Sleep deprivation during pregnancy and its consequence on pregnancy outcome and maternal cognition; any role for genistein?

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Funmileyi Olubajo Awobajo, Tolulope Johnson Ajanaku, Simbiat Akinremi, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8052489/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Sleep deprivation in pregnancy is a recognized risk factor for adverse maternal and fetal outcomes, yet the mechanisms and potential interventions remain unclear. This study evaluated genistein, a phytoestrogen with neuroprotective actions, for its role in modulating the effects of gestational sleep deprivation in rats. Thirty-two pregnant Sprague-Dawley rats were divided into four groups: non-sleep deprived controls, sleep deprived (REM deprivation, GD6–18), genistein-treated non-sleep deprived, and genistein-treated sleep deprived (2 mg/kg oral, GD1–18). Outcomes included maternal weight gain, implantation count, embryo resorption, fetal and placental weights, and performance in labyrinth maze and inhibitory avoidance tests for cognition. Sleep deprivation significantly reduced maternal weight gain, fetal and placental weights, and increased embryo resorption and post-implantation loss. Genistein partially improved cognitive outcomes and increased fetal and placental weights, but decreased the number of successful implantations and raised post-implantation losses in both normal and sleep-deprived contexts. Combined genistein and sleep deprivation did not fully reverse physiological impairments and was associated with elevated maternal corticosterone. In conclusion, genistein provided modest neurocognitive protection against sleep deprivation–induced deficits but also posed additional reproductive risks, failing to restore all adverse outcomes. These findings highlight that the effects of phytoestrogen supplementation during pregnancy depend critically on maternal environment, dosage, and timing, and underscore the need for caution in recommending such interventions sleep deprivation pregnancy genistein cognition embryo resorption placental function phytoestrogen rat model Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Pregnancy requires remarkably precise adaptations in maternal hormonal, metabolic, and immune systems to support fetal development and successful gestation ( 1 , 2 ). Maternal sleep loss is now recognized as a significant, prevalent stressor worldwide and is consistently associated with adverse outcomes in both human and animal studies ( 3 – 6 ). Large meta-analyses and cohort studies confirm that insufficient or poor-quality sleep during pregnancy increases the risk for reduced maternal weight gain, metabolic dysregulation, hypertension, fetal growth restriction, preterm birth, and impaired long-term offspring health ( 3 , 4 , 7 – 10 ). Rodent models have shown that maternal sleep restriction disrupts circadian rhythm and stress hormone regulation, leading to placental inflammation, increased embryo resorption, and pregnancy loss ( 2 , 5 , 11 , 12 ). Key mediators of these effects include heightened activity of the maternal hypothalamo-pituitary-adrenal (HPA) axis and excessive glucocorticoid production, which in turn impair placental function and program adverse fetal outcomes during development ( 12 , 13 ). Placental inflammation and neuroendocrine disturbance can ultimately disrupt fundamental neurodevelopmental processes, including hippocampal neurogenesis and long-term cognitive outcomes in the offspring ( 14 – 16 ). Beyond environmental stress, nutritional interventions—particularly the use of phytoestrogens such as genistein, the major isoflavone in soy—have attracted attention for their potential to mitigate gestational stress-induced harm ( 17 , 18 ). Genistein acts as an estrogen receptor modulator, and research in animal models consistently shows anti-inflammatory, antioxidant, and neuroprotective effects, including evidence of improved memory and synaptic plasticity under conditions of developmental challenge or neurodegeneration ( 19 – 23 ). Despite these promising effects, reproductive toxicology studies and reviews also highlight that prenatal genistein exposure can reduce embryo implantation, increase resorption, disrupt placental signaling, and adversely affect fetal growth ( 24 – 27 ). The outcomes of genistein supplementation are thus markedly context-dependent: the effects are critically shaped by dose, timing during development, placental environment, and the degree of concurrent maternal stress or adversity ( 25 – 27 ). Given these dual and sometimes opposing effects, few studies have directly addressed whether genistein supplementation can rescue or exacerbate the consequences of gestational sleep deprivation. The present study examines the independent and combined effects of maternal sleep deprivation and genistein on weight gain, implantation outcomes, placental and fetal growth, and maternal cognition using robust rat models and validated behavioral assays. This work aims to define the mechanistic balance between neuroprotection and reproductive risk in the context of prenatal adversity. Materials and Methods Animals used and grouping Thirty-two female Sprague-Dawley rats (190–200 g) and seven proven breeder males were obtained from the College of Medicine, University of Lagos. Females were housed in standardized polypropylene cages (143 square inches, 7-inch height) with wood shavings as bedding, maintained at controlled room temperature (22 ± 2°C) and humidity (60 ± 5%), under a 12-hour light/dark cycle (lights off at 5:00 pm) to stabilize circadian rhythms. Following one week of acclimation, females were housed with two males (2:1) during the proestrus phase and monitored daily for vaginal cytology indicative of estrus. Pregnancy was confirmed by detection of sperm in vaginal smears and persistent diestrus over two consecutive days, with day of sperm detection designated as gestational day (GD) 0 (28). Pregnant rats were randomly assigned to one of four experimental groups (n=8 per group): NSLD (non-sleep deprived control); SLD (sleep deprived); G+NSLD (genistein, non-sleep deprived); and G+SLD (genistein, sleep deprived). Throughout gestation, animals had ad libitum access to phytoestrogen-free pelleted rat chow and filtered water. Sleep deprivation Sleep deprivation used a validated multiple platform water tank to selectively disrupt REM sleep for 9 hours daily on GD6–GD18. Platforms were ~1 cm above water level; muscle atonia during REM led to water contact and awakening (29,30). Behavioral testing was performed during light hours. Administration of genistein Genistein (98.2%, Chengdu Biopurify) was administered at 2 mg/kg by oral gavage to G+NSLD and G+SLD groups from GD1–GD18, based on efficacy and safety seen in rodent neurodevelopmental and reproductive studies (31–33). Controls received vehicle only. Classical Labyrinth Maze Test for Spatial Learning On gestational day 21 (GD21), spatial learning and working memory were evaluated using a classical labyrinth maze following established protocols (34). The maze was constructed from opaque acrylic sheets (height: 45 cm) and comprised multiple interconnected arms and dead-ends leading to a single exit. Training was conducted once daily for four consecutive days (GD17–GD20), during which animals were familiarized with the maze layout and response strategy. Freshly baked rat chow cubes saturated with a distinctive odor cue were placed at the maze exit to encourage rapid task acquisition, but were withheld during testing to exclude olfactory guidance (34). On the test day, each rat was placed at the maze entry point in a well-lit, isolated room with minimal distractions. The maximum trial duration was 20 minutes (10 minutes initially, extended once if necessary); failure to locate the exit within this period resulted in removal, a three-hour rest, and one retest. The maze was sanitized with 70% ethanol between trials to eliminate residual scent cues. Performance variables included total escape latency, path efficiency, and behavioral strategy (direct, random, or perseverative). All task observations were made live and recorded for subsequent behavioral scoring as needed. Inhibitory Avoidance Test for Learning and Memory Assessment The inhibitory avoidance method was employed to assess contextual associative learning and long-term memory in rats, as previously described (35). The test was conducted in a dedicated behavioral suite with low illumination and minimal noise during the light phase to minimize environmental confounds. The apparatus consisted of two equal-sized adjoining chambers: one brightly lit and non-electrified, and the other dark and equipped with an electrifiable grid floor (35). During two habituation sessions (GD14–GD15), animals were allowed to explore both chambers freely for five minutes without shock. On GD16 (training day), each animal was placed in the illuminated chamber and allowed to enter the dark compartment. Upon all four paws entering the dark chamber, a mild foot-shock (0.35 mA, 62 Hz, three seconds) was automatically delivered via the grid floor, upon which the animal typically escaped back to the illuminated chamber. This session established an aversive association between the dark environment and the shock stimulus. Memory retention was tested 48 hours later (GD18) by placing the animal in the lit compartment and recording the latency to re-enter the dark electrified chamber. The number of entries was also observed during a five-minute trial. Higher latency and fewer entries were interpreted as better memory retention (35). Animals in sleep deprivation groups resumed their deprivation schedule following training. Sacrifice and tissue sample collection and processing Half the animals in each group were sacrificed on gestation day 21. Blood, brain, pituitary, fetuses, resorbed embryos, and placentas were harvested. Tissue collection was performed aseptically and on ice. Blood was processed for serum and stored at -4°C until ELISA analysis. Tissues were fixed for histological analysis. Serum corticosterone and dopamine analysis Serum corticosterone and dopamine levels were analyzed by ELISA assay technique according to the manufacturer instructions. The corticosterone and dopamine ELISA assay kits were purchased from Elabscience Biotechnology Inc., China. Briefly, all reagents were taken to room temperature prior to use while optimal results for intra and inter assay reproducibility was obtained when incubating samples at various steps were at 37 o C. While one well was used as the blank with no sample, 50 µL of serially titrated standards, diluted samples or blank were added to the wells containing anti-corticosterone or anti-dopamine microplates. All samples were prepared in duplicates. 50 µL of 1x corticosterone-biotin or dopamine-biotin complex were added to each corresponding well excluding only the blank well. The plates were covered with the plate sealers and incubated at 37 o C for 60 minutes as required by the manufacturer. The liquids in the wells were thereafter discarded by rigorously flicking them into a receptacle. Any remaining liquid in the wells was removed by gently tapping inverted on the bench top unto a clean paper towel provided in the assay kit while avoiding completely drying the plates. The plates were thereafter washed three times with the wash buffer provided and drained as earlier explained. The plates were covered with the sealer and incubated again at 30 o C for 25 minutes. A stop solution was added to the wells and the optical density read at an absorbance of 450 nm with a standard ELISA microplate reader. The corresponding concentration was derived from the standard curve provided. Statistical analysis All data were analyzed with one-way analysis of variance (ANOVA) with GraphPad Prism8. Results were presented as mean ± standard error of mean (SEM) and the differences between means were accepted as significant at p<0.05 . Bar chats and line graph were used for graphical presentation. RESULTS Maternal body weight under sleep deprivation and genistein administration (Figure 1) The SLD group gained weight towards the end of the second week while they lost significant weight at first and third week of gestation. The NSLD group gained their highest weight towards the end of first and second week and towards the 19 th day of pregnancy. The G+NSLD group gained maximum weight towards the end of third week while they recorded a weight loss toward the fourth week of gestation. The G+SLD gained maximum weight towards the end of the third week and towards the end of gestation but lost weight shortly towards the 19 th day of pregnancy. Between GD-19 and GD-6, the NSLD gained the highest weight compared to the rest of the group. The weight gained in the SLD, G+NSLD and the G+SLD were all significantly lowered that of the NSLD group. In addition, the weight gained between GD-6 and GD-19 in all genistein treated groups were significantly lowered compared to that in SLD group. Number of embryonic implant, resorbed embryo and post implantation loss (Figure 2) There was a significant decrease in the number of growing fetus at GD-19 in the G+SLD compared to NLSD, while a significant increase in the number was recorded in SLD group compared with NSLD group. Also a significant increase in the number of resorbed embryo was recorded in the SLD, G+NSLD and G+SLD groups compared with that obtained in the NSLD group. Post implantation loss was significantly reduced in all the groups compare with the NSLD group. The post implantation loss was also significantly higher in all genistein treated groups (sleep deprived and non-sleep deprived) compared with the NSD group. Fetal and placenta weight (Figure 3) While a significant reduction in the relative fetal weight was recorded in the SLD, a significant increase was recorded in all the 2 mg genistein treated groups (both sleep deprived and non-sleep deprived compared with the NSLD at GD-19. The relative placenta weight recorded similar pattern with a significant decrease in the SLD and a significant increase in both G+SLD and G+NSLD groups compared with that of NSLD group at GD-19. Litter weight during gestation and litter birth weight at delivery (Figure 4) There was a significant increase in the fetal weight in all the groups compared with the NSLD group at GD-8. A significant increase was also recorded in the fetal weight at SLD at GD-18 compared with the NSD group. The fetal weight in all genistein treated groups were significantly reduced compared with NSLD at GD-18. A significant increase was recorded in the fetal weight in all groups compared with the NSLD group at GD-21. There was also a significant decrease in fetal weight in 2 mg genistein treated groups compare with the SLD group at GD-2. A significant decrease in litter size was recorded in the SLD Maternal brain and pituitary weight (Figure 5) There was a significant increase in the maternal relative brain weight in the SLD compare with the NSLD. There was also a significant decrease recorded in the maternal relative brain weight in all 2 mg genistein treated groups compared with the NSLD group. A significant increase was recorded in the relative pituitary weight in G+SLD group compared to the NSLD and the SLD groups at GD-19. The cognitive and memory test (Figure 6) There was a significant decrease in the time taken to locate the exit in the maze labyrinth test in all groups compared with the NSLD group. specifically, The NSLD group latent time in locating the exit was significantly lower than the G+NSLD and SLD latent time also was lower than that of G+SLD, however it was not significant. The result of the inhibition avoidance test showed a significant decrease in the latent time taken to return to the shock compartment in SLD and G+SLD group compare with the NSLD group. Serum corticosterone and Plevel (Figure 7) A significant increase in serum corticosterone level was recorded in all the groups compared with the NSLD group. At GD-19. A significant decrease in serum corticosterone level was recorded in the G+SLD and G+NSLD groups compared with the SLD group at GD-19. The dopamine recorded no significant difference across all the groups. Discussion This study confirms that not getting enough sleep during pregnancy harms maternal health and reproductive success ( 3 , 5 , 11 , 12 ). Maternal sleep deprivation resulted in reduced maternal weight gain and increased fetal/implantation loss, matching meta-analyses and animal models showing that poor sleep disrupts gestational metabolism and fetal viability ( 3 , 4 , 7 – 10 ). Genistein supplementation in this context led to even less maternal weight gain and amplified reproductive loss, consistent with reports that phytoestrogens can interfere with normal gestational adaptation and placental function ( 24 – 27 ). Mechanistically, both sleep deprivation and genistein produced marked reductions in fetal and placental weights, suggesting impaired placental nutrient transfer—the same effect seen in prior rodent and clinical research ( 12 , 13 , 14 – 16 ). Cognitive outcomes were nuanced: genistein-treated mothers showed improved performance in maze tasks regardless of sleep deprivation, in line with preclinical evidence that phytoestrogens support memory and neuroplasticity under challenge ( 19 – 23 ). However, avoidance memory was only fully preserved in genistein-treated, non-sleep-deprived mothers, while combined stressors blunted cognitive resilience. An important mechanistic finding is that, while genistein alone aggravated reproductive loss, it partially reduced embryo resorption rates in the face of severe maternal stress, highlighting the context-dependence of genistein’s effects ( 25 – 27 ). This is consistent with systematic reviews indicating that genistein outcomes depend on dose, timing, the placental environment, and concurrent stress exposure ( 25 – 27 ). Corticosterone assays confirmed that stress and genistein both increased HPA axis activity, paralleling hormonal findings in stressed pregnancies across animal and human literature ( 12 , 13 ). Conclusion In summary, both maternal sleep deprivation and genistein supplementation independently impaired maternal health and reproductive outcomes in pregnant rats, with additive or context-dependent effects observed when both stressors were combined. Maternal sleep loss consistently reduced weight gain and gestational success, while genistein further exacerbated fetal loss and placental insufficiency under standard conditions, but showed a modest protective effect against resorption in the context of severe stress. Although genistein improved some aspects of maternal cognitive function, it did not fully offset the deleterious effects of sleep deprivation. These findings underscore the risks of both gestational sleep loss and indiscriminate phytoestrogen use in pregnancy, emphasizing that the impact of nutritional supplements is highly dependent on dose, timing, and maternal environment. Careful consideration is warranted before recommending genistein or similar agents for neuroprotection in pregnancy, and further research is needed to determine safe contexts and mechanisms for their use. Declarations Ethics Declaration All procedures performed in this study were approved by the Animal Care and Use Research Ethics Committee of the College of Medicine, University of Lagos (CMUL/ACUREC), approval number: CMUL/ACUREC/07/25/2020. Funding This piece of research does not enjoy any direct funding. However, the University of Lagos provided the laboratory space and facility where the study was conducted. Author Contribution Tolulope J. Ajanaku contributed primarily to the manuscript writing, participated in experimental investigation including behavioral assessments, estrous cycle monitoring, and data collection. Funmileyi Olubajo Awobajo conceived the experimental design, conducted most statistical analyses, contributed to manuscript drafting, and provided technical oversight. Simbiat Akinremi participated in experimental investigation, including hands-on animal experimentation, sample collection, and contributed to sections of the manuscript. 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Learning and memory: basic mechanisms, animal models, and memory disorders. Brain Res Brain Res Rev. 2002;39(2-3):109-129. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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07:52:44","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":17291,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8052489/v1/ef63622a3858725f2669fbe4.png"},{"id":96444836,"identity":"8e51b2b5-20c4-40a0-b440-f0f161d854fc","added_by":"auto","created_at":"2025-11-21 07:52:44","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":17516,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8052489/v1/395c57e16ff71bd4c22b4439.png"},{"id":96444839,"identity":"876d7eb7-31b2-4701-8049-3f0504288a49","added_by":"auto","created_at":"2025-11-21 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10:04:12","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":17357,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8052489/v1/d285d8037e2f2d3ec117ba16.png"},{"id":96455374,"identity":"e58adb81-cd2b-4685-8649-3e80758be3df","added_by":"auto","created_at":"2025-11-21 10:04:01","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":45018,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8052489/v1/6208aaa52b57ce3aa82f5470.png"},{"id":96455546,"identity":"efed912f-7cf0-4bae-a961-76be28b5f950","added_by":"auto","created_at":"2025-11-21 10:04:19","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":17746,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8052489/v1/8ada8af03fa17d6f055bd6bf.png"},{"id":96444850,"identity":"92a2b915-eed8-4fc9-9304-177ad346b89f","added_by":"auto","created_at":"2025-11-21 07:52:45","extension":"xml","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":69411,"visible":true,"origin":"","legend":"","description":"","filename":"04652538e8a94f78983758350ca8cdcb1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8052489/v1/571b9a38e7652ae4c232988a.xml"},{"id":96444849,"identity":"1539925c-8d35-4ed2-a58f-e9bda89bd2c2","added_by":"auto","created_at":"2025-11-21 07:52:44","extension":"html","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":77342,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8052489/v1/3b490c2bea3acc3728a21add.html"},{"id":96444824,"identity":"57761bed-2800-4f5e-9547-a2d279e5d648","added_by":"auto","created_at":"2025-11-21 07:52:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":125461,"visible":true,"origin":"","legend":"\u003cp\u003ePercent body weight gain per week in sleep-deprived pregnant rats treated with genistein (2 mg/kg) compared with non–sleep-deprived (NSLD), sleep-deprived (SLD), genistein-treated non–sleep-deprived (G + NSLD), and genistein-treated sleep-deprived (G + SLD) rats.\u003c/p\u003e\n\u003cp\u003eValues represent mean ± SEM (n = 8 per group); p \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8052489/v1/9d040c5e4765afa17b7601ce.png"},{"id":96455205,"identity":"1933c596-340b-4eb5-af3f-29251fc25791","added_by":"auto","created_at":"2025-11-21 10:03:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":97449,"visible":true,"origin":"","legend":"\u003cp\u003eNumber of implanted embryo, number of resorbed embryo and the post implantation loss on GD-19 in sleep deprived pregnant rats treated with genistein (2 mg/kg) compared with the non sleep deprived and sleep deprived and genistein 2mg/kg treated non sleep deprived rats.\u003c/p\u003e\n\u003cp\u003eResults are presented as mean ± SEM\u003cem\u003e, p\u0026lt;0.05\u003c/em\u003e, n=8. Non sleep deprived (NSLD), sleep deprived (SLD), 2 mg genistein treated non sleep deprived (G+NSLD), 2 mg genistein treated sleep deprived (G+SLD)\u003c/p\u003e\n\u003cp\u003e* = significant ¢ to NSLD; † = significant ¢ to SLD\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8052489/v1/9854e9bb8337a6b62fb4478d.png"},{"id":96444825,"identity":"4701d342-31bb-495e-b7e5-9eeb2a6d66dc","added_by":"auto","created_at":"2025-11-21 07:52:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":79874,"visible":true,"origin":"","legend":"\u003cp\u003eRelative GD-19 fetal and placenta weights from pregnant rats dosed with genistein and deprived of sleep compared with the non sleep deprived and sleep deprived\u003c/p\u003e\n\u003cp\u003eResults are presented as mean ± SEM\u003cem\u003e, p\u0026lt;0.05\u003c/em\u003e, n=8. Non sleep deprived (NSLD), sleep deprived (SLD), 2 mg genistein treated non sleep deprived (G+NSLD), 2 mg genistein treated sleep deprived (G+SLD)\u003c/p\u003e\n\u003cp\u003e* = significant ¢ to NSLD; † = significant ¢ to SLD\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8052489/v1/740155eaef793d9ea7ead197.png"},{"id":96455299,"identity":"6c0b9017-773b-42a6-b8c8-98eeb9493506","added_by":"auto","created_at":"2025-11-21 10:03:54","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":84972,"visible":true,"origin":"","legend":"\u003cp\u003eLitter size at birth and the litter weights from pregnant rats dosed with genistein and deprived of sleep compared with the non sleep deprived and sleep deprived\u003c/p\u003e\n\u003cp\u003eResults are presented as mean ± SEM\u003cem\u003e, p\u0026lt;0.05\u003c/em\u003e, n=8. Non sleep deprived (NSLD), sleep deprived (SLD), 2 mg genistein treated non sleep deprived (G+NSLD), 2 mg genistein treated sleep deprived (G+SLD)\u003c/p\u003e\n\u003cp\u003e* = significant ¢ to NSLD; † = significant ¢ to SLD\u003c/p\u003e\n\u003cp\u003ewhile a significant increase was recorded in the litter size at birth in all the genestein treated groups.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8052489/v1/51fa19daf51d38d9639bf0b1.png"},{"id":96444829,"identity":"6b2be18c-01e4-4f1f-a328-37322150b8df","added_by":"auto","created_at":"2025-11-21 07:52:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":38518,"visible":true,"origin":"","legend":"\u003cp\u003eBrain and pituitary weights on GD-19 in sleep deprived pregnant rats treated with genistein (2mg/kg) compared with the non sleep deprived and sleep deprived only\u003c/p\u003e\n\u003cp\u003eResults are presented as mean ± SEM\u003cem\u003e, p\u0026lt;0.05\u003c/em\u003e, n=8. Non sleep deprived (NSLD), sleep deprived (SLD), 2 mg genistein treated non sleep deprived (G+NSLD), 2 mg genistein treated sleep deprived (G+SLD)\u003c/p\u003e\n\u003cp\u003e* = significant ¢ to NSLD; † = significant ¢ to SLD\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8052489/v1/a21ee75e11869df119a5d543.png"},{"id":96444832,"identity":"10bd3c3a-361e-428e-ad9b-3ae51dafaed9","added_by":"auto","created_at":"2025-11-21 07:52:44","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":35739,"visible":true,"origin":"","legend":"\u003cp\u003eClassic Maze test result and Passive avoidance or Inhibitory avoidance test result on GD 19 from sleep deprived rats treated with genistein (2 mg/kg) compared with pregnant non sleep deprived\u003cem\u003e, \u003c/em\u003ePregnant sleep deprived and genistein treated non sleep deprived rats\u003c/p\u003e\n\u003cp\u003eResults are presented as mean ± SEM\u003cem\u003e, p\u0026lt;0.05\u003c/em\u003e, n=8. Non sleep deprived (NSLD), sleep deprived (SLD), 2 mg genistein treated non sleep deprived (G+NSLD), 2 mg genistein treated sleep deprived (G+SLD)\u003c/p\u003e\n\u003cp\u003e* = significant ¢ to NSLD; † = significant ¢ to SLD\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8052489/v1/fcb495e96cc4fe03e5d3b025.png"},{"id":96444834,"identity":"053f91df-38eb-4f82-8204-743d671c5a58","added_by":"auto","created_at":"2025-11-21 07:52:44","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":50273,"visible":true,"origin":"","legend":"\u003cp\u003eSerum corticosterone and dopamine level on GD-19 in pregnant rats dosed with genistein and deprived of sleep compared with the non sleep deprived and sleep deprived only\u003c/p\u003e\n\u003cp\u003eResults are presented as mean ± SEM\u003cem\u003e, p\u0026lt;0.05\u003c/em\u003e, n=8. Non sleep deprived (NSLD), sleep deprived (SLD), 2 mg genistein treated non sleep deprived (G+NSLD), 2 mg genistein treated sleep deprived (G+SLD)\u003c/p\u003e\n\u003cp\u003e* = significant ¢ to NSLD; † = significant ¢ to SLD\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8052489/v1/7ba6be7867b22043ec52280b.png"},{"id":99791021,"identity":"7a528c26-822c-49ca-90ab-89586d1cf2d0","added_by":"auto","created_at":"2026-01-08 12:58:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1136366,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8052489/v1/d6f23aa4-ceac-4749-a545-8e4e4b78a550.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Sleep deprivation during pregnancy and its consequence on pregnancy outcome and maternal cognition; any role for genistein?","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePregnancy requires remarkably precise adaptations in maternal hormonal, metabolic, and immune systems to support fetal development and successful gestation (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Maternal sleep loss is now recognized as a significant, prevalent stressor worldwide and is consistently associated with adverse outcomes in both human and animal studies (\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Large meta-analyses and cohort studies confirm that insufficient or poor-quality sleep during pregnancy increases the risk for reduced maternal weight gain, metabolic dysregulation, hypertension, fetal growth restriction, preterm birth, and impaired long-term offspring health (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Rodent models have shown that maternal sleep restriction disrupts circadian rhythm and stress hormone regulation, leading to placental inflammation, increased embryo resorption, and pregnancy loss (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eKey mediators of these effects include heightened activity of the maternal hypothalamo-pituitary-adrenal (HPA) axis and excessive glucocorticoid production, which in turn impair placental function and program adverse fetal outcomes during development (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Placental inflammation and neuroendocrine disturbance can ultimately disrupt fundamental neurodevelopmental processes, including hippocampal neurogenesis and long-term cognitive outcomes in the offspring (\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eBeyond environmental stress, nutritional interventions\u0026mdash;particularly the use of phytoestrogens such as genistein, the major isoflavone in soy\u0026mdash;have attracted attention for their potential to mitigate gestational stress-induced harm (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Genistein acts as an estrogen receptor modulator, and research in animal models consistently shows anti-inflammatory, antioxidant, and neuroprotective effects, including evidence of improved memory and synaptic plasticity under conditions of developmental challenge or neurodegeneration (\u003cspan additionalcitationids=\"CR20 CR21 CR22\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eDespite these promising effects, reproductive toxicology studies and reviews also highlight that prenatal genistein exposure can reduce embryo implantation, increase resorption, disrupt placental signaling, and adversely affect fetal growth (\u003cspan additionalcitationids=\"CR25 CR26\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). The outcomes of genistein supplementation are thus markedly context-dependent: the effects are critically shaped by dose, timing during development, placental environment, and the degree of concurrent maternal stress or adversity (\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eGiven these dual and sometimes opposing effects, few studies have directly addressed whether genistein supplementation can rescue or exacerbate the consequences of gestational sleep deprivation. The present study examines the independent and combined effects of maternal sleep deprivation and genistein on weight gain, implantation outcomes, placental and fetal growth, and maternal cognition using robust rat models and validated behavioral assays. This work aims to define the mechanistic balance between neuroprotection and reproductive risk in the context of prenatal adversity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAnimals used and grouping\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThirty-two female Sprague-Dawley rats (190\u0026ndash;200 g) and seven proven breeder males were obtained from the College of Medicine, University of Lagos. Females were housed in standardized polypropylene cages (143 square inches, 7-inch height) with wood shavings as bedding, maintained at controlled room temperature (22 \u0026plusmn; 2\u0026deg;C) and humidity (60 \u0026plusmn; 5%), under a 12-hour light/dark cycle (lights off at 5:00 pm) to stabilize circadian rhythms. Following one week of acclimation, females were housed with two males (2:1) during the proestrus phase and monitored daily for vaginal cytology indicative of estrus. Pregnancy was confirmed by detection of sperm in vaginal smears and persistent diestrus over two consecutive days, with day of sperm detection designated as gestational day (GD) 0 (28).\u003c/p\u003e\n\u003cp\u003ePregnant rats were randomly assigned to one of four experimental groups (n=8 per group): NSLD (non-sleep deprived control); SLD (sleep deprived); G+NSLD (genistein, non-sleep deprived); and G+SLD (genistein, sleep deprived). Throughout gestation, animals had ad libitum access to phytoestrogen-free pelleted rat chow and filtered water.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSleep deprivation\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSleep deprivation used a validated multiple platform water tank to selectively disrupt REM sleep for 9 hours daily on GD6\u0026ndash;GD18. Platforms were ~1 cm above water level; muscle atonia during REM led to water contact and awakening (29,30). Behavioral testing was performed during light hours.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdministration of genistein\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenistein (98.2%, Chengdu Biopurify) was administered at 2 mg/kg by oral gavage to G+NSLD and G+SLD groups from GD1\u0026ndash;GD18, based on efficacy and safety seen in rodent neurodevelopmental and reproductive studies (31\u0026ndash;33). Controls received vehicle only.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClassical Labyrinth Maze Test for Spatial Learning\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOn gestational day 21 (GD21), spatial learning and working memory were evaluated using a classical labyrinth maze following established protocols (34). The maze was constructed from opaque acrylic sheets (height: 45 cm) and comprised multiple interconnected arms and dead-ends leading to a single exit. Training was conducted once daily for four consecutive days (GD17\u0026ndash;GD20), during which animals were familiarized with the maze layout and response strategy. Freshly baked rat chow cubes saturated with a distinctive odor cue were placed at the maze exit to encourage rapid task acquisition, but were withheld during testing to exclude olfactory guidance (34).\u003c/p\u003e\n\u003cp\u003eOn the test day, each rat was placed at the maze entry point in a well-lit, isolated room with minimal distractions. The maximum trial duration was 20 minutes (10 minutes initially, extended once if necessary); failure to locate the exit within this period resulted in removal, a three-hour rest, and one retest. The maze was sanitized with 70% ethanol between trials to eliminate residual scent cues. Performance variables included total escape latency, path efficiency, and behavioral strategy (direct, random, or perseverative). All task observations were made live and recorded for subsequent behavioral scoring as needed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInhibitory Avoidance Test for Learning and Memory Assessment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe inhibitory avoidance method was employed to assess contextual associative learning and long-term memory in rats, as previously described (35). The test was conducted in a dedicated behavioral suite with low illumination and minimal noise during the light phase to minimize environmental confounds. The apparatus consisted of two equal-sized adjoining chambers: one brightly lit and non-electrified, and the other dark and equipped with an electrifiable grid floor (35). During two habituation sessions (GD14\u0026ndash;GD15), animals were allowed to explore both chambers freely for five minutes without shock.\u003c/p\u003e\n\u003cp\u003eOn GD16 (training day), each animal was placed in the illuminated chamber and allowed to enter the dark compartment. Upon all four paws entering the dark chamber, a mild foot-shock (0.35 mA, 62 Hz, three seconds) was automatically delivered via the grid floor, upon which the animal typically escaped back to the illuminated chamber. This session established an aversive association between the dark environment and the shock stimulus. Memory retention was tested 48 hours later (GD18) by placing the animal in the lit compartment and recording the latency to re-enter the dark electrified chamber. The number of entries was also observed during a five-minute trial. Higher latency and fewer entries were interpreted as better memory retention (35). Animals in sleep deprivation groups resumed their deprivation schedule following training.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSacrifice and tissue sample collection and processing\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHalf the animals in each group were sacrificed on gestation day 21. Blood, brain, pituitary, fetuses, resorbed embryos, and placentas were harvested. Tissue collection was performed aseptically and on ice. Blood was processed for serum and stored at -4\u0026deg;C until ELISA analysis. Tissues were fixed for histological analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSerum corticosterone and dopamine analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSerum corticosterone and dopamine levels were analyzed by ELISA assay technique according to the manufacturer instructions. The corticosterone and dopamine ELISA assay kits were purchased from Elabscience Biotechnology Inc., China. Briefly, all reagents were taken to room temperature prior to use while optimal results for intra and inter assay reproducibility was obtained when incubating samples at various steps were at 37 \u003csup\u003eo\u003c/sup\u003eC. While one well was used as the blank with no sample, 50 \u0026micro;L of serially titrated standards, diluted samples or blank were added to the wells containing anti-corticosterone or anti-dopamine microplates. All samples were prepared in duplicates. 50 \u0026micro;L of 1x corticosterone-biotin or dopamine-biotin complex were added to each corresponding well excluding only the blank well. The plates were covered with the plate sealers and incubated at 37 \u003csup\u003eo\u003c/sup\u003eC for 60 minutes as required by the manufacturer. The liquids in the wells were thereafter discarded by rigorously flicking them into a receptacle. Any remaining liquid in the wells was removed by gently tapping inverted on the bench top unto a clean paper towel provided in the assay kit while avoiding completely drying the plates. The plates were thereafter washed three times with the wash buffer provided and drained as earlier explained. The plates were covered with the sealer and incubated again at 30 \u003csup\u003eo\u003c/sup\u003eC for 25 minutes. A stop solution was added to the wells and the optical density read at an absorbance of 450 nm with a standard ELISA microplate reader. The corresponding concentration was derived from the standard curve provided.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStatistical analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data were analyzed with one-way analysis of variance (ANOVA) with GraphPad Prism8. Results were presented as mean \u0026plusmn; standard error of mean (SEM) and the differences between means were accepted as significant at \u003cem\u003ep\u0026lt;0.05\u003c/em\u003e. Bar chats and line graph were used for graphical presentation.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMaternal body weight under sleep deprivation and genistein administration (Figure 1)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe SLD group gained weight towards the end of the second week while they lost significant weight at first and third week of gestation. The NSLD group gained their highest weight towards the end of first and second week and towards the 19\u003csup\u003eth\u003c/sup\u003e day of pregnancy. The G+NSLD group gained maximum weight towards the end of third week while they recorded a weight loss toward the fourth week of gestation. The G+SLD gained maximum weight towards the end of the third week and towards the end of gestation but lost weight shortly towards the 19\u003csup\u003eth\u003c/sup\u003e day of pregnancy. Between GD-19 and GD-6, the NSLD gained the highest weight compared to the rest of the group. The weight gained in the SLD, G+NSLD and the G+SLD were all significantly lowered that of the NSLD group. In addition, the weight gained between GD-6 and GD-19 in all genistein treated groups were significantly lowered compared to that in SLD group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eNumber of embryonic implant, resorbed embryo and post implantation loss (Figure 2)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere was a significant decrease in the number of growing fetus at GD-19 in the G+SLD compared to NLSD, while a significant increase in the number was recorded in SLD group compared with NSLD group. Also a significant increase in the number of resorbed embryo was recorded in the SLD, G+NSLD and G+SLD groups compared with that obtained in the NSLD group. Post implantation loss was significantly reduced in all the groups compare with the NSLD group. The post implantation loss was also significantly higher in all genistein treated groups (sleep deprived and non-sleep deprived) compared with the NSD group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFetal and placenta weight (Figure 3)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhile a significant reduction in the relative fetal weight was recorded in the SLD, a significant increase was recorded in all the 2 mg genistein treated groups (both sleep deprived and non-sleep deprived compared with the NSLD at GD-19. The relative placenta weight recorded similar pattern with a significant decrease in the SLD and a significant increase in both G+SLD and G+NSLD groups compared with that of NSLD group at GD-19.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eLitter weight during gestation and litter birth weight at delivery (Figure 4)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere was a significant increase in the fetal weight in all the groups compared with the NSLD group at GD-8. A significant increase was also recorded in the fetal weight at SLD at GD-18 compared with the NSD group. The fetal weight in all genistein treated groups were significantly reduced compared with NSLD at GD-18. A significant increase was recorded in the fetal weight in all groups compared with the NSLD group at GD-21. There was also a significant decrease in fetal weight in 2 mg genistein treated groups compare with the SLD group at GD-2. A significant decrease in litter size was recorded in the SLD\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMaternal brain and pituitary weight (Figure 5)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere was a significant increase in the maternal relative brain weight in the SLD compare with the NSLD. There was also a significant decrease recorded in the maternal relative brain weight in all 2 mg genistein treated groups compared with the NSLD group. A significant \u0026nbsp; \u0026nbsp;increase was recorded in the relative pituitary weight in G+SLD group compared to the NSLD \u0026nbsp; and the SLD groups at GD-19.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eThe cognitive and memory test (Figure 6)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere was a significant decrease in the time taken to locate the exit in the maze labyrinth test in all groups compared with the NSLD group. specifically, The NSLD group latent time in locating the exit was significantly lower than the G+NSLD and SLD latent time also was lower than that of G+SLD, however it was not significant. The result of the inhibition avoidance test showed a significant decrease in the latent time taken to return to the shock compartment in SLD and G+SLD group compare with the NSLD group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSerum corticosterone and Plevel (Figure 7)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA significant increase in serum corticosterone level was recorded in all the groups compared with the NSLD group. At GD-19. A significant decrease in serum corticosterone level was recorded in the G+SLD and G+NSLD groups compared with the SLD group at GD-19. The dopamine recorded no significant difference across all the groups.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study confirms that not getting enough sleep during pregnancy harms maternal health and reproductive success (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Maternal sleep deprivation resulted in reduced maternal weight gain and increased fetal/implantation loss, matching meta-analyses and animal models showing that poor sleep disrupts gestational metabolism and fetal viability (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Genistein supplementation in this context led to even less maternal weight gain and amplified reproductive loss, consistent with reports that phytoestrogens can interfere with normal gestational adaptation and placental function (\u003cspan additionalcitationids=\"CR25 CR26\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMechanistically, both sleep deprivation and genistein produced marked reductions in fetal and placental weights, suggesting impaired placental nutrient transfer\u0026mdash;the same effect seen in prior rodent and clinical research (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Cognitive outcomes were nuanced: genistein-treated mothers showed improved performance in maze tasks regardless of sleep deprivation, in line with preclinical evidence that phytoestrogens support memory and neuroplasticity under challenge (\u003cspan additionalcitationids=\"CR20 CR21 CR22\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). However, avoidance memory was only fully preserved in genistein-treated, non-sleep-deprived mothers, while combined stressors blunted cognitive resilience.\u003c/p\u003e\u003cp\u003eAn important mechanistic finding is that, while genistein alone aggravated reproductive loss, it partially reduced embryo resorption rates in the face of severe maternal stress, highlighting the context-dependence of genistein\u0026rsquo;s effects (\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). This is consistent with systematic reviews indicating that genistein outcomes depend on dose, timing, the placental environment, and concurrent stress exposure (\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCorticosterone assays confirmed that stress and genistein both increased HPA axis activity, paralleling hormonal findings in stressed pregnancies across animal and human literature (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, both maternal sleep deprivation and genistein supplementation independently impaired maternal health and reproductive outcomes in pregnant rats, with additive or context-dependent effects observed when both stressors were combined. Maternal sleep loss consistently reduced weight gain and gestational success, while genistein further exacerbated fetal loss and placental insufficiency under standard conditions, but showed a modest protective effect against resorption in the context of severe stress. Although genistein improved some aspects of maternal cognitive function, it did not fully offset the deleterious effects of sleep deprivation. These findings underscore the risks of both gestational sleep loss and indiscriminate phytoestrogen use in pregnancy, emphasizing that the impact of nutritional supplements is highly dependent on dose, timing, and maternal environment. Careful consideration is warranted before recommending genistein or similar agents for neuroprotection in pregnancy, and further research is needed to determine safe contexts and mechanisms for their use.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eEthics Declaration\u003c/h2\u003e\u003cp\u003e All procedures performed in this study were approved by the Animal Care and Use Research Ethics Committee of the College of Medicine, University of Lagos (CMUL/ACUREC), approval number: CMUL/ACUREC/07/25/2020.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis piece of research does not enjoy any direct funding. However, the University of Lagos provided the laboratory space and facility where the study was conducted.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eTolulope J. Ajanaku contributed primarily to the manuscript writing, participated in experimental investigation including behavioral assessments, estrous cycle monitoring, and data collection. Funmileyi Olubajo Awobajo conceived the experimental design, conducted most statistical analyses, contributed to manuscript drafting, and provided technical oversight. Simbiat Akinremi participated in experimental investigation, including hands-on animal experimentation, sample collection, and contributed to sections of the manuscript. John Ihayi Ogbu reviewed the manuscript, contributed to interpretation of results, and assisted with statistical analysis. All authors read and approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003edata can be made available from the corresponding author upon reasonable request\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eCermakian N, Gagnidze K. Circadian clocks and pregnancy. Prog Mol Biol Transl Sci. 2013;119:181-202.\u003c/li\u003e\n \u003cli\u003eBaratta AM, Kanyuch NR, Cole CA, et al. Sleep deprivation during pregnancy elevates placental and fetal inflammation. Neurobiol Stress. 2020;12:100215.\u003c/li\u003e\n \u003cli\u003eSedov ID, Cameron EE, Madigan S, Tomfohr-Madsen LM. Sleep quality during pregnancy: a meta-analysis. Sleep Med Rev. 2018;38:168-176.\u003c/li\u003e\n \u003cli\u003eFacco FL, Grobman WA, Kramer J, Ho KH, Zee PC. Sleep disturbances in pregnancy: A longitudinal study. Obstet Gynecol. 2010;115(1):77-83.\u003c/li\u003e\n \u003cli\u003eYu Y, Hu K, Yang H, Wang Y, Yang J. Sleep and adverse pregnancy outcomes: a meta-analysis. BMC Pregnancy Childbirth. 2022;22(1):874.\u003c/li\u003e\n \u003cli\u003eWang H, Li N, Zhu X, et al. Sleep quality and duration during pregnancy and risk for preterm birth: a meta-analysis. Eur J Obstet Gynecol Reprod Biol. 2022;276:35-42.\u003c/li\u003e\n \u003cli\u003eZhang L, Zhang X, Zhang Y, et al. Sleep duration and quality and adverse pregnancy outcomes: a systematic review and meta-analysis. Obstet Gynecol. 2024;144(3):491-507.\u003c/li\u003e\n \u003cli\u003eOkun ML, Buysse DJ, Hall MH. Identifying insomnia in early pregnancy. J Clin Sleep Med. 2015;11(6):645-654.\u003c/li\u003e\n \u003cli\u003eMoisiadis VG, Matthews SG. Glucocorticoids and fetal programming part 2: Mechanisms. Nat Rev Endocrinol. 2014;10(7):403-11.\u003c/li\u003e\n \u003cli\u003eRinaudo P, Wang E. Fetal programming and metabolic syndrome. Annu Rev Physiol. 2012;74:107-130.\u003c/li\u003e\n \u003cli\u003e11.Wang H, et al. Sleep deprivation during pregnancy leads to poor fetal outcomes. Sci Rep. 2023;13:6724.\u003c/li\u003e\n \u003cli\u003e12. Zhao Q, Peng C, Wu X, et al. Maternal sleep deprivation inhibits hippocampal neurogenesis. Neurobiol Dis. 2014;68:57-65.\u003c/li\u003e\n \u003cli\u003eBrown AS, Meyer U. Maternal immune activation and neuropsychiatric development. Am J Psychiatry. 2018;175(4):319-328.\u003c/li\u003e\n \u003cli\u003eElovitz MA, et al. Intrauterine inflammation reduces hippocampal neurogenesis. J Neuroimmunol. 2018;319:112-118.\u003c/li\u003e\n \u003cli\u003eBurd I, et al. Inflammation, neurodevelopment, and brain injury. Pediatr Res. 2019;85(2):147-155.\u003c/li\u003e\n \u003cli\u003eDuffy R, Wiseman H, File SE. Improved cognitive function after isoflavone-rich diet in rodents. Pharmacol Biochem Behav. 2003;75(3):721-729.\u003c/li\u003e\n \u003cli\u003ePatisaul HB. Effects of phytoestrogen exposure on reproductive function. J Steroid Biochem Mol Biol. 2020;202:105721.\u003c/li\u003e\n \u003cli\u003eJefferson WN, Williams CJ. Circulating genistein concentrations in the neonatal mouse. Reprod Toxicol. 2011;31(2):209-212.\u003c/li\u003e\n \u003cli\u003eParamanik V, Kurrey K, Singh P, Tiwari S, Nisha. Roles of genistein in learning and memory during aging and neurological disorders. Biogerontology. 2023;24:329-346.\u003c/li\u003e\n \u003cli\u003eLi Y, Wang J, Zhang H, et al. Genistein improves memory and neuroplasticity in a model of neurodegeneration. Front Neurol. 2022;13:917683.\u003c/li\u003e\n \u003cli\u003eShi J, Wang L, Zhang P, et al. Genistein attenuates neuroinflammation and synaptic loss after hypoxic injury. Brain Res. 2021;1755:147287.\u003c/li\u003e\n \u003cli\u003eDuffy R, Wiseman H, File SE. Improved cognitive function after isoflavone-rich diet in rodents. Pharmacol Biochem Behav. 2003;75(3):721-729.\u003c/li\u003e\n \u003cli\u003eJefferson WN, Padilla-Banks E, Newbold RR. Reproductive consequences of developmental phytoestrogen exposure. Reproduction. 2011;142:905\u0026ndash;916.\u003c/li\u003e\n \u003cli\u003ePatisaul HB, Jefferson WN. The pros and cons of phytoestrogens. Front Neuroendocrinol. 2010;31(4):400-419.\u003c/li\u003e\n \u003cli\u003eLee JS, Kim JH, Kim JY. High intake of genistein suppresses reproductive performance. J Reprod Dev. 2014;60(4):303-307.\u003c/li\u003e\n \u003cli\u003eAwobajo FO, Nandedkar TD, Balasinor NH. Genistein disrupts implantation and pregnancy. Niger Q J Hosp Med. 2013;23(3):188-193.\u003c/li\u003e\n \u003cli\u003eAjayi AF, Akhigbe RE. Anim Reprod Update. 2020;5(5):1-15.\u003c/li\u003e\n \u003cli\u003eAjayi AF, Akhigbe RE. Staging of the estrous cycle and induction of estrus in experimental rodents: an update. Anim Reprod Update. 2020;5(5):1-15.\u003c/li\u003e\n \u003cli\u003eMachado RB, Hip\u0026oacute;lide DC, Benedito-Silva AA, Tufik S. Sleep deprivation induced by the modified multiple platform technique: quantification of sleep loss and recovery. Brain Res. 2004;1004(1-2):45-51.\u003c/li\u003e\n \u003cli\u003eSuchecki D, Tufik S. REM sleep deprivation in rats: an update on the multiple platform method. Sleep Res Online. 2000;3(4):147-53.\u003c/li\u003e\n \u003cli\u003eAwobajo FO, Nandedkar TD, Balasinor NH. Genistein alters oestrous cyclicity, oocyte fertilization and implantation process in rats. Niger Q J Hosp Med. 2013;23(3):188-193.\u003c/li\u003e\n \u003cli\u003eLee JS, Kim JH, Kim JY. High intake of genistein suppresses reproductive performance and increases embryo resorption in rats. J Reprod Dev. 2014;60(4):303-307.\u003c/li\u003e\n \u003cli\u003ePatisaul HB, Jefferson WN. The pros and cons of phytoestrogens. Front Neuroendocrinol. 2010;31(4):400-419.\u003c/li\u003e\n \u003cli\u003eLin PJ, Markakis EA, Munk A, McHugh TJ. Multiple platform water maze for testing spatial learning and memory in rodents. J Vis Exp. 2020;(156):e60846.\u003c/li\u003e\n \u003cli\u003eCammarota M, Bevilaqua LR, Kerr DS, Medina JH, Izquierdo I. Learning and memory: basic mechanisms, animal models, and memory disorders. Brain Res Brain Res Rev. 2002;39(2-3):109-129.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"sleep deprivation, pregnancy, genistein, cognition, embryo resorption, placental function, phytoestrogen, rat model","lastPublishedDoi":"10.21203/rs.3.rs-8052489/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8052489/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSleep deprivation in pregnancy is a recognized risk factor for adverse maternal and fetal outcomes, yet the mechanisms and potential interventions remain unclear. This study evaluated genistein, a phytoestrogen with neuroprotective actions, for its role in modulating the effects of gestational sleep deprivation in rats. Thirty-two pregnant Sprague-Dawley rats were divided into four groups: non-sleep deprived controls, sleep deprived (REM deprivation, GD6\u0026ndash;18), genistein-treated non-sleep deprived, and genistein-treated sleep deprived (2 mg/kg oral, GD1\u0026ndash;18). Outcomes included maternal weight gain, implantation count, embryo resorption, fetal and placental weights, and performance in labyrinth maze and inhibitory avoidance tests for cognition. Sleep deprivation significantly reduced maternal weight gain, fetal and placental weights, and increased embryo resorption and post-implantation loss. Genistein partially improved cognitive outcomes and increased fetal and placental weights, but decreased the number of successful implantations and raised post-implantation losses in both normal and sleep-deprived contexts. Combined genistein and sleep deprivation did not fully reverse physiological impairments and was associated with elevated maternal corticosterone. In conclusion, genistein provided modest neurocognitive protection against sleep deprivation\u0026ndash;induced deficits but also posed additional reproductive risks, failing to restore all adverse outcomes. These findings highlight that the effects of phytoestrogen supplementation during pregnancy depend critically on maternal environment, dosage, and timing, and underscore the need for caution in recommending such interventions\u003c/p\u003e","manuscriptTitle":"Sleep deprivation during pregnancy and its consequence on pregnancy outcome and maternal cognition; any role for genistein?","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-21 07:52:39","doi":"10.21203/rs.3.rs-8052489/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8ba9a70d-b3c4-4058-b52d-4e2baf302b50","owner":[],"postedDate":"November 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-04T16:38:59+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-21 07:52:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8052489","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8052489","identity":"rs-8052489","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}