Luteal Phase Days Research Articles

#129 : The Ratio of Luteal Phase Serum Progesterone and Estradiol Can be Used to Predict Pregnancy in Frozen Embryo Transfer Cycles

Background and Aims: Low serum progesterone (P) levels on day of embryo transfer (ET) and luteal phase have been associated with poorer pregnancy outcome. This study aimed to know if serum estradiol (E2) and P levels during mid and late luteal phase are related with ongoing pregnancy. Method: The serum E2 and P levels from mid luteal phase (5 days after ET) to the day of the [Formula: see text]-hCG check (11 days after ET) in both fresh and frozen embryo transfer (FET) cycles was investigated between January 2019 and December 2021. Eligible patients were reproductive aged women with a normal uterus. Single blastocyst transfer cycles were included. Estradiol valerate (2 mg/bid) was started from day 3 of cycle as hormone replacement and vaginal micronized P (400 mg/bid) was started 5 days before ET in all FET cycles. The 2-degree polynomial fitted data were analysed and logistic linear model and ROC analysis were performed to assess E2 and P4 polynomial coefficients as a predictive test for ongoing pregnancy. Results: A total of 388 patients were included. The overall pregnancy rate was 43.8% and 31.3% for FET and fresh ET, respectively. On day 5 post ET, the serum P levels correlated significantly with ongoing pregnancy and live birth rates, especially in FET cycles. The ROC curve showed that there is a significant day 5 post ET predictive value of serum P4/E2 levels for pregnancy rate, being the AUC (95% CI) = 0.596 (0.539–0.653) for the combination of both fresh and FET cycles; and 0.611 (0.548–0.674) for FET cycles, with best cut-off values of 0.079 and 0.077, respectively. Conclusion: Serum P levels in mid and late luteal phase days are associated with pregnancy outcome both in fresh and frozen ET cycles. Ongoing pregnancies can be predicted using the day 5 post ET serum P4/E2 levels.

SAT371 Gavaging Adult Female Rats with Rothia sp. Leads to Profound Disruption of the Estrous Cycles and Metabolic Consequences

Abstract Disclosure: R. Santos: None. G. Parodi: None. G. Leite: None. W. Morales: None. S.R. Weitsman: None. G. Barlow: None. I. Rivera: None. M. Sanchez: None. D. Flor: None. L. Choi: None. S.L. Gonzales: None. F. Faria: None. M. Villanueva-Millan: None. M. Pimentel: None. R. Mathur: None. Polycystic ovary syndrome (PCOS) is a major endocrine disorder affecting ∼12% of reproductive age women. It has diverse clinical manifestations, including hyperandrogenism, ovarian dysfunction and enlargement, infertility, insulin resistance, and others. The role of the gut microbiome in PCOS is poorly understood. We previously found higher testosterone (T) levels in PCOS stool samples vs. controls and identified bacterial candidates that correlated with testosterone levels. Here, we further investigate the role of one candidate microbe, Rothia sp., in the development of PCOS-like phenotypes. Methods: The presence of androgen biosynthesis associated genes in Rothia sp. was confirmed by RT-PCR. Rothia sp. was cultured in M9 minimal medium plus cholesterol. T production in the supernatant was measured by LC-MS/MS. Next, reproductive-age female rats were gavaged with 106 CFU/mL Rothia or with PBS (controls) daily for 2 weeks. H&E stained slides from daily vaginal lavages were used to assess estrous cycles. Serum T and progesterone (P) levels were measured by LC-MS/MS, and insulin levels by ELISA. Ovaries, uteri, and cardiac blood were harvested. Results: Genes encoding proteins associated with T biosynthesis (cytochrome P450, 3-beta hydroxysteroid dehydrogenase/isomerase family protein and NAD(P)H steroid dehydrogenase-like protein) were detected in Rothia sp. T production in Rothia sp. cultures was confirmed, with levels from 3.2±0.01 pg/mL after 48 hrs to 8.9±0.02 pg/mL after 6 days. T was not detected in negative controls (P<0.001). In gavaged rats, analysis of vaginal slides (controls n=10; Rothia n=10) revealed a disruption of estrous cycles in Rothia rats, with 68% of days in luteal phase (metestrus & diestrus) vs. 46% in controls (P=0.001). The predominance of luteal phase days in Rothia rats was driven by longer time in diestrus (1.96 vs 1.67 days in controls, P=0.04), and shorter time in estrus (1.24 vs 1.91 days in controls, P=0.001). Peak P and T levels occurred in diestrus in both groups. Rothia rats spent more days in diestrus, and the cumulative exposure times to P (P=0.046) and T (P=0.036) in the diestrus phase were significantly higher in Rothia rats vs controls. Final serum insulin levels were higher in Rothia rats (1.8±0.5 ng/dL) vs controls (1.0±0.1 ng/dL; P=0.07). Rothia rats also had heavier ovaries vs controls (0.128±0.007g vs 0.097±0.01g; P=0.07), and higher uterus weights (0.48±0.04g vs. 0.36±0.04g; P=0.04). Conclusion: Here we show that a specific Rothia sp. can produce testosterone in vitro. Gavaging female rats with this microbe results in PCOS-like phenotypes, including higher insulin levels, enlarged ovaries, significant disruption of reproductive cycles and longer exposure to peak progesterone and testosterone. This longer exposure to peak progesterone and testosterone may result in reproductive and metabolic sequelae that warrant further investigation. Presentation Date: Saturday, June 17, 2023

Relationship Between Ovulation and Body Temperature in the Mare: A Preliminary Study

In the equine industry, monitoring of the reproduction cycle is key to be able to produce one foal per mare and per year. Ovulation detection is difficult partly due to the variability of the estrus length. Currently, the most reliable method for ovulation detection is transrectal ultrasonography. This technique, however, implies handling of the mare as well as veterinary costs. The aim of this experimentation is to study body temperature variations around ovulation. Nine reproduction cycles were monitored around ovulation. Transrectal ultrasonographies were performed each day as well as blood sampling to dose estradiol-17β and progesterone to confirm ultrasonographic results. Body temperature was automatically recorded every 10 minutes using an identification chip equipped with a temperature sensor implanted in the mares’ neckline. Data were analyzed using linear mixed models. Daily body temperature pattern did not vary between the phases of the reproductive cycle (follicular, ovulatory and luteal). Temperature differences between phases, however, were identified and appeared hourly-specific. There was an increase of temperature at ovulation compared to the end of the follicular phase ranging from 0.51°C ± 0.21°C to 0.92°C ± 0.26°C and occurring between 04:30 and 08:00. Moreover, a significant increase of body temperature was measured during the first days of luteal phase, ranging from 0.29°C ± 0.17°C to 0.60°C ± 0.16°C, between 10:30 and 16:00. Body temperature varied around ovulation and it might be a promising tool for mare reproduction monitoring. A more complete study, however, focusing on the whole cycle is required.

The effect of local endometrial injury on the success of intrauterine insemination

To determine the effect of local endometrial injury on implantation success in patients diagnosed with unexplained infertility and undergoing intrauterine insemination (IUI) after ovulation induction with gonadotropins. In this prospective randomized controlled trial, 82 infertile patients underwent IUI following ovulation induction with gonadotropin. In the study group (n:40), local endometrial injury (stratch) was performed to the posterior side of the endometrial cavity with a biopsy catheter between the 21-26th days of luteal phase of the cycle preceding ovarian stimulation. There was no statistically significant difference between the study and the control groups in terms of age of female, age of male, duration of infertility, BMI, serum FSH and LH levels, mean dose of gonadotropin and mean duration of ovulation induction (p>0.05). Clinical pregnancy was achieved in two patients (4.76%) in control group and four (10%) patients in the study group, with no significant difference between groups (p=0.18). All pregnancies achieved in the control and the study groups passed 12th gestational weeks and continued. Ectopic pregnancy, multiple pregnancy and abortion was not observed in any patient in both groups. In the study group, pain level immediately after endometrial biopsy procedure was evaluated with visual analog scale (VAS) and it was established that only one (2.5%) patient experienced severe pain after the procedure. Although local endometrial damage in the menstural period before ovulation induction and IUI cycle increases clinical pregnancy rates in the infertile patients, this increase is not statistically significant. Multi-center randomized controlled studies are needed for local endometrial damage to be recommended routinely in clinical practice.

The effects of long- or short-term increased feed allowance prior to first service on litter size in gilts.

Replacing stock is costly in any pig production. In addition, it takes time for young animals to reach the same level of productivity as more mature animals. Therefore, the aim of this study was to investigate the effects of long- or short-term increased feed allowance (covering the luteal and follicular phases) prior to service in the second estrus on first parity performance. In order to achieve this, altrenogest was used to synchronize the gilts cycle to allow a precise feeding strategy, and only gilts inseminated 0–10 d after altrenogest withdrawal were included in the study. Altrenogest was given at days 0–18 to control the luteal phase and, therefore, treatments covered different feeding strategies in either or both the luteal phase (days 0–18) and follicular phase (days 18–25). High feed allowance (H) was induced using 0.97 kg more feed per day compared to the low feed allowance (L) given 2.33 kg/d. Four feeding strategies, low–low (LL), high–high (HH), high–low (HL), and low–high (LH), were included. Once gilts had been inseminated, feed allowance was reduced to 2.23 kg/d to prevent the loss of embryos in early gestation. A tendency was observed between feeding strategy and backfat thickness before altrenogest treatment, showing that total born piglets were positively correlated to backfat in the LL and LH (no increased feed allowance or short-term increased feed allowance), treatments (P = 0.076), compared to when gilts had longer periods with high feed allowance (HH and HL). High feed allowance in the follicular phase (LH) tended to increase the number of total born piglets compared to the other groups (P = 0.069) when applied in the follicular phase of the second standing estrus after the gilts were given altrenogest. This would be equivalent to the last 5–7 d of a 21-d cycle in gilts. The three other feeding strategies, comprising either the luteal and follicular phases (HH) or the luteal phase (HL) or none (LL), did not increase litter size. The weight of the gilt when entering the insemination section also had an effect on total born piglets (P < 0.001) with an increase in litter size with increased weight of the sow, but no differences between treatments. In conclusion, the weight of the gilt had an influence on the total litter size and gilts with low backfat tended to respond more positively to a longer period with high feed allowance than fatter gilts.

Changes in Aquaporin 1, 5 and 9 Gene Expression in the Porcine Oviduct According to Estrous Cycle and Early Pregnancy.

Aquaporins (AQPs) are a group of small, integral membrane proteins which play an important role in fluid homeostasis in the reproductive system. In our previous study, we demonstrated AQP1, 5 and 9 protein expression and localization in the porcine oviduct. The presence of these isoforms could suggest their role in the transport of the ovum to the uterus by influencing the epithelial cells’ production of oviductal fluid. The aim of this study was to evaluate the expression of AQP1, AQP5 and AQP9 in the infundibulum, ampulla and isthmus in the porcine oviduct during the estrous cycle (early luteal phase, days 2–4, medium luteal phase, days 10–12, late luteal phase days 14–16, follicular phase days 18–20) and pregnancy (period before implantation, days 14–16 and after the implantation, days 30–32) using the Real-Time PCR technique. As clearly demonstrated for the first time, AQP1, 5, and 9 gene expression is influenced by the estrus cycle and pregnancy. Furthermore, expression of AQPs in the porcine oviduct may provide the physiological medium that sustains and enhances fertilization and early cleavage-stage embryonic development. Overall, our study provides a characterization of oviduct AQPs, increasing our understanding of fluid homeostasis in the porcine oviduct to successfully establish and maintain pregnancy.

Associations between the menstrual cycle, lifestyle factors and clinical assessment of the ocular surface: a prospective observational study

BackgroundLittle is known about the ocular surface changes over the menstrual cycle in young women and the interactions with lifestyle factors. Therefore, the purpose of this study was to explore the associations between modifiable lifestyle factors and menstrual cycle phases on the ocular signs and symptoms of dry eye in young healthy women.MethodsThis was a prospective 1-month observational study. Thirty young healthy women with regular, 24 to 32-day menstrual cycles were recruited. Participants attended three visits at day 7, 14, and 21 (± 1) of their menstrual cycle. At baseline, general health questionnaire was conducted. At each visit, symptomology was quantified using Ocular Surface Disease Index (OSDI) and overall ocular comfort (OOC, visual analogue scale). Ocular signs were assessed using Efron scales, tear break-up time (TBUT) and phenol red thread (PRT). Pearson’s correlation was used to determine associations between variables at each visit.ResultsA total of 26 participants (mean age = 22.3 ± 3.7 years) with an average menstrual cycle of 28.3 ± 1.3 days completed the 3 visits. The interaction between signs/symptoms and lifestyle factors changed over the cycle. At the follicular phase (day 7), lifestyle factors such diet and levels of stress were correlated with PRT and OSDI, (r = − 0.4, p = 0.022; r = 0.4, p = 0.045 respectively). At the ovulation phase (day 14), the general health score was correlated with OOC scores (r = 0.4, p = 0.047). At day 14, exercise frequency correlated with PRT (r = − 0.4, p = 0.028) and caffeine intake was positively correlate with both; TBUT (r = 0.5, p = 0.020) and PRT (r = 0.5, p = 0.014). At the luteal phase (day 21), we found no correlations between lifestyle factors and dry eye signs or symptoms.ConclusionsThe associations between lifestyle factors and objective and subjective ocular surface assessment appeared to be more pronounced during the ovulation phase of the menstrual cycle compared to the follicular and luteal phases. Misalignment of these factors with the ocular health during the luteal phase could be attributed to central sensitization and changes in levels of luteinising hormone. Natural hormonal changes during menstrual cycle should be considered for diagnosis and treatment of dry eye in young healthy women.

The Effect of the Menstrual Cycle on Daily Measures of Heart Rate Variability in Athletic Women

Abstract. Heart rate variability (HRV) is a biomarker used to reflect both healthy and pathological state(s). The effect of the menstrual cycle and menstrual cycle phases (follicular, luteal) on HRV remains unclear. Active eumenorrheic women free from exogenous hormones completed five consecutive weeks of daily, oral basal body temperature (BBT) and HRV measurements upon waking. Descriptive statistics were used to characterize shifts in the HRV measures: standard deviation of NN intervals (SDNN), root mean square of successive difference (rMSSD), high (HF) and low frequency (LF) across the menstrual cycle and between phases. All HRV measures were assessed by medians ( Mdn), median difference of consecutive days ( Mdn∆) and variance. Seven participants ( M ± SD; age: 28.60 ± 8.40 year) completed the study with regular menstrual cycles (28.40 ± 2.30 days; ovulation day 14.57 ± 0.98 day). Median rMSSD displayed a nonlinear decrease across the menstrual cycle and plateau around the day of ovulation. A negative shift before ovulation in Mdn∆, rMSSD, SDNN, and LF as well as peak on luteal phase Day 4 in rMSSD and SDNN was observed. Median variance increased in rMSSD (150.06 ms2) SDNN (271.12 ms2), and LF variance (0.001 sec2/Hz) from follicular to luteal phase. Daily HRV associated with the parasympathetic nervous system was observed to decrease nonlinearly across the menstrual cycle.

Postprandial energy metabolism and substrate oxidation in response to the inclusion of a sugar- or non-nutritive sweetened beverage with meals differing in protein content

BackgroundThe macronutrient composition of the diet may play a more important role in maintaining a healthy body weight and preventing obesity than previously thought. The primary goal of this research was to determine the extent to which the simple addition of a small serving of a sugar-sweetened beverage (SSB) to meals with different macronutrient compositions impacts appetite, energy metabolism and substrate oxidation.MethodsAppetite, energy metabolism and substrate oxidation were measured in 27 healthy weight adults (age = 23 ± 5 y; BMI = 23 ± 2 kg/m2) on two occasions in a room calorimeter after consuming a SSB or a non-nutritive-sweetened beverage (NNSB) with a standard (15%E) or high- (30%E) protein meal. Meal carbohydrate (CHO) content was adjusted to maintain equivalent calories for both study visits. All meals were composed of the same foods and provided 17 g of fat and 500 non-beverage calories. Study visits were separated by at least 1 week and menstruating females were studied during the luteal phase (Days 15–20). The effects of sex, protein level and beverage type and their interactions on satiety, appetite for foods with specific taste profiles, diet-induced thermogenesis (DIT) and rates of substrate oxidation were assessed using a 3-way Repeated Measures Analysis of Variance.ResultsIncreasing dietary protein decreased hunger and increased satiety. Males were hungrier and less satisfied with the meals than females. Increasing dietary protein also decreased the desire to eat something savory, salty and fatty and the males had a greater appetite for food with these taste profiles. Interestingly, there was no effect of sex, dietary protein or beverage type on the desire to eat something sweet. The inclusion of a SSB markedly suppressed DIT (2.42% ± 5.91%) and fat oxidation (9.87 ± 11.09 g).ConclusionAppetite sensations, food preferences, energy expenditure and substrate oxidation are significantly altered in response to changes in meal macronutrient composition produced by modifications in the protein content of a meal and consumption of a SSB. Most notably, consumption of a SSB during a meal markedly reduces energy efficiency and fat oxidation independent of macronutrient composition.Trial registrationsClinicalTrials.gov: NCT02211599, registered August 05, 2014.

Relaxin Influences Knee Laxity Changes Across the Menstrual Cycle

Greater knee laxity is associated with an increased risk for anterior-cruciate ligament (ACL) injury, particularly in women. Research suggests that the hormone relaxin may influence the structural integrity of the ACL, rendering a weaker and more lax ligament. PURPOSE: To assess the impact of relaxin on knee laxity once progesterone and testosterone (potential endocrine antagonists) are accounted for. METHODS: College-aged females (166.0±5.7cm, 65.9±8.5kg, 20.8±2.9yr) provided blood samples for the first 6 days of menses and first 10 days of luteal phase of one menstrual cycle. Knee laxity was recorded as anterior knee laxity (AKL; mm), genu recurvatum (GR; °), and general joint laxity (GJL; score, 0-9), and dependent variables were calculated as mean (X), cyclic Δ (max – min), coefficient of variation (CV), and standard deviation (SD). Progesterone (P; ng/ml), testosterone (T; ng/dl), and relaxin (R; pg/ml) were analyzed via ELISA assays. The sum of the 6 greatest hormone concentrations during menses (M) and luteal phase (L) and the change in exposure from M to L (MLΔ) were calculated. Only subjects with captured R and P peak were included in analysis (n=18). M and MLΔ for each hormone were entered into backward stepwise (in: p=.05, out: p=.20) multiple linear regression models to predict each laxity measure. RESULTS: R and P were significantly different between M and L (p<.001, RM=22.2±29.4 vs. RL=147.0±96.3; p<.001, PM=5.8±1.8 vs. PL=89.7±36.6), whereas T was less variable (p=.07, TM=203.8±63.0 vs. TL=222.7±82.6). All laxity showed variation across the menstrual cycle (range [min-max]: AKL=5.9-7.6; GR=2.6-5.6; GJL=.8-2.1). Significant models were observed for GRX (p=.014; R2=.418; GRX=3.501+.1.024RMLΔ-.757RM-.395PMLΔ), GJLMLΔ (p=.044; R2=.253; GJLMLΔ=-.469RMLΔ-.440TM), and GJLX (p=.035; R2=.379; GJLX=-.317RM-.353PM-.440PMLΔ-.507TM). Models for AKLMLΔ, AKLCV, GRΔ, and GJLSD approached significance (all p<.075). CONCLUSION: Once controlling for P and T, R was a significant predictor of knee laxity mean and cyclic changes. These findings would suggest that R, combined with other sex hormones, may affect the structural integrity of the ligament and impact injury risk. Further study is needed to explore potential mechanisms for this association.

80 EFFECT OF BOVINE OVIDUCTAL FLUID ON DNA METHYLATION OF BOVINE BLASTOCYSTS PRODUCED IN VITRO

During the transit through the oviduct, the early embryo undergoes an epigenetic reprogramming of its genome, which induces changes in DNA methylation pattern. Given that epigenetic modifications are susceptible to environmental influence, the oviducal milieu may affects DNA methylation marks in the developing embryo. The aim of this study was to evaluate whether bovine oviducal fluid (OF) exerts an effect on methylation status of genomic regions at different time points of embryo development. In vitro-produced zygotes were cultured in SOF + 3 mg mL−1 BSA (control, C) or in SOF + 1.25% OF at 3 different time points: until 98 h post-insemination (hpi) (OF1–16: 1–16 cell), 52 hpi (OF1–8: 1–8 cell), or from 52 until 98 hpi (OF8–16: 8–16 cell). The OF used was acquired from Embryocloud (Murcia, Spain) from cow oviducts at the early luteal phase (Day 1–4). After, embryo culture took place in control medium up to Day 8. For all the groups, the speed of development was considered, and normal developing embryos that reached ≥6 cells at 52 hpi and ≥16 cells at 98 hpi were selected and separately cultured from slow developing embryos. Cleavage (52 hpi) and blastocyst yield (Day 7–8) were analysed by ANOVA (8 replicates). Expanding blastocysts (Day 7–8) from the normal developing groups were collected for bisulfite sequencing analysis. The DNA bisulfite conversion was performed with a MethylEdge Bisulfite Conversion System kit (Promega, WI, USA) in groups of 20 blastocysts obtained from 5 replicates. Methylation status was analysed on regions localised in 4 developmental important genes (MTERF2, ABCA7, OLFM1, and GMDS) and within 2 LINE L1 elements located on chromosomes 9 (L9) and 29 (L29). Methylation percentages (10 sequenced clones/group) were compared using statistical z-test. No significant differences were found on cleavage rate (C: 89.7 ± 1.0, OF1–16: 84.9 ± 1.7; OF1–8: 85.4 ± 1.9; OF8–16: 89.1 ± 1.9%) and blastocyst yield between normal developing embryos (C: 36.8 ± 5.3; OF1–16: 34.7 ± 3.7; OF1–8: 41.0 ± 3.8; OF8–16: 43.9 ± 5.1%). Blastocysts derived from all OF groups showed the CpG region of MTERF2 hypomethylated compared with C group (20.0, 26.2, and 32.9% v. 56.2%, respectively; P &lt; 0.001). The CpG sequence of ABCA7 exhibited significant hypomethylation in embryos from OF1–16 group compared with OF1–8, OF8–16, and C groups (31.1 v. 56.8, 57.9, and 65.8%, respectively; P &lt; 0.001). Although the methylation of the CpG region within OLFM1 did not differ between OF1–16 and C groups (24.1 v. 19.4%, respectively), embryos from OF1–8 group showed a highly methylated region (47.1%) compared with OF1–16 and C groups (P &lt; 0.001). The CpG sequence on L9 showed a high methylation level in blastocysts derived from OF1–16 group compared with OF8–16 and C groups (36.4 v. 14.5 and 20.0%, respectively; P &lt; 0.05). There were no differences in methylation marks between groups examined for CpG regions of GMDS and L29. These results indicated that embryos exhibit a temporal sensitivity to OF at early embryonic stages, which is reflected by DNA methylation changes of specific genes at blastocyst stage. This is the first report describing that OF could modify specific epigenetic marks of the bovine embryonic genome.

Human fallopian tube epithelium co-culture with murine ovarian follicles reveals crosstalk in the reproductive cycle.

Do interactions between human fallopian tube epithelium and murine follicles occur during an artificial reproductive cycle in a co-culture system in vitro? In a co-culture system, human fallopian tissues responded to the menstrual cycle mimetic by changes in morphology and levels of secreted factors, and increasing murine corpus luteum progesterone secretion. The entire fallopian tube epithelium, including ciliated and secretory cells, can be regulated in the reproductive cycle. Currently, there are no in vitro culture models that can monitor fallopian tissues in real time in response to factors produced by the ovary. In addition, there are no reports on the impact of fallopian tissue on ovarian function during the menstrual cycle. Human fallopian tissue (n=24) was obtained by routine hysterectomies from women (aged 26-50 years, mean age=43.6) who had not undergone exogenous hormonal treatment for at least 3 months prior to surgery. CD1 female mice were used for ovarian follicle isolation. The human fallopian epithelium layers were either co-cultured with five murine multilayer secondary follicles (150-180 μm follicles, encapsulated in one alginate gel bead) for 15 days or received stepwise steroid hormone additions for 13 days. The fallopian tissue morphology and cilia beating rate, as measured by an Andor Spinning Disk Confocal, were investigated. Oviduct-specific glycoprotein 1 (OVGP1), human insulin-like growth factor 1 (hIGF1), vascular endothelial growth factor A (VEGF-A) and interleukin 8 (IL8) as biological functional markers were measured either by ELISA or western blot to indicate dynamic changes in the fallopian epithelium during the reproductive cycle generated by mouse follicles or by stepwise steroid hormone induction. Three or four patients in each experiment were recruited for replicates. Data were presented as mean±SD and further analyzed using one-way ANOVA followed by Tukey's multiple comparisons test. The cultured fallopian tube epithelium responded to exogenous steroid hormone stimulation, as demonstrated by enhanced cilia beating rate (~25% increase, P=0.04) and an increase in OVGP1 secretion (P=0.02) in response to 1 nM estradiol (E2) treatment when compared with 0.1 nM E2. Conversely, 10 nM progesterone plus 1 nM E2 suppressed cilia beating rate by ~30% (P=0.008), while OVGP1 secretion was suppressed by 0.1 nM E2 plus 50 nM progesterone (P=0.002 versus 1 nM E2 alone). Human fallopian tube epithelium was co-cultured with murine secondary follicles to mimic the human menstrual cycle. OVGP1 and VEGF-A secretion from fallopian tissue was similar with stepwise hormone treatment and when cultured with murine follicles. However, the secretion patterns of hIGF1 and IL8 differed in the luteal phase when comparing steroid treatment with follicle co-culture. In co-culture, hIGF1 secretion was suppressed in the luteal versus follicular phase (P=0.005) but stepwise hormone treatment had no effect on hIGF1. In co-culture, IL8 secretion was also suppressed on luteal phase day 15 (P=0.013) versus follicular phase day 7, but IL8 secretion increased continuously under high E2/progesterone treatment (P=0.003 for D13 versus D3). In the co-culture system, the corpus luteum continuously produced progesterone in the presence of fallopian tube tissue until Day 18 while, without fallopian tissue, progesterone started to drop from Day 13. One limitation of this study is that murine follicles were used to mimic the human menstrual cycle. However, although secretion patterns of peptide hormones such as inhibins and activins differ in mice and humans, the co-culture system used here did reveal interactions between the tissues that govern reproductive function. In vitro co-culture models of fallopian reproductive tissues with ovarian follicles can provide an important tool for understanding fertility and for uncovering the mechanisms responsible for reduced fertility. In addition, the role of oviductal secretions and how they influence ovarian function, such as the production of progesterone during the menstrual cycle, can be uncovered using this model. None. This work was funded by grants from the NIH (UH3TR001207), the American Cancer Society (RSG-12-230-01-TBG) and NIH (R01EB014806). The authors declare no competing financial interest.

Can steroidal ovarian suppression during the luteal phase after oocyte retrieval reduce the risk of severe OHSS?

BackgroundOvarian stimulation in IVF cycle results in luteal supraphysiological steroid concentrations especially for high response patients. The aim of this study was to evaluate the efficacy of ovarian steroid hormone suppression in luteal phase after oocyte retrieval for preventing severe ovarian hyperstimulation syndrome (OHSS) in high-risk patients with embryo cryopreservation.Methods281 patients with high risk of OHSS were enrolled in this study among 4735 infertile women undergoing their first IVF treatment. The subjects were allocated into treatment and control group. The treatment group (n = 161) received letrozole (n = 43), mifepristone (n = 51), cetrotide (n = 39) and three-drug combinations (n = 28) during the luteal phase after oocyte retrieval, respectively. The control group (n = 120) received no medicine. Fertilization rate, good embryo rate, serum steroid concentration, clinical outcome, and incidence of severe OHSS were compared between the two groups.ResultsOn days 2, 5 and 8 after oocyte retrieval, serum estradiol levels in the letrozole and three-drug combination therapy group were significantly lower than in the other three groups at the same time (P < 0.001, respectively). There were no significantly difference of serum luteinizing hormone concentration on days 2, 5 and 8 and progesterone concentration on day 8 after oocyte retreival among the five groups (P > 0.05, respectively). Compared with the control group, the incidence of severe OHSS, the paracentesis rate, the duration of hospitalization and the days of luteal phase in each subgroup of treatment groups was not significantly decreased (P > 0.05, respectively).ConclusionsOur findings indicate that steroidal ovarian suppression in luteal phase after oocyte retrieval seems to be unable to prevent severe OHSS in high-risk patients with embryo cryopreservation.

Luteal phase support with estrogen in addition to progesterone increases pregnancy rates in in vitro fertilization cycles with poor response to gonadotropins

In this study, our objective was to determine the effect of adding estradiol hemihydrate (E2) to progestin (P) for luteal phase support on pregnancy outcome in in vitro fertilization (IVF) cycles with poor response to gonadotropins. Ninety-five women with poor ovarian response who underwent controlled ovarian hyperstimulation (COH) with gonadotropin releasing hormone (GnRH) agonist or GnRH antagonist plus gonadotropin protocol for IVF were prospectively randomized into three groups of luteal phase support after oocyte retrieval. Group 1 (n = 33) received only intravaginal progesterone gel (Crinone 8% gel). Group 2 (n = 27) and Group 3 (n = 35) received intravaginal progesterone plus oral 2 and 6 mg estradiol hemihydrate, respectively. Main outcome measures were overall and clinical pregnancy rates (PRs) per patient. Serum LH, E2 and P levels at 7th and 14th days of luteal phase were also measured. Overall and clinical PRs were significantly higher in 2 mg E2 + P than P-only group (44% versus 18% and 37% versus 12.1%, respectively). There were no statistically significant differences between 6 mg E2 + P versus P-only and 2 mg E2 + P versus 6 mg E2 + P groups regarding PRs. Addition of 2 mg/day E2 in addition to P for luteal support significantly increase overall and clinical PRs in cycles with poor response to gonadotropins after IVF.

Ultrasonographic endometrial thickness measurement is predictive for treatment response in simple endometrial hyperplasia without atypia

We sought to determine the predictors of treatment response in simple endometrial hyperplasia without atypia. We prospectively treated 67 women with simple endometrial hyperplasia without atypia who were administered cyclic oral medroxyprogesterone acetate 10 mg/day for 12 days of luteal phase for 3 months and underwent control endometrial sampling after treatment. All subjects were evaluated in terms of age, gravidity, parity, body mass index (BMI), menstrual cycle, endometrial thickness, uterine fibroids, ovarian cysts, serum CA 125 levels, systemic disorders and cigarette smoking. All parameters were used to predict treatment success. Persistent hyperplasia was observed in 11 subjects. Endometrial thickness was significantly correlated with treatment failure (r=0.293, p=0.015). In ROC analysis, endometrial thickness was found to be predictive for persistent hyperplasia (area under curve: 0.724, P=0.019). Optimal cut off value was calculated to be 16.5 mm with 64% sensitivity, 72% specificity and 91% negative predictive value. The number of persistent hyperplasia in women with and without endometrial thickness greater than 16.5 mm was significantly different (7/23 vs. 4/45, p=0.029). Odds ratio of endometrial thickness higher than 16.5 mm for treatment failure was 4.4 (95% CI, 1.2-17.4, p=0.03). Results of this study suggest treatment modification according to the baseline endometrial thickness in patients with simple endometrial hyperplasia without atypia.

57 DIETARY CLA SUPPLEMENTATION AFFECTS LUTEAL GENE EXPRESSION AND PERIPHERAL BLOOD PROGESTERONE CONCENTRATION IN CATTLE

Shortly after parturition the metabolic situation of high-yielding dairy cows is often dominated by a negative energy balance. These effects affect the whole animal and may especially be detected in the reproductive tract, where they result in reduced fertility. An oral supplementation with dietary fats is often used to counteract by reducing milk fat content and, thus, supplying the individual animal with an increased amount of energy. The focus of the present study was to analyse the effects of an oral supplementation with conjugated linoleic acids (CLA) on corpus luteum (CL) function. Healthy Holstein-Friesian cows and heifers were randomly allocated to 2 treatment groups (Group 1: 50 g of CLA day–1 per animal, 2 heifers, 6 cows; Group 2: 100 g of CLA day–1 per animal, 2 heifers, 6 cows) and 1 control group (Ctl; 0 g of CLA day–1 per animal, 3 heifers, 4 cows). Feeding of the supplement began shortly after calving. After calving, all animals were subjected to a standard synchronisation protocol and experienced AI on Day 59 ± 3. Following AI, transvaginal biopsies of the corpus luteum were obtained of pregnant (Group I: n = 4; Group II: n = 4; Ctl: n = 4) and nonpregnant (Group I: n = 4; Group II: n = 4; Ctl: n = 3) animals on Days 6, 13 and 20 post-AI. Animals deemed pregnant on Day 28 were again biopsied on Day 42. Additionally, blood samples were taken from the vena sacralis mediana at the time of each biopsy. The biopsies were analysed regarding the relative abundance of 8 gene transcripts (VEGF, ECE1, PLA2G4A, PTGS2, PTGFR, PPARG, STAR and HSD3B1) via RT-qPCR. Blood samples were analysed for their concentration of progesterone through a radioimmunoassay (RIA). Statistical analysis for both datasets was performed via a 3-way ANOVA with adjoining Tukey test. The expression of 7 of these genes was affected by 1, 2, or all 3 of the following factors: day of cycle (VEGF, ECE1, PLA2G4A, PTGFR, STAR and HSD3B1), pregnancy status (ECE1, PTGFR and HSD3B1) and CLA supplementation (ECE1, PTGS2, PTGFR, STAR and HSD3B1). The effects of the CLA supplementation could be seen as a down-regulation in the mentioned gene transcripts. Progesterone concentrations differed significantly in dependency of the pregnancy status (significantly higher in pregnant vs nonpregnant individuals) of the animals, as well as during the days of the oestrous cycle (physiological progesterone curve with highest values on Day 13 of these samples). An effect of the oral supplementation with CLA could be detected during the early luteal phase (Day 6) where animals that had received 100 g of CLA day–1 had a significantly lower blood progesterone concentration than those receiving 50 g of CLA day–1 or no CLA. In conclusion, dietary CLA supplementation has an effect on luteal gene expression and functionality. The authors thank the DFG (German Research Foundation) for their financial support (PAK286/1; WR154/1-1).

How to determine symptom severity in premenstrual syndrome: A combination of daily symptom ratings and interviews

Objective To investigate how premenstrual symptoms are experienced and affect daily life, and to see if there is an agreement in reported symptom severity based on interviews compared to ratings on a symptom rating scale. Study design Twenty-two women with different degree of premenstrual symptoms were interviewed about their symptoms. Based on the luteal-phase interviews, they were categorized in four different severity groups: severe ( n = 5), moderate ( n = 3), mild ( n = 8), and no symptoms/cyclicity ( n = 6). The interviews were then compared with rated symptom scores, number of expressed symptoms per day, number of days with symptoms, and daily life impairment. Main outcome measures Agreement between rated symptom scores and reported symptoms in the interviews. Results Comparing seven days in luteal phase scorings with interview data the group with no symptoms/cyclicity showed high agreement between severities reported in the interviews and daily rated scores. Among women who reported severe symptoms, an agreement was seen in three out of five. In the mild/moderate group, the agreement was less conclusive. The day of interview there was a high agreement between data from the reported symptom ratings and symptoms reported in the interview. Conclusion Rated symptom scores the day of interview reflects well symptoms reported in the interviews. Mean symptom scores for seven luteal phase days showed an agreement between symptom ratings and symptoms expressed in interviews among women with severe symptoms and no symptoms/cyclicity. In the group with mild/moderate symptoms, data was less conclusive.

Effects of GnRH or Progesterone Treatment on Day 5 Post‐AI on Plasma Progesterone, Luteal Blood Flow and Leucocyte Counts During the Luteal Phase in Dairy Cows

This study was designed to test the effects of progesterone or GnRH treatment on day 5 post-AI on fertility and luteal function in dairy cows and heifers. Five days after AI, 32 animals were randomly assigned to a control, intravaginal progesterone for 14 days progesterone releasing intravaginal device (PRID) or GnRH treatment group. On days 5, 7, 12, 14, 17 and 19 post-AI, each animal underwent colour Doppler ultrasonography of the corpus luteum and blood samples were collected for cell counts and plasma progesterone determination. Through general linear model repeated measures analysis of variance, significant effects were observed of treatment, parity, inseminating bull, reduced vascularization of the CL and pregnancy on plasma progesterone concentrations, whereas mean plasma progesterone and time luteal phase day, and treatment and plasma progesterone concentration on day 5 post-AI were found to, respectively, affect neutrophil and lymphocyte counts throughout the luteal phase. Moreover, two binary logistic regression analyses were performed. Based on the odds ratio, the likelihood of pregnancy by days 26-32 post-AI was 23.4 times higher in animals with high mean progesterone levels throughout the study period, compared with animals with low mean progesterone. The likelihood of reduced CL vascularization was 14 times higher in animals treated with PRID, compared with control and GnRH-treated animals. In conclusion, our results indicate that treatment on day 5 post-AI with PRID reduced subsequent CL vascularization, whereas GnRH treatment increased plasma progesterone concentrations on day 12 post-AI, although an effect was identified of the inseminating bull on plasma progesterone levels. Pregnant animals showed higher mean plasma progesterone concentrations than in nonpregnant ones and heifers higher than in lactating cows, whereas blood cell counts differed depending on the treatment and on the mean plasma progesterone concentration on day 5 post-AI.

The ability of subordinate follicles of the second follicular wave to become dominant is lost by day 15 of the estrous cycle in cattle

Generally, unilateral ovariectomy before a critical period in the latter part of the estrous cycle induces a transitory increase in plasma FSH, which causes subordinate follicles to develop and maintain ovulation rates characteristic of the species. A limiting period for subordinate follicles to assume dominance and from which ovulation occurs has not been shown for cattle. Growth and/or regression of subordinate follicles were characterized following removal of the dominant follicle at different days of the luteal phase of the estrous cycle in cattle in this study. In the mid-luteal phase (Day 13 or 15), the ovary with the dominant follicle of the second wave was ablated via unilateral ovariectomy; the corpus luteum also was removed. In the late luteal phase (Day 17 or 19), the dominant follicle was ablated with an ultrasonically guided 20 gauge needle. When the dominant follicle was removed on Day 13, the largest subordinate follicle of the second wave of follicular development became dominant and ovulation occurred from this follicle in 4 of 4 animals. However, when the dominant follicle was removed on Day 15, 17 or 19, a new wave of follicular development was induced in 14 of 15 animals. Moreover, the recovered subordinate follicle of the second wave of follicular development had similar growth characteristics to naturally occurring dominant follicles. In conclusion, the subordinate follicle in the second follicular wave in cattle retained the ability to become dominant, but this ability was lost by Day 15 of the estrous cycle. However, cattle then were able to maintain ovulation by developing a new wave of follicular growth.

Comparison of expression levels of candidate genes in endometrium of dairy heifers and lactating dairy cows

Pretheeban, T., Gordon, M. B., Singh, R. and Rajamahendran, R. 2011. Comparison of expression levels of candidate genes in endometrium of dairy heifers and lactating dairy cows. Can. J. Anim. Sci. 91: 255–264. Pregnancy rates (PR) in high-producing lactating dairy cows have declined drastically over the past several decades, but those of heifers have remained constant. Reduced PR could be due to multiple causes, and the underlying pathophysiological mechanisms are still unclear. A compromised maternal uterine environment could be one of factors that could affect the PR. This study was performed to compare the nature of the uterine environment in dairy heifers and lactating dairy cows (2nd/3rd parity) by analyzing the expression levels of selected endometrial genes. Estrus was synchronized in heifers (n=5) and lactating dairy cows (n=5) and endometrial biopsies were performed during the mid luteal phase (day 11) of the estrous cycle. Real-time polymerase chain reaction (Q-RT PCR) and immunohistochemistry were performed to analyse the mRNA and protein levels of genes respectively. Relative abundance of BCL2, HSPA1A, IL1A, TNF, IGF1, FGF2 and SERPINA14 transcripts and the protein expression of IL1A, TNF and FGF2 were significantly higher in heifers in comparison with lactating dairy cows. Our findings suggest an altered endometrial environment in lactating dairy cows compared with heifers. However, whether these differences play a role in pregnancy outcomes should be further investigated.