Allopregnanolone Concentrations Research Articles

Peripartum allopregnanolone blood concentrations and depressive symptoms: a systematic review and individual participant data meta-analysis.

Neuroactive steroids including allopregnanolone are implicated in the pathophysiology of peripartum depressive symptoms (PDS). We performed a systematic review searching PubMed/Embase/PsychInfo/Cinhail through 08/2023 (updated in 07/2024), and conducted a random-effects meta-analysis of studies comparing allopregnanolone blood concentrations in women with versus without PDS at various timepoints during the 2nd and 3rd trimester and the postpartum period, calculating standardized mean differences (SMDs) and 95% confidence intervals (CIs). Meta-regression and subgroup analyses included age, diagnoses of affective disorders before pregnancy, antidepressant treatment, analytical methods, and sample type. Study quality was assessed using the Newcastle-Ottawa-scale. The study protocol was registered on PROSPERO (registration number CRD42022354495). We retrieved 13 studies with 2509 women (n = 849 with PDS). Allopregnanolone concentrations did not differ between women with versus without PDS at any timepoint (p > 0.05). Allopregnanolone concentrations assessed during pregnancy did not differ for women with versus without PDS at postpartum follow-up (p > 0.05). Subgroup analyses indicated higher allopregnanolone concentrations in women with versus without PDS at gestational weeks 21-24 and 25-28 (SMD = 1.07, 95% CI = 0.04, 2.11 and SMD = 0.92, 95% CI = 0.26, 1.59 respectively). Moreover, we reported differences between studies using mass-spectrometry combined with chromatography versus immunoassays at gestational weeks 25-28 (p = 0.01) and plasma versus serum samples at gestational weeks 21-24 (p = 0.005). Study quality was rated as poor, good, and fair for two, one and ten studies respectively. PDS were not associated with differences for allopregnanolone concentrations. The use of heterogenous peripartum time points, study cohorts, depression symptom measures and analytical methods has hampered progress in elucidating neuroactive steroid signaling linked to PDS.

Allopregnanolone and progesterone in relation to a single electroconvulsive therapy seizure and subsequent clinical outcome: an observational cohort study

BackgroundElectroconvulsive therapy (ECT) is an important treatment for several severe psychiatric conditions, yet its precise mechanism of action remains unknown. Increased inhibition in the brain after ECT seizures, mediated by γ-aminobutyric acid (GABA), has been linked to clinical effectiveness. Case series on epileptic patients report a postictal serum concentration increase of the GABAA receptor agonist allopregnanolone. Serum allopregnanolone remains unchanged after a full ECT series, but possible transient effects directly after a single ECT seizure remain unexplored. The primary aim was to measure serum concentrations of allopregnanolone and its substrate progesterone after one ECT seizure. Secondary aims were to examine whether concentrations at baseline, or postictal changes, either correlate with seizure generalization or predict clinical outcome ratings after ECT.MethodsA total of 130 participants (18–85 years) were included. Generalization parameters comprised peak ictal heart rate, electroencephalographic (EEG) seizure duration, and prolactin increase. Outcome measures were ratings of clinical global improvement, perceived health status and subjective memory impairment. Non-parametric tests were used for group comparisons and correlations. The prediction analyses were conducted with binary logistic and simple linear regression analyses.ResultsAllopregnanolone and progesterone remained unchanged and correlated neither with seizure generalization nor with clinical outcome. In men (n = 50), progesterone increased and allopregnanolone change correlated negatively with EEG seizure duration. In a subgroup analysis (n = 62), higher baseline allopregnanolone and progesterone correlated with postictal EEG suppression.ConclusionsECT seizures have different physiologic effects than generalized seizures in epilepsy. Progesterone might have implications for psychiatric illness in men.

Conversion of 5α-Androstane-3α,17β-diol to bis[(4-dimethylamino)phenyl carbamate] derivative for sensitive determination of its rat brain level by LC/ESI-MS/MS.

5α-Androstane-3α,17β-diol (3α,5α-Adiol) is a testosterone-derived neurosteroid and has anxiolytic and analgesic effects via γ-aminobutyric acid type A receptors as with the progesterone-derived neurosteroid, allopregnanolone (AP). Although the psychotropic drug-evoked changes in the brain AP concentration have been intensively studied, those in the brain 3α,5α-Adiol concentration remain poorly understood. One of the causes for this is the limited availability of a validated method for quantifying the brain 3α,5α-Adiol with a sufficient sensitivity and specificity, which is described in this study. To enhance the detectability of 3α,5α-Adiol by electrospray ionization-tandem mass spectrometry (ESI-MS/MS), derivatization with 4-dimethylaminobenzoyl azide was employed. The brain sample was purified by solid-phase extraction and the recovered 3α,5α-Adiol and the deuterated internal standard were derivatized, then measured by liquid chromatography (LC)/ESI-MS/MS with selected reaction monitoring. The derivatized 3α,5α-Adiol, i.e., the bis[(4-dimethylamino)phenyl carbamate] derivative, provided the intense doubly-protonated molecule as the precursor ion, then the specific product ion containing the 3α,5α-Adiol-skeleton by collision-induced dissociation. The detectability of 3α,5α-Adiol was eventually increased 1000-fold by derivatization. Separation of the derivatized 3α,5α-Adiol from its stereoisomers and interfering brain components was achieved using a SunShell Biphenyl column with an isopropyl alcohol-containing mobile phase. A good linearity in the sufficient concentration range, acceptable precision and accuracy, and negligible matrix effect were demonstrated by the validation tests. The animal (rat) study using this method revealed that the brain 3α,5α-Adiol levels were unaffected by the administration of fluoxetine (FLX) and clozapine (CLZ), in contrast to the significant increase of the AP levels. An LC/ESI-MS/MS method capable of quantifying 3α,5α-Adiol in the rat brain using a 20-mg tissue was developed and validated. The brain levels of 3α,5α-Adiol had an entirely different behavior from those of AP due to FLX and CLZ administration.

Bidirectional associations between perinatal allopregnanolone and depression severity with postpartum gray matter volume in adult women.

Perinatal depression (PND) is a debilitating condition affecting maternal well-being and child development. Allopregnanolone (ALLO) is important to perinatal neuroplasticity, however its relationship with depression severity and postpartum structural brain volume is unknown. We examined perinatal temporal dynamics and bidirectional associations between ALLO and depression severity and the association between these variables and postpartum gray matter volume, using a random intercept cross-lagged panel model. We identified a unidirectional predictive relationship between PND severity and ALLO concentration, suggesting greater depression severity early in the perinatal period may contribute to subsequent changes in ALLO concentration (β = 0.26, p = 0.009), while variations in ALLO levels during the perinatal period influences the development and severity of depressive symptoms later in the postpartum period (β = 0.38, p = 0.007). Antepartum depression severity (Visit 2, β = 0.35, p = 0.004), ALLO concentration (Visit 2, β = 0.37, p = 0.001), and postpartum depression severity (Visit 3, β = 0.39, p = 0.031), each predicted the right anterior cingulate volume. Antepartum ALLO concentration (Visit 2, β = 0.29, p = 0.001) predicted left suborbital sulcus volume. Antepartum depression severity (Visit 1, β = 0.39, p = 0.006 and Visit 2, β = 0.48, p < 0.001) predicted the right straight gyrus volume. Postpartum depression severity (Visit 3, β = 0.36, p = 0.001) predicted left middle-posterior cingulate volume. These results provide the first evidence of bidirectional associations between perinatal ALLO and depression severity with postpartum gray matter volume.

ShuYu capsule alleviates emotional and physical symptoms of premenstrual dysphoric disorder: Impact on ALLO decline and GABAA receptor δ subunit in the PAG area

Premenstrual dysphoric disorder (PMDD) is a severe subtype of premenstrual syndrome in women of reproductive age, with its pathogenesis linked to the heightened sensitivity of type A γ -aminobutyric acid receptors (GABAAR) to neuroactive steroid hormone changes, particularly allopregnanolone (ALLO). While a low dose of fluoxetine, a classic selective serotonin reuptake inhibitor, is commonly used as a first-line drug to alleviate emotional disorders in PMDD in clinical settings, its mechanism of action is related to ALLO-GABAA receptor function. However, treating PMDD requires attention to both emotional and physical symptoms, such as pain sensitivity. This study aims to investigate the efficacy of ShuYu capsules, a traditional Chinese medicine, in simultaneously treating emotional and physical symptoms in a rat model of PMDD. Specifically, our focus centres on the midbrain periaqueductal grey (PAG), a region associated with emotion regulation and susceptibility to hyperalgesia. Considering the underlying mechanisms of ALLO-GABAA receptor function in the PAG region, we conducted a series of experiments to evaluate and define the effects of ShuYu capsules and uncover the relationship between the drug's efficacy and ALLO concentration fluctuations on GABAA receptor function in the PAG region. Our findings demonstrate that ShuYu capsules significantly improved oestrous cycle-dependant depression-like behaviour and reduced stress-induced hyperalgesia in rats with PMDD. Similar to the low dose of fluoxetine, ShuYu capsules targeted and mitigated the sharp decline in ALLO, rescued the upregulation of GABAAR subunit function, and activated PAG neurons in PMDD rats. The observed effects of ShuYu capsules suggest a central mechanism underlying PMDD symptoms, involving ALLO_GABAA receptor function in the PAG region. This study highlights the potential of traditional Chinese medicine in addressing both emotional and physical symptoms associated with PMDD, shedding light on novel therapeutic approaches for this condition.

Reduced urine pregnenolone concentration after clinical response in patients with depression: An open-label short-term prospective study

BackgroundIdentifying biological alterations in patients with depression, particularly those that differ between responders and non-responders, is of interest to clinical practice. Biomarker candidates involve neuroactive steroids, including pregnenolone (PREG) and allopregnanolone (ALLO). However, alterations in PREG and ALLO associated with treatment response are understudied. This study's main aim was to evaluate the effects of antidepressant treatment, clinical response, and treatment duration on PREG and ALLO in depression. Materials and MethodsIn a 4-week, open-label trial, participants were allocated randomly to the venlafaxine (n = 27) or mirtazapine (n = 30) group. Urine concentrations of PREG and ALLO were assessed through gas chromatography-mass spectrometry. Participants collected night urine between 10:30 p.m. and 8:00 a.m. Two primary outcomes were analyzed. Firstly, the effect of treatment (mirtazapine or venlafaxine), clinical response (operationalized through the Hamilton Depression Rating Scale), and time (baseline compared to 28 days) on the urine concentrations of PREG or ALLO in depression. Finally, the effect of clinical response and time on the urine concentration of PREG or ALLO, independently of the antidepressant given (mirtazapine or venlafaxine). Linear mixed models were carried out. ResultsThere was no significant difference in PREG and ALLO concentrations between baseline and 28 days in responders and non-responders when investigating the venlafaxine or the mirtazapine group. However, we found a significant reduction of urine PREG concentration after 28 days of treatment in responders who received either venlafaxine or mirtazapine (estimate = −0.56; p = 0.016; 95CI [−1.003; −0.115]; Cohen’s d = −0.61). ConclusionsOur main results indicate that responders in depression show reduced urinary PREG concentrations after 4-weeks of therapy, independently of the antidepressant used. More studies are needed to confirm these findings.

(075) Exposing the Unsung Cycle: A Proposed Study to Examine the Connection Between Allopregnanolone and Adverse Mood Effects of Hormonal Contraception

Abstract Introduction Allopregnanolone is a GABAnergic neurosteroid. Reduced concentration of allopregnanolone in the cerebrospinal fluid (CSF) and serum are closely associated with several neuropsychiatric conditions, including Alzheimer’s disease, anxiety disorders, premenstrual dysphoric disorder, and postpartum depression. Allopregnanolone is a progesterone metabolite predominantly synthesized in women in the ovaries and prior literature shows the menstrual cycle directly affects serum and CSF allopregnanolone concentrations. We hypothesize that allopregnanolone’s biosynthesis is similarly affected by exogenous hormonal contraceptives. In the United States, 65% of reproductive-aged women use hormonal contraceptives and their discontinuation is commonly attributed to adverse changes in mood; yet, little is understood when it comes to what and how specific components of hormonal contraceptives affect mood and how to manage such effects. Objective We aim to review current literature and propose a study to understand the connection between allopregnanolone and the adverse mood effects of hormonal contraceptives in order to improve women’s health. Methods We conducted a semi-systematic literature search, using PubMed Central and other databases, compiling peer-reviewed material using keywords including allopregnanolone AND hormonal contraceptives; combined oral contraceptive; progestin; suppression; levonorgestrel, ethinyl estradiol, medroxyprogesterone. All languages of publication were included where reliable translations were available. Studies were excluded if they did not include serum and/or CSF measurements of allopregnanolone. Results Rodent studies demonstrated that exogenous hormonal contraceptives reduce both the serum and CSF concentration of allopregnanolone. Human studies analyzing only serum allopregnanolone concentrations showed the same results, but no human studies analyzing CSF allopregnanolone concentrations were identified. Only two human studies have examined serum allopregnanolone levels and mood, and no studies have done so in the context of exogenous hormonal contraceptive use. Conclusions While exogenous sex hormones, namely progestins, affect allopregnanolone levels in rodents, their impact on both allopregnanolone levels and mood remains inconclusive and under-researched in humans. We propose a large multi-arm study on humans analyzing CSF and serum allopregnanolone and mood effects following administration of hormonal contraceptives to assess how contraceptives affect allopregnanolone metabolism in connection to adverse mood effects. This research will allow clinicians to better tailor contraceptive selection. Disclosure No

Validation of an ELISA kit to measure allopregnanolone in human and equine hair.

In humans, allopregnanolone plays important roles in a number of different neurodegenerative disorders, and it has been proposed for use in some therapies. Horses are commonly used as animal models for human neurodegenerative diseases, mental and behavioral disorders, and neuropsychiatric disorders, and there is interest in using hair as a biological sample to study hormones in these conditions. We validated the use of a commercial ELISA kit (DetectX allopregnanolone kit; Arbor Assays), which was designed for serum, plasma, feces, urine, and tissue samples, to assess allopregnanolone in hair samples from 30 humans and 63 horses. The ELISA kit had good precision (intra- and inter-assay CVs: 6.4% and 11.0% for equine hair; 7.3% and 11.0% for human hair, respectively), sensitivity (50.4 pg/mL for both equine and human hair), and accuracy (parallelism and recovery tests) in determining allopregnanolone concentrations in hair from both species. The allopregnanolone concentrations in human hair were 7.3-79.1 pg/mg; the concentrations were 286 ± 141 pg/mg (x̄ ± SD) in mares on the day of parturition and 16.9 ± 5.5 pg/mg in nonpregnant mares. The DetectX ELISA kit offered a simple and accessible analysis of allopregnanolone in human and equine hair samples.

Tolerability and pharmacokinetics of intravenous allopregnanolone with and without midazolam pretreatment in two healthy dogs.

The neurosteroid allopregnanolone (ALLO) is under investigation as a treatment for benzodiazepine-refractory status epilepticus (SE). Here we assess the cardiopulmonary safety of intravenous ALLO by itself and after a clinically recommended dose of midazolam (MDZ) in two healthy adult beagles. Each dog received ALLO (1 mg/kg, IV), and after a washout period of 2 weeks, the dog was dosed with MDZ (0.2 mg/kg, IV) followed 10 min later by ALLO. Behavioral state, vital signs, arterial blood gasses, blood chemistries, and plasma ALLO concentrations were monitored for up to 6 h after dosing. The dogs appeared sleepy but were fully responsive after both treatments. No depression of mean arterial pressure or respiratory rate was noted. Blood gas measurements failed to show evidence of drug-induced acute respiratory acidosis. Estimated maximum plasma ALLO concentrations were in the range of 1500 to 3000 ng/mL. The results indicate that intravenous ALLO can be used safely to treat benzodiazepine-refractory SE, even when administered shortly after a benzodiazepine.

Trajectories of Allopregnanolone and Allopregnanolone to Progesterone Ratio across the Six Subphases of Menstrual Cycle.

Background: Allopregnanolone is one of the most studied neuroactive steroids; yet, despite its relevance to neuropsychiatric research, it is not known how it, as well as its ratio to progesterone, varies across all six subphases of the menstrual cycle. Two enzymes-5α-dihydroprogesterone and 5α-reductase-convert progesterone to allopregnanolone, and, based on immunohistochemical studies in rodents, the activity of 5α-reductase is considered the rate-limiting step in the formation of allopregnanolone. It is not clear, however, whether the same phenomenon is observed across to the menstrual cycle, and, if so, at what point this takes place. Methods: Thirty-seven women completed the study during which they attended eight clinic visits across one menstrual cycle. We analyzed their allopregnanolone and progesterone serum concentrations using ultraperformance liquid chromatography-tandem mass spectrometry, and we implemented a validated method to realign the data from the original eight clinic study visits, following which we imputed the missing data. Hence, we characterized allopregnanolone concentrations, and the ratio of allopregnanolone:progesterone at six menstrual cycle subphases: (1) early follicular, (2) mid-follicular, (3) periovulatory, (4) early luteal, (5) mid-luteal, and (6) late luteal. Results: There were significant differences in allopregnanolone levels between (1) early follicular and early luteal, (2) early follicular and mid-luteal, (3) mid-follicular and mid-luteal, (4) periovulatory and mid-luteal, and (5) mid-luteal and late luteal. We detected a sharp drop in allopregnanolone:progesterone ratio in the early luteal subphase. Within the luteal subphase, the ratio was the lowest in the mid-luteal subphase. Conclusions: Allopregnanolone concentrations are the most distinct, relative to the other subphases, in the mid-luteal subphase. The shape of the allopregnanolone trajectory across the cycle is similar to that of progesterone; however, the proportion of the two neuroactive steroid hormones is drastically different due to enzymatic saturation, which takes place at the start of the early luteal subphase, but continuing through, and peaking, in the mid-luteal subphase. Hence, the estimated activity of 5α-reductase decreases, but does not cease, at any point across the menstrual cycle.

Prenatal Stress Induces Translational Disruption Associated with Myelination Deficits

Disruptions to neurodevelopment are known to be linked to behavioral disorders in childhood and into adulthood. The fetal brain is extremely vulnerable to stimuli that alter inhibitory GABAergic pathways and critical myelination processes, programing long-term neurobehavioral disruption. The maturation of the GABAergic system into the major inhibitory pathway in the brain and the development of oligodendrocytes into mature cells capable of producing myelin are integral components of optimal neurodevelopment. The current study aimed to elucidate prenatal stress-induced mechanisms that disrupt these processes and to delineate the role of placental pathways in these adverse outcomes. Pregnant guinea pig dams were exposed to prenatal stress with strobe light exposure for 2 h/day on gestational age (GA) 35, 40, 45, 50, 55, 60, and 65, and groups of fetuses and placentae were collected after the stress exposure on GA40, GA50, GA60, and GA69 (term). Fetal plasma, placental, and brain tissue were collected for allopregnanolone and cortisol quantification with ELISA. Relative mRNA expression of genes of specific pathways of interest was examined with real-time PCR in placental and hippocampal tissue, and myelin basic protein (MBP) was quantified immunohistochemically in the hippocampus and surrounding regions for assessment of mature myelin. Prenatal stress in mid-late gestation resulted in disruptions to the translational machinery responsible for the production of myelin and decreased myelin coverage in the hippocampus and surrounding regions. The male placenta showed an initial protective increase in allopregnanolone concentrations in response to maternal psychosocial stress. The male and female placentae had a sex-dependent increase in neurosteroidogenic enzymes at term following prenatal stress. Independent from exposure to prenatal stress, at gestational day 60 – a critical period for myelin development, the placentae of female fetuses had increased capability of preventing cortisol transfer to the fetus through expression of 11-beta-hydroxysteroid dehydrogenase types 1 and 2. The deficits early in the process of maturation of myelination indicate that the reduced myelination observed at childhood equivalence in previous studies begins in fetal life. This negative programing persists into childhood, potentially due to dysregulation of MBP translation processes. Expression patterns of neurosteroidogenic enzymes in the placenta at term following stress may identify at-risk fetuses that have been exposed to a stressful in utero environment.

Progesterone and contraceptive progestin actions on the brain: A systematic review of animal studies and comparison to human neuroimaging studies

In this review we systematically summarize the effects of progesterone and synthetic progestins on neurogenesis, synaptogenesis, myelination and six neurotransmitter systems. Several parallels between progesterone and older generation progestin actions emerged, suggesting actions via progesterone receptors. However, existing results suggest a general lack of knowledge regarding the effects of currently used progestins in hormonal contraception regarding these cellular and molecular brain parameters. Human neuroimaging studies were reviewed with a focus on randomized placebo-controlled trials and cross-sectional studies controlling for progestin type. The prefrontal cortex, amygdala, salience network and hippocampus were identified as regions of interest for future preclinical studies. This review proposes a series of experiments to elucidate the cellular and molecular actions of contraceptive progestins in these areas and link these actions to behavioral markers of emotional and cognitive functioning. Emotional effects of contraceptive progestins appear to be related to 1) alterations in the serotonergic system, 2) direct/indirect modulations of inhibitory GABA-ergic signalling via effects on the allopregnanolone content of the brain, which differ between androgenic and anti-androgenic progestins. Cognitive effects of combined oral contraceptives appear to depend on the ethinylestradiol dose.

Acute stress impairs sensorimotor gating via the neurosteroid allopregnanolone in the prefrontal cortex

Ample evidence indicates that environmental stress impairs information processing, yet the underlying mechanisms remain partially elusive. We showed that, in several rodent models of psychopathology, the neurosteroid allopregnanolone (AP) reduces the prepulse inhibition (PPI) of the startle, a well-validated index of sensorimotor gating. Since this GABAA receptor activator is synthesized in response to acute stress, we hypothesized its participation in stress-induced PPI deficits. Systemic AP administration reduced PPI in C57BL/6J mice and Long-Evans, but not Sprague-Dawley rats. These effects were reversed by isoallopregnanolone (isoAP), an endogenous AP antagonist, and the GABAA receptor antagonist bicuculline and mimicked by AP infusions in the medial prefrontal cortex (mPFC). Building on these findings, we tested AP's implication in the PPI deficits produced by several complementary regimens of acute and short-term stress (footshock, restraint, predator exposure, and sleep deprivation). PPI was reduced by acute footshock, sleep deprivation as well as the combination of restraint and predator exposure in a time- and intensity-dependent fashion. Acute stress increased AP concentrations in the mPFC, and its detrimental effects on PPI were countered by systemic and intra-mPFC administration of isoAP. These results collectively indicate that acute stress impairs PPI by increasing AP content in the mPFC. The confirmation of these mechanisms across distinct animal models and several acute stressors strongly supports the translational value of these findings and warrants future research on the role of AP in information processing.

The Pharmacokinetic Study of Progesterone and Allopregnanolone in Patients with Drug-Resistant Epilepsy: A Phase II Study

Background: Progesterone, one of a class of neurosteroids, has been linked to seizure control. However, the pharmacokinetics of progesterone and its active metabolite, allopregnanolone, have never been investigated among patients with drug-resistant epilepsy (DRE). Objective: To investigate the pharmacokinetics of progesterone, 400 mg micronized progesterone per oral every 12 hours for three months, among patients with DRE as an add-on therapy for seizure control. The pharmacokinetic parameters were examined using Phoenix WinNonlin® Software. Results: Twelve patients were recruited. The biochemical measurements of the serum progesterone and allopregnanolone levels after taking the first dose of micronized progesterone were characterized by a time to maximum concentration (Tmax) median of 1.0 and 2.5 hours, a maximum concentration (Cmax) median of 274.97 and 3.81 ng/mL and a minimum concentration (Cmin) median of 56.93 and 1.06 ng/mL, respectively. The median values of the pharmacokinetic parameters of progesterone and allopregnanolone during the steady state were as follows: half-life (t1/2) of 2.4 and 2.1 hours, a Cmax of 964.35 and 8.92 ng/mL and a Cmin of 64.67 and 1.86 ng/mL, respectively. By examining the relationship between progesterone or allopregnanolone concentrations with seizure-controlling ability, the authors were able to identify a responder patient group with 5- to 7-fold higher serum concentrations of progesterone and 2- to 4-fold in allopregnanolone than the non-responders. Conclusion: The present study revealed the pharmacokinetic parameters of progesterone and allopregnanolone. Moreover, the authors could establish higher serum levels of both progesterone and allopregnanolone, which could consequently relate to lowering the seizure frequency among patients with DRE. Keywords: Neurosteroids; Dose regimens; Epilepsy; Pharmacokinetics; Drug-resistant epilepsy

Pregnant women with more seizures have lower allopregnanolone concentrations

Neuroactive steroids have rapid, nongenomic effects on neuronal excitability. The effects in humans are less clear. We compared seizure control and concentrations of neuroactive steroids, known to influence neuroexcitability in animal studies, in pregnant women. Participants were prospectively followed throughout pregnancy with seizure-medication diaries and blood samples, assayed for steroid concentrations with gas chromatography-mass spectrometry. Baseline seizure frequency was calculated for the preconception year, and it was determined if seizure frequency was increased in each trimester. The Wilcoxon rank-sum test was used to compare neuroactive steroid concentrations in between the group with increased frequency to the group without, as calculated for the respective trimester, with the Holm-Bonferroni method to correct for multiple comparisons.Among eighty-three pregnancies included, twenty-eight had increased seizure frequency during at least one trimester (15, 18 and 10, respectively) compared to preconception seizure frequency. Allopregnanolone concentrations were lower in the 3rd trimester (p < 0.001), with a similar trend in the 1st (p = 0.08), for pregnancies with increased compared to those with stable seizure frequency. Other neuroactive steroid concentrations were similar. Our findings suggest that lower allopregnanolone concentrations are associated with increased seizure frequency during pregnancy. Validation of these finding in a larger cohort has potential important clinical applications.

Involvement of neurosteroids in the control of hypothalamic-pituitary-adrenal axis activity in pregnant sheep under basal and stressful conditions

Neurosteroids are synthesized locally in the brain, where they can modify neuronal functionality depending on the physiological state. A high correlation was demonstrated between the increasing activity of the hypothalamic-pituitary-adrenal (HPA) axis and allopregnanolone (AL) concentration in the cerebrospinal fluid in sheep during pregnancy. Therefore, the present study tested the hypothesis that blocking neurosteroid synthesis in the brain of a pregnant sheep would affect HPA axis activity under both basal and stressful conditions. Two groups of sheep in the fourth month of gestation (n = 7 each) were subjected to the following treatments: 1) intracerebroventricular (icv) infusion of vehicle for three days (C) and then icv infusion of finasteride (a total of 100 μg/240 μL/day) for three days (F), one week apart, and 2) icv infusion of vehicle for three days and application of stressful stimuli (isolation and partial movement restriction) on the third day (S), and subsequently icv infusion of finasteride for three days and application of stressful stimuli on the third day (SF), one week apart. On the third days of the experiment, a 4-h push-pull perfusion of the infundibular nucleus/median eminence and blood sampling were performed. Mean perfusate corticotropin-releasing hormone (CRH), plasma adrenocorticotropin (ACTH) and cortisol concentrations were significantly higher in sheep treated with finasteride, stress and finasteride in combination with stress compared to controls. The highest hormone concentrations in Groups F, S and SF, were recorded during the first 60 min; however, significant increases in CRH and ACTH levels were observed in Group SF towards the end of the experiment. It can be concluded that neurosteroids may be an essential component of the mechanism controlling HPA axis activity in pregnant sheep, not only under stress-free conditions, but more importantly, also by inhibiting the neuroendocrine response to stressors.

Development and validation of an LC-MS/MS assay for the quantification of allopregnanolone and its progesterone-derived isomers, precursors, and cortisol/cortisone in pregnancy.

Neuroactive steroids are potent neuromodulators that play a critical role in both maternal and fetal health during pregnancy. These stress-responsive compounds are reportedly low in women with perinatal depression and may be associated with poor pregnancy outcomes in animal models. Chronic stress is a risk factor for adverse birth outcomes. Simultaneous quantification of neuroactive steroids, in combination with stress hormones cortisol/cortisone, provides an opportunity to investigate the synergistic relationship of these analytes within the convenience of one assay. A simple, reliable, and sensitive method for quantifying these endogenous compounds is necessary for further research with the potential to advance clinical diagnostic tools during pregnancy. Analytes were extracted from serum with a simple protein precipitation using methanol and then separated and quantified using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). After online extraction, analytes were separated using an Agilent Poroschell 120, 50 × 4.6mm, 2.7μm particle size, EC-C18 analytical column. The reliable quantification range was from 0.78 to 1000ng/mL. QC sample inter- and intraday trueness was between 90 and 110% while inter- and intraday imprecision was less than 10%. Extracted samples were stable up to 7days at 4°C and extraction recovery was above 95%. Serum samples from 54 women in pregnancy were analyzed using this method. Here, we provide a validated, fast, and specific assay with sufficient sensitivity that allows for simultaneous quantification of blood serum concentrations of allopregnanolone (3α-hydroxy-5α-pregnan-20-one), pregnanolone (3α-hydroxy-5β-pregnan-20-one), epipregnanolone (3β-hydroxy-5β-pregnan-20-one), pregnenolone, progesterone, cortisol, and cortisone in pregnancy for clinical study samples and clinical diagnostics.

A Potential Mechanism Underlying the Therapeutic Effects of Progesterone and Allopregnanolone on Ketamine-Induced Cognitive Deficits.

Ketamine exposure can model cognitive deficits associated with schizophrenia. Progesterone (PROG) and its active metabolite allopregnanolone (ALLO) have neuroprotective effects and the pathway involving progesterone receptor membrane component 1 (PGRMC1), epidermal growth factor receptor (EGFR), glucagon-like peptide-1 receptor (GLP-1R), phosphatidylinositol 3 kinase (PI3K), and protein kinase B (Akt) appears to play a key role in their neuroprotection. The present study aimed to investigate the effects of PROG (8,16 mg kg−1) and ALLO (8,16 mg kg−1) on the reversal of cognitive deficits induced by ketamine (30 mg kg−1) via the PGRMC1 pathway in rat brains, including hippocampus and prefrontal cortex (PFC). Cognitive performance was evaluated by Morris water maze (MWM) test. Western blot and real-time quantitative polymerase chain reaction were utilized to assess the expression changes of protein and mRNA. Additionally, concentrations of PROG and ALLO in plasma, hippocampus and PFC were measured by a liquid chromatography-tandem mass spectrometry method. We demonstrated that PROG or ALLO could reverse the impaired spatial learning and memory abilities induced by ketamine, accompanied with the upregulation of PGRMC1/EGFR/GLP-1R/PI3K/Akt pathway. Additionally, the coadministration of AG205 abolished their neuroprotective effects and induced cognitive deficits similar with ketamine. More importantly, PROG concentrations were markedly elevated in PROG-treated groups in hippocampus, PFC and plasma, so as for ALLO concentrations in ALLO-treated groups. Interestingly, ALLO (16 mg kg−1) significantly increased the levels of PROG. These findings suggest that PROG can exert its neuroprotective effects via activating the PGRMC1/EGFR/GLP-1R/PI3K/Akt pathway in the brain, whereas ALLO also restores cognitive deficits partially via increasing the level of PROG in the brain to activate the PGRMC1 pathway.

Progesterone's Effects on Cognitive Performance of Male Mice Are Independent of Progestin Receptors but Relate to Increases in GABAA Activity in the Hippocampus and Cortex.

Progestogens' (e.g., progesterone and its neuroactive metabolite, allopregnanolone), cognitive effects and mechanisms among males are not well-understood. We hypothesized if progestogen's effects on cognitive performance are through its metabolite allopregnanolone, and not actions via binding to traditional progestin receptors (PRs), then progesterone administration would enhance performance in tasks mediated by the hippocampus and cortex, coincident with increasing allopregnanolone concentrations, brain derived neurotrophic factor (BDNF) and/or muscimol binding of PR knock out (PRKO) and wild-type PR replete mice. Experiment 1: Progesterone (4 mg/kg, subcutaneously (SC; n = 12/grp), or oil vehicle control, was administered to gonadally-intact adult male mice PRKO mice and their wild-type counterparts and cognitive behaviors in object recognition, T-maze and water maze was examined. Progesterone, compared to vehicle, when administered post-training increased time investigating novel objects by the PRKO and wild-type mice in the object recognition task. In the T-maze task, progesterone administration to wild-type and PRKO mice had significantly greater number of spontaneous alternations compared to their vehicle-administered counterparts. In the water maze task, PRKO mice administered vehicle spent significantly fewer seconds in the quadrant associated with the escape platform on testing compared to all other groups. Experiment 2: Progesterone administered to wild-type and PRKO mice increased plasma progesterone and allopregnanolone levels (n = 5/group). PRKO mice had higher allopregnanolone levels in plasma and hippocampus, but not cortex, when administered progesterone and compared to wild-type mice. Experiment 3: Assessment of PR binding revealed progesterone administered wild-type mice had significantly greater levels of PRs in the hippocampus and cortex, compared to all other groups (n = 5/group). Wild-type mice administered progesterone, but not vehicle, had increased BDNF levels in the hippocampus, but not the cortex, compared to PRKOs. Wild-type as well as PRKO mice administered progesterone experienced significant increases in maximal GABAA agonist, muscimol, binding in hippocampus and cortex, compared to their vehicle-administered counterparts. Thus, adult male mice can be responsive to progesterone for cognitive performance, and such effects may be independent of PRs trophic actions of BDNF levels in the hippocampus and/or increases in GABAA activity in the hippocampus and cortex.

Association of serum allopregnanolone with restricted and repetitive behaviors in adult males with autism

Autism spectrum disorder (ASD) has been associated with imbalance between excitatory and inhibitory (E/I) neurotransmission systems, as well as with neuroinflammation. Sitting at the crossroads between E/I imbalance and neuroinflammation is a class of endogenous hormones known as neurosteroids. Current literature points to dysregulated steroid metabolism and atypical neurosteroid levels in ASD as early as in utero. However, due to the complexity of neurosteroid metabolomics, including possible sex differences, the impact of neurosteroids on ASD symptomatology remains unclear. In this study, we assessed neurosteroid levels and ASD symptom severity of 21 males with ASD and 20 full-scale-IQ-matched typically developing (TD) males, all aged 18–39. Using liquid chromatography-tandem mass spectrometry, concentrations of allopregnanolone, cortisol, dehydroepiandrosterone, progesterone, and testosterone were measured in saliva and serum. With the exception of cortisol’s, all neurosteroids’ concentrations were found to have ASD vs. TD group differences in distribution, where one group was normally distributed and the other non-normally distributed. Serum allopregnanolone levels in males with ASD were found to negatively correlate with clinician-rated measures of restricted and repetitive behavior measures (ADOS-2 RRB and ADI-R RRSB domain scores). Additionally, lower serum allopregnanolone levels were found to predict more negative camouflaging scores, which represent greater differences in self- and clinician-rated symptom severity, of both ASD symptomatology overall and repetitive behaviors in particular. Taken together, our findings demonstrate that in adult males with ASD, decreased serum allopregnanolone levels are associated with more severe restricted and repetitive behaviors and with less insight into the severity of these behaviors.