Objective

2

To observe the effect of "Zhibian" (BL54)-to-"Shuidao" (ST28) needling technique on the reproductive function of the mice with poor ovarian response (POR) and explore the molecular mechanism of acupuncture on early embryos after in vitro fertilization-embryo transfer (IVF-ET) in POR mice.

Methods

2

Of 70 female C57BL/6 mice

60 mice with regular estrous cycle were screened and 30 of them were randomly divided into a blank group

a model group

and an acupuncture group

10 mice in each one. Mice in the model and acupuncture groups were administered with tripterygium glycosides suspension (50 mg·kg

-1

·d

-1

) via gavage for 2 weeks to prepare POR models; while in the blank group

the mice received an equal volume of 0.9% sodium chloride solution via gavage. After successful modeling

mice in the acupuncture group underwent "Zhibian" (BL54)-to-"Shuidao" (ST28) needling technique

once daily

for 20 min each time and lasting 2 consecutive weeks. After intervention completion

subsequently

all the three groups underwent ovulation induction

orbital blood collection

and ovary extraction and fresh denuded oocyte collection. Denuded oocytes

after incubated

together with the sperms from 15 male C57BL/6 mice

were transferred into the oviducts of 30 donor pseudopregnant C57BL/6 female mice via IVF-ET; and the embryonic tissue was collected on day 7 of implantation. After successful modeling and intervention completion

the general conditions of mice in each group were observed

and the estrous cycle disorder rate was compared among the groups. After intervention completion

the numbers of oocyte

ovarian wet weight and ovarian index were recorded in each group; the levels of anti-Müllerian hormone (AMH)

follicle-stimulating hormone (FSH)

luteinizing hormone (LH)

and estradiol (E2) in the serum were measured using ELISA; and ovarian morphology and mitochondrial ultrastructure were examined using HE staining and transmission electron microscopy. The transcriptome sequencing technology was employed to identify differentially expressed genes (DEGs) in early embryos of each group

followed by bioinformatics analysis; and the reversed DEGs with significant difference were v

erified using quantitative real-time PCR (qRT-PCR).

Results

2

After intervention

compared with the blank group

the mice in the model group presented poor spirits and declined water and food intake

reduced activity

the higher rate of estrous cycle disorder (

P

<

0.01); the decrease of the numbers of oocyte

ovarian wet weight

and ovarian index (

P

<

0.01)

reduced AMH and E2 in the serum (

P

<

0.01)

elevated FSH and LH in the serum (

P

<

0.01); more atresia follicles in ovarian tissue

disorganized granulosa cells

reduced the numbers of mitochondria

vacuoles appeared and cristae breakage. When compared with the model group

in the acupuncture group

the spirits of mice

food intake and activity were improved

the rate of estrous cycle disorder was reduced (

P

<

0.01); the numbers of oocyte

ovarian wet weight

and ovarian index were higher (

P

<

0.01

P

<

0.05)

the levels of AMH and E2 in the serum increased (

P

<

0.01)

the levels of FSH and LH in the serum decreased (

P

<

0.01); the atresia follicles were reduced

the numbers of follicle increased at all levels

the numbers of mitochondria got higher

vacuoles and cristae breakage were declined. In comparison between the blank group and the model group

and between the model group and the acupuncture group

a total of 258 intersecting DEGs were identified

and 194 DEGs of them exhibited a trend of reversion before and after intervention. The top 5 reversed DEGs with significant difference included dexamethasone-induced Ras-related protein 1 (Rasd1)

gene regulated by estrogen in breast cancer 1 (Greb1)

leucine-rich repeat-containing G protein-coupled receptor 6 (Lgr6)

Fraser syndrome 1 (Fras1)

and apolipoprotein D (Apod). Compared with the blank group

the mRNA expression of Rasd1

Greb1

Lgr6

Fras1 and Apod in embryonic tissues decreased in the model group (

P

<

0.01

P

<

0.05); and when compared with the model group

the mRNA expression of Rasd1

Greb1

Lgr6

Fras1 and Apod in embryonic tissues increased in the acupuncture group (

P

<

0.05

P

<

0.01). These findings were consistent with the DEGs screening results. Gene ontology (GO) analysis revealed that the reversed genes of co-expression were primarily participated in the biological processes such as myoblast differentiation

endocardial cushion development

and cardiac morphogenesis. Kyoto encyclopedia of genes and genomes (KEGG) analysis indicated that the reversed genes of co-expression are enriched in the Wnt signaling pathway

and they were associated with various types of cardiac diseases.

Conclusion

2

The "Zhibian" (BL54)-to-"Shuidao" (ST28) needling technique may ameliorate the decline of ovarian response in mice with POR

promote normal follicle development and ovulation

thereby improve embryo quality and reduce the risk of developmental defects and deformity in the organs such as the embryonic heart. The underlying mechanism may be related to the regulation of the expression pattern of embryonic key genes through the Wnt signaling pathway.