C57BL/6 wild-type female mice (8–10 weeks old, weighing 18–20 g) were used to establish COH models. C57BL/6 wild-type male mice (12–16 weeks old, weighing 25–30 g) were castrated and employed for mating with the females. All mice were obtained from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd. and housed in a specific pathogen-free facility under controlled environmental conditions: temperature maintained at 22 ± 2 °C, relative humidity at 40-60%, and a 12: 12 h light-dark cycle (lights on at 08:00 and off at 20: 00). Mice had free access to food and water and underwent a 2-week acclimation period before the experiment. All experimental procedures were reviewed and approved by the Animal Ethics Committee of Nanjing University of Chinese Medicine (Approval No. 202309A067). Female mice were monitored daily for estrous cycles via vaginal cytology. Only mice exhibiting two consecutive regular estrous cycles were included in subsequent experiments. In Experiment 1, female mice were assigned to three groups: CTR, COH, and EA. Each group was further divided into six subgroups according to sampling time: ZT12 (20: 00), ZT16 (0: 00), ZT20 (04: 00), ZT0 (08: 00), ZT4 (12: 00), ZT8 (16: 00). In Experiment 2, animals were allocated into five groups: CTR, COH, EA, AG, and AT+EA.

The COH protocol was conducted following established experimental methods described in previous studies (22, 23). Female C57BL/6 mice in the COH, EA, AG, and AT+EA groups received intraperitoneal injections of PMSG (5 U/mouse, Ningbo Second Hormone Factory, Veterinary Drug Registration No. 110254564) at 17: 00 during the diestrus. After 48 hours, the mice were administered an intraperitoneal injection of HCG (5 U/mouse, Ningbo Second Hormone Factory, Veterinary Drug Registration No. 110251282). Mice in the CTR group received an equal volume of 0.9% saline at the corresponding time points. Subsequently, female mice were housed overnight with vasectomized males in individual cages for mating. To eliminate the influence of embryos on the uterine endometrium after conception, males were vasectomized prior to pairing, and successful copulation induced a pseudopregnant state in the females. Mating success was confirmed by the presence of a vaginal plug at 08:00 the following morning, which was designated as gestation day 1 (D1). Only female mice that successfully mated were included in the experiment.

Female rats in the AG group received intraperitoneal injections of the AVP Receptor 1A (AVPR1A) agonist: (Phe2, Orn8)-Oxytocin (1.0 mg/kg, MCE, HY-P4678), starting on the day of PMSG administration and given once daily at 17:30 for 7 consecutive days (24, 25). Female rats in the AT+EA group received intraperitoneal injections of the AVPR1A antagonist: SR49059 (1.0 mg/kg, MCE, HY-18345) at the same time as (Phe2, Orn8)-Oxytocin (26). For the EA and AT+EA groups, mice were anesthetized before receiving EA at bilateral SP6 and CV4 acupoints using disposable acupuncture needles (0.18 × 13 mm; Beijing Zhongyan Taihe Medical Devices Co., Ltd). Needles were inserted perpendicularly to a depth of approximately 5 mm. EA was delivered via an electronic acupuncture device (Suzhou Medical Supplies Factory Co., Ltd, SDZ-II) with a sparse-dense waveform at a 2 Hz/15 Hz frequency and intensity adjusted to induce mild twitching of the hind limbs. During each electroacupuncture session, electrical stimulation was applied between CV4 and one-sided SP6, while the contralateral SP6 was needled without electrical stimulation. Start electroacupuncture at 21:00 on the day of PMSG injection, once daily for 15 minutes each time, and continue for 7 consecutive days. Mice in all other groups were anesthetized at the same time points without further intervention (Figure 1A). As mice are nocturnal animals, 21:00 falls within their active period, making electroacupuncture at this time more consistent with their natural living habits.

Experimental mice were dissected at designated time points according to their subgroup assignments between D4-5. Sampling was performed at six ZT points: ZT12, ZT16, ZT20, ZT0, ZT4, and ZT8. Following anesthesia, blood samples were collected from the retinal vein, and mice were subsequently euthanized via cervical dislocation. Serum was separated by centrifugation and stored for enzyme-linked immunosorbent assay (ELISA) to determine E2 and P4 levels. Tissue samples, including the hypothalamus, pituitary gland, ovaries, and unilateral uterus, were rapidly harvested and stored at −80 °C for subsequent molecular analyses. The contralateral uterus was fixed in 4% paraformaldehyde and stored at 4 °C for HE staining and immunohistochemical analysis. In Experiment 2, mice were sacrificed at ZT4 (12: 00) on D5. After anesthesia, animals underwent cardiac perfusion, and the whole brain was fixed in 4% paraformaldehyde. The brains were then processed for sectioning and subsequent immunofluorescence staining.

Fresh uterine tissue was immediately fixed in 4% paraformaldehyde for more than 24 hours. Embed the tissue in paraffin blocks, section it into 5-micrometer slices, mount the sections onto slides, and air-dry them for later use. The tissue sections underwent a series of histological procedures, including dewaxing, staining, dehydration, and sealed. Observe the sections under an optical microscope (Olympus Corporation, BX53) and collect images.

Serum concentrations of E2 and P4 were determined using the ELISA method. The following commercial kits were used: Mouse Estradiol ELISA Kit (Afantibody, AF02457-A) and Mouse Progesterone ELISA Kit (Afantibody, AF02568-A). All procedures were performed strictly in accordance with the manufacturer’s instructions. After completing the assays, absorbance values were measured using a microplate reader, and hormone concentrations were calculated based on the corresponding standard curves.

Paraffin sections are deparaffinized in xylene, then placed in gradient concentrations of ethanol for antigen retrieval. Endogenous peroxidase activity was quenched using 3% hydrogen peroxide (H₂O₂) in deionized water for 10 minutes. Antigen retrieval was performed in citrate buffer using heat-induced epitope retrieval for 23 minutes, followed by blocking with 5% bovine serum albumin at room temperature for 30 minutes to reduce nonspecific binding. Primary antibodies were applied and incubated overnight at 4 °C: Homeobox A10 (HOXA10, 1: 100 dilution, Abclonal, A8550) and Secreted Phosphoprotein 1 (SPP1, 1: 100 dilution, Abmart, T55333). The following day, sections were incubated with a biotin-labeled goat anti-rabbit IgG secondary antibody (1: 200 dilution, Boster, BA1003) at room temperature for 30 minutes. Subsequently, an SABC signal amplification reagent was added and incubated for 30 minutes at room temperature. Visualization was achieved using a DAB chromogen reaction for 10 minutes, followed by hematoxylin counterstaining for 3 minutes. Finally, the sections are dehydrated, cleared, and sealed. Stained slides were observed under a light microscope, and digital images were captured. The staining intensity and positive area were quantitatively analyzed using ImageJ software to evaluate protein expression levels.

Paraffin sections were first dewaxed in xylene and rehydrated through a series of graded ethanol solutions for antigen retrieval. The sections were then treated with citrate buffer for antigen retrieval. After antigen retrieval, peroxidase inhibitor solution was applied to block endogenous peroxidase activity, followed by incubation with normal goat serum to block non-specific binding sites. The sections were incubated overnight at 4 °C with the primary antibody AVP (1: 2000 dilution, Immunostar, 20069). Following primary antibody incubation, a Polymer-HRP secondary antibody was added and incubated for 30 minutes at room temperature in the dark. Subsequently, add TYR-520 fluorescent dye dropwise, and incubate at room temperature for 5–10 minutes. Finally, a fluorescence quenching mounting medium containing DAPI was added, and the slides were allowed to stand for approximately 5 minutes. The stained sections were examined under a fluorescence microscope, and digital images were captured. Staining intensity and positive area were analyzed quantitatively using ImageJ software to assess the expression levels of AVP. All reagents used for this procedure were obtained from the Single Label Signal Amplification Fluorescent Staining Kit (Afantibody, AFIHC022).

Tissue samples were minced and lysed in RIPA buffer for 10 minutes on ice. Lysates were then centrifuged at 12,000 rpm for 15 minutes at 4 °C, and the supernatant was collected. Protein concentrations were quantified using a microplate reader. Equal amounts of protein were mixed with SDS-PAGE loading buffer and denatured by boiling. Proteins were separated by SDS-PAGE electrophoresis and subsequently transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% bovine serum albumin at room temperature for 1 hour to prevent nonspecific binding. Membranes were incubated overnight at 4 °C with primary antibodies against HOXA10 (1: 1000 dilution, Abclonal, A8550), SPP1 (1: 1000 dilution, Abmart, T55333), and β-actin (1: 5000, Proteintech, 20536-1-AP). After washing, membranes were incubated with appropriate HRP-conjugated secondary antibodies at room temperature for 1 hour. Protein bands were visualized using a chemiluminescent imaging system (VILBER BIO IMAGING, FUSION-FX6.MINI), and band intensities were quantified by ImageJ software to calculate the relative expression levels of target proteins normalized to β-actin.

Tissue samples were processed with Trizol to extract total RNA. Chloroform was then used to remove proteins and lipids, followed by isopropanol precipitation of RNA. The RNA pellets were washed with ethanol, and RNA concentration and purity were assessed using a spectrophotometer. cDNA was synthesized using a reverse transcription kit (Yeasen, 11141ES60). PCR amplification was carried out with a pre-mixed solution kit (Yeasen, 11202ES08) on a real-time quantitative PCR system (Thermo Fisher Scientific, ViiA 7). Gene-specific primers are listed in Table 1. Relative gene expression was calculated using the 2^−ΔΔCT method, with CT values normalized to the reference gene Gapdh.

Statistical analysis was conducted using GraphPad Prism 10.4.1 software. Data are expressed as mean ± standard error. The Shapiro-Wilk test was employed to assess the normality of the data. For comparisons between groups at a single time point, One-way analysis of variance was used. If the data met the assumption of homogeneous variances, Tukey’s multiple comparison test was applied to determine pairwise differences. If variances were found to be heterogeneous, Brown-Forsythe and Welch’s analysis of variance tests were used to reanalyze the data. For comparisons across multiple time points, two-way analysis of variance was conducted, followed by Dunnett’s multiple comparison test to assess specific group differences. For data that did not follow a normal distribution, nonparametric tests were applied. A P < 0.05 was considered statistically significant.

To evaluate the effect of EA on uterine morphology during the WOI in COH mice, we first examined the gross appearance of the uterus and then performed HE staining to assess histological changes, including quantitative analysis of endometrial thickness and gland number. Compared with the CTR group, uteri from COH mice appeared markedly thickened and edematous with a pale coloration. The endometrial architecture was disorganized, the endometrium was thinner, and both the number of glands and the glandular lumen diameter were reduced. In contrast, relative to the COH group, uteri from EA-treated mice were slimmer with attenuated edema, and showed significantly increased endometrial thickness and gland numbers (Figures 1B–E). These findings indicate that EA ameliorates COH-induced uterine morphological abnormalities.

Endometrial thickness and gland number are key histological parameters for evaluating endometrial receptivity. The histological findings described above suggested that EA may improve endometrial receptivity. To further clarify the effect of EA on endometrial receptivity during the WOI in COH mice, we assessed uterine mRNA levels of the receptivity markers Hoxa10, Spp1, and Monoamine Oxidase A (Maoa) at six time points from ZT12 to ZT8. Compared with the CTR group, COH mice showed reduced Hoxa10, Spp1, and Maoa mRNA levels across ZT12-ZT8. In contrast, relative to the COH group, EA-treated mice exhibited increased Hoxa10, Spp1, and Maoa mRNA levels at the same time points (Figure 2A). These data indicate that COH impairs endometrial receptivity, whereas EA can partially restore it.

To determine the time point at which EA exerts the most pronounced effect on WOI endometrial receptivity in COH mice, we compared, for each of the six Zeitgeber times from ZT12 to ZT8, the absolute differences in Hoxa10, Spp1, and Maoa mRNA levels between the CTR and COH groups, as well as between the COH and EA groups. Both sets of absolute differences reached their maximum at ZT4 (Figure 2B). This finding suggests that endometrial receptivity during the WOI is most severely impaired by COH at ZT4, and that the beneficial effect of EA on COH-induced impairment is also greatest at this time point. Accordingly, ZT4 was selected as the observation time point for subsequent experiments.

P4-induced secretory transformation is a central mechanism underlying the acquisition of endometrial receptivity (27). The P4/E2 ratio during the WOI reflects the degree of synchronization between the uterine microenvironment and embryonic development and provides a more accurate predictor of pregnancy outcome (28, 29). To evaluate the influence of EA on P4 and E2 levels during the WOI in COH mice, we measured serum P4 and E2 concentrations at ZT12-ZT8 and calculated the P4/E2 ratio. Compared with the CTR group, COH mice showed significantly increased serum P4 levels and P4/E2 ratios across ZT12-ZT8. EA treatment markedly reduced serum P4 levels and P4/E2 ratios relative to the COH group, suggesting that EA may help normalize sex steroid hormone levels in COH mice. In contrast, no significant differences in serum E2 levels were observed among the three groups (Figure 2C).

Circadian variation in core clock genes plays a critical role in regulating endometrial receptivity during the WOI (30). To investigate the effect of EA on the circadian expression of clock genes along the WOI-HPOU axis in COH mice, we measured mRNA levels of the clock genes Period Circadian Regulator 1 (Per1), Cryptochromes 1 and 2 (Cry1, Cry2), and their key regulatory factors F-box and Leucine-Rich Repeat Protein 3 (Fbxl3) and Casein Kinase 1ϵ (Csnk1e) in the hypothalamus, pituitary, ovary, and uterus at six Zeitgeber time points from ZT12 to ZT8.

The results showed that in mice from the CTR group, hypothalamic and pituitary mRNA expression of Per1, Cry1, Cry2, Fbxl3, and Csnk1e exhibited regular circadian rhythmicity. In contrast, in the COH group, the circadian rhythmicity of these genes in the hypothalamus and pituitary was disrupted; notably, the rhythmic expression of hypothalamic Fbxl3 and Csnk1e mRNA was lost and the amplitude was markedly reduced. In the EA group, hypothalamic and pituitary Per1, Cry1, Cry2, Fbxl3, and Csnk1e mRNA expression regained clear circadian rhythmicity, and the phases of the peak and trough of Cry1 mRNA rhythms were consistent with those in the CTR group (Figures 3A–D). This indicates that circadian rhythmicity of hypothalamus and pituitary-related clock genes is disrupted in COH mice during the WOI stage, while EA can improve and partially restore these rhythmic changes.

In line with the changes observed in the hypothalamus and pituitary, ovarian and uterine Per1, Cry1, Cry2, Fbxl3, and Csnk1e mRNA expression in the CTR group displayed regular circadian rhythmicity. In COH mice, however, the circadian rhythmicity of these genes in the ovary and uterus was disrupted, whereas EA treatment restored their rhythmic expression. In the ovaries of CTR and EA mice, Per1, Cry1, Cry2, Fbxl3, and Csnk1e mRNA levels showed similar peak and trough phases, with peak expression occurring between ZT16 and ZT20 and trough expression between ZT4 and ZT8 (Figures 4A–D). Taken together, these findings indicate that COH disrupts the circadian rhythmicity of Per1, Cry1, Cry2, Fbxl3, and Csnk1e expression along the HPOU axis during the WOI, whereas EA restores their circadian rhythmic patterns.

The central circadian clock in the SCN is a network composed of neurons and glial cells, among which AVP-expressing neurons act as the principal pacemakers of the SCN clock network (31). To examine the effect of EA on the central clock at WOI-ZT4 in COH mice, we performed immunofluorescence staining for AVP neuropeptide in the SCN of the hypothalamus to assess AVP neuronal activity. The results showed that, compared with the CTR group, AVP neuropeptide expression at WOI-ZT4 was markedly reduced in the COH group. Relative to the COH group, AVP neuropeptide expression intensity at WOI-ZT4 was increased in both the EA and AG groups. In contrast, AVP neuropeptide expression at WOI-ZT4 was reduced in the AT+EA group compared with the EA group (Figures 5A, B).

In parallel, we measured serum P4 levels and the P4/E2 ratio at WOI-ZT4 to evaluate the hormonal status of COH mice. Compared with the CTR group, COH mice exhibited elevated serum P4 concentrations and P4/E2 ratios at WOI-ZT4. Relative to the COH group, both the EA and AG groups showed reduced serum P4 levels and P4/E2 ratios. Notably, serum P4 levels and the P4/E2 ratio were higher in the AT+EA group than in the EA group (Figures 5C, D). Collectively, these findings demonstrate that COH mice exhibit reduced AVP neuropeptide expression and hormone levels in the hypothalamic SCN at WOI-ZT4. EA and AVPR1A agonist treatment can improve these alterations, while AVPR1A antagonist combined with EA treatment attenuates the modulatory effect of EA.

Because AVP neurons in the central clock of the SCN can regulate peripheral clocks, we next examined mRNA expression of the clock genes Per1, Cry1, and Cry2 in the hypothalamus, pituitary, ovary, and uterus at WOI-ZT4. Compared with the CTR group, COH mice showed decreased Per1 and Cry2 mRNA expression in the hypothalamus and pituitary, and reduced Cry1 mRNA expression in the ovary and uterus at WOI-ZT4, whereas the other clock genes were upregulated. These findings indicate that COH disrupts the normal expression pattern of key clock genes along the HPOU axis at this time point.

By contrast, in the EA and AG groups, Per1 and Cry2 mRNA levels in the hypothalamus and pituitary, as well as Cry1 mRNA levels in the ovary and uterus, were increased relative to the COH group, while the other clock genes were altered in the opposite direction. Compared with the EA group, the AT+EA group exhibited reduced Per1 and Cry2 mRNA expression in the hypothalamus and pituitary, and decreased Cry1 mRNA levels in the ovary and uterus at WOI-ZT4, accompanied by opposite changes in the remaining clock genes (Figures 6A–D).

Taken together, these results show that in COH mice at WOI-ZT4, Per1 and Cry2 mRNA levels are reduced in the hypothalamus and pituitary, and Cry1 mRNA levels are reduced in the ovary and uterus, whereas hypothalamic and pituitary Cry1, and ovarian and uterine Per1 and Cry2 mRNA levels are increased. EA and AVPR1A agonist treatment can improve the abnormal expression of HPOU axis clock genes in COH mice during the WOI-ZT4 period, whereas co−administration of an AVPR1A antagonist attenuates this regulatory effect of EA on gene expression.

HOXA10 and SPP1 exhibit dynamic changes in expression in endometrial epithelial and stromal cells (32, 33). To examine the expression of HOXA10 and SPP1 in the endometrium of mice at WOI-ZT4, we performed immunohistochemical staining. The results showed that at WOI-ZT4, HOXA10 and SPP1 were positively expressed in endometrial glandular epithelial cells, stromal cells, and luminal epithelial cells (Figure 7A).

Quantitative immunohistochemical analysis revealed that compared with the CTR group, HOXA10 and SPP1 protein levels at WOI-ZT4 were reduced in the COH group. Compared with the COH group, HOXA10 and SPP1 protein expression at WOI-ZT4 was increased in the EA and AG groups. Compared with the EA group, the AT+EA group showed decreased expression of the endometrial receptivity markers HOXA10 and SPP1 at WOI-ZT4 (Figures 7B, C).

Western blotting and RT-qPCR analyses confirmed that the expression patterns of HOXA10 and SPP1 protein and mRNA in each group were consistent with the immunohistochemical findings (Figures 7D–H). These results indicate that COH mice exhibit impaired endometrial receptivity at WOI-ZT4, while both EA and AVPR1A agonist treatment improve endometrial receptivity-related markers. Co−administration of the AVPR1A antagonist with EA attenuates this ameliorative effect.