P4 Levels Research Articles

Dan'e fukang decoction reduces hemorrhage in a rat model of mifepristone induced incomplete abortion and may correlate with cell adhesion molecule signaling interference

Ethnopharmacological relevanceDan'e fukang decoction(DFD) is a traditional Chinese medicine formula. DFD obtains 10 herbs, including Salvia yunnanensis C.H.Wright (Zidanshen) , Curcuma zedoaria (Christm.) Roscoe (Ezhu), Angelica sinensis (Oliv.) Diels (Danggui), Cyperus rotundus L. (Xiangfuzi), Corydalis yanhusuo (Y.H.Chou & Chun C.Hsu) W.T.Wang ex Z.Y.Su & C.Y.Wu, Bupleurum marginatum Wall. ex DC. (Yanhusuo), Sparganium stoloniferum (Buch.-Ham. ex Graebn.) Buch.-Ham. ex Juz. (Sanleng), Panax notoginseng (Burkill) F.H.Chen (Sanqi), Paeonia lactiflora Pall. (Shaoyao) and Glycyrrhiza uralensis Fisch. (Gancao). DFD is now clinically used for the treatment of menstrual irregularities, dysmenorrhea and menstrual discomfort caused by blood stasis and easing of endometriosis. Based on this, it is reasonable to presume that DFD may be effective in treating incomplete abortion and reducing postpartum bleeding, but no specific studies have been reported so far. Aim of the studyTo investigate the efficacy of Dan'e fukang decoction (DFD) in reducing prolonged vaginal bleeding followed by mifepristone induced incomplete abortion and explore the mechanisms of action of DFD in treating incomplete abortion. MethodsAn incomplete abortion model of rat was established by single intragastrically administered 8.5 mg/kg mifepristone on the 7th day of pregnancy. From the 8th day of pregnancy, the abortive rats were administered solvent, a positive control drug, or different doses of DFD, respectively for seven consecutive days. The efficacy of DFD was assessed by measuring the vaginal bleeding volume of the rats. Four coagulation parameters and platelet counts were measured. Hematoxylin and eosin (HE) staining was performed to evaluate pathological changes in the uterine embryos. Serum levels of progesterone and estrogen were measured using ELISA. Network pharmacology and transcriptomics were used to predict potential targets and pathways for DFD to reduce hemorrhage. The levels of mRNA related to cell adhesion molecules (CAMs) were detected by RT-qPCR. The levels of progesterone and estrogen receptors and the proteins associated with CAMs pathway in uterine tissues were detected by Western Blot. ResultsDFD significantly reduced the volume of vaginal bleeding of the abortive rats and significantly downgraded the pathological scores of uterine embryos. DFD significantly increased serum levels of E2, and had no impact on serum levels of P4 and the protein expression of ER and PR in the uteri of the abortive rats. Pathways in cancer, lipid, focal adhesion and immune-related signaling were predicted to be influenced by DFD via the analysis of network pharmacology. The CAMs signaling was found the most critical pathway regulated by both mifepristone and DFD via RNA-seq assay, followed by axon guidance, basal cell carcinoma, hippo signaling pathway and neuroactive ligand-receptor interaction. Combining the two analytical methods, ICAM-1 was predicted likely the key targeted gene by DFD. Finally, DFD was validated to decrease the protein expression of ICAM-1, ITGB2, ITGB7 and RASSF5 in the uterine tissues, which correlated to suppress the CAMs signaling pathway. ConclusionDFD significantly reduced hemorrhage. DFD significantly increased the serum levels of E2 and inhibited CAMs signaling pathway, which was likely to be involved in the mechanism of action of DFD facilitating residual uterine embryo expulsion in the rat model of incomplete abortion.

Higher serum progesterone level has no negative impact on live birth rate in frozen embryo transfer

Optimal synchronisation between the endometrium and the embryo is key to the success of frozen embryo transfers (FET). It is achieved by administering different doses of exogenous oestrogen and progesterone (P4). The negative impact of low levels of P4 on FET has been reported, but there is a lack of knowledge about which levels are most appropriate. We aimed to evaluate whether high serum P4 levels measured on FET day affect the probability of having a baby at home. This retrospective cohort study includes 7546 FET cycles performed with hormone replacement therapy (HRT) for artificial endometrial preparation. Patients were divided into three groups according to P level on FET day (ng/mL): P4 < 20(n = 6623), P4 ≥ 20–40 (n = 770) and P > 40 (n = 146). Female age was per group P4 < 20 = 38.7 ± 3.1, P ≥ 20–40 = 39.0 ± 3.0, and P4 > 40 = 38.1 ± 2.9 years old (p = 0.53). The group with the highest progesterone levels found lower ongoing pregnancy rates without statistical significance (56.3 % (P4 < 20) vs 56.8 % (P4 ≥ 20-<40) vs 51.4 % (P4 > 40); p = 0.5). Miscarriage rate was also higher, although not significantly: 16.2 % (P4 < 20) vs 15.0 % (P4 ≥ 20-<40) vs 18.0 % (P4 > 40) (p = 0.6). These findings were explored with an adjusted analysis. A higher, but not significant, odds of miscarriage was observed when P4 > 40 ng/ml, aOR = 1.14 (0.75–1.74) (p = 0.55) whereas no such association was found with P4 ≥ 20–40 ng/ml (aOR = 0.92 (0.74–1.15) (p = 0.46)). The probability of livebirth is similar when the patient was P4 > 40 (aOR = 0.77 (0.51–1.15) (p = 0.20)) than when P4 ≥ 20-<40 (aOR = 0.94 (0.79–1.11) (p = 0.47). In conclusion, women with elevated serum P4 levels on the day of FET after HRT do not find their probability of live birth impaired.

Vascular Alterations in Uterine and Ovarian Hemodynamics and Hormonal Analysis throughout Pregnancy Loss in Cows under Heat Stress.

This current study examined the ovarian (OA) and middle uterine arteries (MUA) blood flow under heat stress conditions regarding hormonal status (progesterone (P4), estradiol (E2), and nitric oxide metabolites (NOMs) assays). Eighteen pluriparous cows were examined, with twelve only being subjected to the natural mating as the other six animals were not bred. Pregnancy diagnosis was confirmed at day 30 by embryonic heartbeat and CL graviditatis (n = 6; pregnant), but some animals (n = 6) showed early embryonic death (EED), with the presence of control cows (n = 6). In the pregnant group, luteal diameter (cm) increased after mating, while in the pregnancy loss group it increased (p < 0.05) until reaching day 38 (1.41 ± 0.03), then decreased again. Luteal vascularity was declined in cows with EED after day 36 (p < 0.05) and reached its lowest level at day 50. P4 levels elevated in cows with EED until day 36 (13.64 ± 0.11) then declined. Both ipsilateral OA and MUA Doppler indices were declined in both groups except in cows who suffered from EED; both were elevated from day 38 until day 50 after mating. Ipsilateral peak systolic point (PSV cm/sec) of OA and MUA was elevated in both groups (p < 0.05), but in cows with EED this parameter was declined. E2 and NOMs levels were declined in cows with EED from day 40 and day 38 after mating (p < 0.05), respectively. This study provided novel data on the relations among the luteal diameter, E2, P4, and NOM levels, and luteal hemodynamics that predicts the amount of blood supply, which acts as a sensitive parameter to detect the alterations in luteal function during the first 50 days after mating.

Differences in perinatal complications and serum hormone levels due to uterine endometrial preparation methods in frozen-thawed embryo transfer.

In frozen-thawed embryo transfer (FET), differences in endometrial preparation methods affect the incidence of perinatal complications. However, the underlying causes are unclear. We aimed to investigate whether serum E2, P4 levels are associated with perinatal complications. This is a retrospective cohort study, involving 306 successful FET pregnancies from 2017 to 2022. Participants were divided into Natural Cycle (NC) and Hormone Replacement Cycle (HRC) group. We compared serum hormone levels, maternal backgrounds, and perinatal outcomes and complications. Furthermore, within the HRC group, serum hormone levels were compared for perinatal complications previously reported to show differences in incidence rates depending on the method of endometrial preparation. HRC exhibited significantly higher serum E2 levels during the implantation period, but lower P4 levels during ovulation, implantation, and pregnancy test period compared with NC. HRC also had significantly higher rates of postpartum hemorrhage (PPH) and placenta accreta spectrum (PAS). There was no association found between perinatal complications more likely to occur in HRC and serum E2, P4 levels. In HRC, there were more occurrences of PPH and PAS. Although serum E2, P4 levels during FET did not correlate with perinatal complications.

Comparative analysis of reproductive hormones, serum biochemical indexes and ovarian metabolites in Muscovy breeder duck at different laying stages

The hypothalamic-pituitary-gonadal (HPG) axis regulates egg laying through control hormones secretion in poultry. In this study, the serum hormones (12 samples per stage), serum biochemical indexes (12 samples per stage), and ovarian metabolites (8 samples per stage) of Muscovy breeder ducks were detected at prelaying stage (PT), start of laying stage (ST), high laying stage (HT), and the end of laying stage (ET). The serum hormones of Muscovy ducks were measured at 8:00, 13:00, 18:00, 23:00, and 4:00 within 1 d. The TG, TP, ALB, and GLB were significantly increased, while HDL-C was significantly decreased at ST as compared to PT (P < 0.05). Serum Na, Cl, Ca, P, and K showed significant rise at ST as compared to PT. Serum Na, Cl, Ca, and K were significantly declined, while P was significantly increased at ET as compared to HT (P < 0.05). Serum FSH, LH, PRL, E2, P4 levels peaked at ST (P < 0.05) with only FSH and LH fluctuated significantly within 1 ovulation cycle at ST (P < 0.05). Differential metabolites showed continued ovarian aging. The decline of nucleic acid metabolism occured in ST, the decline of sugar metabolism occurred in ET, and the decline of amino acid metabolism continued at all stages. Temporal expression patterns and correlation analyses indicated a high correlation between ovarian cAMP and serum reproductive hormone levels across different reproductive stages. In conclusion, this study revealed the changes in serum hormones, serum biochemical indicators, and ovarian metabolites, as well as the relationship between serum hormones and ovarian metabolites.

Effects of progesterone administered to maternal rats during the teratogenesis-sensitive period through different routes on neurobehavior and gut microbiota in offspring

Objective: Progesterone (P4) is the optimal agent for luteal support in assisted reproductive technologies. Despite the availability of various formulations, the impacts of P4 treatment administered through different routes on offspring growth remain unevaluated. This study aimed to investigate and compare the effects of P4 administration through three different routes in pregnant rats during the teratogenesis-sensitive period on fertility outcomes and the behavior, plasma P4 and pregnenolone levels, and alterations in gut microbiota in the filial generation (F1) of offspring. Methods: Female rats that mated successfully were randomly classified into four groups: Group 1, the normal control group; Group 2, the IG group, in which utrogestan was administered via intragastric gavage; Group 3, the IM group, in which P4 was administered via intramuscular injection; and Group 4, the VAGIN group, in which P4 sustained-release vaginal gel was administered through the vagina. Except for the control group, P4 was administered in other groups through their respective routes for 13 consecutive days daily during the teratogenic-sensitive gestation period (days 6–18). The fertility outcomes of the maternal rats were observed, and open-field and three-chamber social preference tests were conducted to assess anxiety and social behavior, along with an exhaustive swimming experiment to evaluate the motor endurance of the offspring. Plasma P4 and pregnenolone levels in the offspring were measured, and fecal samples were assessed using gut microbiota sequencing and subsequent bioinformatics analysis. Results: No significant alterations in fertility outcomes were observed through three P4 administration routes—intragastric gavage, intramuscular injection, and vaginal administration—in rats. However, in behavioral experiments in offspring, a significant increase was observed in swimming time in the IG group than in the control group (P = 0.013). Furthermore, the offspring in the IG group exhibited a marked decrease in non-social behaviors compared with the control group (P = 0.030). No significant behavioral changes were observed in the F1 offspring in the open-field test. Additionally, the offspring in the IG group exhibited a significant elevation in plasma pregnenolone levels compared with the control group (P = 0.019) 30 days after birth. No notable differences in the α-diversity of gut microbiota were observed among the groups. However, significant differences in β-diversity were observed in both the IG and IM groups compared with the control group, indicating that P4 treatment via the two routes exhibited a significant impact on the gut microbiota composition of F1 offspring. Distinct differences were observed in the gut microbiota of the offspring among the three P4 treatment groups, with 22, 28, and 33 differential bacterial taxa identified in the IG, IM, and VAGIN groups, respectively, using linear discriminant effect size analysis, whereas Olivella appeared exclusively in the control group rather than in the P4 treatment groups. Conclusion: Oral administration of P4 to maternal rats during the teratogenesis-sensitive period enhanced motor endurance and plasma pregnenolone levels and altered the composition of the gut microbiota in the offspring. Our findings demonstrated different effects on offspring growth when P4 is administered orally, intramuscularly, or vaginally.

Electro-responsive Molecularly Imprinted Polymers (E-MIPs) for Rapid Detection of Progesterone in Human Serum Samples.

Rapid and reagent-free detection of progesterone (P4) is crucial in point-of-care (POC) measurement due to its important role in the human endocrine and central nervous system. Currently available technologies for P4 detection are often not rapid or require reagents, limiting their use in POC settings. In this work, a self-signaling electrochemical sensing platform for rapid detection of P4 is developed by using electroactive molecularly imprinted polymers (E-MIPs). E-MIPs possess the ability to transduce the molecular recognition event into a measurable electrochemical signal. The E-MIPs are prepared by bulk copolymerization of acrylic acid (AA) and ferrocenylmethyl methacrylate (FcMA) in the presence of P4 followed by its removal from the polymer matrix. By incorporation of ferrocene moieties, the MIP gains intrinsic electroactivity, enabling label-free and direct electrochemical detection of P4 levels. Electrochemical techniques, including cyclic voltammetry and electrochemical impedance spectroscopy, were employed to elucidate the electron transport reaction responsible for the MIP's intrinsic electroactivity. Additionally, the predetermined complementary binding sites confined to the small working area of the screen-printed carbon electrode (SPCE) within the E-MIP matrix facilitate rapid P4 binding, achieving completion within 120 s. The developed E-MIP sensing platform was used for the rapid detection of P4 levels in human serum samples. The short incubation times reported for the E-MIP sensing platform would be beneficial in minimizing the nonspecific binding in the real-world samples. We believe that the proposed E-MIP sensing platform can be applied to clinical samples for detecting fluctuations in P4 levels.

Exogenous estrogen partially rescues progesterone deficiency and autophagosome enlargement in Mcoln1 −/− mouse model with lysosomal storage disorder

Objective: Female Mcoln1 −/− mice exhibit progressive progesterone (P4) deficiency, luteal cell degeneration, and premature embryo implantation failure at five months old. We attempted to rescue embryo implantation in non-virgin Mcoln1 −/− mice (5–6 months old) with exogenous P4 treatment on days 1.5 post-coitum (D1.5), D2.5, and D3.5, and observed partially restored luteal cell morphology on D4.5, but unexpectedly found 17β-estradiol (E2) contamination in the P4 working solution. In this study, we aim to investigate exogenous P4 and/or E2 for the partial recovery of luteal cell morphology in infertile Mcoln1 −/− mice. Methods: Control and non-virgin Mcoln1 −/− mice (5–6 months old) were treated with newly ordered vehicle, P4, E2, or P4+ E2 on D1.5 and D2.5 and dissected on D3.5 for P4 and E2 measurements, ovary histology, immunofluorescence, lipid droplet staining, and transmission electron microscopy. Results: E2 treatment significantly increased serum P4 levels in D3.5 Mcoln1 −/− mice. E2 and P4+E2 treatments, but not P4 treatment alone, largely improved the morphology of D3.5 Mcoln1 −/− corpora lutea, indicated by a more contiguous web-like collagen IV expression pattern, increased heat shock protein 60 expression, and reduced accumulation of large lipid droplets. Transmission electron microscopy revealed extremely enlarged autophagosomes and lipid droplets, lysosomes with lamellar structures, and mitochondria with reduced cristae in vehicle-treated D3.5 Mcoln1 −/− luteal cells, while in E2-treated D3.5 Mcoln1 −/− luteal cells, extremely enlarged autophagosomes and lipid droplets were reduced, indicating improved luteal cell ultrastructure. Conclusion: These findings reveal protective effects of high levels of exogenous E2 on P4 production and lysosomal function in Mcoln1 −/− luteal cells.

Unraveling the impact of hyperleptinemia on female reproduction: insights from transgenic pig model

BackgroundInfertility is a growing global health concern affecting millions of couples worldwide. Among several factors, an extreme body weight adversely affects reproductive functions. Leptin is a well-known adipokine that serves as an endocrine signal between adiposity and fertility. However, the exact mechanisms underlying the effects of high leptin level on female reproduction remain unclear.MethodsTransgenic pigs overexpressing leptin (♀) were produced by backcrossing and screened for leptin overexpression. The growth curve, fat deposition, reproductive performance, apoptosis, serum hormones and cholesterol production, RNA sequencing, and single-nucleus RNA sequencing (snRNA-seq) of the leptin-overexpressing pigs and wild-type group were evaluated.ResultsTransgenic pigs overexpressing leptin (♀) were obtained, which exhibited significantly reduced body weight, body size, and back fat thickness. These pigs manifested a late onset of puberty (330 ± 54.3 vs. 155 ± 14.7 days), irregular estrous behavior characterized by increased inter-estrous interval (29.2 ± 0 vs. 21.3 ± 0.7 days), and more number of matings until pregnancy (at least 3 times). This reproductive impairment in leptin pigs was related to hormonal imbalances characterized by increased levels of FSH, LH, prolactin, E2, P4, and TSH, altered steroidogenesis such as increased levels of serum cholesterol esters along with steroidogenic markers (StAR, CYP19A), and ovarian dysfunctions manifested by neutrophilic infiltration and low expression of caspase-3 positive cells in the ovaries. Moreover, bulk RNA sequencing of the ovaries also revealed neutrophilic infiltration followed by upregulation of inflammation-related genes. Furthermore, snRNA-seq reflected that leptin overexpression triggered immune response, suppressed follicle development and luteinization, resulting in metabolic dysfunction and hormone imbalance in the ovary.ConclusionsLow body weight in leptin overexpressing pigs adversely affects the reproductive performance, causing delayed puberty, irregular estrous cycles, and reduced breeding efficiency. This is linked to metabolic imbalances, an increased immune response, and altered ovarian functions. This study provides a theoretical basis for the complex mechanisms underlying leptin, and infertility by employing leptin-overexpressing female pigs.

Association between serum hormone levels in early pregnancy and risk of hypertensive diseases of pregnancy in women undergoing assisted reproduction.

We examined the association between progesterone (P4), estradiol (E2), and human chorionic gonadotropin (hCG) levels in early pregnancy and the development of hypertensive diseases of pregnancy among women undergoing assisted reproduction. Retrospective study including patients who underwent frozen embryo transfer (FET), ovarian stimulation (OS), or unassisted conception (UC) and had a live singleton birth. The primary outcome was the development of hypertensive diseases of pregnancy (gestational hypertension, preeclampsia, HELLP, or eclampsia). Secondary outcomes were the development of fetal intrauterine growth restriction (IUGR), gestational diabetes mellitus, birth weight, and pre-term birth. Hormone levels and the development of the outcomes were correlated. A total of 681 patients were included; 189 had FET, 193 had OS, and 299 had UC. Patients undergoing FET or OS were not more likely to develop hypertensive diseases of pregnancy compared with UC patients. While median levels of E2 and P4 were significantly different between P-FET and NC-FET patients (E2: 252 vs 317pg/mL, P4: 64 vs 29ng/mL, respectively; both p < 0.01), rates of hypertensive diseases of pregnancy did not significantly differ between those two groups. In the multivariate analyses, P4, E2, and hCG were not associated with the development of hypertensive diseases of pregnancy, but progesterone levels were significantly higher among those with IUGR. This remained consistent when the analysis was limited to FET patients. P4, E2, and hCG levels did not correlate with the development of hypertensive diseases of pregnancy but elevated progesterone levels did correlate with the development of IUGR.

Preovulatory progesterone levels are the top indicator for ovulation prediction based on machine learning model evaluation: a retrospective study

BackgroundAccurately predicting ovulation timing is critical for women undergoing natural cycle-frozen embryo transfer. However, the precise predicting of the ovulation timing remains challenging due to the lack of consensus among different clinics regarding the definition of this significant event.ObjectiveTo compare the effectiveness of preovulatory serum progesterone levels (P4) versus luteinizing hormone levels (LH) in predicting ovulation time using two machine learning models.Methods771 patients who underwent autologous natural cycle-frozen embryo transfer between January 2015 and February 2022 were recruited. Utilizing variables including follicle diameters, preovulatory serum levels of LH, E2, and P4, two machine learning models were constructed to predict the ovulation time, the importance of the variables in predicting ovulation timing was further ranked.ResultsTwo machine learning models have the capability to accurately predict the timing of ovulation, specifically within 72, 48, or 24 h. The overall accuracy rates of the validation dataset, as determined by the classification trees and random forest models, were found to be 78.83% and 85.28% respectively. Notably, when predicting ovulation within 24 h, the accuracy rate of P4 ≥ 0.65ng/ml exceeded 92%. Furthermore, it was important to consider LH or E2 levels in conjunction with P4 when assessing ovulation timing in cases where P4<0.65ng/ml.ConclusionsPreovulatory serum P4 levels are better predictors of ovulation timing than LH levels and could be used as an alternative in clinical settings, and the model we developed can be used to pinpoint the day of ovulation. Ongoing research and advancements in technology are anticipated to enhance and refine the ovulation method.

The annual ovarian cycle in Northern pit vipers (Bothrops atrox) using serum steroids and ultrasound of the female reproductive tract

Knowledge on hormonal regulation of reproductive cycles in viperid snakes is still incipient, especially when it comes to females and tropical species. There is an urgent need to understand the reproduction of venomous snakes to improve assisted reproduction techniques and optimize the maintenance of these animals in captivity. With this in mind, we monitored Northern pit viper females year-round throughout different seasons via serum levels of progesterone (P4) and estradiol (E2) in conjunction with ultrasound examinations. Ovarian follicles were classified according to their size and stage of vitellogenesis in F-I and F-II (non-vitellogenic phase) or in F-III and F-IV (vitellogenic phase). During autumn and winter, five adult males were rotated among these females for reproductive pairing, which resulted in 17 copulations and 2 pregnancies in the first year and 12 copulations and 5 pregnancies in the second year. Then, we assessed changes in P4 and E2 levels according to seasons, predominant ovarian structures and the presence of embryos or eggs in the oviduct. Our findings showed high levels of E2 when a greater number of vitellogenic follicles were detected, indicating a possible influence of E2 on vitellogenesis and higher levels of P4 whenever eggs and embryos were visualized in the oviduct, implying its role in maintaining pregnancy. Descriptive analysis of the vipers’ ovarian cycles revealed a greater number of vitellogenic follicles during winter, probably as a result of increases in E2; whereas pregnancies occurred predominantly in spring, under the influence of P4. The use of ultrasound images, as a minimally invasive methodology, associated with serum steroid levels has proven to be an efficient approach in the reproductive monitoring of Northern pit vipers in vivo. In addition, these data suggest that female pit vipers under human care display a seasonal reproductive cycle, despite earlier studies involving captive males of the species indicating a lack of seasonality in sperm production and quality.

Integrated Transcriptomic-Metabolomic Analysis Reveals the Effect of Different Light Intensities on Ovarian Development in Chickens.

Light is a key environmental factor regulating reproduction in avians. However, the mechanism of light intensity regulating ovarian development is still unclear. In this study, 5-week-old (5 wk) partridge broiler breeders were randomly divided into a low-light-intensity group (LL group) and a natural-light-intensity group (NL group) (n = 100). In the rearing period (5 wk to 22 wk), the light intensity of the LL group and NL group were 0.41 ± 0.05 lux and 45.39 ± 1.09 lux, and in the laying period (23 wk to 32 wk) they were 23.92 ± 0.06 lux and 66.93 ± 0.76 lux, respectively. Samples were collected on 22 wk and 32 wk. The results showed that the LL group had a later age at first egg and a longer laying period than the NL group. Serum P4 and LH levels in the LL group were higher than in the NL group on 22 wk (p < 0.05). On 32 wk, P4, E2, LH and FSH levels in the LL group were lower than in the NL group (p < 0.05). Ovarian transcriptomics and metabolomics identified 128 differentially expressed genes (DEGs) and 467 differential metabolites (DMs) on 22 wk; 155 DEGs and 531 DMs on 32 wk between two groups. An enrichment analysis of these DEGs and DMs identified key signaling pathways, including steroid hormone biosynthesis, neuroactive ligand-receptor interaction. In these pathways, genes such as CYP21A1, SSTR2, and NPY may regulate the synthesis of metabolites, including tryptamine, triglycerides, and phenylalanine. These genes and metabolites may play a dominant role in the light-intensity regulation of ovarian development and laying performance in broiler breeders.

Is it possible to replace eCG by GnRH in the hormonal protocol for timed artificial insemination in ewes during the breeding season?

Hormonal protocols based on progestogens and equine chorionic gonadotrophin (eCG) are efficient for estrus and ovulation synchronization in ewes. Although eCG is indispensable during seasonal anestrus, it may not be necessary during the breeding season. Thus, we tested the hypothesis that GnRH is effective in replacing eCG during the breeding season allowing satisfactory ovulation rate, luteal function and conception rates after timed artificial insemination (TAI). Ewes (n = 134) with a minimum body condition score of 2.5 (0–5 scale) were treated with intravaginal devices (IVD) containing 60 mg of medroxyprogesterone acetate (MPA) for seven days and received 0.26 mg of sodium cloprostenol at the time of IVD removal. In Exp. 1, at IVD removal, ewes (n = 29) were allocated to three groups: eCG (200 IU at IVD removal; n = 10); eCG+GnRH (200 IU eCG at IVD removal and 4 µg of buserelin 36 h later; n = 10); or GnRH (buserelin 36 h after IVD removal; n = 9). Blood samples were collected 2, 6 and 12 days after TAI moment (54 h after IVD removal), for progesterone (P4) analysis. In Exp 2, the ewes were allocated to eCG (n = 10) or GnRH (n = 10) groups, as above described, and ovulation moment was evaluated 54, 66 and 78 h after IVD removal. In Exp 3, TAI was performed in ewes from eCG (n = 45) and GnRH (n = 40) groups using 100 × 106 motile spermatozoa from a pool of semen collected from four rams. In Exp. 1, based on P4 levels, we confirmed that all the ewes ovulated (29/29) and there was no significant effect of group (P = 0.89) or group x day (P = 0.18) on P4 concentration, being observed a significant effect of day (P = 0.0001). In Exp. 2, the maximum DF diameter (P = 0.26) and ovulation moment (P = 0.69) did not differ between groups. In Exp. 3, pregnancy rate was significantly lower (P = 0.02) in GnRH (22.5 %; 9/40) compared to eCG (46.7 %; 21/45). The results indicate that, although ovulation and luteal function were not altered after eCG, eCG+GnRH or GnRH treatment, GnRH alone before TAI cannot be used to replace eCG treatment during the breeding season.

The natural menstrual cycle revisited – can natural cycle be trusted

BackgroundThe serum progesterone (P4) level during the luteal phase (LP) plays a crucial role in the initiation and maintenance of pregnancy. However, it is unclear whether the natural cycle consistently provides the best endocrine profile and whether mid-luteal serum P4 levels are always sufficient to support implantation and early pregnancy. The question has become more relevant in relation to fertility treatment, as more frozen embryo transfer cycles are performed in the natural cycle. Moreover, can serum hormone levels and covariates measured during the follicular phase (FP), such as Follicle Stimulation Hormone (FSH), Luteinizing Hormone (LH), Estradiol (E2), Anti-Mullerian Hormone (AMH) and Antral Follicle Count (AFC), be used to predict P4 levels during the luteal phase (LP)?ResultsThis observational prospective cohort study analysed 26 healthy women with a cycle length between 21–35 days and a body mass index (BMI) < 30 kg/m2. Blood sampling started on the fifth day of the menstrual cycle and continued every fifth day until the next cycle. The procedure was repeated for a total of three cycles. The study found that only ten women had a P4 level greater than 30 nmol/L on cycle day 20 or 25 in all three cycles. In total, only 45 cycles out of 77 cycles had serum P4 levels ≥ 30 nmol/L.The E2 level ≥ 345 pmol/L on cycle day 10 proved to be predictive of a P4 level of ≥ 30 nmol/L on either day 20 or day 25 with a sensitivity of 57% and a specificity of 89%. No other covariates, including the FSH level cycle day 5, LH levels during the follicular phase, age, weight, AFC and AMH cycle day 5 correlated with LP P4 levels.ConclusionsA significant correlation between FP E2 levels cycle day 5 (> 131pmol/L) and cycle day 10 (> 345pmol/L) and a LP P4 level ≥ 30 nmol/l was found; thus, the FP E2 level is a predictor of corpus luteum competence. Our findings highlight the existence of suboptimal P4 levels during the LP and a significant inter-individual and intra-cycle variation in P4 levels during the LP in regular menstruating women.

Triclosan affects steroidogenesis in mouse primary astrocytes in vitro with engagement of Sirtuin 1 and 3

Triclosan (TCS) is a widely used antimicrobial, antifungal, and antiviral agent. To date, it has been reported that TCS can enter the human body and disrupt hormonal homeostasis. Therefore, the aim of our paper was to evaluate the impact of TCS on astrocytes, i.e. a crucial population of cells responsible for steroid hormone production. Our data showed that, in mouse primary astrocyte cultures, TCS can act as an endocrine disrupting chemical through destabilization of the production or secretion of progesterone (P4), testosterone (T), and estradiol (E2). TCS affects the mRNA expression of enzymes involved in neurosteroidogenesis, such as Cyp17a1, 17β-Hsd, and Cyp19a1. Our data showed that a partial PPARγ agonist (honokiol) prevented changes in Cyp17a1 mRNA expression caused by TCS. Similarly, honokiol inhibited TCS-stimulated P4 release. However, rosiglitazone (classic PPARγ agonist) or GW9662 (PPARγ antagonist) had a much stronger effect. Therefore, we believe that the changes observed in the P4, T, and E2 levels are a result of dysregulation of the activity of the aforementioned enzymes, whose expression can be affected by TCS through a Pparγ-dependent pathway. TCS was found to decrease the aryl hydrocarbon receptor (AhR) and Sirtuin 3 protein levels, which may be the result of the activation of the these proteins. Since our study showed dysregulation of the production or secretion of neurosteroids in astrocytes, it can be concluded that TCS reaching the brain may contribute to the development of neurodegenerative diseases in which an abnormal amount of neurosteroids is observed.

P-429 Measuring serum progesterone levels on the day of a fresh blastocyst transfer is debatable as there is no evidence for correlation with ongoing pregnancy results

Abstract Study question Does serum progesterone (P4) level on the day of a fresh blastocyst transfer relate to ongoing pregnancy rate (OPR)? Summary answer OPR after a fresh single blastocyst transfer seems not to be associated with serum P4 level on the day of transfer. What is known already Several studies have suggested an optimal serum P4 threshold for embryo transfers in frozen embryo transfer cycles. However, for fresh embryo transfers, data are scarce and conflicting. Study design, size, duration A retrospective single-center cohort study was performed on 598 complete data cycles, retained out of 631 consecutive fresh single blastocyst transfer cycles. Data were collected from patients undergoing a fresh IVF/ICSI cycle with autologous oocytes at a tertiary fertility center between June 2022 and August 2023. Participants/materials, setting, methods A total of 482 unique patients underwent one or more single fresh blastocyst transfers using micronized vaginal progesterone (MVP) 800mg BID for luteal phase support starting day after oocyte retrieval. Serum P4 levels were determined on day of transfer. The primary endpoint was OPR, positive hCG-test and early pregnancy loss (EPL) were secondary endpoints. Unadjusted and adjusted generalized estimating equations (GEE) assessed P4 level association with the endpoints, using an exchangeable working correlation matrix. Main results and the role of chance No association was identified between P4 levels on day of transfer and OPR after accounting for maternal age, blastocyst quality, P4 and estradiol level at last ultrasound, BMI and endometrial thickness. Data were grouped according to 10/50/90 and 25/50/75 percentiles, resulting in groups &amp;lt; = or &amp;gt; 27ng/ml and &amp;lt; = or &amp;gt; 40ng/ml, with omission of 90th and 75th percentile groups due to right-skewed distribution. With serum P4 levels higher than 40ng/ml, the probability of a positive hCG-test significantly increased (OR 1.79, CI 1.11-2.91; p = 0.018). No significant difference was observed in patients with serum P4 &amp;lt; = or &amp;gt; 27ng/ml (OR 1.23, CI 0.66-2.30; p = 0.506). Regarding OPR, no significant differences were observed when comparing patients with serum P4 &amp;lt; = or &amp;gt; 27/ng/ml or &amp;lt; = or &amp;gt; 40ng/ml (OR 0.88, CI 0.42-18.85; p = 0.736 and OR 1.41, CI 0.83-2.37; p = 0.203, respectively). Moreover, EPL did not significantly differ between serum P4 level groups (OR 1.93, CI 0.61-6.13; p = 0.265 and OR 1.2, CI 0.51-2.84; p = 0.674). Age (p &amp;lt; 0.001) and embryo quality (p &amp;lt; 0.001) are significantly related with OPR. Additionally, late follicular progesterone levels and OPR are significantly associated (OR 2.39, CI 1.16-4.95 in patients with P4 &amp;lt; = or &amp;gt; 27/ng/ml and OR 2.21, CI 1.07-4.55 in patients with &amp;lt; =40ng/ml and &amp;gt;40ng/ml). Limitations, reasons for caution Limitations include the retrospective design and the lack of live birth outcome data. Progesterone levels exceeding 60 ng/ml were not further analyzed to their precise value but grouped as 60 plus which did not allow to identify potential detrimental effects of very high P4 levels on treatment outcome. Wider implications of the findings Our data suggest that measuring P4 on the day of a single blastocyst transfer with MVP luteal phase support may not be indicative of OPR or the risk of EPL. This raises questions regarding the necessity for routine P4 measurements as well as the development of rescue strategies. Trial registration number ONZ-2023-0628

P-418 What is the optimal midluteal phase circulating progesterone level during a fresh IVF cycle? The answer differs from what one expects in a natural cycle

Abstract Study question Do midluteal phase circulating progesterone (P4) levels relate to the fresh embryo transfer clinical outcome and is rescue P4 useful in such cases? Summary answer Live birth rate (LBR) was significantly lower when P4 levels were below 20ng/ml and did not seem to benefit from rescue intramuscular P4. What is known already Preceding literature on the predictive value of midluteal P4 for LBR has shown conflicting results and frequently included small sample sets and heterogeneous ovarian stimulation protocols. The findings reported ranged from the lack of an association between P4 serum levels and clinical outcome to worse success rates seen either in the groups with the highest or the lowest P4 in midluteal phase. Furthermore, while rescue P4 has been empirically proposed, it has lacked formal assessment thus far. Study design, size, duration In total, 652 patients who underwent a fresh single blastocyst transfer in an IVF cycle in our center between 2014 and 2021 were included in this retrospective cohort study. All were GnRH-antagonist cycles using hCG trigger and luteal support with micronized vaginal progesterone 200mg twice daily. Rescue intramuscular progesterone was systematically administered whenever low midluteal P4 (below 1 standard deviation of the center’s mean) levels were detected. Participants/materials, setting, methods The cycles were subdivided into three groups according to the midluteal P4 levels: &amp;lt;10ng/ml, 10-20ng/ml and &amp;gt;20ng/ml. The primary outcome assessed was LBR, with secondary outcomes including clinical pregnancy (CPR) and pregnancy loss rates. Maternal age and body mass index (BMI), year of treatment, ovarian response, endometrial thickness, cause of infertility and embryo quality were controlled for using multivariable logistic regression. Main results and the role of chance The mean age ± standard deviation of patients was 36.8±3.8 years, while mean weight and BMI were 63.2±11.5kg and 23.3±3.9kg/m2, respectively. LBR in the group with P4&amp;gt;20ng/ml (46.5%) was significantly higher than in the groups with low and intermediate P4 serum levels (23.1% and 28.1%, respectively, p &amp;lt; 0.001). The same was true for CPR (54.5%, 30.8% and 37.9%, respectively, p &amp;lt; 0.001). Conversely, the rate of pregnancy loss in the group with P4&amp;gt;20ng/ml (23.8%) was significantly lower than in the groups with low and intermediate P4 levels (42.6% and 37.5%, respectively, p = 0.018). The multivariable logistic regression confirmed that, after controlling for the confounding variables described above, P4&amp;gt;20ng/ml remained significantly associated with a higher LBR (OR: 2.285; 95% CI: 1.506-3.469) and a lower pregnancy loss rate (OR: 0.488; 95% CI: 0.273-0.873), irrespective of the use of rescue IM P4. Finally, a multivariable linear regression analysis of the patients baseline characteristics suggested that the only parameter predictive of midluteal P4 levels was the female patients’ weight, with an inverse relationship (adjusted regression coefficient: -0,012; 95% CI: -0,016/-0,009). Limitations, reasons for caution As the pituitary suppression, ovulation trigger and luteal support protocols used in the cycles included in the study were homogeneous (in order to maximize the internal validity of the results), this limits the extrapolibility of the results to other stimulation protocols. Furthermore, we caution that this is a retrospective study. Wider implications of the findings The usefulness of both midluteal P4 serum measurements and rescue P4 should continue to be assessed. The reasons behind the need for higher levels of P4 in a fresh cycle, when compared to natural cycles, must also be investigated. Luteal support may require personalization according to female body weight. Trial registration number Not Applicable

P-653 Impact of dydrogesterone use in cycles with low progesterone levels on the day of frozen embryo transfer

Abstract Study question Assessing if Frozen Embryo Transfer (FET) outcomes in hormonal replacement treatment with dydrogesterone (DYD) for low progesterone levels match those with normal progesterone (P4) values Summary answer The live birth rate showed no significant difference between patients with low and normal serum P4 levels in FET, utilizing DYD as rescue therapy What is known already The number of IVF/ICSI cycles and frozen embryo transfers (FET) is increasing. The preparation for FET can occur in natural or artificial cycles, showing no significant differences in clinical pregnancy and live birth rates. Artificial cycles involve exogenous estradiol supplementation, starting after menses, and progesterone (P4) addition post-endometrial proliferation. Numerous studies consistently indicate that diminished P4 levels are correlated with inferior outcomes in hormonal replacement treatment, showing a potential decrease of up to 30% in ongoing pregnancy rates. Despite a suggested rescue strategy, evidence supporting its efficacy using an oral route is insufficient. Study design, size, duration This was a retrospective cohort study, of single embryo transfers (SET) from FET cycles conducted between July 2019 and March 2022, after artificial endometrial preparation with valerate-estradiol and micronized vaginal P4. In cases of low serum P4 (up to 12,5ng/dl) on the planned transfer day, 10 mg of dydrogesterone (DYD) three times daily was added. Solely blastocyst single embryo transfers (SET) were included. The primary endpoint was the live birth rate&amp;gt; 24 weeks. Participants/materials, setting, methods 535 FET-SET cycles were analyzed. Patients with an endometrial layer thinner than 7 mm, endocavitary pathology or serum P4 values&amp;lt; 4ng/dl were excluded. The explanatory variables evaluated were: relevant female characteristics, previous IVF/ICSI cycle and relevant FET cycle parameters (endometrial thickness, serum P4 value on the FET day, days in culture before blastocyst freezing) and use of DYD as rescue therapy. Statistical analysis was performed using IBM®SPSS® statistics v22.0 and R4.2.2 for Windows. Main results and the role of chance Out of the 535 FET cycles analyzed, 136(25.4%) underwent treatment with DYD. There were 337 pregnancies (63%), 207 live births (38.6%) and 130 miscarriages (38.5%). Serum P4 varied from 4.2-32.3 ng/dl, with an average of 14.93 ng/dl (SD 4.52). The best model to explain serum P4 distribution considered a gamma distribution for P4. Female age on the FET day, female ethnicity and female weight seemed to explain P4 variation. A sensitivity analysis on the P4 values was performed. The data were divided into 5 groups according to P4 levels and the rate of live birth per quintile was evaluated. There were no statistically significant differences between the groups. Patients with low P4 which had DYD added had no statistical differences regarding deliveries, 40% vs 36% (p = 0,354). The multivariable model revealed that older females at pick up, a thinner endometrial measure and transferring a day 6 blastocyst were negativey associated with live birth (p &amp;lt; 0,05), but not P4 levels or medication with DYD. A sub-group analyses was performed according to the median value of P4 (9.6 ng/ml) of those to whom DYD was added. No statistically significant differences were identified for biochemical pregnancy 59% vs 60% (p = 1) and live birth rate 34%vs38% (p = 0,653). Limitations, reasons for caution This retrospective study lacks an untreated control group, causing inherent heterogeneity. It’s unclear if there’s an upper limit for P4 levels to restrict DYD supplementation or a minimum level rendering this strategy unsuitable. Monitoring DYD’s fetal/neonatal safety is imperative. Wider implications of the findings The supplementation with oral DYD in hormonal replacement FET cycles is an easy and cheap strategy in patients with for low P4 levels. The duration of DYD administration should be further evaluated. Trial registration number not applicable

P-297 Intensive luteal phase support regimens for endometriosis and adenomyosis patients in hormone replacement therapy frozen embryo transfer cycles – subcutaneous or intramuscular progesterone treatment?

Abstract Study question Does subcutaneous (SC) progesterone treatment provide progesterone levels comparable to intramuscular (IM) progesterone when using a cut-off level of 118nmol/l(37.1ng/ml) during intensive luteal phase support? Summary answer As part of an intensive luteal phase support regimen SC progesterone provides higher P4 levels compared to the use of IM progesterone. What is known already Low serum progesterone (P4) levels in HRT-FET cycles negatively impact reproductive outcomes. This effect is particularly pronounced in patients with endometriosis and adenomyosis, due to reduced progesterone actions and progesterone resistance at the receptor level. Therefore, higher serum P4 levels are required to maintain an optimal reproductive outcome during HRT-FET. A P4 cut-off of 118nmol/l (37.1ng/ml) or above has been suggested, a level four times higher than that of the non-endometriosis patient. To achieve high P4 levels, intensive luteal phase support (LPS) regimen has been suggested. This includes vaginal progesterone administration and additional treatment via IM, SC or rectal administration. Study design, size, duration This cohort study includes 380 HRT-FET cycles in endometriosis/adenomyosis patients. Patients were treated from January 2016 until August 2019 with one type of intensive LPS regimen (n = 262) and from January 2000 to January 2023 with two different LPS regimens depending on whether serum P4 levels were lower or higher than 118nmol/l on the day of blastocyst transfer (BT) (n = 118). P4 was measured in a standardised manner. Participants/materials, setting, methods Patients received treatment in a public university affiliated fertility clinic. Endometrial priming involved the use of estradiol followed by vaginal progesterone. From the fourth day of progesterone, one of the following intensive LPS regimens were added: A) 50mg IM progesterone, B) 25mg SC progesterone BD, or C) 25mg SC progesterone BD plus 400mg rectal rescue progesterone BD if P4&amp;lt;118nmol/l. Blastocyst transfer (BT) and P4 measurement were performed on the sixth day of progesterone administration. Main results and the role of chance The overall pregnancy rate, live birth rate (LBR) and total pregnancy loss rates for the whole cohort were 63%, 41% and 36%, respectively. For the three groups A, B and C the mean serum P4 levels on the day of BT were 103.1 ±44.4nmol/l, 170.5 ±62.9nmol/l and 90.1 ±21.5nmol/l. In Group A (IM) 67% of patients (176/262) had P4 levels &amp;lt;118 nmol/l whereas only 36% (43/118; p &amp;lt; 0.001) had P4 &amp;lt;118 nmol/l in the groups of patients treated with SC progesterone (Group B and C). The unadjusted LBR was not significantly different between groups, 39%, 39%, 51%, (p = 0.32). However, a sub-group analysis showed that comparing patients with serum P4 levels &amp;lt;118nmol/l on the BT day in Group A with patients with P4 levels &amp;lt;118nmol/l in Group C a significantly higher LBR was found if intensive LPS included both SC and rectally administered progesterone compared to IM progesterone, only: 51% (44/86) vs 34% (59/176), p = 0.03. The adjusted odds ratio for live birth was 2.04 (95% CI [1.00, 4.16], p = 0.05) after adjusting for age, vitrification day 5 or 6, blastocyst quality and numbers of blastocysts transferred, if intensive LPS included SC and rectal rescue compared to IM, only in patients with P4 levels &amp;lt;118nmol/l. Limitations, reasons for caution This cohort study was conducted over two different time periods. Single embryo transfer was performed only during the later period. The bio-pharmacological profiles of SC and IM progesterone differ, which can influence serum P4 levels. However, in the present study, all blood samples were collected in a standardised manner. Wider implications of the findings This study is the first to report reproductive outcomes following different intensive LPS regimens in a cohort of endometriosis/adenomyosis patients. To increase reproductive outcomes, individualisation of the HRT-FET cycle may be beneficial and, importantly, rectal progesterone administration can be used as a rescue regimen in addition to SC progesterone. Trial registration number Due to the retrospective study design no ethical approval was needed.