The overall effect of mild-warm moxibustion on ovarian function in mice with premature ovarian insufficiency (POI) was observed by orthogonal design and multi-model comprehensive evaluation with different moxibustion parameters (MP).

The experiment was conducted using an L₉(3⁴) orthogonal array, with duration (factor A), distance (factor B), and frequency (factor C) as the three factors, each set at three levels. This arrangement resulted in mild-warm moxibustion intervention groups Ⅰ-Ⅸ. SPF female C57BL/6J mice were randomly assigned to these groups using a random number table method, including a blank and model group, with 15 mice per group. The POI model was established via continuous gavage of Tripterygium wilfordii multiglucoside (75 mg/kg) for 14 days. In accordance with the MP specified in the orthogonal table, each group received alternating mild-warm moxibustion on both sides of the "Shenshu" (BL23), or "Zhongwan" (CV12) and "Guanyuan" (CV4) acupoints every other session for 14 days. The estrous cycle disorder of the mice was monitored by vaginal smear. Serum estradiol (E₂), follicle-stimulating hormone (FSH), luteinizing hormone (LH), anti-Müllerian hormone (AMH), and testosterone (T) levels were measured using an enzyme-linked immunosorbent assay. Ovarian morphology and follicle count were assessed using hematoxylin-eosin staining. The pregnancy rate, implantation count, live fetuses, and post-implantation mortality rate were evaluated via cohabitation observation. The estrous cycle disorder rate and FSH level were defined as the primary efficacy indicators, with the remaining indicators serving as secondary indicators. The effects of single indicators were analyzed through range and variance analyses. The Criteria Importance Through Intercriteria Correlation (CRITIC) method and the entropy weight method were used to determine indicator weights, which were then combined with the Technique for Order Preference by Similarity to Ideal Solution (TOPSIS) and grey relational analysis for comprehensive evaluation to identify the preference MP.

Compared with the blank group, the model group showed an increase in the estrous cycle disorder rate, decreased serum E₂ and AMH levels, increased FSH, LH, and T levels, reduced numbers of primordial, primary, secondary, and antral follicles, increased numbers of atretic follicles, decreased pregnancy rate, implantation count, and number of live fetuses, and increased post-implantation mortality (P < 0.05). Compared with the model group, all intervention groups showed varying degrees of improvement in the indicators mentioned above. The orthogonal experiment, CRITIC-TOPSIS, entropy weight-TOPSIS, and grey relational analysis identified Ⅳ group (10 min duration, 1 cm distance, once daily) as the preference parameter combination. This combination significantly reduced the estrous cycle disorder rate, upregulated serum E₂ and AMH levels, downregulated FSH, LH, and T levels, increased the numbers of primordial, primary, and secondary follicles, decreased the number of atretic follicles, and improved the pregnancy rate, implantation count, and number of live fetuses (P < 0.05).

The preferred MP for treating POI is 10 min duration, 1 cm distance, and once daily, which improves ovarian function and fertility in POI mice.

To investigate the effect of moxibustion on delaying ovarian aging in naturally aging mice and to explore the underlying mechanisms.

Fifteen 2-month-old female SPF ICR mice with regular estrous cycles were included in a 2-month-old group. Thirty 10-month-old female SPF ICR mice were randomly divided into a 10-month-old group and a moxibustion group, with 15 mice in each group. The moxibustion group received alternating moxibustion on bilateral "Shenshu"(BL23) and "Zhongwan" (CV12), "Guanyuan" (CV4) for 10 min every other day, for 21 consecutive days. The vaginal smears were used to observe the estrous cycle irregularities of mice. The serum contents of estradiol (E₂), follicle-stimulating hormone (FSH), and anti-Müllerian hormone (AMH) were measured by ELISA. The ovarian morphology was observed by hematoxylin-eosin staining. The contents of adenosine triphosphate (ATP) in granulosa cells were measured by luminescence-based ATP assay. The levels of mitochondrial membrane potential (MMP) of granulosa cells were measured by JC-1 staining. The real-time fluorescence quantitative PCR, Western blot, and immunohistochemistry were used to assess the mRNA and protein expression levels of mitochondrial unfolded protein response (UPR^mt)-related signaling pathway components, including activating transcription factor 5 (ATF5), caseinolytic protease P (ClpP), and Lon protease 1 (LONP1) in the ovarian tissues.

Compared with the 2-month-old group, the 10-month-old group exhibited an increased rate of estrous cycle irregularity (P < 0.01), elevated serum FSH contents (P < 0.01), decreased E₂ and AMH contents (P < 0.01), reduced number of follicles at all stages (P < 0.01), reduced granulosa cell ATP content and MMP level (P < 0.01), decreased positive expression levels of ATF5, ClpP, and LONP1 protein, and diminished expression levels of ATF5, ClpP, and LONP1 mRNA and protein in ovarian tissue (P < 0.01). Compared with the 10-month-old group, the moxibustion group showed a lower rate of estrous cycle irregularity (P < 0.01), decreased serum FSH content (P < 0.01), increased E₂ and AMH contents (P < 0.01), increased the number of primordial, growing and antral follicles (P < 0.05, P < 0.01), elevated granulosa cell ATP content and MMP level (P < 0.01), increased ATF5, ClpP, and LONP1 protein positive expression levels, and enhanced ATF5, ClpP, and LONP1 mRNA and protein expression levels in the ovarian tissue (P < 0.01, P < 0.05).

Moxibustion can delay ovarian dysfunction related to natural aging in mice and modulate hormonal levels. Its mechanism may involve regulating the expressions of UPR^mt-related signaling molecules such as ATF5, ClpP and LONP1, and enhancing mitochondrial function in granulosa cells.