- Email: [email protected]
Increased Oocyte Production after Acupuncture Treatment during Superovulation Process in Mice
Journal of Reproduction & Contraception
http://www.RandC.cn
2009 Mar.; 20(1):35-44
[email protected]
Increased Oocyte Production after Acupuncture Treatment during Superovulation Process in Mice Chun-lan JIN1, Kazuo Tohya2, Koichi Kuribayashi3, Michio Kimura2, Yuki-hisa Hirao2 1. Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences, Beijing 100700, China 2. Department of Anatomy, Kansai University of Health Sciences, Kumatori, Osaka 5900482, Japan 3. Department of Immunology and Pathology, Kansai University of Health Sciences, Kumatori, Osaka 5900482, Japan
Objective To investigate whether acupuncture treatment during superovulation process improves ovarian response and increases egg production. Methods ICR female mice aged 1215 weeks were divided into control group, anaesthesia group and acupuncture group. Female mice were injected intraperitoneally with pregnant mare’s serum gonadotropin (PMSG), followed by human chorionic gonadotropin (hCG) injection 56 h later. Anaesthesia group and acupuncture group were anaesthetized three times by injecting 10% nembutal solution according to 7.5 10.0 Pl/g weight. Acupuncture group was treated three times with puncture Sanyinjiao Points(SP6) under anaesthesia. After 17 h of hCG injection, eggs were recovered and ovaries were isolated. Matured eggs were counted, ovarian histology and expression of follicle stimulating hormone receptor(FSH-R) in ovary were analyzed. Results Acupuncture treatment statistically increased the number of ovulated eggs. Histological section showed that some matured follicles left in the ovaries of control and anaesthesia groups after ovulation. On the contrary, all matured follicles ruptured and converted into corpus lutea in Acupuncture group. Expression of FSH-R in ovary was decreased in acupuncture group compared with those of the two others. Conclusion Acupuncture treatment during superovulation process improves ovarian response so as to increase egg production. The positive effect of acupuncture may be associated with regulated FSH-R expression. Key words: acupuncture; superovulation; egg production; ovarian response; FSH-R expression This study was supported in part by the Japan-China Sasakawa Medical Fellowship Corresponding author: Chun-lan JIN; E-mail: [email protected] 35
With environmental pollution being aggravated and mental pressure increasing gradually accompanied by the social economic development, infertility has been a significant public health problem which families as well as the whole society have to take seriously. According to the most recent estimates of infertility in the United States, 10% of women of reproductive age (1544 years) reported a previous infertility-associated health-care visit[1], 7% of married couples in which the woman was of reproductive age, reported they had not conceived after 12 months of unprotected intercourse[2]. Since the first in vitro fertilization (IVF) infant was born in England in 1978, the practice of assisted reproductive technology (ART) has steadily increased. The average pregnancy rate, however, is still only 20%25% per IVF cycle[3]. Controlled ovarian hyperstimulation (COH) is an important process closely associated with IVF success rates. Scarce numbers of ovulated eggs give rise in shortage of the quantity of embryo available for transfer, and the success rates will be declining accordingly. Therefore, increasing the ovulation rates in COH is the key factor for improving success rates of IVF-ET. COH is a process employing external hormone to induce the continued development of multiple ovarian follicles from the initially recruited cohort of follicles. Follicular development depends on many regulators, among which follicle stimulating hormone(FSH) plays a central role in the process of maturation of ovarian follicles. It triggers the maturation of follicles, and plays a pivotal role in the recruitment of the dominant follicle[4]. FSH action is mediated by the FSH receptor(FSH-R), which is transmembrane proteins present on granulosa cells of growing follicles[5]. FSH binding to its receptor in the dominant follicle directly increases estradiol(E2) output and also potentiates progesterone production by granulose cells[6]. In addition to stimulating granulose cell mitosis, FSH induces the expression of luteinizing hormone (LH) receptors in the dominant follicle, which are necessary for ovulation[7]. Acupuncture therapy takes the theories of traditional Chinese medicine as guidance to treat patients by stimulating certain points of the human body to regulate the flow of Qi and blood in the meridian. Acupuncture has been widely used in many diseases due to ovulation disorders, like infertility, premature incipient ovarian failure, irregular menstruation, polycystic ovary syndrome(PCOS) and so on[8]. Various investigators have shown that acupuncture influenced the levels of FSH, LH and E2 in plasma so as to promote ovulation[9-11]. In recent years, acupuncture as an adjuvant to conventional treatment in IVF-ET has increased in popularity. Studies have proved that acupuncture may reduce pain intensity during oocyte aspiration[12,13], improve the success rate of IVF [14-16] and the quality of life (QOL) of patients undergoing IVF[13,17]. However, there are few observations on the effect of acupuncture during superovulation. One case report has shown that acupuncture treatment and dehydroepiandrosterone (DHEA) significantly improved ovarian reserve and increased oocyte production in one patient undergoing IVF[18]. What interaction will occur between acupuncture and hormonal medicine on the superovulation and whether acupuncture as an adjuvant to conventional treatment in 36
ovulation induction will increase oocyte production remain unclear. In the present study, we investigated the application of acupuncture therapy on the superovulation stage of ART in female mice.
Materials & Methods Animals SPF female ICR mice aged 1215 weeks were obtained from Japan SLC, Inc (Sizuoka, Japan). The animals were maintained under a 12 h light and 12 h dark regimen (light on at 8ĩ00 am). Mice were fed a standard pellet diet ad libitum, and had free access to drink water. All animals had been observed two estral cycles continually to confirm that they had normal sexual cycle. It was designated to day 1 when animals were at diestrus stage by vaginal smear examination. Mice were divided into 3 groups (n=10 in each group): superovulation (control group); superovulation+anaesthesia (anaesthesia group); superovulation+anaesthesia+ acupuncture (acupuncture group). Superovulation Mice were induced superovulation by injection of hormones, 10 IU of pregnant mare’s serum gonadotropin (PMSG, Aska Pharmaceutical Co., Ltd. Japan) was injected intraperitoneally to female mice of three groups on the morning of day 1. Fifty-six hours after the injection of PMSG, mice were given 10 IU of human chorionic gonadotropin (hCG, ASKA Pharmaceutical Co. Ltd. Japan) with peritoneal injection on the evening of day 3. Seventeen hours after hCG injection, animals were killed and their oviducts were removed[19]. Anaesthesia and acupuncture In order to anaesthetize animals, 10% nembutal solution according to 7.510.0 Pl/g weight was injected into body cavity. Anaesthesia group was anaesthetized by above mentioned on the morning of day 1, day 2 and day 3, respectively. Acupuncture group was designed as followed: after the injection of PMSG on the morning of day 1, followed by anaesthesia, the stainless steel needles (Carbo, Japan; size 0.16 mm h 13 mm) were inserted intramuscularly into the points SP6 (Sanyinjiao) to a depth of 35 mm and remained 10 min. With the same methods, acupuncture stimulation was given at the same time on day 2 and day 3, respectively. The location of points SP6 was presumed according to the description in the book of Experimental Acupuncture and Moxibustion[20]. Animal body weight and ovarian weight At day 4, 17 h after hCG injection, animals were weighed and then killed by ether. Ovaries were isolated and immediately weighed. The sum of the both ovaries was regarded for ovarian weight. For each female mouse, one ovary was used for histological examination, and the other one was subjected to RT-PCR as described below. 37
Counting for the number of eggs Cumulus-enclosed eggs were retrieved from the ampullae of oviducts by rupturing with thin needles and incubated in 0.1% hyaluronidase (Sigma) for about 10 min to disperse the cumulus cells. After rinsing, the number of cumulus free-eggs was counted under stereomicroscope. HE staining Ovaries were fixed in Bouin’s solution for over night at room temperature, sectioned in wax, and stained with hemotoxylin and eosin under standard conditions. Ovarian expression of FSH receptor Extraction of RNA was performed according to AGPC method[21]. Reverse transcription (RT) was carried out using advantage RT-for-PCR kit (Toyobo, Japan). Master mix per sample was prepared as follows: regulated as 4 Pg RNA in 20 Pl DEPC-W solution and added 5 U/ml reverse transcriptase, 10 mol/L dNTP, 5hRT Buffer, and 25 mmol/L random primer. Once all components were mixed, the samples were incubated at 37ć for 1 h and then heated at 94ć for 5 min. Primers for PCR were searched in the Gen Bank database and designed by using the Primer 3 Software. The FSH-receptor sense primer was 5'-GAGATGGGAGCTGGGTTGTA3', the antisense FSH-R primer was 5'-GCCTGTCTGTCCTCTTCCAG-3', and the size of amplification product was 345 bp. The E-actin sense primer was 5'-GACGATATCG CTGCGCTGGT-3', antisense E-actin primer was 5'-ATCTTTTCACGGTTGGCCT-3', and the size of amplification product was 353 bp. In each PCR reaction, a total of 4 Pl from each RT tube was mixed with 10hPCR buffer, 25 mmol/L MgCl2, 10 mmol/L dNTP, 5 U/Pl AmpliTaq DNA polymerase(Applied Biosystems, Branchburg, NJ, USA), and 25 Pmol/L FSH-R primer or E-actin primer to a final volume of 50 Pl. The cDNA was amplified for 32 cycles with denaturation at 94ć for 90 s, annealing at 58ć for 60 s, and extension at 72ć for 120 s. The products of PCR were analyzed on a 2.5% agarose gel stained with ethidium bromide. Image J software was used to analyze the concentration of PCR products. The ratio of FSH-R/E-actin concentration in each sample was accounted for FSH-R mRNA expression in the ovary. Statistical analysis Data were expressed as xf s. Statistical calculations were performed using SPSS11.0, and One-way Anova and correlation analysis were conducted. P<0.05 was considered statistically significant.
Results The number of eggs The average number of superovulated eggs was 45.6 in acupuncture group, 31.5 in 38
control group and 26.3 in anaesthesia group (n=9, one sample was deleted due to the failure to retrieve eggs). Eggs production of acupuncture group was significantly increased compared with control group (P=0.030) and anaesthesia group (P=0.005). The weight of ovary showed significant difference in the three groups, being higher in acupuncture group than in both control group (P=0.028) and anaesthesia group (P=0.006). Ovary/body weight ratio was compared in the three groups, also showing higher in acupuncture group than in the other two groups, P=0.005 and P=0.020, respectively. Both ovarian weight and ovary/body weight ratio were positively correlated with the number of eggs, P=0.000, R=0.679 and P=0.000, R=0.634, respectively (Table 1). Table 1 A comparison in superovulated eggs, and body and ovarian weight between the three groups No. of
No. of
Weight of
ovulated
superovulated
mouse body
eggs
eggs
(g)
10
315
31.5*
38.69 f 3.92
32.4 f 4.2*
0.84 f 0.086**
Anaesthesia
9
237
26.3
35.70 f 3.38
31.0 f 2.8
0.87 f 0.076*
Acupuncture
10
456
45.6
37.33 f 4.11
37.4 f 6.4
Group
Control
No. of mice treated
**
Weight of
Weight ratio of
mouse ovary (mg)
ovary/body (10-3)
**
1.00 f 0.172
*:P<0.05, **: P<0.01, compared with acupuncture group
The results of HE staining In the all three groups, a lot of corpus lutea were formed and follicles of primary or secondary stage were mainly situated at cortex of the ovary. Several matured follicles, however, were found to leave in the ovaries of control and anaesthesia group, but not in acupuncture group. This fact indicated that ovulation occurred completely and the ovulation rate was improved in acupuncture group compared with the other two groups (Figure1).
It was found that several large matured follicles(
) remained in both ovaries of control group (A) and
anesthesia group (B), but not in acupuncture group (C) Figure 1 A comparison of histology of ovaries between the three groups
The level of FSH receptor The result showed that there was no difference in FSH-R expression between control and anaesthesia mice (Figure 2). FSH-R expression in acupuncture group, however, was significantly decreased to almost half of the control group (P=0.029) and anaesthesia group (P=0.023)(Figure 3). 39
Figure 2 Expression of the FSH-R in the ovaries of the treated mice (n=4)
1.6
*: P <0.05, compared with other two groups
FSHR/ȕ-actin ratio
1.4 1.2 1.0 0.8 *
0.6 0.4 0.2 0.0 Control group
Anesthesia group
Acupuncture group
Figure 3 Expression of the FSH-R in the ovaries of the treated mice
Discussion It is well known that female aging is associated with the outcome of COH. Oocyte quality has been shown to decline with age in the studies of the effects of age on the response to human menopausal gonadotropin(hMG) stimulation and IVF outcome[22,23]. In animal studies, SD rats’ COH outcome is best during 45 weeks aging and decreases with age[24], and mice aging is associated with decreased number of ovulated oocytes and increased percentage of abnormal oocytes as well[25]. It is generally considered that the sexual maturity and lifespan in the laboratory for mice is around 6 weeks to 1 year. In the present study, we used 1215 weeks aged mice, which belong to a period of stable fertility and are suitable for a research of the effects of acupuncture during the superovulation. In addition, by using the mice aged 1215 weeks, it can also prevent some issues such as animals’ over-response or poor response to hormone, which could interfere with the outcome of experimentation. Acupuncture therapy has been extensively applied in many diseases due to ovulation disorders. Recent studies show that acupuncture has the same effect on ovulation as clomiphene, but can promote pregnancy rate and reduce abortion rate, compared with 40
clomiphene[26]. According to Traditional Chinese Medicine (TCM), it is considered that there are mainly three organs related to the reproductive physiology: the kidney storing essence and dominating development and reproduction; the spleen governing transportation and transformation and considered to be the main organ for the manufacture of Qi and blood; the liver maintaining the free flow of Qi and storing blood. Etiology of infertility mainly includes three factors: kidney deficiency, Qi stagnation in the liver, and damp or phlegm stagnation due to the weakened function of the spleen, all of which can cause innutrition or stagnation of Qi and blood in the uterine vessels and result in infertility. The principle of the treatment for infertility usually aims to tonify the kidney, fortify the spleen, and to regulate Qi stagnation in the Liver[27]. The point of SP 6 used in this study, which belongs to spleen channel and connect with kidney channel and liver channel, is one of the main point to treat infertility[28]. To puncture the point could benefit the kidney and liver, strengthen the spleen to eliminate phlegm, and regulate the flow of Qi and blood. We investigated the effect of acupuncture treatment in superovulation process. As we expected, acupuncture on the points SP6 considerably enhanced animals’ ovulation, and the number of eggs increased to half as much again as the control animals. Furthermore, the histological observation of ovary in acupuncture group has shown that there are not any large follicles found in the ovary, which indicated that acupuncture treatment played a positive role in the ovulation process and improved the ovulation rate cooperating with hCG. Interestingly, this inference seemed to be supported by the PCR outcome of ovarian tissue as well. It is well established that FSH receptors are present in granulosa cells of growing follicles at preantral and graafian stages[29]. Recent report had proved that FSH-R gene was not expressed in primordial follicles until after they enter their growth phase and seemed to be enhanced with their growth: FSH-R was negative in the primordial follicles, and positive in the 1/3 of the primary and 2-layer follicles, but dedicated in all multilaminar preantral follicles[30]. In the present study, at 17 h later of hCG injection, expression of FSH-R mRNA showed lower level in the Acupuncture group, which may be related to the fact that the ovary was filled up with a large number of corpus lutea and small primary or secondary follicles. In contrast, in control and anaesthesia groups, there were some large follicles still left in the ovary, which could result in high levels of FSH-R expression. The maturation of ovarian follicles involves several sequential stages: initiation, growth, recruitment, selection and ovulation, and that process is regulated by endocrine factor from hypothalamic-pituitary-ovarian axis. FSH plays an important role during the growth of follicle. As the dominant follicle matures, E2 and FSH act to up-regulate LH and FSH in anticipation of the LH/FSH surge preceding ovulation[31]. As we took the samples at a few hours later of ovulation when the surge of FSH and LH had gone away, it was difficult to confirm how the acupuncture promoted the ovulation process. However, based on the previous studies and the present result, it is considered that there are some conceivable mechanisms as follows: 41
1) Promoting follicle development. Acupuncture can promote FSH release by regulating hypothalamic-pituitary-ovarian axis[9,32]. Acupuncture may also influence FSH-R expression at the granulose cells to enhance FSH action so that more follicles will be recruited and developed. 2) Inhibiting follicle atresia. Acupuncture may inhibit follicle atresia undergoing developing stages by promoting release of FSH. It has been known that granulosa cell apoptosis is a direct and important inducement of leading follicles to atresia. Apoptosis can occur at all stages of follicle development. In rodents, the preantral to early antral transition is most susceptible to atresia[33]. However, FSH is the predominant survival factor at early antral follicle development[34]. It has been proved in the latest study that acupuncture can inhibit granulosa cell apoptosis so as to reduce follicle atresia by regulating expression of apoptosis-related genes[35]. 3) Promoting follicle rupture. It was reported that the surge of LH induces to rupture the follicle wall in the rabbit and that the change in the follicle preceding rupture is due to the release of collagenase enzymes by the cells lining the follicle[36]. Some studies showed that the thinning of the follicle wall and overlying surface epithelium result in a transparent area or stigma, so that follicle rupture ultimately occurs, and the egg is released. Acupuncture may interact with hCG to regulate the appearance of LH surge and then result in the increase in ovulation rate. By using acupuncture as a surrogate for hCG in ovulation induction, ovulation was successfully realized 2472 h after treatment[37]. Another report showed that patients who suffered anovulation due to failure of follicular rupture were ovulated after 13 times of the acupuncture treatment [38]. In the present study, it was suspected that acupuncture may strengthen the action of exogenous hCG, regulate the occurrence of LH surge and promote the rupture of follicle wall so as to increase ovulation rate. In the study we also observed that after 3 times of anaesthesia egg numbers were decreased at a certain extent although there was no significant difference. For practical reason, it is necessary to use anaesthesia before acupuncture stimulation is conducted on animals. The study may remind investigators to pay more attention to the influence of anaesthesia when they design and analyze experiment. In conclusion, the present study shows for the first time that acupuncture used in superovulation process can interact with hormonal medication to increase egg production significantly. It will not only provide a new concept for the improvement in the skill of supervulation in the assisted reproductive technology, but also will open new frontiers for the application of acupuncture as an adjuvant in the modern reproductive medicine. Furthermore, it is believed that it will serve as a useful reference for the research on other animals’ reproduction in the animal husbandry. As for such issues as the quality of eggs from superovulation, the mechanism of acupuncture effect, and whether acupuncture will increase the risk of the appearance of ovarian hyperstimulation syndrome (OHSS) accompanied by the increase in eggs, they require verification in the further studies. 42
References 1. Chandra A, Martinez GM, Mosher WD, et al. Fertility, family planning, and reproductive health of U.S. women: data from the 2002 National Survey of Family Growth. Vital Health Stat, 2005, 25(1):1-160. 2. Wright VC, Chang J, Jeng G, et al. Assisted reproductive technology surveillance-United States, 2004. MMWR Surveill Summ, 2007, 56(6):1-22. 3.
Freeman ML,Blacker CM. Tubal surgery versus in vitro fertilization: where do we draw the line? /Ransom SB, Dombrowski MP, Evans MI, et al. Contemporary Therapy in Obstetrics and Gynecology. U.S.A: W.B. Saunders Company, 2002:459-64.
4. Simoni M, Gromoll J, Nieschlag E. The follicle-stimulating hormone receptor: biochemistry, molecular biology, physiology, and pathophysiology. Endocr Rev, 1997, 18(6):739-73. 5.
Greenwald GS, Roy SK. Follicular development and its control. /Knobil E, Neill JD(eds). The Physiology of Reproduction, Vol. 1. New York: Raven Press, 1994:629-724.
6. Erickson G. Physiologic basis of ovulation induction. Semin Reprod Endocrinol, 1996, 14(4):287-97. 7. Mcnatty KP, Madris A, Osathamondh R. Effects of luternizing hormone on steroidogenesis by thecal tissue from human ovarian follicles in vitro. Steoids,1980, 36(1):53-63. 8.
Peng Y, Hou L, Wu X. Advances of modern studies of acupuncture and moxibustion for treatment of ovulation disorders. Chin Acupunct Moxib (in Chinese), 2006, 26(10):756-9.
9. Yu J, Zheng H, Ping S. Changes in serum FSH, LH and ovarian follicular growth during electroacupuncture for induction of ovulation. J Integr Tradit West Med (in Chinese), 1989, 9(4):199-202. 10. Mo X, Li D, Pu Y, et al. Clinical studies on the mechanism of acupuncture stimulation of ovulation. J Tradit Chin Med (in Chinese), 1993, 13(2):115-9. 11. Ma R, Ji P, Sha G, et al. Clinical observation of acupuncture and moxibustion for treatment of polycystic ovary syndrome. Chin Acupunct Moxib (in Chinese), 1996, 16(11):18. 12. Stener-Victorin E, Waldenst-m U, Nilsson L, et al. A prospective randomized study of electro-acupuncture versus alfentanil as anaesthesia during oocyte aspiration in in-vitro fertilization. Hum Reprod, 1999,14(10): 2 480-4. 13. Stener-Victorin E, Waldenstr-m U, Wikland M, et al. Electro-acupuncture as a peroperative analgesic method and its effects on implantation rate and neuropeptide Y concentrations in follicular fluid. Hum Reprod, 2003, 18(7):1 454-60. 14. Paulus WE, Zhang M, Strehler E, et al. Influence of acupuncture on the pregnancy rate in patients who undergo assisted reproduction therapy. Fertil Steril, 2002, 77(4):721-4. 15. Dieterle S, Ying G, Hatzmann W, et al. Effect of acupuncture on the outcome of in vitro fertilization and intracytoplasmic sperm injection: a randomized, prospective, controlled clinical study. Fertil Steril, 2006, 85 (4):1 347-51. 16. Westergaard LG, Mao Q, Krogslund M, et al. Acupuncture on the day of embryo transfer significantly improves the reproductive outcome in infertile women: a prospective, randomized trial. Fertil Steril, 2006, 85 (4):1 341-6. 17. Gejervall AL, Stener-Victorin E, Möller A, et al. Electro-acupuncture versus conventional analgesia: a comparison of pain levels during oocyte aspiration and patients’ experiences of well-being after surgery. Hum Reprod, 2005, 20(3):728-35. 18. Barad DH, Gleicher N. Increased oocyte production after treatment with dehydroepiandrosterone. Fertil Steril, 2005, 84(3):756. 19. Hiraoka J, Hirao Y. Fate of sperm tail components after incorporation into the hamster egg. Gamete Res, 1988, 19(4):369-80. 43
20. Li Z. Experimental acupuncture and moxibustion (in Chinese). Beijing: China Press of Traditional Chinese Medicine, 2002:327-9. 21. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction. Anal Biochem, 1987, 162(1):156-9. - D F R E V 6 / 0
H W ] J H U '
$
'
R G V R Q :
&
et al. Effect of age on response to human menopausal gonadotropin
stimulation. J Clin Endocrinol Metab, 1990, 71(6):1 525-30. 23. Van Kooij RJ, Looman CWN, Habbema JD, et al. Age dependent decrease in embryo implantation rate after in vitro fertilization. Fertil Steril, 1996, 66(5):769-75. 24. Zhang CY, Liu LJ, Di M, et al. Effects of the PMSG dosages on superovulation and oocyte quality in immature rats. Chin J Comp Med (in Chinese), 2007, 17(6):338-40, 344. 25. Tar1´n JJ, Pérez-Albalá S, Cano A. Cellular and morphological traits of oocytes retrieved from aging mice after exogenous ovarian stimulation. Biol Reprod, 2001, 65(1):141-50. 26. Song F, Zheng S, Ma D. Clinical observation on acupuncture for treatment of infertility of ovulatory disturbance. Chin Acupunct Moxib (in Chinese), 2008, 28(1):21-3. 27. Zhang F, Li C. The pattern identification and treatment of acumoxatherapy (in Chinese). Beijing: China Pharmaceutical Technology Publisher, 2000:349. 28. Yan H, Liu Y. A clinical overview on acupuncture-stimulating ovulation. Shanghai J Acupunct Moxib (in Chinese), 2005, 24(2):40-2. 29. Peters H. Some aspects of early follicular development. /Midgley Jr AR, Sandler WA(eds). Ovarian Follicular Development and Function. New York: Raven Press, 1979:1-13. 30. Oktay K, Briggs D, Gosden RG. Ontogeny of follicle stimulating hormone receptor gene expression in isolated human ovarian follicles. J Clin Endocrinol Metab, 1997, 82(11):3 748-51. 31. Irianni F, Hodgen GD. Mechanism of ovulation. Endocrinol Metab Clin North Am, 1992, 21(1):19-38. 32. Chen BY. Acupuncture normalizes dysfunction of hypothalamic-pituitary-ovarian axis. Acupunct Electrother Res, 1997, 22(2):97-108. 33. Hirshfield AN. Development of follicles in the mammalian ovary. Int Rev Cytol, 1991, 124:43-101. 34. Chun SY, Eisenhauer KM, Minami S, et al. Hormonal regulation of apoptosis in early antral follicles: follicle stimulating hormone as a major survival factor. Endocrinology, 1996, 137(4):1 447-56. 35. Ma XP, Dai M, Wu HG, et al. Effects of acupuncture on granulocyte apoptosis and expressions of apoptosisassociated genes in the ovary of perimenopausal rats. Chin Acupuct Moxib (in Chinese), 2007, 27(5):357-61. 36. David TB. The ovary. /Austin CR, Short FRS RV (eds). Reproduction in Mammals. Book 3: Hormonal Control of Reproduction. 2nd. ed. New York: Cambridge University Press, 1984:91-114. 37. Chen L. 42 clinical observations on the acupunture-stimulating follicle rupture. Chin Acupunct Moxib (in Chinese), 1997, 17(8):483. 38. Yang L, Zuo F. The clinical observation on acupuncture at Luanchao points for treatment of anovulation. Chin Acupunct Moxib (in Chinese), 1998, 18(10):585-6. (Received on December 9, 2008)
44
http://www.RandC.cn
2009 Mar.; 20(1):35-44
[email protected]
Increased Oocyte Production after Acupuncture Treatment during Superovulation Process in Mice Chun-lan JIN1, Kazuo Tohya2, Koichi Kuribayashi3, Michio Kimura2, Yuki-hisa Hirao2 1. Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences, Beijing 100700, China 2. Department of Anatomy, Kansai University of Health Sciences, Kumatori, Osaka 5900482, Japan 3. Department of Immunology and Pathology, Kansai University of Health Sciences, Kumatori, Osaka 5900482, Japan
Objective To investigate whether acupuncture treatment during superovulation process improves ovarian response and increases egg production. Methods ICR female mice aged 1215 weeks were divided into control group, anaesthesia group and acupuncture group. Female mice were injected intraperitoneally with pregnant mare’s serum gonadotropin (PMSG), followed by human chorionic gonadotropin (hCG) injection 56 h later. Anaesthesia group and acupuncture group were anaesthetized three times by injecting 10% nembutal solution according to 7.5 10.0 Pl/g weight. Acupuncture group was treated three times with puncture Sanyinjiao Points(SP6) under anaesthesia. After 17 h of hCG injection, eggs were recovered and ovaries were isolated. Matured eggs were counted, ovarian histology and expression of follicle stimulating hormone receptor(FSH-R) in ovary were analyzed. Results Acupuncture treatment statistically increased the number of ovulated eggs. Histological section showed that some matured follicles left in the ovaries of control and anaesthesia groups after ovulation. On the contrary, all matured follicles ruptured and converted into corpus lutea in Acupuncture group. Expression of FSH-R in ovary was decreased in acupuncture group compared with those of the two others. Conclusion Acupuncture treatment during superovulation process improves ovarian response so as to increase egg production. The positive effect of acupuncture may be associated with regulated FSH-R expression. Key words: acupuncture; superovulation; egg production; ovarian response; FSH-R expression This study was supported in part by the Japan-China Sasakawa Medical Fellowship Corresponding author: Chun-lan JIN; E-mail: [email protected] 35
With environmental pollution being aggravated and mental pressure increasing gradually accompanied by the social economic development, infertility has been a significant public health problem which families as well as the whole society have to take seriously. According to the most recent estimates of infertility in the United States, 10% of women of reproductive age (1544 years) reported a previous infertility-associated health-care visit[1], 7% of married couples in which the woman was of reproductive age, reported they had not conceived after 12 months of unprotected intercourse[2]. Since the first in vitro fertilization (IVF) infant was born in England in 1978, the practice of assisted reproductive technology (ART) has steadily increased. The average pregnancy rate, however, is still only 20%25% per IVF cycle[3]. Controlled ovarian hyperstimulation (COH) is an important process closely associated with IVF success rates. Scarce numbers of ovulated eggs give rise in shortage of the quantity of embryo available for transfer, and the success rates will be declining accordingly. Therefore, increasing the ovulation rates in COH is the key factor for improving success rates of IVF-ET. COH is a process employing external hormone to induce the continued development of multiple ovarian follicles from the initially recruited cohort of follicles. Follicular development depends on many regulators, among which follicle stimulating hormone(FSH) plays a central role in the process of maturation of ovarian follicles. It triggers the maturation of follicles, and plays a pivotal role in the recruitment of the dominant follicle[4]. FSH action is mediated by the FSH receptor(FSH-R), which is transmembrane proteins present on granulosa cells of growing follicles[5]. FSH binding to its receptor in the dominant follicle directly increases estradiol(E2) output and also potentiates progesterone production by granulose cells[6]. In addition to stimulating granulose cell mitosis, FSH induces the expression of luteinizing hormone (LH) receptors in the dominant follicle, which are necessary for ovulation[7]. Acupuncture therapy takes the theories of traditional Chinese medicine as guidance to treat patients by stimulating certain points of the human body to regulate the flow of Qi and blood in the meridian. Acupuncture has been widely used in many diseases due to ovulation disorders, like infertility, premature incipient ovarian failure, irregular menstruation, polycystic ovary syndrome(PCOS) and so on[8]. Various investigators have shown that acupuncture influenced the levels of FSH, LH and E2 in plasma so as to promote ovulation[9-11]. In recent years, acupuncture as an adjuvant to conventional treatment in IVF-ET has increased in popularity. Studies have proved that acupuncture may reduce pain intensity during oocyte aspiration[12,13], improve the success rate of IVF [14-16] and the quality of life (QOL) of patients undergoing IVF[13,17]. However, there are few observations on the effect of acupuncture during superovulation. One case report has shown that acupuncture treatment and dehydroepiandrosterone (DHEA) significantly improved ovarian reserve and increased oocyte production in one patient undergoing IVF[18]. What interaction will occur between acupuncture and hormonal medicine on the superovulation and whether acupuncture as an adjuvant to conventional treatment in 36
ovulation induction will increase oocyte production remain unclear. In the present study, we investigated the application of acupuncture therapy on the superovulation stage of ART in female mice.
Materials & Methods Animals SPF female ICR mice aged 1215 weeks were obtained from Japan SLC, Inc (Sizuoka, Japan). The animals were maintained under a 12 h light and 12 h dark regimen (light on at 8ĩ00 am). Mice were fed a standard pellet diet ad libitum, and had free access to drink water. All animals had been observed two estral cycles continually to confirm that they had normal sexual cycle. It was designated to day 1 when animals were at diestrus stage by vaginal smear examination. Mice were divided into 3 groups (n=10 in each group): superovulation (control group); superovulation+anaesthesia (anaesthesia group); superovulation+anaesthesia+ acupuncture (acupuncture group). Superovulation Mice were induced superovulation by injection of hormones, 10 IU of pregnant mare’s serum gonadotropin (PMSG, Aska Pharmaceutical Co., Ltd. Japan) was injected intraperitoneally to female mice of three groups on the morning of day 1. Fifty-six hours after the injection of PMSG, mice were given 10 IU of human chorionic gonadotropin (hCG, ASKA Pharmaceutical Co. Ltd. Japan) with peritoneal injection on the evening of day 3. Seventeen hours after hCG injection, animals were killed and their oviducts were removed[19]. Anaesthesia and acupuncture In order to anaesthetize animals, 10% nembutal solution according to 7.510.0 Pl/g weight was injected into body cavity. Anaesthesia group was anaesthetized by above mentioned on the morning of day 1, day 2 and day 3, respectively. Acupuncture group was designed as followed: after the injection of PMSG on the morning of day 1, followed by anaesthesia, the stainless steel needles (Carbo, Japan; size 0.16 mm h 13 mm) were inserted intramuscularly into the points SP6 (Sanyinjiao) to a depth of 35 mm and remained 10 min. With the same methods, acupuncture stimulation was given at the same time on day 2 and day 3, respectively. The location of points SP6 was presumed according to the description in the book of Experimental Acupuncture and Moxibustion[20]. Animal body weight and ovarian weight At day 4, 17 h after hCG injection, animals were weighed and then killed by ether. Ovaries were isolated and immediately weighed. The sum of the both ovaries was regarded for ovarian weight. For each female mouse, one ovary was used for histological examination, and the other one was subjected to RT-PCR as described below. 37
Counting for the number of eggs Cumulus-enclosed eggs were retrieved from the ampullae of oviducts by rupturing with thin needles and incubated in 0.1% hyaluronidase (Sigma) for about 10 min to disperse the cumulus cells. After rinsing, the number of cumulus free-eggs was counted under stereomicroscope. HE staining Ovaries were fixed in Bouin’s solution for over night at room temperature, sectioned in wax, and stained with hemotoxylin and eosin under standard conditions. Ovarian expression of FSH receptor Extraction of RNA was performed according to AGPC method[21]. Reverse transcription (RT) was carried out using advantage RT-for-PCR kit (Toyobo, Japan). Master mix per sample was prepared as follows: regulated as 4 Pg RNA in 20 Pl DEPC-W solution and added 5 U/ml reverse transcriptase, 10 mol/L dNTP, 5hRT Buffer, and 25 mmol/L random primer. Once all components were mixed, the samples were incubated at 37ć for 1 h and then heated at 94ć for 5 min. Primers for PCR were searched in the Gen Bank database and designed by using the Primer 3 Software. The FSH-receptor sense primer was 5'-GAGATGGGAGCTGGGTTGTA3', the antisense FSH-R primer was 5'-GCCTGTCTGTCCTCTTCCAG-3', and the size of amplification product was 345 bp. The E-actin sense primer was 5'-GACGATATCG CTGCGCTGGT-3', antisense E-actin primer was 5'-ATCTTTTCACGGTTGGCCT-3', and the size of amplification product was 353 bp. In each PCR reaction, a total of 4 Pl from each RT tube was mixed with 10hPCR buffer, 25 mmol/L MgCl2, 10 mmol/L dNTP, 5 U/Pl AmpliTaq DNA polymerase(Applied Biosystems, Branchburg, NJ, USA), and 25 Pmol/L FSH-R primer or E-actin primer to a final volume of 50 Pl. The cDNA was amplified for 32 cycles with denaturation at 94ć for 90 s, annealing at 58ć for 60 s, and extension at 72ć for 120 s. The products of PCR were analyzed on a 2.5% agarose gel stained with ethidium bromide. Image J software was used to analyze the concentration of PCR products. The ratio of FSH-R/E-actin concentration in each sample was accounted for FSH-R mRNA expression in the ovary. Statistical analysis Data were expressed as xf s. Statistical calculations were performed using SPSS11.0, and One-way Anova and correlation analysis were conducted. P<0.05 was considered statistically significant.
Results The number of eggs The average number of superovulated eggs was 45.6 in acupuncture group, 31.5 in 38
control group and 26.3 in anaesthesia group (n=9, one sample was deleted due to the failure to retrieve eggs). Eggs production of acupuncture group was significantly increased compared with control group (P=0.030) and anaesthesia group (P=0.005). The weight of ovary showed significant difference in the three groups, being higher in acupuncture group than in both control group (P=0.028) and anaesthesia group (P=0.006). Ovary/body weight ratio was compared in the three groups, also showing higher in acupuncture group than in the other two groups, P=0.005 and P=0.020, respectively. Both ovarian weight and ovary/body weight ratio were positively correlated with the number of eggs, P=0.000, R=0.679 and P=0.000, R=0.634, respectively (Table 1). Table 1 A comparison in superovulated eggs, and body and ovarian weight between the three groups No. of
No. of
Weight of
ovulated
superovulated
mouse body
eggs
eggs
(g)
10
315
31.5*
38.69 f 3.92
32.4 f 4.2*
0.84 f 0.086**
Anaesthesia
9
237
26.3
35.70 f 3.38
31.0 f 2.8
0.87 f 0.076*
Acupuncture
10
456
45.6
37.33 f 4.11
37.4 f 6.4
Group
Control
No. of mice treated
**
Weight of
Weight ratio of
mouse ovary (mg)
ovary/body (10-3)
**
1.00 f 0.172
*:P<0.05, **: P<0.01, compared with acupuncture group
The results of HE staining In the all three groups, a lot of corpus lutea were formed and follicles of primary or secondary stage were mainly situated at cortex of the ovary. Several matured follicles, however, were found to leave in the ovaries of control and anaesthesia group, but not in acupuncture group. This fact indicated that ovulation occurred completely and the ovulation rate was improved in acupuncture group compared with the other two groups (Figure1).
It was found that several large matured follicles(
) remained in both ovaries of control group (A) and
anesthesia group (B), but not in acupuncture group (C) Figure 1 A comparison of histology of ovaries between the three groups
The level of FSH receptor The result showed that there was no difference in FSH-R expression between control and anaesthesia mice (Figure 2). FSH-R expression in acupuncture group, however, was significantly decreased to almost half of the control group (P=0.029) and anaesthesia group (P=0.023)(Figure 3). 39
Figure 2 Expression of the FSH-R in the ovaries of the treated mice (n=4)
1.6
*: P <0.05, compared with other two groups
FSHR/ȕ-actin ratio
1.4 1.2 1.0 0.8 *
0.6 0.4 0.2 0.0 Control group
Anesthesia group
Acupuncture group
Figure 3 Expression of the FSH-R in the ovaries of the treated mice
Discussion It is well known that female aging is associated with the outcome of COH. Oocyte quality has been shown to decline with age in the studies of the effects of age on the response to human menopausal gonadotropin(hMG) stimulation and IVF outcome[22,23]. In animal studies, SD rats’ COH outcome is best during 45 weeks aging and decreases with age[24], and mice aging is associated with decreased number of ovulated oocytes and increased percentage of abnormal oocytes as well[25]. It is generally considered that the sexual maturity and lifespan in the laboratory for mice is around 6 weeks to 1 year. In the present study, we used 1215 weeks aged mice, which belong to a period of stable fertility and are suitable for a research of the effects of acupuncture during the superovulation. In addition, by using the mice aged 1215 weeks, it can also prevent some issues such as animals’ over-response or poor response to hormone, which could interfere with the outcome of experimentation. Acupuncture therapy has been extensively applied in many diseases due to ovulation disorders. Recent studies show that acupuncture has the same effect on ovulation as clomiphene, but can promote pregnancy rate and reduce abortion rate, compared with 40
clomiphene[26]. According to Traditional Chinese Medicine (TCM), it is considered that there are mainly three organs related to the reproductive physiology: the kidney storing essence and dominating development and reproduction; the spleen governing transportation and transformation and considered to be the main organ for the manufacture of Qi and blood; the liver maintaining the free flow of Qi and storing blood. Etiology of infertility mainly includes three factors: kidney deficiency, Qi stagnation in the liver, and damp or phlegm stagnation due to the weakened function of the spleen, all of which can cause innutrition or stagnation of Qi and blood in the uterine vessels and result in infertility. The principle of the treatment for infertility usually aims to tonify the kidney, fortify the spleen, and to regulate Qi stagnation in the Liver[27]. The point of SP 6 used in this study, which belongs to spleen channel and connect with kidney channel and liver channel, is one of the main point to treat infertility[28]. To puncture the point could benefit the kidney and liver, strengthen the spleen to eliminate phlegm, and regulate the flow of Qi and blood. We investigated the effect of acupuncture treatment in superovulation process. As we expected, acupuncture on the points SP6 considerably enhanced animals’ ovulation, and the number of eggs increased to half as much again as the control animals. Furthermore, the histological observation of ovary in acupuncture group has shown that there are not any large follicles found in the ovary, which indicated that acupuncture treatment played a positive role in the ovulation process and improved the ovulation rate cooperating with hCG. Interestingly, this inference seemed to be supported by the PCR outcome of ovarian tissue as well. It is well established that FSH receptors are present in granulosa cells of growing follicles at preantral and graafian stages[29]. Recent report had proved that FSH-R gene was not expressed in primordial follicles until after they enter their growth phase and seemed to be enhanced with their growth: FSH-R was negative in the primordial follicles, and positive in the 1/3 of the primary and 2-layer follicles, but dedicated in all multilaminar preantral follicles[30]. In the present study, at 17 h later of hCG injection, expression of FSH-R mRNA showed lower level in the Acupuncture group, which may be related to the fact that the ovary was filled up with a large number of corpus lutea and small primary or secondary follicles. In contrast, in control and anaesthesia groups, there were some large follicles still left in the ovary, which could result in high levels of FSH-R expression. The maturation of ovarian follicles involves several sequential stages: initiation, growth, recruitment, selection and ovulation, and that process is regulated by endocrine factor from hypothalamic-pituitary-ovarian axis. FSH plays an important role during the growth of follicle. As the dominant follicle matures, E2 and FSH act to up-regulate LH and FSH in anticipation of the LH/FSH surge preceding ovulation[31]. As we took the samples at a few hours later of ovulation when the surge of FSH and LH had gone away, it was difficult to confirm how the acupuncture promoted the ovulation process. However, based on the previous studies and the present result, it is considered that there are some conceivable mechanisms as follows: 41
1) Promoting follicle development. Acupuncture can promote FSH release by regulating hypothalamic-pituitary-ovarian axis[9,32]. Acupuncture may also influence FSH-R expression at the granulose cells to enhance FSH action so that more follicles will be recruited and developed. 2) Inhibiting follicle atresia. Acupuncture may inhibit follicle atresia undergoing developing stages by promoting release of FSH. It has been known that granulosa cell apoptosis is a direct and important inducement of leading follicles to atresia. Apoptosis can occur at all stages of follicle development. In rodents, the preantral to early antral transition is most susceptible to atresia[33]. However, FSH is the predominant survival factor at early antral follicle development[34]. It has been proved in the latest study that acupuncture can inhibit granulosa cell apoptosis so as to reduce follicle atresia by regulating expression of apoptosis-related genes[35]. 3) Promoting follicle rupture. It was reported that the surge of LH induces to rupture the follicle wall in the rabbit and that the change in the follicle preceding rupture is due to the release of collagenase enzymes by the cells lining the follicle[36]. Some studies showed that the thinning of the follicle wall and overlying surface epithelium result in a transparent area or stigma, so that follicle rupture ultimately occurs, and the egg is released. Acupuncture may interact with hCG to regulate the appearance of LH surge and then result in the increase in ovulation rate. By using acupuncture as a surrogate for hCG in ovulation induction, ovulation was successfully realized 2472 h after treatment[37]. Another report showed that patients who suffered anovulation due to failure of follicular rupture were ovulated after 13 times of the acupuncture treatment [38]. In the present study, it was suspected that acupuncture may strengthen the action of exogenous hCG, regulate the occurrence of LH surge and promote the rupture of follicle wall so as to increase ovulation rate. In the study we also observed that after 3 times of anaesthesia egg numbers were decreased at a certain extent although there was no significant difference. For practical reason, it is necessary to use anaesthesia before acupuncture stimulation is conducted on animals. The study may remind investigators to pay more attention to the influence of anaesthesia when they design and analyze experiment. In conclusion, the present study shows for the first time that acupuncture used in superovulation process can interact with hormonal medication to increase egg production significantly. It will not only provide a new concept for the improvement in the skill of supervulation in the assisted reproductive technology, but also will open new frontiers for the application of acupuncture as an adjuvant in the modern reproductive medicine. Furthermore, it is believed that it will serve as a useful reference for the research on other animals’ reproduction in the animal husbandry. As for such issues as the quality of eggs from superovulation, the mechanism of acupuncture effect, and whether acupuncture will increase the risk of the appearance of ovarian hyperstimulation syndrome (OHSS) accompanied by the increase in eggs, they require verification in the further studies. 42
References 1. Chandra A, Martinez GM, Mosher WD, et al. Fertility, family planning, and reproductive health of U.S. women: data from the 2002 National Survey of Family Growth. Vital Health Stat, 2005, 25(1):1-160. 2. Wright VC, Chang J, Jeng G, et al. Assisted reproductive technology surveillance-United States, 2004. MMWR Surveill Summ, 2007, 56(6):1-22. 3.
Freeman ML,Blacker CM. Tubal surgery versus in vitro fertilization: where do we draw the line? /Ransom SB, Dombrowski MP, Evans MI, et al. Contemporary Therapy in Obstetrics and Gynecology. U.S.A: W.B. Saunders Company, 2002:459-64.
4. Simoni M, Gromoll J, Nieschlag E. The follicle-stimulating hormone receptor: biochemistry, molecular biology, physiology, and pathophysiology. Endocr Rev, 1997, 18(6):739-73. 5.
Greenwald GS, Roy SK. Follicular development and its control. /Knobil E, Neill JD(eds). The Physiology of Reproduction, Vol. 1. New York: Raven Press, 1994:629-724.
6. Erickson G. Physiologic basis of ovulation induction. Semin Reprod Endocrinol, 1996, 14(4):287-97. 7. Mcnatty KP, Madris A, Osathamondh R. Effects of luternizing hormone on steroidogenesis by thecal tissue from human ovarian follicles in vitro. Steoids,1980, 36(1):53-63. 8.
Peng Y, Hou L, Wu X. Advances of modern studies of acupuncture and moxibustion for treatment of ovulation disorders. Chin Acupunct Moxib (in Chinese), 2006, 26(10):756-9.
9. Yu J, Zheng H, Ping S. Changes in serum FSH, LH and ovarian follicular growth during electroacupuncture for induction of ovulation. J Integr Tradit West Med (in Chinese), 1989, 9(4):199-202. 10. Mo X, Li D, Pu Y, et al. Clinical studies on the mechanism of acupuncture stimulation of ovulation. J Tradit Chin Med (in Chinese), 1993, 13(2):115-9. 11. Ma R, Ji P, Sha G, et al. Clinical observation of acupuncture and moxibustion for treatment of polycystic ovary syndrome. Chin Acupunct Moxib (in Chinese), 1996, 16(11):18. 12. Stener-Victorin E, Waldenst-m U, Nilsson L, et al. A prospective randomized study of electro-acupuncture versus alfentanil as anaesthesia during oocyte aspiration in in-vitro fertilization. Hum Reprod, 1999,14(10): 2 480-4. 13. Stener-Victorin E, Waldenstr-m U, Wikland M, et al. Electro-acupuncture as a peroperative analgesic method and its effects on implantation rate and neuropeptide Y concentrations in follicular fluid. Hum Reprod, 2003, 18(7):1 454-60. 14. Paulus WE, Zhang M, Strehler E, et al. Influence of acupuncture on the pregnancy rate in patients who undergo assisted reproduction therapy. Fertil Steril, 2002, 77(4):721-4. 15. Dieterle S, Ying G, Hatzmann W, et al. Effect of acupuncture on the outcome of in vitro fertilization and intracytoplasmic sperm injection: a randomized, prospective, controlled clinical study. Fertil Steril, 2006, 85 (4):1 347-51. 16. Westergaard LG, Mao Q, Krogslund M, et al. Acupuncture on the day of embryo transfer significantly improves the reproductive outcome in infertile women: a prospective, randomized trial. Fertil Steril, 2006, 85 (4):1 341-6. 17. Gejervall AL, Stener-Victorin E, Möller A, et al. Electro-acupuncture versus conventional analgesia: a comparison of pain levels during oocyte aspiration and patients’ experiences of well-being after surgery. Hum Reprod, 2005, 20(3):728-35. 18. Barad DH, Gleicher N. Increased oocyte production after treatment with dehydroepiandrosterone. Fertil Steril, 2005, 84(3):756. 19. Hiraoka J, Hirao Y. Fate of sperm tail components after incorporation into the hamster egg. Gamete Res, 1988, 19(4):369-80. 43
20. Li Z. Experimental acupuncture and moxibustion (in Chinese). Beijing: China Press of Traditional Chinese Medicine, 2002:327-9. 21. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction. Anal Biochem, 1987, 162(1):156-9. - D F R E V 6 / 0
H W ] J H U '
$
'
R G V R Q :
&
et al. Effect of age on response to human menopausal gonadotropin
stimulation. J Clin Endocrinol Metab, 1990, 71(6):1 525-30. 23. Van Kooij RJ, Looman CWN, Habbema JD, et al. Age dependent decrease in embryo implantation rate after in vitro fertilization. Fertil Steril, 1996, 66(5):769-75. 24. Zhang CY, Liu LJ, Di M, et al. Effects of the PMSG dosages on superovulation and oocyte quality in immature rats. Chin J Comp Med (in Chinese), 2007, 17(6):338-40, 344. 25. Tar1´n JJ, Pérez-Albalá S, Cano A. Cellular and morphological traits of oocytes retrieved from aging mice after exogenous ovarian stimulation. Biol Reprod, 2001, 65(1):141-50. 26. Song F, Zheng S, Ma D. Clinical observation on acupuncture for treatment of infertility of ovulatory disturbance. Chin Acupunct Moxib (in Chinese), 2008, 28(1):21-3. 27. Zhang F, Li C. The pattern identification and treatment of acumoxatherapy (in Chinese). Beijing: China Pharmaceutical Technology Publisher, 2000:349. 28. Yan H, Liu Y. A clinical overview on acupuncture-stimulating ovulation. Shanghai J Acupunct Moxib (in Chinese), 2005, 24(2):40-2. 29. Peters H. Some aspects of early follicular development. /Midgley Jr AR, Sandler WA(eds). Ovarian Follicular Development and Function. New York: Raven Press, 1979:1-13. 30. Oktay K, Briggs D, Gosden RG. Ontogeny of follicle stimulating hormone receptor gene expression in isolated human ovarian follicles. J Clin Endocrinol Metab, 1997, 82(11):3 748-51. 31. Irianni F, Hodgen GD. Mechanism of ovulation. Endocrinol Metab Clin North Am, 1992, 21(1):19-38. 32. Chen BY. Acupuncture normalizes dysfunction of hypothalamic-pituitary-ovarian axis. Acupunct Electrother Res, 1997, 22(2):97-108. 33. Hirshfield AN. Development of follicles in the mammalian ovary. Int Rev Cytol, 1991, 124:43-101. 34. Chun SY, Eisenhauer KM, Minami S, et al. Hormonal regulation of apoptosis in early antral follicles: follicle stimulating hormone as a major survival factor. Endocrinology, 1996, 137(4):1 447-56. 35. Ma XP, Dai M, Wu HG, et al. Effects of acupuncture on granulocyte apoptosis and expressions of apoptosisassociated genes in the ovary of perimenopausal rats. Chin Acupuct Moxib (in Chinese), 2007, 27(5):357-61. 36. David TB. The ovary. /Austin CR, Short FRS RV (eds). Reproduction in Mammals. Book 3: Hormonal Control of Reproduction. 2nd. ed. New York: Cambridge University Press, 1984:91-114. 37. Chen L. 42 clinical observations on the acupunture-stimulating follicle rupture. Chin Acupunct Moxib (in Chinese), 1997, 17(8):483. 38. Yang L, Zuo F. The clinical observation on acupuncture at Luanchao points for treatment of anovulation. Chin Acupunct Moxib (in Chinese), 1998, 18(10):585-6. (Received on December 9, 2008)
44