Articles

Those who received acupuncture increased their chances of conceiving by 65 percent.

February 8, 2008

Vittorio Hernandez – AHN News Writer

London, England (AHN) – Seven scientific trials among 1,366 women of different ages who found it difficult to conceive showed that having acupuncture at the same time the embryo was placed inside the womb during an in vitro fertilization procedure more than doubles the chance of the woman becoming pregnant.

The study was made by researchers at the University Of Maryland School Of Medicine and the VU University Amsterdam. It compared results of women who underwent acupuncture, those who were given fake needle treatments and those who had no extra therapy.

Those who received acupuncture increased their chances of conceiving by 65 percent, the study said. The British Medical Journal published the result of the medical breakthrough Friday.

While the study did not clearly explain how acupuncture aids fertility, experts theorized it could possible be the relaxing effect of acupuncture on the IVF procedure, considered extremely stressful.

Compared with repeated fertility treatment cycles which cost $7,785 (4,000 pound) per cycle in Britain, the acupuncture therapy is easier on the pocket.

One percent of births in the U.K. or 11,000 babies out of 32,000 IVF procedures are born every year in the U.K. The findings will be particularly significant for many western nations grappling with dwindling populations.

CONTROVERSY: ACUPUNCTURE

Acupuncture on the day of embryo transfer significantly improves the reproductive outcome in infertile women: a prospective, randomized trial

Lars G. Westergaard, M.D., Ph.D.,a Qunhui Mao, M.D.,b Marianne Krogslund,a Steen Sandrini,c Suzan Lenz, M.D., Ph.D.,a and Jørgen Grinsted, M.D., Ph.D.a

a Fertility Clinic Trianglen, Hellerup; b Holistic Acupuncture Clinic, Frederiksberg C; and c Sandrini Acupuncture I/S, Varde, Denmark

Objective: To evaluate the effect of acupuncture on reproductive outcome in patients treated with IVF/ intracytoplasmic sperm injection (ICSI). One group of patients received acupuncture on the day of ET, another group on ET day and again 2 days later (i.e., closer to implantation day), and both groups were compared with a control group that did not receive acupuncture.

Design: Prospective, randomized trial.

Setting: Private fertility center.

Patient(s): During the study period all patients receiving IVF or ICSI treatment were offered participation in the study. On the day of oocyte retrieval, patients were randomly allocated (with sealed envelopes) to receive acupuncture on the day of ET (ACU 1 group, n 95), on that day and again 2 days later (ACU 2 group, n 91), or no acupuncture (control group, n 87).

Intervention(s): Acupuncture was performed immediately before and after ET (ACU 1 and 2 groups), with each session lasting 25 minutes; and one 25-minute session was performed

Main Outcome Measure(s): Clinical pregnancy and ongoing pregnancy rates in the three groups.

Result(s): Clinical and ongoing pregnancy rates were significantly higher in the ACU 1 group as compared with controls (37 of 95 [39%] vs. 21 of 87 [26%] and 34 of 95 [36%] vs. 19 of 87 [22%]). The clinical and ongoing pregnancy rates in the ACU 2 group (36% and 26%) were higher than in controls, but the difference did not reach statistical difference.

Conclusion(s): Acupuncture on the day of ET significantly improves the reproductive outcome of IVF/ICSI, compared with no acupuncture. Repeating acupuncture on ET day ±2 provided no additional beneficial effect. (Fertil Steril 2006;85:1341- 6. ©2006 by American Society for Reproductive Medicine.)

Key Words: Acupuncture, ET day, IVF, pregnancy

Acupuncture is an ancient therapeutic art, which has been given renewed attention in light of recent scientific research and current integration with modern medical practice in the treatment of a wide range of diseases, including infertility.

The mechanisms through which acupuncture influence female fertility are believed to involve [1] central stimulation of -endorphin secretion (1), which in turn impacts on the GnRH pulse generator and thereby on gonadotrophin and steroid secretion (2, 3), and [2] a general sympathoinhibitory effect through increased blood flow to the uterus and ovaries (4), resulting in uterine conditions favoring implantation (for a recent review, see Chang et al. [5]).

Many reports in the literature claim positive effects of acupuncture in the treatment of female infertility, but only a few of them satisfy the requirements of rigorously conducted prospective, randomized trials (6). In a prospective, randomized study comparing electro-acupuncture and alfentanil as anesthesia during oocyte aspiration in IVF, Stener-Victorin et al. (7) found, unexpectedly, a significantly higher implantation rate and “take-home baby” rate per ET in the electroacupuncture group. Later and larger studies using electroacupuncture on the day of oocyte retrieval, however, did not confirm these positive effects on reproductive outcome (8, 9). Using conventional manual acupuncture on the day of ET, Paulus et al. (10) demonstrated a significantly increased clinical pregnancy rate in a group receiving acupuncture

TABLE 1

a) No ET owing to total failure of fertilization or poor embryo development.
b) Declined participation after randomization.
Westergaard. Acupuncture on ET day improves IVF outcome. Fertil Steril 2006.

(n = 80) as compared with a control group (n = 80) not receiving acupuncture.

Accepting the above notion that the positive effects of acupuncture on the day of oocyte retrieval and on the day of ET might be mediated through effects on local ovarian and uterine blood flow, enhancing the quality of the endometrium, we hypothesized that applying acupuncture 5 days after oocyte retrieval (i.e. closer to the day of implantation, 6-12 days after oocyte retrieval) might further optimize endometrial conditions for the embryo to implant.

In the present prospective study, women undergoing IVF/ intracytoplasmic sperm injection (ICSI) treatment were randomly allocated to one of three groups: [1] no acupuncture (control group), [2] acupuncture on the day of ET (i.e., 3 days after oocyte retrieval) (ACU 1 group), and [3] acupuncture on the ET day as above and again 2 days later (i.e., 5 days after oocyte retrieval) (ACU 2 group).

The aims of the study were to evaluate the effects of acupuncture on the reproductive outcome of IVF/ICSI treatment by comparing the rates of positive pregnancy tests, clinical pregnancy, and ongoing pregnancy/delivery in these three groups.

MATERIALS AND METHODS
Setting and Design
This prospective, randomized trial was carried out in a large, private IVF clinic in Copenhagen, Denmark from March 1, 2003 to June 30, 2004. During that period, all couples admitted to the clinic for IVF or ICSI treatment of infertility were consecutively invited to participate. At the start of hormonal stimulation for IVF/ICSI, all patients were informed orally and in writing about the aims and practical details of the project, and willingness to participate was confirmed in writing. The study was approved by the institutional review board of the cities of Copenhagen and Frederiksberg (no. 01-203/02).

By design, the study population comprised an unselected average of couples seeking infertility treatment in our clinic. The only inclusion criterion for participation in the study was the couples’ consent to be randomized to one of three groups (see below), independent of infertility diagnosis, number of previous ART attempts, and hormonal treatment in the actual cycle. Patients who after randomization did not achieve ET or who for personal reasons did not want to participate further were excluded from the study (Table 1).

On the basis of the data previously published by Paulus et al. (9), combined with an average 25% clinical pregnancy rate per ET in our clinic during the previous 5 years, power calculations (Medcalc software, Mariakerke, Belgium) anticipated that a significant difference in clinical pregnancy rate of 11% between no acupuncture and acupuncture would require approximately 100 patients in the control group and 200 patients in the acupuncture groups.

Randomization

During the study period of 16 months, a total of approximately 1000 couples underwent IVF or ICSI treatment in our clinic. Of these, a total of 300 couples accepted participation in the study and were randomized to one of three groups by the drawing of a sealed envelope on the day of oocyte retrieval. After randomization, 27 patients were excluded for various reasons (Table 1). Of the remaining 273 patients, 87 were allocated to no acupuncture (control group), 95 to acupuncture on the day of ET (ACU 1 group), and 91 to receive acupuncture on the day of ET and 2 days later.

Acupuncture The acupuncture points used in the present study were, as in the study by Paulus et al. (10), chosen in agreement with the concepts of traditional Chinese medicine. According to these, the kidney system dominates the reproductive system, the liver (LR) regulates Qi (vital force and energy), and spleen (SP) and stomach (ST) are sources of Qi and blood. Spleen 6 (SP6) is the crossing point of the spleen, kidney, and liver meridians and is considered the key point in treating infertility. Needling SP6, SP8, SP10, ST36, and ST29 aims to provide improved blood perfusion and more energy to the uterus. Large intestine 4 (LI4) and LR3 are the so-called “four gates points,” which are commonly used to open relevant meridians and calm the mind. Combining them with pericardium 6 (PC6) and DU20 would relax the patient.

In the ACU 1 group, acupuncture was given on the day of ET (i.e., 3 days after oocyte retrieval) in two sessions lasting 25 minutes immediately before and after ET. Acupoints before ET included DU20 (Baihui), ST29, SP8, PC6, and LR3. Acupoints after ET were ST36, SP6, SP10, and LI 4.

Needles were inserted into the above points and manipulated until needle-sensation was obtained, (i.e., Deqi-a feeling of, for example, soreness or numbness, distension or pain). After 10 minutes of retention, the needles were again manipulated to maintain Deqi. Fifteen minutes later, the needles were removed.

For the ACU 2 group, the same acupuncture protocol as for the ACU 1 group was applied on the day of ET. In addition, this group received one acupuncture session of 25 minutes’ duration 2 days after ET (i.e., 5 days after oocyte retrieval), to the following acupoints: DU20, Ren 3, ST29, SP10, SP6, ST36, and LI 4. The acupoints chosen for this session aimed at general relaxation and improvement of uterine blood perfusion, to further enhance endometrial receptivity for implantation. Manipulation and retention was the same as in the previously described procedure.

The patients in the control group followed the clinic’s routine procedure (i.e., had bed rest for 1 hour after ET before leaving the clinic).

All acupuncture procedures in the present study were administered by nurses who, before the initiation of the project, were instructed carefully by two professional acupuncture practitioners (Q.M. and S.S.), who supervised the procedures by frequent visits throughout the study period. One of the nurses (M.K.), who was working daily in the clinic, performed approximately half of all acupunctures (94 of 186), whereas the other eight nurses, assisting during weekends and holidays, performed from 3 to 24 acupunctures each.

Hormone Treatments and IVF/ICSI Procedures
Apart from the acupuncture, all patients were treated according to well-established standard regimens of the clinic. These included [1] long protocol GnRH agonist down-regulation from the midluteal phase, followed by gonadotropin stimulation after down-regulation had been ascertained by ultrasound and serum E2 levels

200 pmol/L, or [2] a short protocol including gonadotropin stimulation from day 2 of the cycle combined with a flexible antagonist protocol, or [3] in a few cases, no hormone stimulation at all.

In all cases, an ovulatory dose of hCG (Pregnyl; Organon, Skovlunde, Denmark) was administered 36 hours before oocyte retrieval. Oocytes were retrieved by ultrasoundguided transvaginal aspiration with automated suction. In cases of male factor or idiopathic infertility, ICSI was used for fertilization. A maximum of three embryos was transferred back to the uterus after 3 days of culture. Surplus transferable embryos (i.e. more than six even blastomeres and

20% fragmentation) were cryopreserved.

Luteal support was given to all patients, administered as intravaginal P pessaries (Cyclogest; Alpharma, Barnstaple, United Kingdom; 400 mg three times daily) and oral E2 tablets (Nycomed Danmark, Roskilde, Denmark; 2 mg twice daily) from the day of ET until 12 to 13 days after ET, when a pregnancy test was performed by measurement of serum hCG. Patients with a positive pregnancy test result (serum hCG

10 IU/L) were scanned by ultrasound 3 weeks later, and a clinical pregnancy was diagnosed by the presence of at least one intrauterine gestational sac on that occasion. An ongoing pregnancy was defined as the presence of a viable intrauterine fetus beyond 12 weeks’ gestation.

Statistical Methods
Data were expressed as mean ± SEM. Student’s t-test was used to test for possible imbalances between the groups regarding the following variables: patient age, body mass index, duration of infertility, mean number of stimulation days, consumption of FSH during stimulation, and mean number of oocytes retrieved, fertilized, cryopreserved, and transferred, and number of transferable embryos. Fisher’s exact test was applied to compare frequencies between groups, such as rates of pregnancy, clinical pregnancy, and ongoing pregnancy and delivery. A P value

.05 was considered significant.

RESULTS
A total of 273 women were included in the study (Table 1). Of these, 87 were allocated to no acupuncture (control group), 95 to acupuncture on the day of ET only (ACU 1 group), and 91 to acupuncture on the day of ET day and again 2 days later (ACU 2 group). Demographic characteristics were comparable among the groups, with no significant differences with regard to age (median, 37 years; range, 24-45 years), body mass index, duration of infertility, proportion of primary fertility, number of previous IVF/ICSI attempts, and cause of infertility (Table 2). The distribution of ovarian stimulation regimens (i.e., no hormonal stimulation or short [antagonist] protocol or long [agonist] protocol) was not significantly different among the three groups (no stimulation: 2%, 1%, and 3%; short protocol: 24%, 19%, and 18%; long protocol: 74%, 80%, and 79% in the control, ACU 1, and ACU 2 groups, respectively). In addition, the mean (±SEM) number of stimulation days and mean (±SEM) total consumption of gonadotropin in the three groups was similar (stimulation days: 11.2 ± 0.3, 11.5 ± 0.2, and 11.6 ± 0.3; total consumption of gonadotropin (IU): 2543 ± 118, 2598±103, and 2660±123, respectively, in the control, ACU 1, and ACU 2 groups).

Table

Westergaard. Acupuncture on ET day improves IVF outcome. Fertil Steril 2006.

Table 3 shows the mean (±SEM) number of oocytes retrieved and fertilized and the number of transferred and transferable embryos in the three groups. There were no significant differences between the groups.

The reproductive outcomes in the three groups are shown in Table 4. Of the 273 women, 100 (37%) became pregnant; of these, 91 (33%) had clinical pregnancy and 77 (28%) had ongoing pregnancy (beyond 12 weeks’ gestation) or delivery. The rates of positive pregnancy test results, clinical pregnancies, and ongoing pregnancy or delivery were all significantly higher in the ACU 1 group than in the control group (positive pregnancy test: 40 of 95 (42%) vs. 24 of 87 (28%), P.044; clinical pregnancy: 37 of 95 (39%) vs. 21 of 87 (24%), P.038; ongoing pregnancy or delivery: 34 of 95 (36%) vs. 19 of 87 (22%), P.049). The numbers and rates of positive pregnancy tests, clinical pregnancy, and ongoing pregnancy or delivery in the ACU 2 group were all higher than in the control group, but none of these differences were statistically significant. The rate of early pregnancy loss (expressed as percentage of positive pregnancy tests) was higher in the ACU 2 group (33%) than in the control (21%) and ACU 1 (15%) groups, but the differences were not statistically significant.

DISCUSSION
This prospective, randomized study demonstrates that acupuncture administered on the day of ET significantly improves the reproductive outcome in women undergoing IVF or ICSI treatment for infertility. Thus, our results confirm and extend those of the only comparable prospective, randomized study previously reported (10). The acupuncture procedures used in the ACU 1 group of the present study

TABLE 3

Note: Data are mean ± SEM unless otherwise noted.
Westergaard. Acupuncture on ET day improves IVF outcome. Fertil Steril 2006.

TABLE 4

Data are n (%), unless otherwise noted. Fisher’s exact test (two-tailed):
[a] P = .044.
[b] P = .038.
[c] P = .049.

Westergaard. Acupuncture on ET day improves IVF outcome. Fertil Steril 2006.

were very similar to those used in the Paulus et al. study (10), except for the additional use of auricular acupuncture in the latter.

In the present study, an additional group of patients were randomized to receive acupuncture twice, on the day of ET and on ET day ±2 (ACU 2 group). Although the clinical and ongoing pregnancy rates were higher in the ACU 2 group than in the control group, the differences did not reach statistical significance. However, this might relate to the relatively small size of the groups. Combining the acupuncture groups resulted in a significant improved reproductive outcome as compared with the control group, suggesting a beneficial effect on the day of ET, whereas acupuncture on ET day ±2 (i.e. closer to the day of implantation) was without additional beneficial effect.

With the application of modern Western scientific principles, the underlying physiologic mechanisms of acupuncture are now increasingly being documented (5). Effects of acupuncture in relation to female infertility might be mediated through central effects on the release of neurotransmitters, including -endorphin and serotonin, which in turn influence GnRH release and thereby impact on pituitary gonadotropin secretion, ovarian follicular growth, ovulation, and fertility (2, 3, 5).

In addition to this central effect on the hypothalamic- pituitary- ovarian axis, acupuncture exerts a general sympathoinhibitory effect, which locally might reduce uterine artery impedance and thus increase uterine and ovarian blood flow. With Doppler ultrasound, this effect of serial electroacupuncture was documented in 10 infertile women who were down-regulated by GnRH analogue to avoid the effect of endogenous hormones (4). It was proposed that the effect on uterine blood flow might improve the growth and thickness of the endometrium, rendering it more receptive to implantation of the early embryo (4). This notion seemed supported by the results of a later prospective, randomized study by the same group, in which electro-acupuncture was compared with alfentanil for analgesia during oocyte retrieval. Although the effect on fertility was not the primary objective of that study, a significantly higher implantation rate and ongoing pregnancy rate was found in the electroacupuncture group as compared with the alfentanil group (7). Later and larger prospective, randomized studies by the same group, however, were not able to confirm this positive effect on the reproductive outcome of electro-acupuncture administered on the day of oocyte retrieval (8, 9).

Measuring uterine artery pulsatility index on the day of acupuncture (i.e. the day of ET), Paulus et al. (10) could not demonstrate significant differences between the acupuncture and control groups, although as mentioned above there was a significant difference in reproductive outcome between the two groups (10). The discrepancy between this finding and the above might relate to differences in setup (i.e., electroacupuncture vs. manual technique and administration of acupuncture on the day of oocyte retrieval vs. the day of ET).

In the present study, we did not try to measure uterine blood flow during ET or before, and the present results therefore do not contribute to the discussion regarding whether the positive effects of acupuncture could be attributed to improved endometrial blood flow (and oxygen tension?). Another possibility could be that acupuncture indirectly, through effects on ovarian and endometrial blood flow, or directly impacts on local humoral factors (hormones, peptide growth factors) that are involved in the regulation of implantation. Experiments with administration of acupuncture during the preovulatory phase of the menstrual cycle have shown that the amount of LH and P in the circulation is increased after needling, and 2-6 hours later the LH peak occurs (11). Whether such effects of acupuncture are also reflected in variations in the circulating levels of hormones and other substances in the luteal phase is not known, but the question is being approached in an ongoing study in our clinic.

The above-described physiologic, neuroendocrine effects of acupuncture do not rule out that psychological factors (reduction of stress) or placebo effects of the acupuncture procedure might significantly contribute to improve female infertility. Acupuncture certainly has the attributes of a good placebo (Oriental mystique, skin penetration, novelty). Use of placebo acupuncture has been controversial owing to difficulties in designing a method not affecting the acupoints (12). Recently, however, a promising placebo acupuncture technique validated in a prospective, randomized study was described (13), but to our knowledge it has so far not been used in studies on acupuncture effects in female infertility.

In the present study, placebo acupuncture was not used in the control group, and it could be argued that the improvement of the reproductive outcomes found in the acupuncture group could be ascribed to a placebo effect. A number of observations in this study, however, tend to argue against placebo effects explaining the significant differences in reproductive outcomes between the acupuncture and control groups. For instance, if placebo effects were significantly associated with the atmosphere around the acupuncture procedures, one might expect that administering acupuncture in more sessions on different days, as in the ACU 2 group, would further improve the reproductive outcome, but this was not the case. In addition, in a post hoc analysis of our data, we related the reproductive outcomes to acupuncture and to the age of the patients below and above the median of the whole population (37 years). We found that the significantly higher clinical and ongoing pregnancy rates in the ACU 1 group compared with the control group were restricted to patients younger than 38 years (clinical and ongoing pregnancy rates in controls 23% (8 of 35) and 20% (7 of 35) vs. 49% (26 of 53) and 47% (25 of 53) in the ACU 1 group; P.015 and .012, respectively). By contrast, in women aged ±38 years the outcomes were not significantly different (controls: 25% (13 of 52) and 23% (12 of 52) vs. 26% (11 of 42) and 21% (9 of 42), respectively). Although these age-related differences are difficult to explain, they can hardly be ascribed to placebo effects of acupuncture.

Notwithstanding these arguments against a significant influence of placebo on our results, we acknowledge that these beneficial effects of acupuncture in assisted reproductive technologies ought to be confirmed in future prospective, randomized trials including a control group subjected to a reliable, reproducible placebo acupuncture technique, for instance the one recently reported by Park et al. (13).

In conclusion, the present study confirms that acupuncture administered on the day of ET significantly improves the reproductive outcome of IVF/ICSI. It is also concluded that adding acupuncture on ET day ±2 (i.e., closer to the day of implantation) does not further improve the reproductive outcome.

To finally settle the role and relevance of acupuncture in fertility treatment, future prospective, randomized trials including use of a good placebo acupuncture technique are needed.

REFERENCES
1. Petti F, Bangrazi A, Liguori A, Reale G, Ippoliti F. Effects of acupuncture on immune response related to opoid-like peptides. J Tradit Chin Med 1998;18:55- 63.

2. Ferin M, Van de Wiele R. Endogenous opioid peptides and the control of the menstrual cycle. Eur J Obstet Gynecol Reprod Biol 1984;18: 365-73.

3. Petraglia F, Di Meo G, Storchi R, Segre A, Facchinette F, Szalay S, et al. Proopiomelanocortin-related peptides and methionin enkephalin in human follicular fluid: changes during the menstrual cycle. Am J Obstet Gynecol 1987;157:142- 6.

4. Stener-Victorin E, Waldenström U, Andersson SA, Wikland M. Reduction of blood flow impedance in uterine arteries of infertile women with electro-acupuncture. Hum Reprod 1996;11:1314 -7.

5. Chang R, Chung PH, Rosenwaks Z. Role of acupuncture in the treatment of female fertility. Fertil Steril 2002;78:1149 -53.

6. White AR. A review of controlled trials of acupuncture for women’s reproductive healthcare. J Fam Plan Reprod Health Care 2003;29: 233-6.

7. Stener-Victorin E, Waldenström U, Nilsson L, Wikland M, Jansson P. A prospective randomised study of electro-acupuncture versus alfentanil as anaesthesia during oocyte aspiration in in-vitro fertilization. Hum Reprod 1999;14:2480-4.

8. Stener-Victorin E, Waldenström U, Wikland M, Nilsson L, Hägglund L, Lundeberg T. Electro-acupuncture as a peroperative analgesic method and its effects on implantation rate and neuropeptide Y concentrations in follicular fluid. Hum Reprod 2003;18:1454-60.

9. Humaidan P, Stener-Victorin E. Pain relief during oocyte retrieval with a new short duration electro-acupuncture technique-an alternative to conventional analgesic methods. Hum Reprod 2004;19:1367-72.

10. Paulus WE, Zhang M, Strehler E, El-Danasouri I, Sterzik K. Influence of acupuncture on the pregnancy rate in patients who undergo assisted reproduction therapy. Fertil Steril 2002;77:721- 4.

11. She Y. Research on mechanism of acupuncture and herbs promoting ovulation. J Combination TCM Western Med 1985;4:210.

12. Stener-Victorin E, Wikland M, Waldenström U, Lundeberg T. Alternative treatments in reproductive medicine: much ado about nothing. Hum Reprod 2002;17:1942- 6.

13. Park J, White A, Stevinson C, Ernst E, James M. Validating a new non-penetrating sham acupuncture device: two randomised controlled trials. Acupunct Med 2002;20:168 -74.

Acupuncture Normalizes Dysfunction of Hypothalamic-Pituitary-Ovarian Axis By Bo-Ying Chen M.D. Professor of Neurobiology

Institute of Acupuncture and Department of Neurobiology
Shanghai Medical University, Shanghai 200032, P.R. China
(Received June 3, 1997; Accepted with revisions June 30,1997)

ABSTRACT

This article summarizes the studies of the mechanism of electroacupuncture (EA) in the regulation of the abnormal function of hypothalamic pituitary-ovarian axis (HPOA) in our laboratory. Clinical observation showed that EA with the effective acupoints could cure some anovulatory patients in a highly effective rate and the experimental results suggested that EA might regulate the dysfunction of HPOA in several ways, which rneans EA could influence some gene expression of brain, thereby, normalizing secretion of some hormones, such as GnRH, LH and E2. The effects of EA might possess a relative specificity on acupoints.

KEY WORDS: Electroacupuncture, ß-Endorphin, GnRH, LH, Estradiol, Estrogen receptor, Ovariectomized rat, Hypothalamic-pituitary-ovarian axis

INTRODUCTON

Acupuncture is a treasure of Chinese traditional medicine, which is employed in the treatment of different diseases, especially in relief of all kinds of pain [1, 2] over the world. Since 1960s we have used acupuncture with appropriate electro-stimulation to cure patients with anovulation disorder (sterility), the rate of EA induction of ovulation was increased from 50% initially to 80% presently. Other authors in China also reported that acupuncture was successfully to treat patients with sterility [3] and the lying-in woman with subnormal contraction of uterus [4]. All the above research demonstrates that acupuncture may be an effective curative method of some woman’s diseases. However, many questions, such as “why”, “how to” and “which” about the mechanism of EA effect are unknown. To address these problems we supposed that EA might influence the production and secretion of hormones, neurotransmitters or neuro-modulators of HPOA leading to the normalization of hormone status. We also noticed certain artides reported that EA might affect the blood levels of LH, FSH, estradiol (E2) and prolactin in the female patients [4, 5, 6] and EA may be related to long term changes in gene expression [7, 8]. These results are all significant, yet insufficient to explain the mechanism of EA in the regulation of the function of HPOA. To obtain more data, a series of experimental studies in human and animal models has been performed in our laboratory.

MATERIALS AND METHODS

Selection and treatment of cases
Ten cases of chronically anovulatatory patients including eight cases of polycystic ovarian disease (POCA), one case of hypogonadotropic amenorrhoea and one case of oligomenorrhea were treated with EA in 13 menstruation cycles. They were all of productive age and the courses of disease were 3 to 12 years. On the 10th day of each menstruation cycle, the patients accepted the EA treatment. “Guanyuan(RN4),” “Zhongji(RN3),” “Sanyinjiao(SP6),” and bilateral “Zigong(EXCA1)” points were stimulated for 30 min at 8:00 AM, Q.D. for 3 days. The stimulation parameters were 7-8mA and 4-5 Hz with G6805 model generator. The electric current of EA was bearable well for every patient. The blood samples were collected from forearm of the patients one time per 15 min for detection of FSH.LH and ß-endorphin (ß-E).

Five health volunteers of a productive age with normal menstruation cycle were selected as controls, which were undergone the same treatment as above mentioned.

Animals and treatments
Wistar female rats weighting 200-250g were used. The half of animals were undergone ovariectomy and fed in the same environment with the intact rats at least for 15 days and vaginal smears were examined per day for 3 times. No exfoliative epithelium cell was found in the smears as an index for successfill ovariectomy. The ovariectomized rats and intact rats were randomly divided into two groups respectively: ovariectomized rat group (OVX), ovariectomized rat accepted EA treatment group (OVX+EA), intact rat group (INT) and intact rat accepted EA treatment group (INT+EA). The animals in OVX+EA and INT+EA received EA at the experimental acupoints of Guanyuan (RN4), Zhongji (RN3), Sanyinjiao (SP6) and bilateral Zigong (EXCA1) by EA apparatus (Model G6805-2, SMIF, Shanghai, China) with the frequency of 3 Hz and an intensity to produce a slight twitch of the limbs. After 3 days’ treatment animals were given EA at Waiguan (SJ5) and Huatuojiaji (EXTRA21) as the control acupoints in the same way (Fig 1). By the end of last experiment, animals were sacrificed and their adrenals, brains and pituitaries were taken out for detection of nucleolar oganizer regions (AgNORs) and hormones.

Pushpull perfusion in hypothalamic preoptic area (POA) and elution of pituitary and LH and ß-endorphin (ß-EP)

The technique of brain pushpull perfusion was processed as previously described by our laboratory [1]. The perfusate from hypothalamic POA was kept at -70°C for GnRX and ß-EP RIA.

The pituitaries were retrieved and put into 4°C cooled saline. Afterward, each pituitary was homogenized with 500µl of 70% acetone aqueous solution at 4°C. The homogenate was centrifugalized (2,000xg for 15 min at 4°C) and the supernatant was freeze-dried for LH and ß-EP RIA.

Radioimmunoassay (RIA) of hormones GnRH IRA: GnRH content in the perfusate from rat hypothalamus was determined by RIA method developed by Nett in 1973 [9]. GnRH was iodinated by the modified chlomine-T technique[10]. Na125 I was manufactured by Radiochemical Center, Amersham.

ß-EP RIA: The sensitive radioimmunoassay was a routine in our laboratory [1]. The standards of human and rat ß-EP was synthesized by Peninsula Laboratories, Inc. and the rabbit antiserum of both ß-EP was developed in our laboratory. The cross-reaction from human ß-EP and camel ß-EP was detected about 20%. The sensitivity of this method was 10pg/tube.

LH, E2 and corticosterone RIA: LH, E2 and corticosterone RIA kits were bought from Shanghai Institute of Biologic Products, the Ministry of Health, P.R. China. All procedures of RIA were performed as described in the kit manuals.

Fig. 1 A: Sketch of ventral view (left) and dorsal view (right) of rat shows the acupoints we used
B:Diagram shows the electroacupuncture procedures in conscious rat

Staining techniques: Vaginal smears were fixed by 100% ethyl alcohol, then stained with HE method. Adrenal sections were cut in 4µm thickness from paraffin blocks and processed with silver nitrate staining technique[11]. In each case, one hundred cells in zona fascicula were examined randomly under 100-fold oil immersion lens. Numbers and sizes of AgNOR dots were counted and measured.

C-fos protein immunohistochemistry: The inmunohistochemical analysis of c-fos expression in rat brain was perforrned as previously described[11].

Estrogen receptor (ER) protein immunohistochemistry (ABC method): Under sodium pentobarbital anesthesia (50 mg/kg, ip), the animals were perfused via left cardiac ventricle with 100ml of phosphate-buffered saline (PBS), followed by 300ml ice-cold fixative containing 4% paraformaldehyde in 0.1 M phosphate buffer (pH7.4). Afterwards, brain was removed with the same fixative for one day and immersed in 0. lM phosphate buffer containing 30% sucrose for another day. The hypothalamus blocks were frozen with dry ice and cut into 35 µM thick section by cryostat. The brain sections were washed with 0.01M PBS for 15min x 3 and incubated in 0.01M PBS containing 0.5% Triton 100 and 3% normal goat serum (NGS) at 37°C-for one hour. Afterwards, the sections incubated in 1:1,000 ER monoclonal antibody (H222, Abott Co.) at 37°C for one hour, then at 4°C for two days. The sections, washed in PBS three times, were processed by ABC kit (from Vecot Labs) induding sequential incubation at 20°C in the following solutions with washes between them. (1). second antibody (dilution 1:100), 30min. (2). A+B reagents (dilutionl:100), 60min. (3). 0.05% diaminobenzidine/ 0.02% hydrogen peroxide in 0.1M Tris- HCI buffer (pH 7.2) 10min. The sections were washed in tap water, mounted and examined under light microscope. The certain areas of typical immunoreactive positive neurons were measured by computer image analysis system (Vecta PC).

ER mRNA hybridization: The total mRNA of brain was eluted by the modified phenol method [12]. ER cDNA probe (244bp) was labeled by the DlG-labeling kit (from Bohringman Co., Germany). The dot blot hybridization was processed as the method described by Sambrook J and his colleagues [13]. The dot blot images were analyzed with gray density by computer imaging analysis software (TJTY-300, from Tong -Ji university, Shanghai, China).

Statistics: All data in this paper were treated with analysis of variation (ANOVA), least significant difference (ISD) or student T-test.

RESULTS

Effect of EA on ovulatary induction and curing sterility in woman

After EA the blood ß-EP level of the patients resulting in ovulation either declined or maintain at the levels within the range of the normal levels and the ß-EP levels of those failing to show ovulation were significantly higher than the normal’s’ (table 1). On the other hand, the blood LH and FSH levels of the patients with ovulation after EA treatment tended to be the normal [14].

Table 1. Change of blood ß-EP level before and after EA (pg/ml)

The values in this table are mean±SE, *P<0.05

Effect of EA on dysfunction of HPOA in ovariectomized rats For a further study of the mechanism of EA effect on HPOA a series of experiments in the animal models was performed.

(1). EA induces maturation and exfoliation of vaginal epithelium cell and enhances blood level of E2.
After ovariectomy two weeks late, the exfoliated epithelium cell disappeared from the vaginal smears of the rats, but it reappeared in the smears following EA treatment. The blood level of E2 in OVX was increased significantly (table 2). No obvious change was seen in INT after EA treatment and in OVX following EA treatment with the control acupoints.

Table 2. The level of blood E2 following EA treatment (pg/ml)

*P < 0.05 compared with INT, **P<0.01 compared with before EA

(2). EA promotes enlargement of adrenals and enhances activity of adrenal AgNORs as well as blood level of corticosterone
We found the adrenals of OVX+EA were enlarged and the weight of the adrenals was raised significantly. Using histochemical method, the AgNORs of the cells in inner adrenal cortex were examined. The result shows that the activity of AgNORs of OVX was enhanced (table 3, 4), and the level of blood corticosterone in OVX+EA was also increased (table 5). There were no similar effects in INT following EA treatment and in OVX after EA with control acupoints.

Table 3. AgNORs number in OVX and INT

*P < 0.01 tested with ANOVA

Table 4. Weight of adrenal

*P < 0.01 tested with ANOVA

Table 5. The levels of blood corticosterone in OVX and lNT (mean ± SE, ng/ml)

*P < 0.001 compared with before EA

(3). EA decreases the level of hypothalamic GnRH, pituitary LH and increases the contents of hypothalamic and pituitary ß-endorphin
After EA treatment the levels of GnRH released from hypothalamus was rnarkedly decreased however, the ß-endorphin (ß-EP) secretion in hypothalamus was raised. The pituitary content of LH was also fallen, but the ß-EP of pituitary was increased, as well as peripheral LH and ß-EP level (Fig.2).

Fig. 2Change of hypothalarnic GnRH and ß-EP, pituitary LH and ß-EP, blood LH and ß-EP before and after EA

Effect of EA on brain c-fos expression in ovariectomized rats
The area occupied by FOS protein labeled neuron was detected in medial preoptic nucleus (MPN), lateral preoptic nucleus (LPN), suprachiasmatic nucleus (SCN), paraventricular nucleus of the hypothalamus (PAVN), medial amygdala nucleus (MAN), periventricular nucleus of the hypothaLsmus (PVN), ventromedial nucleus of the hypothalamus (VNH) and arcuate nucleus (AR) 4 hours after ovariectomy (fig. 3a). The C-fos immunoreactive labeled neurons disappeared two weeks later following ovariectomy. The rats recovering for more than two weeks after ovariectomy, were received EA treatment. Many specific FOS labeled cells were observed in LPN, VNH, SCN and especially in POA, ARN, and PVN, but not any labeled neuron could be found in MAN. No obvious C-fos expression was shown in those nuclei in INT and INT+EA (fig. 3b).

Fig. 3a C-fos immunocytochemistry neurons distribution after ovariectomy

Fig. 3b C-fos expression labeled neurons following electroacupuncture

Effect of EA on expression of ER protein and ER mRNA in rat brain Estrogen receptor (ER) immunoreactive neurons were observed widely in rat brain with immunohistochemical technique, especially in MPN, ARN and VNH. The above nuclei were measured by computer image analysis system, and the results show that the mean gray density in OVX+EA was decreased apparently compared with that in OVX. Whereas there were no obvious changes of gray density levels in INT and INT+EA (fig, 4).

Fig. 4 Effect of EA on expression of ER protein in rat brain (Immunohistochernistry of monoclonal antibody) *p < 0.01 compared with OVX

The dot blot indicated that ER mRNA expression was increased about 48.11% in OVX compared with INT. The gray density of OVX was 129.75 ± l2.l3 and that in OVX+EA was 199.25 ± 5.75 attenuated significantly (Fig. 5). The gray density level in INT was 87.60 ± 5.91, and the level in INT+EA was 83.60 ± 4.83. There was no significant difference between INT and INT+EA

Fig. 5Effect of EA on expression of ER mRNA in rat brain (dot blot) *** p < 0.01 compared with OVX

DlSCUSSION

Since 1985 we have observed that the effect of EA ovulatary induction might relate to the hand skin temperature (HST) and the blood level of ß-EP [14]. On the other hand, after EA the blood FSH and LH levels of the patients who successfully ovulated either declined or maintained at normal. In general, provided that body temperature was normal and the environmental temperature was constant round 25°C, the HST may reflect the state of sympathetic system of a patient. These results suggest that in anovulatary cases the hyperactive sympathetic system can be depressed by EA and the function of HPOA can be regulated by EA through central sympathetic system. Moreover, EA may mediate the abnormal function via the influence on the secretion of the hormones in the different Level of HPOA.

To gain more evidences, we designed some animal experiments to explain the mechanism of EA effects on HPOA at the whole, cellular and molecular levels. We found that EA can induce maturation and exfoliation of vaginal epithelium cell in OVX rat. It is known that maturation and exfoliation of vaginal epithelium cells are a reaction dependent on estrogen level. So we determined the level of blood E2 in OVX and OVX+EA. The result shows the level of blood E2 in OVX was lower than that in normal, but it was increased significantly after OVX accepted EA treatment with the experimental acupoints. This result suggests EA might promote the activity of the compensative mechanism to elevate the subnormal level of E2 induced by ovariectomy in rats.

What is this compensative mechanism? To resolve this question, we considered that adrenal is the main organ to secrete sexual hormones except ovarian in females and observed the adrenals of the animals in four groups. The results show that the mean weight of the adrenal in OVX+EA was higher than that in OVX, INT and INT+EA, suggesting the adrenal function might be activated by EA. Subsequently, we detected that the number of AgNORs in zona fasciculata of OVX+EA was significantly increased. Nucleolar organizer regions (NORs) are loops of DNA, which possess ribosomal RNA (rRNA) genes. They are of vital significance in the ultimate synthesis of protein. Thus, the number and configuration of AgNORs (NORs stained by silver staining method) may reflect the activity of cell differentiation and transcription of nucleolar rDNA [15]. In the same time we found the content of blood corticosterone in OVX+EA was raised markedly, but there was no change of blood corticosterone in OVX, INT and INT+EA. This result provided a further evidence that the adrenal cortex cells were initiated in OVX+EA.

The results including the changes of GnRH releasing from hypothalamus and of the pituitary and blood LH contents suggest that the effects of acupuncture in the regulation of HPOA may be exerted via to promote the function of hypothalamic pituitary-adrenal axis (HPAA), increasing the synthesis and secretion of adrenal steroid horrnones, the androgen of which then be transformed into estrogen in other tissues and thereby reset the negative feedback of estrogen to HPOA. Moreover, EA may accelerate the release of brain and pituitary ß-EP to inhibit the overnormal secretion of GnRH and LH that may be normalized.

Recently immunohistochemical analysis of the expression of oncogene c-fos ABl was induced by variety of stimuli [16, 17]. This represents a new method for mapping neuronal activity at the cellular level [18] and thus functionally and systematically tracing neuronal pathway in the nervous system (C NS) [19]. We used this method to examine the distribution of FOS labeled neuron in CNS for recovery of more evidences that EA may alter the neuroendocrine function of HPOA in ovariectomized rats in cellular and gene level. The results show that the specific FOS labeled neurons were observed especially in POA, ARN and PVN in OVX following EA treatment. In above nuclei there were a high concentration of GnRH and ß-EP neuron [20]. These results suggest this fact that the expression of FOS labeled neurons reappeared in above mentioned areas following EA treatment in ovariectomized rats may be related to the changes of GnRH and ß-EP from rat hypothalamus after EA treatment.

The level of estrogen in the body may regulate the expression of ER, which may by down-regulated following increase of estrogen level and up-regulated after decrease of estrogen [22]. Our finding that after decline of blood E2 induced by ovariectomy the expression of ER was increased and the expression of ER was inhibited by EA inducing the elevation of blood E2 are in accordance with these reported results. ER existing in the brain, especially in POA, ARN and VHN may mediate the function of neuroendocrine system [22, 23]. Thus, our observations suggest that the influence of EA on the change of ER expression in brain may be one of further mechanisms of EA normalizing the dysfunction of HPOA.

INT rats as experimental control we adopted were all of in the stage of preestrus and estrus because the animal sexual hormes and brain ER expressions were changed with the sexual cycle [24]. All INT rats were selected to fix in the two stages there may be a relative constant comparability.

Our results show no same effects were seen after EA treatment in INT and following EA with control acupoints in OVX, suggesting that EA may possess a relative specificity on acupoint and the effect of EA may be a kind of normalization.

CONCLUSION

Our observations reveal that acupuncture may regulate the abnormal function of HPOA in many ways, which means that acupuncture may activate C-fos expression of brain, then a long term changes at molecular level would start, following the regulation of gene expression in FOS relative gene, such as ER mRNA and GnRH mRNA involved. On the other hand, EA may promote the activity of the body compensative mechanisms, then the levels of hormones, such as GnRH, LH, estrogen and so on would be normalized. The effect of acupuncture on regulating the function of HPOA may possess a relative specificity of acupoint. Moreover, our clinical and animal experimental results suggest that it is necessary for obtaining a satisfactory effect that proper stimulation should be about thirty minutes Q.D. for three days. This suggestion provides a successful consideration for clinical practice in curing the woman patients with dysfunction of sexual endocrine, such as primary ovarian dysfunction, climacteric syndrom, after-ovariectomy and polycystic ovarian disease etc.

ACKNOWLEDGMENT

The work was supported by National Natural Foundation of China (3880910 and 392708340) and a grant from the State Key Laboratory of Medical Neurobiology of China (92003).

REFERENCES

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Shatina, GV et al, Corretive effect of reflextherapy on the hypophyseal-ovarian and sympathetic-adrenal system after ovariectomy. Akush Ginekol MOsk (in Russian), 10, 58-61,1991

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YU, YH et al, Time course of alteration of proopiomelanocorting mRNA level in rat hypothlamic arcuate nucleus following electroacupuncture. Acta Academiae Medicinae Shanghai, 21(Suppl.), 59-62, 1994

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Stallcup, MR and Washington, LD, Region-specific initiation of mouse mammary tumor virus RNA synthesis by endogenous RNA polymerase II in preparations of cell nuclei. J Biologic Chemisty 258, 2802-2904, 1083

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Omura, Y et al, Simple non-invasive mapping of pain pathway in living humans, and the effect of acute non-invasively induced pain on substance P, oncogen C-FOS Ab1, oncogen C-fos Ab2, dopamine and acetycholine. Acupuncture & Electro-Therapeutics Research The International Journal, 17(4), 291-300, 1992

Morgan, TI et al, Mapping patterns of C-fos expression in the central nervous system after seizure. Science, 237, 192-199, 1984

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Substitution of Acupuncture for HCG in Ovulation Induction

Cai Xuefen
Obstetrical & Gynecological Hospital,
Zhejiang Medical University, Zhejiang Province 310006

Source: Journal of Traditional Chinese Medicine 17 (2):119-121,1997

By using human menopausal gonadotropin (HMG) and human chorionic gonadotropin (HCG), fairly good clinical therapeutic efficacy has been obtained in the treatment of infertility. However, difficulties are brought about due to the ovarian hyperstimulation syndrome (OHSS) easily induced by these two drugs. Therefore, we attempted to use acupuncture instead of HCG in the induction of ovulation from 1989 to 1992, and satisfactory therapeutic effect was achieved as reported in the following.

General Data
Ten patients were hospitalized with confirmed diagnosis of infertility and totally observed for 11 menstrual cycles (one patient had recurrence of OHSS for 2 times). Their ages ranged from 27 to 30 years with an average of 29 years. After treatment by HMG, all patients manifested OHSS in varying degrees. In accordance with the criteria for grading of OHSS issued by WHO, among these 11 menstrual cycles 4 cycles were mild (ovarian slight enlargement less than 5 cm with symptoms of slight malaise of lower abdomen); 7 were moderate (marked enlargement of ovary with nausea, vomiting and abdominal distension); no severe case occurred (extreme enlargement of ovary with hydrothorax, ascites, pycnemia and electrolyte disturbance). In order to prevent the exacerbation of OHSS caused by combined use of HMG and HCG, acupuncture was used after HMG treatment to replace HCG for the ovulation induction in 11 menstrual cycles of these patients.

Therapeutic Method
1.5-3 cun long filiform needles (no. 28-30) were used. The acupoints used for needling were Zigong (Extra 16), Shenshu (UB 23), Ciliao (UB 32), (the above acupoints were used bilaterally) and Guanyuan (Ren 4). Baohuang (UB 53) and Zhongji (Ren 3) were selected according to the signs and symptoms as adjuvant points. The manipulation techniques included twirling, rotating, lifting and thrusting. Reinforcing method was used in Shenshu point and the remaining points were punctured by reducing manipulation. The needling sensation should be transmitted toward both sides of lower abdomen. When arrival of Qi, retained the needles for 15 min. and manipulated the needles intermittently during the retaining period to enhance the stimulation. Moxibustion with moxa stick was used for some of these acupoints.

Observation of Therapeutic Effect
Criteria for assessment of therapeutic effect: Therapeutic effect was appraised mainly by comparison of ultrasonic B examination after needling with that before treatment and referred to the score of cervix uteri and basal body temperature to sit judgment on ovulation. Ovulation occurred within 24 h after 1st needling was considered as marked effect; ovulation within 72 h after 2-3 times of needling was effective; no ovulation occurred after 72 h after more than 3 times of needling was scored as ineffective.

Results of Treatment
Of the 11 menstrual cycles, marked effect was shown in 5 cycles, effective in 5 cycles and failed in 1 cycle. Among the 10 markedly effective and effective cycles, ovulation was induced in 2 cases after needling and diagnosed pregnancy by blood HCG assay and ultrasonography. In 9 of the 10 cycles treated with acupuncture for ovulation induction without using HCG and other drugs, the symptoms of OHSS were significantly remitted or even disappeared. Only in one cycle, HCG (with dosage less than for ovulation) was used after needling to maintain the function of corpus luteum and resulted in exacerbation of OHSS and finally remitted by drug treatment.

Typical Case
Fang, 27-year-old, suffered from polycystic ovary syndrome. She was unpregnant after married 2 years and the menstruation was only 1-2 times a year. The basal body temperature was monophase. No effect was observed using clomiphene and then treated with HMG. From the day 5, for bleeding due to withdrawal of progesterone, intramuscular injection of HMG was given at a dose of 150 U once a day for 8 days. The score of cervix uteri was 12 mark. The ultrasonogram showed that the size of right ovary was 9.6 cm x 7.8 cm x 4.6 cm and the left side was 9.2 cm x 7.2 cm x 4.7 cm. Both sides of ovary had 10-20 follicles with maximum size 1.8 cm. In order to avoid severe OHSS, acupuncture was used instead of HCG for ovulation induction after stopping HMG treatment. On the next day after the first needling, the basal body temperature elevated from 36.3°C to 36.8°C and the score of cervix uteri fell from 12 mark to 9 mark, and ultrasonic B examination suggested that part of the follicles were ovulated. After the l9th day of ovulation, the blood concentration of HCG started rising and after 40 days the blood level of HCG reached to 35.6 ng/ml. The ultrasonogram showed that the diameter of embryonic sac was 1.5 cm and early pregnancy was diagnosed.

Discussion
It was reported in literature that using HMG-HCG in the induction of ovulation, the ovulatory rate was about 70%-90%, but the incidence of OHSS might be 10%-15.4% and even life-threatening in the severe case. At present, there were no satisfactory measures for the prevention and remission of OHSS. In most reports, it is considered that when OHSS inclines to occur, stopping injection of HCG is the effective way to avoid severe OHSS. However, stopping HCG would not only discontinue the ovulation of HCH, but also gave up the already developed follicles. Our clinical practice demonstrated that acupuncture is effective in ovulation induction and also the remission of OHSS induced by HMG. Furthermore, we also noted that in most OHSS patients enlarged ovaries and numerous developed follicles were revealed. As a result of excessive follicles developed, dysplasia of ova and insufficiency of corpus luteum often occurred, thus leading to uneasy pregnancy after ovulation. So it is reasonable to infer that using some Chinese drugs benefiting the function of corpus luteum or using certain amount of progesterone as supplementary treatment after acupuncture, the pregnancy rate could be raised.

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