Objective

2

To explore the possible mechanism by which the "Zhibian" (BL54) toward "Shuidao" (ST28) acupuncture improves ovarian function in mice with poor ovarian response (POR) by observing its effect on gut microbiota.

Methods

2

A total of 35 SPF-grade C57BL/6 female mice were screened for normal estrous cycles using vaginal smears

and 30 mice were selected. Ten mice were assigned to the blank group

while the remaining mice were used to establish the POR model by intragastric administration of tripterygium wilfordii suspension. The successfully modeled mice were randomly divided into a model group and an acupuncture group

with 10 mice in each group. After modeling

the acupuncture group received the "Zhibian" (BL54) toward "Shuidao" (ST28) acupuncture method once daily for 20 minutes per session. Ovulation induction began the day after the intervention

and samples were collected after ovulation induction. Vaginal cytology was used to observe estrous cycle changes

and the number of oocytes obtained

ovarian wet weight

and ovarian index were recorded. Serum levels of follicle-stimulating hormone (FSH)

luteinizing hormone (LH)

estradiol (E2)

and anti-Müllerian hormone (AMH) were detected using ELISA. HE staining was used to observe ovarian histology. Gut microbiota was analyzed using 16S rRNA gene sequencing technology. Western blot was used to detect the relative protein expression levels of Occludin and zonula occludens-1 (ZO-1) in colonic tissue. Correlation analysis was conducted among serum hormone indexes

the number of oocytes obtained

ovarian index and gut microbiota.

Results

2

Compared with the blank group

the model group showed a higher estrous cycle disorder rate (

P

<

0.01)

increased serum FSH and LH levels

and a higher LH/FSH ratio (

P

<

0.01)

while the number of oocytes obtained

ovarian wet weight

ovarian index

and serum E2 and AMH levels were significantly reduced (

P

<

0.01). Compared with the model group

the acupuncture group showed a lower estrous cycle disorder rate (

P

<

0.01)

decreased serum FSH and LH levels

and a lower LH/FSH ratio (

P

<

0.01)

along with an increased number of oocytes obtained

higher ovarian wet weight

ovarian index

and elevated serum AMH and E2 levels (

P

<

0.01

P

<

0.05). The blank group had a large number of well-developed primordial follicles

with abundant and closely arranged follicles at various stages. In the model group

there

was a significant increase in the number of atretic follicles

a reduction in the number of follicles at various stages

and loosely arranged ovarian tissue. Compared with the blank group

the model group showed a significant decrease in the number of normal follicles (

P

<

0.01) and an increase in the number of atretic follicles (

P

<

0.01). The acupuncture group showed a reduction in atretic follicles and an increase in the number of follicles at various stages compared with the model group

with a significant increase in normal follicles (

P

<

0.01) and a decrease in atretic follicles (

P

<

0.01). Compared with the blank group

the model group exhibited reduced gut microbiota diversity and richness

with significantly lower Chao1 and Shannon indices (

P

<

0.01)

and a greater clustering distance from the blank group. The model group also showed an increase in the relative abundance of

Firmicutes

_D

Verrucomicrobiota

Paramuribaculum

Dubosiella

and

Muribaculum

(

P

<

0.01

P

<

0.05)

while the relative abundance of

Firmicutes

_A and the relative protein expression of Occludin and ZO-1 in colonic tissue were decreased (

P

<

0.01). Compared with the model group

the acupuncture group showed improved gut microbiota diversity and richness

with increased Chao1 and Shannon indices (

P

<

0.05)

and a clustering distance closer to the blank group. The acupuncture group exhibited reduced relative abundance of

Firmicutes

_D

Verrucomicrobiota

and

Muribaculum

(

P

<

0.05

P

<

0.01)

while the relative abundance of

Firmicutes

_A and the relative protein expression of Occludin and ZO-1 were significantly increased (

P

<

0.01

P

<

0.05).

Correlation analysis indicated a relationship between gut microbiota and serum hormone indicators

as well as the ovarian index. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis showed that the metabolic pathways of the intersecting species were related to amino acid biosynthesis and nucleotide metabolism.

Conclusion

2

The "Zhibian" (BL54) toward "Shuidao" (ST28) acupuncture method improves ovarian function in POR mice

and its mechanism may be related to regulating gut microbiota structure and maintaining intestinal barrier homeostasis.