit stiffness were seen between the early follicular and early luteal phases. Significant positive correlations were found between the stiffness of the RF and PT (r = 0.608, p = 0.016) and between the VM and PT (r = 0.737, p = 0.002) during the early luteal phase. The present results suggest that the stiffness of leg muscle-tendon units of the anterior thigh and posterior lower leg do not change between the early follicular and early luteal phases and that tendons may be stiffer in those women who have stiffer anterior thigh muscles during the early luteal phase.

Monitoring of sprint and change of direction velocity, vertical jump height, and repeated sprint ability in sub-elite female football players throughout their menstrual cycle

ABSTRACT Aims The aim of this study was to investigate the associations between the early follicular (EF, i.e., menstruation), late follicular (LF), and middle luteal (ML) phases of the menstrual cycle and different factors that may influence football performance. Methods To this end, 11 eumenorrheic sub-elite female football players underwent field tests to assess sprint speed, lower extremity power, repeated sprint ability, velocity on change of direction, and technical skills at each cycle phase. Results Performance during the 15-m change of direction ability test, 15-m ball dribbling test, squat jump height, total sprint time [sum of 7 sprints] and decrement score [(mean sprint time/best sprint time × 100) − 100], maximum and mean heart rate, and perceived exertion did not significantly differ among menstrual cycle phases. Conversely, the linear sprint velocity over 10, 20, 30-m distances was decreased in EF vs LF (10-, 20- and 30-m) and in ML vs LF (10- and 20-m) (p < 0.05). The 40-m sprint velocity did not change in the different menstrual cycle phases. Conclusion Overall, our study suggests that sex hormone fluctuations during the menstrual cycle are not associated with vertical jump, velocity on change of direction, and repeated sprint ability, but may influence linear sprint velocity over short distances (10, 20, and 30 m).

129 Selenium-form affects de novo cholesterol synthesis and catabolism of cholesterol and progesterone in the liver of beef heifers

Abstract A deficiency of selenium (Se) in soils and hence forages poses a challenge to producers in the southeast United States, necessitating supplementation of this trace mineral to the diet of pasture fed cattle. Se occurs in either inorganic (ISe) or organic (OSe) forms; ISe is conventionally available in commercial supplements, while OSe is available in grazed forages. We have investigated supplementation with either ISe or a 1:1 mixture of ISe:OSe (MIX) and demonstrated that MIX increases the relative abundance of mRNA encoding the low-density lipoprotein receptor (LDLR) and enzymes involved in de novo cholesterol synthesis in the corpus luteum, concurrent with a MIX-induced increase in systemic progesterone (P4) during the early luteal phase. We also observed a MIX-induced decrease in circulating cholesterol and low-density/very low-density lipoproteins during early gestation, and a MIX-induced increase in conceptus length at maternal recognition of pregnancy (MRP). The objective herein was to investigate the effect of form of Se (ISe versus MIX) on de novo cholesterol synthesis and cholesterol/steroid catabolism in the liver at MRP. We hypothesized form of Se would affect the abundance of transcripts that regulate synthesis, catabolism, and local concentrations of cholesterol and P4. Angus-cross heifers were supplemented with ISe or MIX for at least 90 d before insemination at observed estrus. On d 17 of pregnancy, heifers (n = 6/TRT) were killed and their reproductive tracts and conceptuses recovered. Hepatic samples were collected for qPCR analysis of mRNA transcripts encoding proteins involved in de novo cholesterol synthesis, regulation of cholesterol availability and steroid catabolism. Additionally, on d 7 and d 17 of pregnancy, we quantified systemic concentrations of high-density lipoproteins (HDL), indicative of cholesterol recycling to the liver. We quantified the relative abundance of 22 mRNAs and observed that heifers supplemented with MIX-form Se had an increase in the relative abundance of mRNA encoding the very low-density lipoprotein receptor (VLDLR, P = 0.01). Unexpectedly, we failed to detect an effect of TRT on the abundance of mRNA encoding the high-density lipoprotein receptor (SCARB1, P &amp;gt; 0.05) which was consistent with our inability to detect an effect on the serum concentration of total or free HDL (P &amp;gt; 0.05) during early pregnancy and at MRP. Of the 11 mRNA transcripts encoding enzymes involved in catabolism of cholesterol and progesterone, including steroid alpha reductases (SRD5A1 and SRD5A3) and cytochrome P450 enzymes (CYP4A2, CYP4A11, CYP11A1, and CYP17A1), we were unable to detect a difference in the relative abundance of any transcript. Overall, it appears that the form of Se is altering circulating concentrations of cholesterol and further affecting whole animal physiology and fertility; however, the mechanism involved remains to be fully elucidated and warrants further investigation.

11 Transcriptomic changes in response to form of selenium on the interferon-tau signaling mechanism in the caruncular tissue of beef heifers at maternal recognition of pregnancy

Abstract It is necessary to supplement selenium (Se) to the diet of beef cattle in regions where the soils and forages are deficient in this trace mineral. Supplementation with Se conventionally occurs using an inorganic form (ISe) although the organic forms (OSe) are available when cattle consume forage. Previously we have investigated the effects of supplemental Se as ISe, or a 1:1 mixture of ISe to OSe (MIX) on grazing beef cows and heifers and have observed significant effects on the corpus luteum (CL) and endometrium. The MIX form of Se increased systemic progesterone (P4) during the early luteal phase of the estrous cycle at a time that can significantly improve early embryonic development. We subsequently investigated the effects of MIX form Se versus ISe on the mRNA transcripts encoding IFNτ- and P4-induced proteins in the caruncular (CAR) tissue of the uterine endometrium at maternal recognition of pregnancy (MRP). MIX-supplemented heifers had a decrease in the relative abundance of mRNAs encoding DGAT2, FGF2, IFIT3, ISG15, MX1, OAS2, and RSAD2 in CAR which was coupled with significantly longer conceptuses at MRP. Presently, we examined transcriptomic changes in the CAR tissue of the endometrium at MRP. Angus-cross heifers (n = 20) were exposed to a 45-d period of Se depletion (no supplemental Se) followed by a 45-day repletion period with ISe alone. Heifers were then randomly assigned to 90 days of treatment with a vitamin-mineral mix containing 35-ppm Se as ISe (n = 10), or 1:1 mixture of ISe and OSe (n = 10, MIX). Following insemination, heifers were killed at MRP (d 17) and their reproductive tracts collected. Only heifers with a fully intact recovered conceptus were utilized for further analyses (n = 6 per treatment). Transcriptomic analysis using RNA-sequencing was conducted using total mRNA from CAR biopsies. Differential gene expression was determined using Integrated Differential Expression and Pathway Analysis (iDEP.96), and DEG were subjected to canonical, functional, and network analyses using QIAGEN’s Ingenuity Pathway Analysis (IPA). RNA sequencing results were validated using qPCR. At P &amp;lt; 0.05, there was a total of 2,029 DEGs with 1,038 transcripts upregulated, and 991 transcripts downregulated in MIX versus ISe heifers. Saliently, the interferon JAK/STAT pathway signaling through the STAT1/2 heterodimer was significantly down regulated in CAR samples from MIX heifers, as were the following interferon-responsive genes: IFIT1, IFIT2, IFIT3, IRF1, IRF9, ISG15, ISG20, OAS2, RSAD2, and STAT2. Additionally, there were significant changes in mRNA contribution to the immunotolerant environment (IDO1, ISG20, SCARA1, and TGFB1) to allow the fetal allograft to closely interact with the maternal interface. In combination, these findings suggest more advanced preparation of the CAR and developing conceptus for placentation and to evade immune rejection by the maternal endometrium during implantation.

Monitoring Contractility of Junctional Zone Endometrium across Menstrual Cycle Using the ElectroUteroGraph (EUG): A Clinical Evaluation

(1) Background: Abnormal uterine contractility for nonpregnant women has been associated with gynecological pathologies and infertility. The objective of this study was to evaluate the ability of a novel monitoring technique to assess the contractility of the nongravid uterus using a simple, standardized, direct, in vivo methodology during the different phases of the menstrual cycle. (2) Methods: Twenty-six healthy women of reproductive age (28–48 years) were recruited. An ElectroUteroGraph (EUG) was used to measure the electrical activity from the contractility of the junctional zone endometrium (JZE) across the menstrual cycle. Derived recordings were separated into the early proliferative (EP) (n = 10), late proliferative (LP) (n = 31), early luteal (EL) (n = 27), and late luteal (LL) (n = 12) phases of the menstrual cycle. EUG recordings were performed by inserting a flexible electrode array into the endometrial cavity. (3) Results: Waveforms that were measured from the electrode closer to the fundus (1 cm distance) were processed. The Root-Mean-Square (RMS) Voltage Amplitude (VJZE-RMS) (in μV) and the Mean Frequency (fJZE-mean) (in cycles/min) of the JZE’s electrical activity, as direct indicators of the intensity and frequency changes in the JZE’s contractions, were extracted from the recorded waveforms. There was a trend in the median values of the VJZE-RMS decreasing from the EP to the LP phase (247–158 μV). During the EL phase, an upward trend was observed (158–374 μV, p &lt; 0.05), reaching its highest value during the LL phase (374–477 μV, p &lt; 0.05) when compared to the LP phase. The fJZE-mean showed the opposite trend, increasing from 2.5 cycles/min during the EP phase to 2.96 cycles/min during the LP phase. During the EL phase, a downward trend was observed (2.96–2.37 cycles/min), continuing to fall to 1.33 cycles/min, in the LL phase, with p &lt; 0.05 when compared to the previous three phases. (4) Conclusions: The novel in vivo monitoring technique has shown clinically, for the first time, significant electrical activity differences in the different sub-phases of the menstrual cycle, recorded in a safe and painless way.

Background Breast Parenchymal Signal During Menstrual Cycle on Diffusion-Weighted MRI: A Prospective Study in Healthy Premenopausal Women.

To prospectively investigate the influence of the menstrual cycle on the background parenchymal signal (BPS) and apparent diffusion coefficient (ADC) of the breast on diffusion-weighted MRI (DW-MRI) in healthy premenopausal women. Seven healthy premenopausal women (median age, 37 years; range, 33-49 years) with regular menstrual cycles participated in this study. DW-MRI was performed during each of the four phases of the menstrual cycle (four examinations in total). Three radiologists independently assessed the BPS visual grade on images with b-values of 800 sec/mm² (b800), 1200 sec/mm² (b1200), and a synthetic 1500 sec/mm² (sb1500). Additionally, one radiologist conducted a quantitative analysis to measure the BPS volume (%) and ADC values of the BPS (ADCBPS) and fibroglandular tissue (ADCFGT). Changes in the visual grade, BPS volume (%), ADCBPS, and ADCFGT during the menstrual cycle were descriptively analyzed. The visual grade of BPS in seven women varied from mild to marked on b800 and from minimal to moderate on b1200 and sb1500. As the b-value increased, the visual grade of BPS decreased. On b800 and sb1500, two of the seven volunteers showed the highest visual grade in the early follicular phase (EFP). On b1200, three of the seven volunteers showed the highest visual grades in EFP. The BPS volume (%) on b800 and b1200 showed the highest value in three of the six volunteers with dense breasts in EFP. Three of the seven volunteers showed the lowest ADCBPS in the EFP. Four of the seven volunteers showed the highest ADCBPS in the early luteal phase (ELP) and the lowest ADCFGT in the late follicular phase (LFP). Most volunteers did not exhibit specific BPS patterns during their menstrual cycles. However, the highest BPS and lowest ADCBPS were more frequently observed in EFP than in the other menstrual cycle phases, whereas the highest ADCBPS was more common in ELP. The lowest ADCFGT was more frequent in LFP.

Luteal tissue characteristics of Morada Nova ewes with hCG application 7.5 days after the end of estrus synchronization protocol in the breeding season

Results with the use of hCG after synchronization protocol are still inconsistent, which may vary according to breed, season, day of application and dose of the drug used. The aim of the present study was to evaluate the functionality of luteal tissue and ovarian perfusion after hCG treatment during early luteal phase. Estrus-synchronized ewes were randomly assigned to receive i.m. injection of 300 IU of hCG (G-hCG; n = 40) or 1 mL of saline (G-Control; n = 32) on Day 7.5 after progesterone withdrawal. Ultrasonographic evaluations of the ovaries and ovarian and iliac arteries were performed on Days 7.5, 10.5, 13.5, and 21.5. The accessory corpus luteum (aCL) formation rate was 52.5% for G-hCG. There was interaction (p > 0.05) for treatment (G-hCG and G-Control), days (7.5, 10.5, 13.5 and 21.5) and PD (Pregnant and Non-pregnant) for the variables of biometric characteristics of the corpus luteum B-Mode and Color Doppler on days 7.5, 10.5, 13.5 and 21.5. There was no difference (p > 0.05) for pregnancy rates and mean fetuses per ewe between the treatment groups. It is concluded that the application of hCG 7.5 days after the hormonal protocol in Morada Nova ewes in a breeding season is efficient in inducing aCL formation and increasing luteal tissue biometry. However, there was no effect on pregnancy rate.

Immunoexpression of vascular endothelial growth factor and vWF-regulating angiogenesis in cyclic corpus luteum of Indian buffalo.

The present study was conducted to localize the immunoexpression of VEGF-A (Vascular Endothelial Growth Factor) and von Willebrand factor (vWF) in corpora lutea of healthy buffaloes (24) collected from local slaughterhouses. CL collected were categorized into early (stage I, 1-5 days, n = 6), mid (stage II, 6-11 days, n = 6), late luteal phase (stage III, 12 to 16 days, n = 6) and regressing phase (stage IV, 17 to 20 days, n = 6). The percent positive immunostaining for VEGF-A was significantly (p < 0.05) higher in mid-luteal phase than the other three stages of CL. However, it was higher in early luteal phase as well indicated intense angiogenesis in both early and mid-luteal phases. The number of capillary endothelium expressing vWF was significantly (p < 0.05) highest in mid-luteal phase among all the phases. However, in late luteal phase, the percent area positive for VEGF-A immunostaining was reduced but it was significantly (p < 0.05) higher than corpus albicans phase. Thus, in regressing phase or corpus albicans, it was lowest and reduced considerably. However, in late luteal phase, the number of capillaries with vWF immunoexpression reduced significantly (p < 0.05) but it was lowest in corpus albicans phase. Therefore, the immunotaining pattern for VEGF-A and vWF concluded that there was a spositive linear correlation between the two, that is, as the VEGF-A expression was increased, the number of vWF positive capillaries also increased and vice versa. The VEGF-A expressed by the luteal parenchyma in different stages of development and regression of corpus luteum was thus observed to be involved in promoting the angiogenesis and luteal cell proliferation as supported by vWF expressed by endothelium of proliferating capillaries in buffalo corpus luteum throughout the estrous cycle.

Physiological Uptake Characteristics of Breast on 68Ga-FAPI-04 PET/CT.

Gallium 68 (68Ga)-labeled fibroblast activation protein inhibitor (FAPI) PET/CT is sensitive on breast cancer staging, but its clinical utility may be limited by the high physiological 68Ga-FAPI-04 uptake in normal breast tissue that can obscure primary tumors. The aim of this study was to elucidate the characteristics of physiological 68Ga-FAPI-04 uptake in normal breast. A total of 143 consecutive women with 68Ga-FAPI-04 PET/CT data were reviewed retrospectively. SUVmax, density and thickness of breast, as well as SUVmax of nipple, were measured. Univariate and multivariate regression analyses were used to identify factors related to the breast and nipple SUVs. Twenty-eight premenopausal, 62 menopausal and 10 post-operative (after bilateral adnexectomy) women with 103 examinations were included. All had a diffuse, symmetrical uptake in breast. There was no difference in 68Ga-FAPI-04 uptake between bilateral breasts (right: median, 1.14[IQR, 0.85-1.54] vs. left: median, 1.09[IQR, 0.86-1.54]; P = 0.253). Patients in menstrual status with expected high estrogen level (late follicular, ovulatory and mid luteal phases) had higher breast SUVs (median SUV, 3.91 [IQR, 2.85-4.35]) than those with expected moderate (early follicular, early luteal and late luteal phases; median SUV, 1.57 [IQR, 1.39-2.08]; P < 0.001) or low level (menopause and post-operation; median SUV, 0.98 [IQR, 0.83-1.21]; P < 0.001). Menstrual status was an independent predictors of breast SUV (r2 = 0.689, P < 0.001). All the patients had a focal, symmetrical uptake in nipples. Nipple SUV did not correlate with menstrual status (P = 0.913).

Embryo-maternal communication mediated by extracellular vesicles in the early stages of embryonic development is modified by in vitro conditions

Extracellular vesicles (EVs) have become important in embryo-maternal communication during early development. The aim of this study was to evaluate the effect of an in vitro system on early bidirectional embryo-maternal communication mediated by EVs. For this purpose, two experiments were performed: one to evaluate the effect of embryonic EVs on maternal cells and the second to determine the effect of maternal EVs on early embryonic development. For the first in vitro (IVP) and in vivo (IVV) experiments, bovine blastocysts were selected and individually cultured for 48 h to collect embryonic EVs secreted during days 7–9 of embryonic development. Embryonic EVs were added to the medium of in vitro-cultured bovine endometrial cells to evaluate their effect on the expression pattern of genes associated with endometrial function and response to interferon tau (IFNT). Non-classical interferon-stimulated genes (ISGs) were only induced by in vitro-derived embryos. In the second experiment, EVs released by endometrial cells cultured in vitro (EVC) and collected from uterine fluid (EV-UF) of cows in the early luteal phase were added to the culture medium of bovine embryos produced in vitro during days 5–9 of development. The effect of maternal in vitro or in vivo-derived EVs differs in the quality of bovine embryos produced in vitro during the pre-implantation period. The expression of IFNT in bovine embryos is increased by the effect of EV-UF treatment. Additionally, EV-UF treatment induces a sustained increase in diameter during embryonic development and a tendency towards a greater number of expanded and hatched blastocysts. However, some genes related to embryo quality are induced by EVC treatment.