Reproductive Physiology Research Articles

Pharmacognostical and Pharmaceutical Evaluation of Rasnadi Basti in the Management of Vandhyatvo w.s.r to Anovulation

Rasnadi basti is mainly indicated for the Vataj disorders i.e., Vibandha, Anaha, Aruchi, Shoola, Vandhyatva, Pradara, Prameha, Yonidosha, Pandu, etc. It is having properties like Sarvaroganivarana, Shukrajanana, Vrushya, and Pumsavana Parama. Acharya Siddhinandan Mishra has mentioned the effectiveness of Rasnadi basti in the treatment of Vandhyatva. Vandhyatva (infertility) has got many etiological factors; one among them is anovulation. Vata is governing factor of the whole reproductive physiology & any vitiation in Vata may lead to anovulation. Basti Karma is the best choice for the treatment of Vata Dosha. In the study, Rasnadi Basti was given to one of the trial group having infertility due to anovulation. So, for the assurance of the quality of herbal compounds used to prepare Rasnadi basti, its pharmacognostic and pharmaceutical analysis was necessary. Methods: Freshly prepared Rasnadi Basti was subjected to microscopic evaluation for pharmacognostic & physiochemical analysis like specific gravity & solid content. Results: Pharmacognostical study showed the presence of certain identifying characteristics of the ingredients of Rasnadi basti i.e., Rasna, Eranda, Patala, Musta, Vacha, Pippli, Agnimantha, Kustha, Yastimadhu, etc. In pharmaceutical study, the preliminary physiochemical analysis showed solid content of 52.53% w/v & specific gravity of 1.0242. Conclusions: Pharmacognostical and physico-chemical observations revealed the specific characteristics of all active constituents of Rasnadi basti and confirmed the purity and genuinity of the formulation.

Relaxin (a regulatory-peptide) enhances cryotolerance, fertility potential and plasma reproductive hormones of Nili Ravi buffalo (Bubalus bubalis) during low breeding season

This study investigated the beneficial effects of relaxin on cryotolerance of buffalo spermatozoa and reproductive hormones during low breeding season. Collected semen was diluted in five aliquots with relaxin addition (0.25 mg/mL, 0.50 mg/mL, 0.75 mg/mL, 1 mg/mL, and control). After gentle dilution (37°C), cooling (4°C, 2 h), equilibration (4°C, 4 h), and packaging (straws, polyvinyl French, 0.5 mL), frozen (cell freezer), and thawed (37°C, 30 s) for analysis. Blood samples were collected at different time intervals i.e., −60, −30 and 0 min (pre-dose) and 30, 60, 90, 120 and 150 min (post-dose) from a jugular vein. This study manifest that adding relaxin (1 mg/ mL) in freezing medium ameliorates sperm motility, functionality (%), and seminal plasma total antioxidant capacity (TAC, μM/L) than control during low breeding season. Furthermore, we found that relaxin supplementation at 1 mg/mL significantly improves seminal plasma ATP concentrations (nmol/million) than control, 0.25 mg/mL, and 0.50 mg/mL, and fertility (control, and 0.75 mg/mL). Further, relaxin injection significantly improves plasma T, LH and IGF-1 levels (150 and 120 min vs. −60, and − 30), and FSH, KP, and GnRH concentrations (150 min vs. −60), during low breeding season. Taken together, this study revealed that relaxin ameliorates motility, functionality, and fertility of buffalo spermatozoa. Moreover, relaxin injection (1 mg/mL) improves essential reproductive hormones levels in buffalo signifying its importance in the field of reproductive physiology. Further studies are required to determine the exact mechanism of action of relaxin in enhancing semen quality, fertility and reproductive hormones.

Common sleep disorders in pregnancy: a review.

In this review, we provide a comprehensive overview of common sleep disorders during pregnancy, including their characterization, prevalence, risk factors, and possible contribution to maternal and fetal outcomes. We conducted a quasi-systematic literature search of the MEDLINE database and identified 744 studies from 1991 through 2021, inclusive, that met our inclusion criteria. We synthesized the existing literature on sleep disorders during pregnancy and highlighted controversies, research gaps, and needed clinical developments. Our review covers a range of sleep disorders, including insomnia, obstructive sleep apnea, restless legs syndrome, and circadian rhythm disorders. We discuss the prevalence of these disorders in pregnancy and their potential impact on maternal and fetal health outcomes. We also explore the relationship between sleep disorders, pre-pregnancy comorbidities such as obesity, and pregnancy-related conditions such as gestational diabetes mellitus and preeclampsia. In addition to summarizing the existing literature on sleep disorders during pregnancy, we also highlight opportunities for further research in this area. We suggest that future studies should strive to employ validated and objective measurement tools for sleep disorders and prioritize utilization of longitudinal methods with participant follow-up through postpartum, mid-life, menopause, and beyond. We also put forward investigation into the impact of circadian rhythm disruption on reproductive physiology and early pregnancy outcomes as an area of important work. Overall, our review provides valuable insights on sleep and reproduction and into common sleep disorders during pregnancy and their potential impact on maternal and fetal health outcomes.

Knock-out of vasotocin reduces reproductive success in female zebrafish, Danio rerio.

The vertebrate nonapeptide vasotocin/vasopressin is evolutionarily highly conserved and acts as neuromodulator and endocrine/paracrine signaling molecule. Circumstantial and mechanistic evidence from pharmacological manipulations of the vasotocin system in several teleost fishes suggest sex- and species-specific reproductive roles of vasotocin. While effects of vasotocin on teleost reproductive physiology involve both courtship behaviors and the regulation of the hypothalamic-pituitary-gonadal (HPG) axes, comprehensive studies investigating behavioral and physiological reproductive consequences of genetic ablation of vasotocin in a genetically tractable fish model, such as the zebrafish, are currently lacking. Here, we report the generation of homozygous CRISPR/Cas9-based vasotocin gene knock-out zebrafish. Breeding pairs of vasotocin knock-out fish produce significantly fewer fertilized eggs per clutch compared to wildtype fish, an effect coincident with reduced female quivering courtship behavior. Crossbreeding experiments reveal that this reproductive phenotype is entirely female-dependent, as vasotocin-deficient males reproduce normally when paired with female wild-type fish. Histological analyses of vasotocin knock-out ovaries revealed an overall reduction in oocytes and differential distribution of oocyte maturation stages, demonstrating that the reproductive phenotype is linked to oocyte maturation and release. Ovarian hormone quantification and gene expression analysis in mutant fish indicated reduced synthesis of Prostaglandin F2α, a hormone involved in ovarian maturation, egg release and regulation of female courtship behavior in some cyprinids. However, acute injection of vasotocin did not rescue the female mutant reproductive phenotype, suggesting a contribution of organizational effects of vasotocin. Together, this study provides further support for emerging roles of vasotocin in female teleost reproduction in an important teleost model species.

Immunoregulatory mechanisms between epithelial clear cells and mononuclear phagocytes in the epididymis.

One of the most intriguing aspects of male reproductive physiology is the ability of the epididymis to prevent the mounting of immune responses against the onslaught of foreign antigens carried by spermatozoa while initiating very efficient immune responses versus stressors. Epithelial clear cells are strategically positioned to work in a concerted manner with region-specific heterogeneous subsets of mononuclear phagocytes to survey the epididymal barrier and regulate the balance between inflammation and immune tolerance in the post-testicular environment. This review aims to describe how clear cells communicate with mononuclear phagocytes to contribute to the unique immune environment in which sperm mature and are stored in the epididymis. A comprehensive systematic review was performed. PubMed was searched for articles specific to clear cells, mononuclear phagocytes, and epididymis. Articles that did not specifically address the target material were excluded. In this review, we discuss the unexpected roles of clear cells, including the transfer of new proteins to spermatozoa via extracellular vesicles and nanotubes as they transit along the epididymal tubule; and we summarize the immune phenotype, morphology, and antigen capturing, processing, and presenting abilities of mononuclear phagocytes. Moreover, we present the current knowledge of immunoregulatory mechanisms by which clear cells and mononuclear phagocytes may contribute to the immune-privileged environment optimal for sperm maturation and storage. Notably, we provide an in-depth characterization of clear cell-mononuclear phagocyte communication networks in the steady-state epididymis and in the presence of injury. This review highlights crucial concepts of mucosal immunology and cellcell interactions, all of which are critical but understudied facets of human male reproductive health.

Physiological impacts of chronic and experimental Plasmodium infection on breeding-condition male songbirds

While Plasmodium parasitism is common in songbirds, its impact on avian reproduction is unclear owing to conflicting reports in the existing literature. Particularly understudied is the impact of phase of infection on variation in host reproductive physiology in wild, breeding-condition birds. However, assessing the full impact of Plasmodium on reproductive success in the wild can be difficult because individuals experiencing severe effects of parasitism may not enter the breeding population and may be less likely to be captured during field studies. To address these factors, we quantified metrics of health and reproductive physiology in wild-caught, breeding-condition male dark-eyed juncos (Junco hyemalis hyemalis) before and after experimental Plasmodium inoculation in a captive setting. Metrics of health and reproductive physiology included activity rate, hematocrit, scaled body mass, testosterone and sperm production. Individuals already infected at capture (i.e., chronically infected) had higher levels of hematocrit than males without chronic infections. Experimentally infected males showed a larger reduction in hematocrit and activity rate as compared to controls. However, chronic infection status did not influence the extent of metric decline. Testosterone production did not vary by treatment and most birds produced sperm following inoculation. Broadly, our results suggest that male juncos exposed to Plasmodium during the breeding season likely experience declines in general health, but Plasmodium infections do not negatively impact reproductive physiology. We conclude that physiological tradeoffs in males may favor maintenance of reproductive function despite infection.

A Critical Review of Apanavata with special reference to Menstrual Physiology

Dosha, Dhatu, and Mala constitute the human body. Doshas are an essential component of our body's health, illness, etc. Only by maintaining these Doshas in balance can the human body be kept in a healthy state, and their imbalance results in disease. There are three Doshas: Vata, Pitta and Kapha. Due to its ability to control the actions of other Doshas as well, Vata is regarded as the most important among them. The body's Nishkramana of Mutra (urine), Purisha (faeces), Shukra (semen), Artava (menstrual fluid and ovulation) and Garbha (foetus) are all activities of the Apanavata subdivision of Vata. Nearly every Acharya in Ayurveda has quoted about Apanavata and its activities, but because its mechanism of action is not elucidated, we only possess an incomplete comprehension of it. With the aid of current theories, the present study has been done to examine the Apanavata and fully comprehend its role (physiological action). Ovulation and menstruation are associated with Artava nishkraman, but parturition or childbirth is associated with Garbha nishkraman. The main objective of this work is to take into consideration Artava Nishkraman in female reproductive physiology.

Common Marmoset Cell Lines and Their Applications in Biomedical Research.

Common marmosets (Callithrix jacchus; CMs) are small New World primates widely used in biomedical research. Early stages of such research often include in vitro experiments which require standardized and well-characterized CM cell cultures derived from different tissues. Despite the long history of laboratory work with CMs and high translational potential of such studies, the number of available standardized, well-defined, stable, and validated CM cell lines is still small. While primary cells and immortalized cell lines are mostly used for the studies of infectious diseases, biochemical research, and targeted gene therapy, the main current applications of CM embryonic stem cells and induced pluripotent stem cells are regenerative medicine, stem cell research, generation of transgenic CMs, transplantology, cell therapy, reproductive physiology, oncology, and neurodegenerative diseases. In this review we summarize the data on the main advantages, drawbacks and research applications of CM cell lines published to date including primary cells, immortalized cell lines, lymphoblastoid cell lines, embryonic stem cells, and induced pluripotent stem cells.

Comparative reproduction of the female horse, elephant and rhinoceros: implications for advancing Assisted Reproductive Technologies (ART).

Recent loss of rhinoceros subspecies has renewed interest in using more advanced assisted reproductive technologies (ART) in rhinoceroses and elephants. Currently, only semen collection, semen preservation and artificial insemination (AI) have been used repeatedly with success in these species. Although ovum pick-up (OPU) and intra-cytoplasmic sperm injection (ICSI) have been reported in rhinoceroses, the techniques are not yet optimised. In contrast, multiple ART applications are routinely used in the horse. Since elephant and rhinoceroses share some reproductive features with equids, we postulate that procedures such as OPU, ICSI, in vitro fertilisation (IVF) and embryo transfer (ET), which are well established in the horse, may represent a basis to develop protocols for endangered pachyderms. In this review, we summarize current knowledge on reproductive physiology relevant to ART. We discuss the current state of ART in all three families and the requirements for the successful implementation of OPU, ICSI, IVF and ET in these species.

Effect of dietary energy source on pregnancy rates and reproductive physiology of pastured beef heifers

Proper production of steroid hormones, such as progesterone (P4), is essential for maintenance of pregnancy. Provision of gluconeogenic substrates from the diet may alter postabsorptive energy supplies and associated hormone signaling in a manner supportive of elevating P4 concentrations. Crossbred Angus heifers (n=27) were balanced for pre-trial bodyweight (BW) and body condition score (BCS), then assigned to isoenergetic diets leveraging starch (ES) or fat (EF) as the primary source of energy (45.7% starch vs 11.5% fat). The heifers were placed on the assigned diets 8 days prior to the initiation of estrous synchronization and continued the diets through the second pregnancy diagnosis, 52 days after the initial insemination. On day 28 post-breeding, pregnancy status was diagnosed via ultrasonography. Measurements of follicle size and number were collected via rectal palpation and ultrasonography on days −10, −8, −4, and day 0 relative to breeding. Blood samples were collected on days −10, 1 through 7, 10, 14, 18, 21, 24, and 28 to analyze plasma for P4 concentrations and placental-associated glycoprotein (PAG) concentrations. Data were analyzed using a linear mixed effects model with a fixed effect for treatment and a random effect for pen. Observations collected over time were analyzed using a repeated measures approach with fixed effects for treatment, time, initial condition, and a random effect for pen. Interactions between treatment and pregnancy status were also tested for PAG and P4 concentrations. No treatment differences were observed in final BCS (P=0.12) and total dry matter intake (DMI; P=0.51), though heifers on the ES treatment tended to reach an increased BW (P=0.10). Although heifers fed ES diets had numerically higher conception rates compared to heifers fed EF diets (71.4% vs. 66.7%), there was no statistically significant difference. Furthermore, there were no significant differences observed among diets in terms of pregnancy outcomes, follicle size, or number. A treatment by pregnancy status interaction was detected for both PAG and P4 concentrations. These results suggest that short-term dietary starch supplementation provided around the time of breeding can result in shifts to reproductive hormone abundance that may be conducive for pregnancy establishment.

The Assessment of the Toxic Effects of Bisphenol A (BPA) on Physiology and Reproduction of the Fish Species Oreochromis mossambicus

The industrialized world produces massive amounts of plastic every year. Recent research information suggests that BPA exhibits hormone-like properties, raising concerns about its suitability in some consumer products and food containers. The most important human exposure route to BPA is diet, which includes consumption of unclean food and water. Exposure to BPA has resulted in adverse effects on reproduction, embryonic development, and the onset of genetic abnormalities in both aquatic and terrestrial animals. However, reports of the effects of BPA on reproductive physiology in fish are limited. This study was aimed to study the adverse effect of BPA on the regulation of reproductive genes in Mozambique tilapia (Oreochromis mossambicus) fish species. The tissue distribution patterns of GnRH1 and GnRH1 in adult Oreochromis mossambicus are shown in predominantly expressed in brain tissues but after the exposure of BPA the gene expression were reported down regulated. When t test was applied on the obtained data we have reported the statically significant down regulation of the GnRH2 (t test P = 0.0395) after the exposure of BPA 5mg BPA/L. This study showed the adverse effect on the reproductive genes of the Oreochromis mossambicus.

Gamma-glutamyltransferase-associated glycoprotein patterns in human seminal plasma of normozoospermic men: a new aspect of biomarker heterogeneity.

Gamma-glutamyltransferase (GGT) is a well-known laboratory biomarker. In spite of high concentration and the possible biomedical importance of estimating GGT in human seminal plasma (hSP), it has not been widely explored in reproductive physiology. This study aimed to complement existing data on its diversity, previously obtained on seminal extracellular vesicles, by analyzing matched soluble fraction of hSP. The GGT-associated patterns of selected glycoproteins were analyzed in order to establish an adjunct referent parameter for differentiation between known high molecular mass forms of GGT. Getting insight into distinct GGT-associated glycoprotein patterns should contribute to define them together as possible multimarkers. GGT forms in soluble, membrane-free-fraction isolated form hSP of normozoospermic men were analyzed using gel filtration and lectin blotting using WGA (wheat germ agglutinin) and Con A (concanavalin A). Widely distributed GGT (with two to three partially resolved peaks), which may correspond to high molecular mass aggregates, were detected. GGT-associated patterns of selected glycoproteins (at position of big, medium, and small-GGT) all comprised high molecular mass WGA-reactive smears, but differed in the presence of Con A-reactive glycans, as well as mucin-associated antigens CA19-9 and CA125. GGT contributes to several molecular patterns that differ between the soluble and extracellular vesicle fractions of hSP. Their glycobiochemical heterogeneity is due to difference in the presence of distinct sialylated and mannosylated glycans. Moreover, GGT-associated glycoprotein patterns differentiate between high molecular mass forms of GGT in the soluble fraction of hSP. They hold promise as possible targets for increasing biomarker potential of GGT.

Partner of neuropeptide bursicon homodimer pburs mediates a novel antimicrobial peptide Ten3LP via Dif/Dorsal2 in Tribolium castaneum

Bursicon is a cystine knot family neuropeptide, composed of two subunits, bursicon (burs) and partner of burs (pburs). The subunits can form heterodimers to regulate cuticle tanning and wing maturation and homodimers to signal different biological functions in innate immunity, midgut stem cell proliferation and energy homeostasis, and reproductive physiology in the model insects Drosophila melanogaster or Tribolium castaneum. Here, we report on the role of the pburs homodimer in signaling innate immunity in T. castaneum larvae. Through transcriptome analysis we identified a set of immune-related genes that respond to pburs RNAi. Treating larvae with recombinant-pburs protein led to up-regulation of antimicrobial peptide (AMP) genes in vivo and in vitro. The upregulation of most AMP genes was dependent on the NF-κB transcription factor Relish. Most importantly, we identified a novel AMP, Tenecin 3-like peptide (Ten3LP), regulated by pburs via NF-κB transcription factor Dorsal-related immunity factor (Dif)/Dorsal2, but not Relish. We conducted Ten3LP RNAi, synthesized recombinant Ten3LP protein for microbial inhibition assays and functionally characterized Ten3LP as an AMP specific for fungi and Gram-positive bacteria. We demonstrate that expression of Ten3LP is activated by pburs via the Toll pathway. These findings identify new molecular targets for development of potential antibiotics for treating microbial infections and perhaps for RNAi based pest management technology.

Egg provisioning explains the penetrance of symbiont-mediated sex allocation distortion in haplodiploids.

Maternally transmitted symbionts such as Wolbachia can alter sex allocation in haplodiploid arthropods. By biasing population sex ratios towards females, these changes in sex allocation may facilitate the spread of symbionts. In contrast to symbiont-induced cytoplasmic incompatibility (CI), the mechanisms that underpin sex allocation distortion remain poorly understood. Using a nuclear genotype reference panel of the haplodiploid mite Tetranychus urticae and a single Wolbachia variant that is able to simultaneously induce sex allocation distortion and CI, we unraveled the mechanistic basis of Wolbachia-mediated sex allocation distortion. Host genotype was an important determinant for the strength of sex allocation distortion. We further show that sex allocation distortion by Wolbachia in haplodiploid mites is driven by increasing egg size, hereby promoting egg fertilization. This change in reproductive physiology was also coupled to increased male and female adult size. Our results echo previous work on Cardinium symbionts, suggesting that sex allocation distortion by regulating host investment in egg size is a common strategy among symbionts that infect haplodiploids. To better understand the relevance that sex allocation distortion may have for the spread of Wolbachia in natural haplodiploid populations, we parametrized a model based on generated phenotypic data. Our simulations show that empirically derived levels of sex allocation distortion can be sufficient to remove invasion thresholds, allowing CI to drive the spread of Wolbachia independently of the initial infection frequency. Our findings help elucidate the mechanisms that underlie the widespread occurrence of symbionts in haplodiploid arthropods and the evolution of sex allocation.

Expression of placental growth factor and a disintegrin and metalloprotease with a thrombospondin type motifs 1-4-8 during the three trimesters of rat pregnancy at the maternal-fetal interface.

The functional roles of the a disintegrin and metalloprotease with a thrombospondin type motifs (ADAMTS) gene family in reproductive physiology, reproductive organs developments and adult reproductive health are still under investigation. The expression of the anti-angiogenic proteases ADAMTS-1, ADAMTS-4 and ADAMTS-8 in placental angiogenesis at various stages of pregnancy also remains unclear. The purpose of this study was therefore to determine the localization and expression of the ADAMTS-1, ADAMTS-4 and ADAMTS-8 proteins during the three stages of pregnancy in rats. Maternal-fetal tissue samples were collected on Days 5, 12 and 19 of each trimester, corresponding to the first, second and third trimesters. The expression of placental growth factor (PlGF) and ADAMTS-1, ADAMTS-4 and ADAMTS-8 at the maternal-fetal interface was examined using immunohistochemistry and western blot at three distinct phases of pregnancy. ADAMTS-1, ADAMTS-4 and ADAMTS-8 were detected in all three trimesters of pregnancy. The relative amount of PIGF increased in the first trimester and decreased significantly in the third trimester (p < 0.05). The expression of ADAMTS-1 and ADAMTS-4 was significantly higher in the second (p < 0.05) and third trimesters (p < 0.01) compared to the first trimester. However, no statistically significant change was observed in ADAMTS-8 expression between trimesters. The ADAMTS exhibiting the highest expression during the first trimester was ADAMTS8. These findings indicate that the expression of ADAMTS-1, ADAMTS-4 and ADAMTS-8 in the three different stages of rat pregnancy may be involved in the modulation of decidualization, morphogenesis and angiogenesis. Periodic changes in ADAMTS expression are thought to be regulated by gonadal steroids.

Assessment of a non-invasive approach to pregnancy diagnosis in gray whales through drone-based photogrammetry and faecal hormone analysis.

Knowledge of baleen whales' reproductive physiology is limited and requires long-term individual-based studies and innovative tools. We used 6 years of individual-level data on the Pacific Coast Feeding Group gray whales to evaluate the utility of faecal progesterone immunoassays and drone-based photogrammetry for pregnancy diagnosis. We explored the variability in faecal progesterone metabolites and body morphology relative to observed reproductive status and estimated the pregnancy probability for mature females of unknown reproductive status using normal mixture models. Individual females had higher faecal progesterone concentrations when pregnant than when presumed non-pregnant. Yet, at the population level, high overlap and variability in progesterone metabolite concentrations occurred between pregnant and non-pregnant groups, limiting this metric for accurate pregnancy diagnosis in gray whales. Alternatively, body width at 50% of the total body length (W50) correctly discriminated pregnant from non-pregnant females at individual and population levels, with high accuracy. Application of the model using W50 metric to mature females of unknown pregnancy status identified eight additional pregnancies with high confidence. Our findings highlight the utility of drone-based photogrammetry to non-invasively diagnose pregnancy in this group of gray whales, and the potential for improved data on reproductive rates for population management of baleen whales generally.

The response of the brood pouch transcriptome to synthetic estrogen exposure in the Gulf pipefish (Syngnathus scovelli)

Endocrine disruptors have devastating impacts on the reproductive physiology of aquatic organisms. The Gulf pipefish, Syngnathus scovelli, is a sexually dimorphic species, which demonstrates predictable morphological, physiological, behavioral, and genetic responses to synthetic estrogen exposure. It has a broad geographic range, spanning freshwater and marine environments, making it a potential sentinel species across a wide range of habitats. In this study, we investigated the effects of ecologically relevant levels of 17α-ethinylestradiol (EE2) exposure on gene expression patterns in the male pipefish’s brood pouch. We also characterized the extent to which EE2-exposed males developed coloration patterns that are normally restricted to females. We identified differentially expressed genes in the brood pouches of pregnant and non-pregnant males when males were exposed to 5ng/L EE2 from the second to eighth day of pregnancy (which normally lasts about 2 weeks). Our result revealed several potential candidate genes that have a role in the brood pouch’s response to environmental estrogens. We also identified genes that were differentially expressed between mid-gestation pregnant males and non-pregnant males. We found an overall greater effect of EE2 exposure in the transcriptomes of non-pregnant males, which may explain why estrogen-exposed males exhibited difficulty receiving eggs in previous studies. The offspring developed similarly in the control and estrogen treatments, highlighting a potential link between the timing of EE2 exposure and its effects on male pregnancy. These results provide insight into how breeding pipefish populations may still exist even though they are found in freshwater and coastal locations where they are periodically exposed to potentially high concentrations of endocrine-disrupting compounds. We also present examples of female-typical coloration development on males due to EE2 exposure and identify candidate brood pouch genes that can be utilized as biomarkers, contributing to the development of the Gulf pipefish as a sentinel model for ecotoxicology.

P-209 Implication of delta opioid receptor in the in vitro maturation of oocytes and its effects on subsequent fertilization and embryo development in mice

Abstract Study question Could opiods modulate in vitro maturation of oocyte and improve fertilization rates? Summary answer Delta opioid receptor was present in mice oocytes, changing its expression pattern depending on the maturation stage and helping oocytes to reach blastocyst stage. What is known already The molecular mechanisms responsible for oocyte maturation are not completely understood and different molecules have been reported to be implicated as modulators of this process. The endogenous opioid peptides could be interesting candidates because they are involved in the regulation of reproductive physiology at multiple sites. When those receptors are activated, the adenylyl cyclase is inhibited, thus reducing cyclic AMP and blocking PKA. Moreover, opioids are also able to activate proteins involved in maturation. There is evidence suggesting a role for opioids during oocyte maturation because the signaling exerted by opioids is similar to that necessary for the meiosis resumption. Study design, size, duration Immature cumulus–oocyte complexes (COCs) from female 8 to 10 week old WT (C57BL/6xCBA) were retrieved after EGF stimulation. Mouse in vitro maturation culture was performed. In vitro grown oocytes, their corresponding cumulus (CC) and granulosa cells (GC) were classified at germinal vesicle stage (GV), at metaphase 1 (MI) and at metaphase 2 (MII). Participants/materials, setting, methods The presence of DOR was analysed in granulosa cells and oocytes at each stage of maturation by qRT-PCR and immunocytochemistry. AKT and MAPK signalling pathway (ERK 1/2) was studied by immunocytochemistry. Similarly, COCs were matured in vitro in the absence and presence of DPDPE and naltrindole at various concentrations. After IVM, in vitro fertilisation (IVF) was performed and development was observed until the blastocyst stage was reached, analysing fertilisation and embryo production percentages (p &amp;lt; 0.05). Main results and the role of chance Our findings showed that OPRD1 was present in mice oocytes and granulosa cells, changing its expression pattern depending on the maturation stage. The evaluation of RT-PCR examination and immunohistochemical (IHC) staining revealed the presence of the transcript for Oprd1 gene in the different stages of the mouse oocyte's maturation, as well as in the granulosa cells at the moment of the extraction from the ovary. The obtained transcript was compared with the transcript for Oprd1 of mouse cerebral cortex as a positive control. The expected 482 bp fragment was detected in the germinal vesicle stage and in granulosa cells, whereas in MI and MII stage the signal was lower. Immunofluorescence analysis revealed that OPRD1 protein was present in mouse oocytes but, its localization differed at the different stages of the maturation process. Furthermore, the selective delta opioid agonist DPDPE, modulating PI3K/Akt and MAPK pathways, helped oocytes to reach blastocyst stage. Finally, we examined that the observed effect of DPDPE during oocyte maturation in the in vitro fertilization and subsequent embryo development could be blocked by the opioid antagonist naltrindole. Limitations, reasons for caution Although we have obtained higher rates of blastocysts, the quality of these embryos still needs to be analysed and transferred to recipient females to evaluate the offspring. Wider implications of the findings Our study has clinical implications for the improvement of oocyte in vitro maturation techniques (IVM). OPRD1 could be a possible therapeutic target for in vitro maturation culture medium, as it could improve the blastocyst rates obtained in the actual reproduction assisted techniques. Trial registration number not applicable

P-571 Endometrial cortisol and estradiol levels as indicators of pregnancy prognosis and their association with State-Trait Anxiety Inventory (STAI) psychological questionnaire punctuations in IVF patients

Abstract Study question How anxiety and stress questionnaires punctuations and endometrial steroid biosynthesis metabolites levels are associated with embryo implantation and pregnancy outcomes in IVF patients? Summary answer Those patients with high cortisol and low estradiol endometrial levels, were stressed according to STAI State punctuations and showed lower rates of implantation and pregnancy. What is known already Although IVF treatments are known to be a source of stress, its relevance in reproductive success remains controversial, mainly due to the lack of accurate biomarkers. Steroid hormones, such as progesterone and estrogens, are essential for reproductive physiology, and directly related to common stress biomarkers such as cortisol. Nowadays, clinical stress evaluation is exclusively performed via highly subjective psychological questionnaires, not being standardized in the clinical setting. In this study, we measured endometrial levels of main steroid hormones -including cortisol-, stress psychological questionnaires, and reproductive outcomes for determining the relationship among potential stress biomarkers and their influence on fertility. Study design, size, duration In this prospective cohort study, a total of 55 IVF patients were included (&amp;lt;45 years old, no uterine or systemic pathologies and good quality embryos). All endometrial biopsies were collected in mid-secretory phase for metabolites measurement between 2019 and 2021. Also, these patients were psychologically evaluated by stress related questionnaires the same day of biopsy collection or days later in the same cycle. Reproductive outcomes were compared between groups established according to stress biomarkers thresholds. Participants/materials, setting, methods Concentration of eleven selected steroid metabolites was measured by ultra-performance liquid chromatography-tandem mass spectrometry. A psychological questionnaire was also assessed to measure patients’ stress and anxiety (STAI). According to metabolites’ concentration distribution, Barnard’s test was applied to compare the proportion of patients with successful and unsuccessful treatments at different thresholds. A Wilcoxon test was performed for mean metabolites’ levels comparison between stressed and unstressed patients. A p-value of 0.05 was stablished as significant. Main results and the role of chance All patients with high endometrial cortisol levels (&amp;gt;12.5 ng/g; n = 3) failed to implant (100% vs 45.8% non-implanted in &amp;gt; 12.5 ng/g and &amp;lt;12.5 ng/g patients respectively, p-value=0.047), while the totality of patients with high endometrial estradiol levels (&amp;gt;0.9 ng/g; n = 5) achieved embryo implantation and on-going pregnancy (100% vs 50% implanted in &amp;gt; 0.9 ng/g and &amp;lt;0.9 ng/g patients respectively, p-value=0.038). Those patients with high cortisol levels, all not pregnant, had also low estradiol levels and high stress state punctuation (STAI State punctuations ≥ 60). Conversely, patients with high estradiol levels, all pregnant, had also low cortisol levels and were not stressed (STAI State punctuations &amp;lt; 60). Stratification of patients into stressed (STAI State&amp;gt;60) and unstressed (STAI State&amp;lt;60) independently of their implantation results, revealed that 27% of stressed women presented high cortisol levels, while 100% of unstressed patients had low cortisol levels. In the same way, 42% of unstressed patients had high estradiol levels, while 100% of stressed patients presented low estradiol values. Hence, mean values differences of cortisol (10.54 vs 3.05 ng/g) and estradiol (0.55 vs 4.10 ng/g) between stressed and unstressed patients were not significant (p-value=0.08862 and p-value=0.2327, respectively). No relevant associations were observed with stress as a trait. Limitations, reasons for caution Due to the heterogeneous and non-normal metabolites’ concentration distribution among patients, this study would need a largest sample size for having a representative population in the different subgroups. Although we have used molecular and psychological validated stress biomarkers, a causal relationship between stress and reproductive outcomes should be further explored. Wider implications of the findings This study highlighted cortisol and estradiol as potential endometrial biomarkers that better reflect pregnancy prognosis. Further investigation is needed to find if other psychological questionnaires molecularly recognize stressed patients as a noninvasive diagnosis associated to reproductive outcomes. These patients would benefit from psychological counseling to improve their reproductive success. Trial registration number Not applicable

Diet impacts the reproductive system’s maturation in the male moth Agrotis ipsilon (Noctuidae, Lepidoptera)

In male moth Agrotis ipsilon, sexual maturation occurs between the third and the fifth day of adult life and is characterized by the development of the reproductive organs such as testes and accessory sex glands. Since sexual maturation requires considerable energy investment, we hypothesized that diet would be an essential regulatory factor in this developmental process. Indeed, the links between the male diet and reproductive physiology have not been described as in females. To test the previous hypothesis, we offered male moths diets corresponding to different flower nectars found in nature, and measured morphological and functional changes in the testes and accessory sex glands. In comparison to a diet composed of sucrose only, males fed with a diet composed of diverse sugars, including glucose, supplemented with sodium led to an earlier increase in the length and the protein content of accessory sex glands, as well as a reduction of the testicular volume accompanied by an acceleration of the sperm bundle transfer from the testes to the duplex. These results show that these specific diets accelerate the maturation of the reproductive system in male moth Agrotis ipsilon.