Effects Of Low-intensity Pulsed Ultrasound On Ovarian Follicular Development And Its Mechanism

Perimenopausal period is an interim between sexual maturity andagedness in women. During this period, due to declining ovarian functionand a lower estrogen level, vasomotor disorders and neuropsychiatricsymptoms may appear such as hot flashes, sweating, anxiety, emotionalinstability, depression or irritability, which were known as thePerimenopausal Syndrome （PMS）. PMS seriously impact on the health andlife quality of women. Therefore, how to improve the life quality inperimenopausal period has become a focus of attention in the contemporarymedical field.At present, the most common clinical treatments for PMS is theHormone Replacement Therapy （HRT）. Most scholars are convinced of itsefficacy and rationality for short-term treatment. Nevertheless, there are stillsome problems:（1） Its long efficacy and complications by long-term use arestill controversial, such as the risk of dementia, venous thrombosis.（2） Thebenefit of HRT on components of metabolic syndrome in perimenopausalwomen is uncertain. Thus it’s necessary to seek a more safer and effective treatment for PMS treatment.The low-intensity pulsed ultrasound （LIPUS） use lower ultrasonic dosewhich can be as low as30mw/cm²and so as to avoid damage to body tissues.Currently, LIPU has been applied to promoting fracture healing, nerveregeneration, soft tissue repair and many other treatments and has obtained apositive curative effect. It is being applied in more and more different fieldsdue to its well tolerant, non-invasive, controllable and without radiationmerits, the research of ovarian follicular development can be one. In thisstudy, we radiated one side ovary of natural perimenopausal rats with LIPUS,in order to investigate its stimulation effect on ovarian folliculardevelopment, thus tried to explore a new method to ease the decline ofovarian function during perimenopausal period.Objectives1. Through establish model of natural aging perimenopausal in rat,study the effects of LIPUS on ovarian follicular development in naturalaging perimenpausal rats at different irradiation intensities and differentradiation time so as to figure out an appropriate dose for promoting ovarianfollicle development.2. To explore primarily the possible mechanism of promotion effects onovarian follicular development in natural aging perimenopausal rats causedby LIPUS, so as to provide theoretical basis for this research.Method We randomly selected female clean-level SD rats of11～14monthsand4～6months of age. Through continuous vaginal smears of15days inmorning, screening out the rats of estrous cycle disorder or extension asperimenopausal rats, and detected the level of serum estradiol.According to the method of vaginal smear,24female SD rats duringperimenopausal period were selected, and the rats were randomly dividedinto0.5W/cm²group,0.8W/cm²group and sham-irradiation group. LIPUSgroups were given10min of pulsed ultrasound daily for10days at theintensity of0.5W/cm²or0.8W/cm²and a frequency of1.02MHz. After10days, serum estrogen was measured by chemiluminescence, ovarianmorphology was detected by HE staining, and estrous cycle was observedby vaginal smear. We selected suitable irradiation intensity for subsequenttrials.According to above method,40SD rats during perimenopausal periodwere selected, and the rats were randomly divided into10min group,20mingroup,30min group and sham-irradiation group. LIPUS groups were givenultrasonic irradiation respectively in10minutes,20minutes,30minutesdaily for10days, with an intensity of0.8W/cm²and a frequency of1.02MHz. After10days, serum estrogen was measured, ovarian morphologywas observed, and weighed ovarian coefficients. So that we could selectsuitable irradiation time.We observed ER β protein expression of LIPUS group （0.8W/cm²,30 min） and sham-irradiation group by immunohistochemmistry （IHC）.Results:The results of vaginal smear showed: The young rats have a regularestrous cycle; Within1011～14-month-old rats,8rats’s estrous cycle weredisordered or prolong, and E₂level were below the young control group（P＜0.05）.Morphology: Compared to sham-irradiation group, LIPUS group’sovary tissue had not significant congestion and swelling. Ovary stain withhematoxylin eosin: Compared to sham-irradiation group, primary folliclequantity of LIPUS groups was significant increased （P＜0.05）, primordialfollicle quantity of0.8W/cm²group was significant decreased （P＜0.05）;secondary follicular quantity and total follicular quantity were no significantdifference between LIPUS groups and the sham-irradiation group （P＞0.05）.E₂: The levels of E₂was higher in the LIPUS groups than that ofsham-irradiation group （P＜0.05）. Estrous cycle：The estrous cycle numberof days of0.8W/cm²group was had decreased than the sham-irradiationgroup and0.5W/cm²group （P＜0.05）.0.8W/cm²irradiation can better topromote follicular development, so it was as the best irradiation intensity inthe follow-up study.Morphology: Compared to sham-irradiation group, LIPUS group’sovary tissue were not significant congestion and swelling. Ovary stain withhematoxylin eosin: Compared to sham-irradiation group,20min group and 10min group, secondary follicular quantity and total follicular quantity of30min group increased （P＜0.05）. primary follicle quantity and primordialfollicle quantity of LIPUS groups were no significant difference betweenLIPUS groups and sham-irradiation group （P＞0.05）. E₂: The levels of E₂was higher in the LIPUS groups than that of sham-irradiation group （P＜0.05）. Ovarian coefficient: ovarian coefficient were no significant differencebetween LIPUS group groups and sham-irradiation group （P＞0.05）.The Immunohistochemical staining showed that the expression of theERβ in rat ovary in LIPUS group is stronger than that of sham-irradiationgroup. The Semi-quantitative image analysis showed a significant differencebetween LIPUS goup and sham-irradiation group（P＜0.05）.Conclusions1. The model of natural aging permenopausal in rats was establishedsuccessfully by method of vaginal smear, such model can be used for thisresearch.2. The LIPUS can promote follicular development in perimenopausalrats, the intensity of0.8W/cm²and radiate30min per day is a appropriateparameter.3. The LIPUS can enhance the expression of estrogen receptor betaprotein, which may be one of the mechanisms how it promote the folliculardevelopment.