Those who received acupuncture increased their chances of conceiving by 65 percent.
CONTROVERSY: ACUPUNCTURE
Effect of acupuncture on the outcome of in vitro fertilization and intracytoplasmic sperm injection: a randomized, prospective, controlled clinical study
Reduction of blood flow impedance in the uterine arteries of infertile women with electro-acupuncture
Acupuncture Normalizes Dysfunction of Hypothalamic-Pituitary-Ovarian Axis By Bo-Ying Chen M.D. Professor of Neurobiology
Acupuncture Treatment For Infertile Women Undergoing Intracytoplasmic Sperm injection
Effects of Electro-Acupuncture on Nerve Growth Factor and Ovarian Morphology in Rats with Experimentally Induced Polycystic Ovaries
Substitution of Acupuncture for HCG in Ovulation Induction
Influence of acupuncture on the pregnancy rate in patients who undergo assisted reproduction therapy
Role of acupuncture in the treatment of female infertility
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 costs $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.
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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
| Patients included or excluded after randomization into the control group, ACU 1, or ACU 2. |
||||
| Group | Total no. randomized |
Included | Excluded a | Excluded b |
| Control group | 100 | 87 | 6 | 7 |
| ACU 1 | 100 | 95 | 5 | 0 |
| ACU 2 | 100 | 91 | 8 | 1 |
| TOTAL | 300 | 273 | 19 | 8 |
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
| Demographic characteristics of the study population. | |||
| Characteristic | Control group (n=87) |
ACU 1 (n=95) |
ACU 2 (n=91) |
| Age (y), median (range) | 37 (27-45) | 37 (24-45) | 37 (27-45) |
| BMI (kg/m2), median (range) | 23 (18-32) | 23 (16-40) | 22 (18-34) |
| Duration of infertility (y), median (range) | 4 (1-9) | 3 (1-9) | 4 (1-10) |
| Primary infertility (%) | 37 | 44 | 45 |
| Previous IVF attempts (%) | |||
| 0 | 36 | 37 | 30 |
| =>1 | 64 | 67 | 70 |
| Causes of infertility (%) | |||
| Tubal | 19 | 15 | 22 |
| Anovulatory | 19 | 11 | 14 |
| Endometriosis | 0 | 1 | 4 |
| Male | 20 | 24 | 14 |
| Mixed | 16 | 14 | 14 |
| Idiopathic | 26 | 30 | 26 |
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
| Reproductive outcomes per ET. | |||
| Reproductive outcome | Control group (n=87) |
ACU 1 (n=95) |
ACU 2 (n=91) |
| Positive pregnancy test | 24 (28)[a] | 40 (42)[a] | 36 (40) |
| Clinical pregnancy | 21 (24)[b] | 37 (39)[b] | 33 (36) |
| Early pregnancy loss, n (% of positive pregnancy tests) | 5 (21) | 6 (15) | 12 (33) |
| Ongoing pregnancy/delivery | 19 (22)[c] | 34 (36)[c] | 24 (26) |
| Implantation rate, % (no. of gestational sacs/no. of transferred embryos)d) | 18 (32/178) | 21 (42/200) | 19 (36/192) |
Note: Data are mean ± SEM unless otherwise noted.
Westergaard. Acupuncture on ET day improves IVF outcome. Fertil Steril 2006.
TABLE 4
| Oocytes and embryos retrieved per cycle | |||
| Variable | Control group (n=87) |
ACU 1 (n=95) |
ACU 2 (n=91) |
| Oocytes retrieved | 10.6 ± 0.7 | 10.4 ± 0.3 | 10.7 ± 0.6 |
| ICSI, n (%) | 36 (37) | 42 (44) | 35 (38) |
| Oocytes fertilized | 7.4 ± 0.6 | 7.0 ± 0.4 | 7.6 ± 0.6 |
| Embryos transferred | 2.0 ± 0.1 | 2.1 ± 0.05 | 2.1 ± 0.06 |
| Embryos cryo preserved | 2.4 ± 0.4 | 1.9 ± 0.3 | 2.4 ± 0.4 |
| Transferable embryos (transferred ± cryopreserved) | 4.3 ± 0.4 | 4.0 ± 0.3 | 4.5 ± 0.4 |
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.
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Effect of acupuncture on the outcome of in vitro fertilization and intracytoplasmic sperm injection: a randomized, prospective, controlled clinical study
Stefan Dieterle, M.D.,a Gao Ying, M.D.,a,b Wolfgang Hatzmann, M.D.,a and Andreas Neuer, M.D.a
a Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, University of Witten/ Herdecke, Dortmund, Germany; and b Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
Objective: To determine the effect of luteal-phase acupuncture on the outcome of IVF/intracytoplasmic sperm injection (ICSI).
Design: Randomized, prospective, controlled clinical study.
Setting: University IVF center.
Patient(s): Two hundred twenty-five infertile patients undergoing IVF/ICSI.
Intervention(s): In group I, 116 patients received luteal-phase acupuncture according to the principles of traditional Chinese medicine. In group II, 109 patients received placebo acupuncture.
Main Outcome Measure(s): Clinical and ongoing pregnancy rates.
Result(s): In group I, the clinical pregnancy rate and ongoing pregnancy rate (33.6% and 28.4%, respectively) were significantly higher than in group II (15.6% and 13.8%).
Conclusion(s): Luteal-phase acupuncture has a positive effect on the outcome of IVF/ICSI. (Fertil Steril 2006; 85:1347-51. ©2006 by American Society for Reproductive Medicine.)
Key Words: Acupuncture, assisted reproduction, pregnancy rate, IVF, ICSI
The scientific clinical significance of acupuncture is the subject of controversy. Acupuncture is an ancient traditional Chinese treatment technique with an empirical basis. Its theory is based on the energy flow of Qi. Imbalances are believed to cause diseases, which can be treated by stimulating specific points on the body surface. However, the scientific rationale has yet to be established. Studies have suggested that the effects of acupuncture might be mediated through neuropeptides in the central nervous system (1, 2).
A National Institutes of Health Consensus Development Panel (3) found effects of acupuncture on nausea, vomiting, and pain. A randomized, placebo-controlled patient and observer blind trial demonstrated the effectiveness of acupuncture on nausea and vomiting (4).
The role of acupuncture in the treatment of female infertility is unclear (5). Options for patients who undergo several IVF/intracytoplasmic sperm injection (ICSI) cycles without success remain unsatisfactory. Various approaches have been suggested to increase the pregnancy rate. It has been shown that the receptivity of the endometrium (6) and the uterine contraction frequency at the time of ET (7) are critical for embryo implantation. In a previous randomized, prospective, controlled study, it was demonstrated that acupuncture before and after ET resulted in a higher pregnancy rate compared with a group without acupuncture (8). Further studies were suggested with a placebo control group (9).
The aim of this study was to investigate the effect of luteal-phase acupuncture on IVF/ICSI outcome. To minimize psychological effects, a group of patients with acupuncture according to the principles of traditional Chinese medicine was compared with a group of patients receiving placebo acupuncture.
MATERIALS AND METHODS
Patients
The present investigation was designed as a randomized, prospective, controlled trial. The study was approved by the institutional review board. Written, informed consent was obtained from each participant. All patients underwent IVF or ICSI and participated only once.
Patients were randomized with sealed randomization envelopes. A total of 225 infertile patients were included: 116 women were randomized into group I (study group), and 109 women were randomized into group II (control group). The random allocation was concealed from the physician performing the ET. All patients received acupuncture by the same physician.
IVF Protocol
All patients were down-regulated according to the long protocol, with a GnRH agonist (nafarelin 0.4 mg daily), beginning on day 21 of the previous cycle until the day of hCG injection. Ovarian stimulation was performed with recombinant FSH or hMG. Ovulation was triggered with hCG (10,000 IU) when at least three follicles had a diameter of 18 mm with an adequate serum E2 concentration. Transvaginal oocyte retrieval was performed under ultrasound guidance 35 hours after hCG administration.
According to the German Embryo Protection Law, a maximum number of three embryos was transferred into the uterus 2 to 3 days after oocyte retrieval. Embryo selection is not allowed in Germany. In addition, the German Board of Physicians recommends a transfer of two embryos for women aged 35 years. Patients in both groups were supplemented with P (200 mg three times daily) starting the day after oocyte retrieval. Biochemical pregnancies were diagnosed by serum hCG measurement 2 weeks after ET. Clinical pregnancies were confirmed by transvaginal ultrasound 4 - 6 weeks after ET demonstrating at least one gestational sac.
Acupuncture Treatment
For acupuncture, 4-cm-long disposable stainless steel needles (Suzhou Acupuncture and Medical Instruments Co. Ltd., Suzhou, Jiangsu, P. R. China) were used. In both groups, acupuncture was applied for 30 minutes immediately after ET and again 3 days later. The needles were inserted to a depth of 15-30 mm, depending on the region of the body. They were rotated to evoke the needle reaction of Deqi sensation (numbness, soreness, and distention around the acupoint). Fifteen minutes later, the needles were rotated again to maintain Deqi sensation. After ET, the following acupoints were used in group I: Guanyuan (ren [RN]4), Qihai (RN6), Guilai (stomach [ST]29), Neiguan (pericardium [PC]6), Xuehai (spleen [SP]10), and Diji (SP8).
At the same time, a special Chinese medical drug (the seed of Caryophyllaceae) was placed on the patient's ear. The following points were used: ear point 55 (Shenmen), ear point 58 (Zigong), ear point 22 (Neifenmi), and ear point 33 (Pizhixia). The seeds remained in place for 2 days and were pressed twice daily for 10 minutes. Three days after ET, all patients received a second acupuncture treatment. The following locations were used: Hegu (large intestine [LI]14), Sanyinjiao (SP6), Zusanli (ST36), Taixi (kidney [KI]3), Taichong (liver [LR]3). In addition, the same ear points were pressed at the opposite ear twice daily. The seeds were removed after 2 days.
In group II, the following acupuncture points were used after ET and again 3 days later: San Jiao [SJ]9 (Sidu), SJ12 (Xiaoluo), gallbladder (GB)31 (Fengshi), GB32 (Zhongdu), and GB34 (Yang ling qua). As in group I, patients received acupuncture treatment for 30 minutes. At the same time, the following ear points were used: ear point 17 (Shangzhi), ear point 14 (Feng si), ear point 8 (Sisheng), and ear point 53 (Jian). Equal numbers of needles were applied to the study and control groups. The placebo acupuncture treatment was designed not to influence fertility.
Statistical Analysis
The primary outcome measure was the clinical pregnancy rate, which was used for sample size calculation. We estimated a required sample size of 110 patients in both groups. This was based on the assumption of a clinical pregnancy rate of 20% in the control group, a minimal detectable difference of clinical pregnancies between study group and control group of 15% at a power of 80% (according to a ± of 20%), and a type I error () of 5%. The sample size calculation assumed a one-sided test situation and was performed with an unconditional exact test (StatXact Version 6; CYTEL Software, Cambridge, MA). Secondary outcome measures were the biochemical and the ongoing pregnancy rates. Student's t-test was performed for comparison of continuous parameters between the study and control groups. Comparison of discrete parameters was made by 2 analysis. A level of significance of 5% was chosen for both tests.
RESULTS
A total of 225 patients with a transfer of at least one embryo was included in the study. All patients were randomized: 116 patients received acupuncture according to the principle of traditional Chinese medicine (group I), and 109 patients received placebo acupuncture (group II). All 225 patients completed the study. No patient was lost to follow-up.
TABLE 1
| Patient characteristics. | |||
| Characteristic | Group I (n = 116) |
Group II (n = 109) |
P |
| Age (y) | 35.1 ± 3.8 | 34.7 ± 4.0 | NS |
| BMI (kg/m2) | 24.5 ± 5.1 | 24.1 ± 4.7 | NS |
| Duration ofinfertility (ys) | 5.4 ± 3.4 | 5.3 ± 3.1 | NS |
| Primary infertility | 93 | 86 | NS |
| Secondary infertility | 23 | 23 | NS |
| Tubal infertility | 38 | 35 | NS |
| Endometriosis | 18 | 11 | NS |
| Male infertility | 58 | 60 | NS |
| Other causes | 13 | 8 | NS |
| Previous IVF/ICSI cycles | |||
| 0 cycles | 19 | 18 | NS |
| 1 cycles | 29 | 30 | NS |
| 2 cycles | 35 | 35 | NS |
| 3 cycles | 22 | 17 | NS |
| 4 cycles | 8 | 4 | NS |
| 5 cycles | 2 | 4 | NS |
| 6 cycles | 1 | 1 | NS |
Note: Data are presented as mean ± SD or n. NS = nonsignificant; BMI = body
Dieterle. Acupuncture in IVF/ICSI. Fertil Steril 2006.
Fifty-six clinical pregnancies were confirmed by ultrasound. The clinical characteristics of the patients in both groups are presented in Table 1. There were no significant differences in terms of age, body mass index, duration of infertility, cause of infertility, and number of previous IVF/ ICSI cycles between groups I and II.
Table 2 shows the outcome of IVF/ICSI in both groups. No differences regarding the days of stimulation, the number of FSH units required, and serum E2 concentrations on the day of hCG injection were observed. The number of oocytes, the fertilization rate, and the number of embryos transferred were similar in both groups. The data demonstrate that the implantation rate was significantly higher in group I than in group II (14.2% vs. 5.9%, P.01). Clinical pregnancy and ongoing pregnancy rates per transfer were significantly higher in group I (33.6% and 28.4%, respectively) than in group II (15.6% and 13.8%, P.01).
The experimental event rates and the control event rates, including 90% confidence intervals, are listed in Table 3. The numbers needed to treat are 5.5 for the clinical and 6.8 for the ongoing pregnancy rate.
Thirty-seven patients (group I 19, group II 18) underwent their first IVF/ICSI cycle, 59 patients (group I 29, group II 30) had their second cycle after failing to achieve a pregnancy in their first attempt, and 129 women (group I 68, group II 61) received more than two previous IVF/ICSI cycles (Table 4).
Clinical pregnancy rates and implantation rates declined with an increasing number of treatment cycles. After the first cycle, the clinical pregnancy and implantation rates were 47.4% and 28.9%, respectively, in group I, and 33.3% and 11.1% in group II; after the second cycle, 34.5% and 15.2% in group I and 23.3% and 8.6% in group II. After three or more cycles, the clinical pregnancy rate was 29.4% in group I and 8.2% in group II (P.01), and the implantation rate was 12.6% in group I and 3.2% in group II (P.01).
DISCUSSION
The physiologic mechanisms and clinical significance of acupuncture have not been completely revealed and have been the subject of controversy (10). Recent studies support the concept that acupuncture activates endogenous opioids in
TABLE 2
| IVF/ICSI data. | |||
| Variable | Group I (n = 116) |
Group II (n = 109) |
P |
| IVF cycles | 34 | 36 | NS |
| ICSI cycles | 67 | 59 | NS |
| ET after cryopreservation | 15 | 14 | NS |
| Previous IVF/ICSI cycles | 1.8 ± 1.3 | 1.8 ± 1.3 | NS |
| No. of ampoules | 42.2 ± 23.5 | 42.7 ± 23.7 | NS |
| Days of stimulation | 10.2 ± 1.9 | 9.9 ± 1.7 | NS |
| E2 at ovulation induction (pg/mL) | 1,818 ± 1,317 | 1,887 ± 1,299 | NS |
| Endometrial thickness (mm | 10.5 ± 1.8 | 10.2 ± 1.7 | NS |
| No. of oocytes retrieved | 11.2 ± 7.2 | 12.7 ± 9.4 | NS |
| Fertilization rate (%) | 50.2 ± 22.3 | 50.6 ± 21.6 | NS |
| IVF (%) | 46.7 ± 21.3 | 52.8 ± 15.5 | NS |
| ICSI (%) | 50.9 ± 24.4 | 46.3 ± 27.8 | NS |
| ET (n) | 116 | 109 | NS |
| No. of embryos transferred | 2.6 ± 0.7 | 2.6 ± 0.7 | NS |
| 3 embryos transferred | 83 | 79 | NS |
| 2 embryos transferred | 22 | 20 | NS |
| 1 embryo transferred | 11 | 10 | NS |
| Implantation rate (%) | 14.2 | 5.9 | <.01 |
| No. of biochemical pregnancies | 41 | 18 | <.01 |
| Biochemical pregnancy rate (%) | 35.3 | 16.5 | <.01 |
| No. of clinical pregnancies | 39 | 17 | <.01 |
| Clinical pregnancy rate (%) | 33.6 | 15.6 | <.01 |
| No. of ongoing pregnancies | 33 | 15 | <.01 |
| Ongoing pregnancy rate (%) | 28.4 | 13.8 | <.01 |
| Miscarriages (%) | 15.4 | 11.8 | NS |
Note: Data are presented as mean ± SD or n. NS nonsignificant.
Dieterle. Acupuncture in IVF/ICSI. Fertil Steril 2006.
TABLE 3
| Pregnancy rates. | |||
| Parameter | Group I (n = 116) |
Group I (n = 109) |
P |
| Biochemical pregnancy rate (%) | 35.3 (28.0-43.3) | 16.5 (11.0-23.5) | <.01 |
| Clinical pregnancy rate (%) | 33.6 (26.4-41.5) | 15.6 (10.2-22.5) | <.01 |
| Ongoing pregnancy rate (%) | 28.4 (21.6-36.1) | 13.8 (8.7-20.4) | <.01 |
Data are presented as experimental event rate (group I) or control event rate (group II), with 95% confidence interval in parentheses.
Dieterle. Acupuncture in IVF/ICSI. Fertil Steril 2006.
the central nervous system, which inhibit central sympathetic neural outflow (11). Functional magnetic resonance imaging, a technique sensitive to changes in regional blood oxygenation as an index of neuronal activity to map human brain functions, has been used for quantitative studies of the correlation between various acupoints and specific functional areas of the brain (12). Wu et al. (13) characterized a pathway in the hypothalamus and limbic system that might mediate acupuncture. Cho et al. (11) demonstrated a correlation between brain activation and corresponding acupoint stimulation. Acupuncture might change the charge and potential of neurons and the concentrations of electrolytes and neuropeptides, such as ±-endorphin (1, 2, 14). In addition, psychological effects of acupuncture have been demonstrated (15). Acupuncture can activate inhibitory systems in the spinal cord, which results in segmental inhibition of sympathetic outflow (16).
Acupuncture has been used in the treatment of female infertility. Although the mechanism of acupuncture in the treatment of female infertility is unknown, studies have demonstrated its potential impact on the hypothalamic-pituitary- ovarian axis and on the uterus (17, 18).
Successful IVF/ICSI demands optimal endometrial receptivity at the time of embryo implantation. Uterine receptivity is regulated by a number of factors, including uterine perfusion (19). Stener-Victorin et al. (20) demonstrated that acupuncture can reduce the uterine artery blood flow impedance. Ayoubi et al. (7) found that a high uterine contraction frequency in IVF at the time of ET comes from a delayed establishment of uteroquiescence after ovulation in IVF in contrast to the menstrual cycle. Fanchin et al. (21) showed that pregnancy rates are affected by uterine contractions at the time of ET. Kim et al. (18) demonstrated that acupuncture of acupoint LI14 can inhibit uterus motility.
Stener-Victorin et al. (22) compared electro-acupuncture analgesia with standard analgesia during oocyte aspiration. Implantation and "take-home baby" rates were significantly higher with electro-acupuncture than without. Paulus et al. (8) compared a group of 80 patients with acupuncture before and after ET with a control group of 80 patients without acupuncture. They found a significantly higher pregnancy rate in the acupuncture group than in the control group.
Infertility can cause stress, leading to a release of stress hormones. It has been suggested that stress reduction might improve fertility (23). Verhaak et al. (24) reported that differences in the emotional status between pregnant and nonpregnant women were present before treatment and became more apparent after the first IVF and ICSI cycle. Women who became pregnant showed lower levels of depression than those who did not. The use of acupuncture to reduce anxiety and stress, possibly through its sympathoinhibitory property and impact on ±-endorphin levels, has been
TABLE 4
| Number of IVF/ICSI cycles and IVF/ICSI outcome. | |||||||
| Group I (n = 116) |
Group II (n = 109) |
||||||
| No. ofART cycles | No. of patients | Pregnancy rate (%) | Implantation rate (%) | No. of patients | Pregnancy rate (%) | Implantation rate (%) | P |
| 1 | 19 | 47.4 | 22.4 | 18 | 33.3 | 11.3 | NS |
| 2 | 29 | 34.5 | 15.2 | 30 | 23.3 | 8.6 | NS |
| =>3 | 68 | 29.4 | 12.6 | 61 | 8.2 | 3.2 | <.01 |
Dieterle. Acupuncture in IVF/ICSI. Fertil Steril 2006.
reviewed (25). Middlekauff (26) found that sympathetic activation during acute mental stress was eliminated after acupuncture.
When evaluating this study, it has to be considered that the pregnancy rates are affected by the German Embryo Protection Law. This law prohibits embryo selection. A maximum number of three oocytes in the pronuclear stage is allowed to develop and to be transferred. In addition, the mean age of 35.1 years in group I and 34.7 years in group II had an influence on the pregnancy rates. According to the German IVF/ICSI register (2003), the average clinical pregnancy rates for this age are 24.6% for IVF and 22.6% for ICSI. To minimize psychological effects, placebo acupuncture was used in the control group, which was designed not to influence fertility. However, it cannot be completely excluded that placebo acupuncture had an adverse effect on the pregnancy rate.
The results of this study support the significance of acupuncture for the outcome of IVF/ICSI. Even if further evidence has to be accumulated, acupuncture might be a complementary option for patients undergoing IVF/ICSI.
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Reduction of blood flow impedance in the uterine arteries of infertile women with electro-acupuncture
Elisabet Stener-Victorin[1.4], Urban Waldenström[2], Sven A. Andersson[3] and Matts Wikland[2]
[1]Department of Obstetrics and Gynaecology [2]Fertility Centre Scandinavia. Department of Obstetrics and Gynaecology and [3]Department of Physiology University of Gothenburg. S-413 45 Gothenburg, Sweden
[4]To whom correspondence should be addressed: Department of Obstetrics and Gynecology. Kvinnokliniken Sahlgrensh sjukhuset, S-413 45 Golhenburg, Sweden
Source: European Society for Human Reproduction and Embryology
In order to assess whether electro-acupuncture (EA) can reduce a high uterine artery blood flow inpedance, 10 infertile but otherwise healthy women with a pulsatility index (PI) =3.0 in the uterine arteries were treated with EA in a prospective, non-randomized study. Before inclusion in the study and throughout the entire study period, the women were down-regulated with a gonadotrophin-releasing hormone analogue (GnRHa) in order to exclude any fluctuating endogenous hormone effects on the PI. The baseline PI was measured when the serum oestradiol was =0.1 nmol/l, and thereafter the women were given EA eight times, twice a week for 4 weeks. The PI was measured again closely after the eighth EA treatment, and once more 10-14 days after the EA period. Skin temperature on the forehead (STFH) and in the lumbosacral area (STLS) was measured during the flrst, fifth and eighth EA treatments. Compared to the mean baseline PI, the mean PI was significantly reduced both shortly after the eighth EA treatment (P < 0.0001) and 10-14 days after the EA period (P < 0.0001). STFH increased significantly during the EA treatments. It is suggested that both ot these effects are due to a central inhibition of the sympathetic activity.
Key words: electro acupuncture/pulsatilily index (PI)/trans-vaginal colour Doppler curve/uterine artery blood flow
Introduction
Successful in-vitro fertilization (IVF) and embryo transfer demand optimal endometrial receptivity at the time of implantation. Blood flow impedance in the uterine arteries, measured as the pulsatility index (PI) using transvaginal ultrasonography with pulsed Doppler curves, has been considered valuable in assessing endometrial receptivity (Goswamy and Steptoe, 1988; Sterzik et al., 1989; Steer et al., 1992, 1995a,b; Coulam et al., 1995; Tekay et al., 1995). Steer et al. (1992) found that a PI =3.0 at the time of embryo transfer could predict 35% of the failures to become pregnant. Coulam et al. (1995) did not observe any significant differences between PI measurements done on the day of oocyte retrieval compared with PI measurements on the day of embryo transfer. This would allow prediction of non-receptive endometria earlier in the cycle.
Previous studies on rats have shown a decreased blood pressure after electro-acupuncture (EA) with low frequency (2 Hz) stimulation of muscle afferents (A-d fibres). The decreased blood pressure was related to reduced sympathetic activity (Yao et al., 1982; Hoffman and Thoren, 1986; Hoffman et al.. 1987, 1990a,b), and was paralleled by an increase in the ß-endorphin concentration in the cerebrospinal fluid (CSF), suggesting a causal relationship to central sympathetic inhibition (Cao et al., 1983; Moriyama 1987; Reid and Rubin, 1987). The cardiovascular effects of acupuncture treatment are probably mediated by central opioid activity via the ß-endorphin system from the hypothalamus.
The aim of this study was to evaluate whether EA can reduce a high impedance in the uterine arteries. There are several conceivable mechanisms which may give this effect.
In addition to central sympathetic inhibition via the endorphin system, vasodilatation may be caused by stimulation of sensory nerve fibres which inhibit the sympathetic outflow at the spinal level, or by antidromic nerve impulses which release substance-P and calcitonin gene-related peptide from peripheral nerve terminals (Jansen et al., 1989; Andersson, 1993; Andersson and Lundeberg, 1995).
It has been assumed that various disorders in the autonomic nervous system, such as hormonal disturbances, may be normalized during auricular acupuncture (Gerhard and Postneck, 1992). It has also been suggested that the concentrations of central opioids may regulate the function of the hypothalamic-pituitary-ovarian axis via the central sympathetic system, and that a hyperactive sympathetic system in anovulatory patients could be normalized by EA (Chen and Yin, 1991).
Materials and Methods
Subjects, design and Pl measurements
The study was approved by the ethics committee of the University of Gothenburg and was conducted at the Fertility Centre Scandinavia, Gothenburg, Sweden, a tertiary private IVF unit. All women attending the clinic for information about the IVF/embryo transfer procedure, had the PI of their uterine arteries measured by transvaginal ultrasonography and pulsed Doppler curves (Aloka SSD 680: Berner Medecinteknik, Stockholm, Sweden). The PI value for each artery was calculated electronically from a smooth curve fitted to the average waveform over three cardiac cycles, according to the formula: Pl = (A - B)/mean, where A is the peak systolic Doppler shift, B is the end diastolic shift frequency and mean is the mean maximum Doppler shifted frequency over the cardiac cycle. A reduction in the value of PI is thought to indicate a reduction in impedance distal to the point of sampling (Steer et al., 1990).
In the routine preparation for their IVF/embryo transfer treatment, all women were down-regulated with a gonadotrophin-releasing hormone analogue (GnRHa) (Suprecur: Hoechst. Germany). When their oestradiol concentration in serum was <0.1 nmol/1, the women were considered down-regulated and the PI of their uterine arteries was again measured in those women showing a mean Pl =3.0 before down-regulation. The measurements were done by two of the authors (M.W. and U.W.) between 08.30 h and 14.30 h. These hours were chosen for practical reasons, and also to reduce the risk that the PI measurements would be affected by the circadian rhythm in blood flow, recently reported by Zaidi et al. (1995). Three measurements were made on the right and three on the left uterine artery of each patient. Before the study was conducted, the observers were well trained in PI measurements with the equipment used. Steer et al. (1995) has shown that in trained hands, the inter-, and intra-observer variations in vaginal colour Doppler ultrasound are sufficiently small to provide a basis for clinically reliable work.
PI measurements were done on all women attending the unit for an IVF/embryo transfer treatment between November 1992 and February 1993. Of these, all infertile but otherwise healthy women, with a mean PI =3.0 in the uterine arteries both before and after down-regulation, were invited to be included in the study.
In all, 10 women accepted after informed consent and they had a mean age of 32.3 years (range 25-40 years). The infertility diagnoses were unexplained infertility (n = 6), tubal factor (n = 3) and polycystic ovarian syndrome (n = 1).
From their inclusion and onwards, the women were kept on the GnRHa and were given no other pharmacological treatment. Consequently, their gonadotrophins and ovarian steroids were kept at a constantly low concentration, both at their inclusion in the study and throughout the whole study period. Thus, PI changes due to hormonal fluctuations were avoided.
EA was then given eight times, twice a week for 4 weeks. The mean PI of the uterine arteries was measured (mean of three PI on each side) directly after the eighth EA treatment and again 10-14 days after the EA period.
Of the 10 women included, two were later excluded. One of them, with tubal factor infertility, was excluded because she started taking medications for her migraine, which could have affected her PI. The other excluded woman, with unexplained infertility, stopped her GnRHa treatment because she preferred IVF/embryo transfer in a natural cycle.
Acupuncture Treatment
The sympathetic outflow may be inhibited at the segmental level and, for this reason, acupuncture points were selected in somatic segments according to the innervation of the uterus (Thl2-L2, S2-S3) (Bonica, 1990).
The needles were inserted i.m. to a depth of 10-20 mm. The aim of the stimulation was to activate group III muscle-nerve afferents. The needles were twirled to evoke `needle sensation,' often described as tension, numbness, tingling and soreness, sometimes radiating from the point of insertion. The needles were then attached to an electrical stimulator (WQ-6F: Wilkris & Co. AB, Stockholm, Sweden) for 30 min. The location of the needles was the same in all women (Table I).
Table 1. Acupuncture points, their anatomical position and their innervation
| Points* | Segmental innervation (afferent muscle) | Muscle localization |
| BL 23 | L1, 2, 3 | Erector spinae thoracolumbale |
| BL 28 | L4, 5, S1, 2, 3 | Erector spinae lumbosacrale |
| SP 6 | L4, 5, S2, 3 | Tibialis posterior at the medial side |
| BL 57 | S1, 2 | Gastrocnemius and m. soleus at the dorsal side |
*All were placed bilaterally.
BL - bladder channel.
SP - spleen channel.
Four needles were located bilaterally at the thoracolumbar and lumbosacral levels of the erector spinae, and were stimulated with high frequency (100 Hz) pulses of 0.5 ms duration. The intensity was low, giving non-painful paraesthesia.
Four needles were located bilaterally in the calf muscles, and were stimulated with low frequency (2 Hz) pulses of 0.5 ms duration. The intensity was sufficient to cause local muscle contractions.
Skin temperature
The skin temperature was measured with a digital infrared thermometer (Microscanner D-series: Exergen, Watertown, MA, USA) between the applied acupuncture needles in the lumbosacral region (25 mm from each needle), skin temperature lumbosacral (STLS), and on the forehead, skin temperature forehead (STFH). The measurements were made during the first, fifth and eighth EA treatments. The first measurements were made after 10 min rest, and just before the EA, these being considered as `baseline.' Thereafter, further measurements of STLS and STFH were done every seventh minute during the EA and immediately after the EA. The room temperature was constant during the three EA treatments.
Statistics
Analysis of variance (ANOVA: Newman-Keul's range test) was used to analyze the data.
Results
Blood flow impedance
Compared to the mean baseline PI, the mean PI was significantly reduced both soon after the eighth EA treatment (P < 0.0001) and 10-14 days after the EA period (P < 0.0001) (Figure 1), at which time six women had a mean PI <2.6 (Table II and Figure 2).
Figure 1. The mean pulsatility index (PI) (n = 8) for all women before the first electro-acupuncture (EA) treatment, immediately after the eighth EA treatment and 10-14 days after the EA period. *** = significant changes (P < 0.0001) compared to the mean PI before the first EA treatment.
Figure 2. The individual mean pulsatility index (PI) before down-regulation, before the first electro-acupuncture (EA) treatment, immediately after the eighth EA treatment and 10-14 days after the EA period.
Table II. The individual mean pulsalility index (PI) before down-regulation, before the first electro-acupunclure (EA) treatment, immedialely after the eighth EA trealment, 10-11 days after the EA period, and average mean values
| lndividual patients | |||||||||
| Pl value | A | B | C | D | E | F | G | H | Mean Value |
| Before down-regulation | 3.00 | 3.00 | 3.30 | 3.75 | 3.90 | 3.25 | 3.14 | 3.33 | 3.34 |
| Before EA | 3.38 | 3.15 | 3.27 | 3.04 | 3.30 | 3.50 | 3.10 | 3.24 | 3.26 |
| After eight EA | 3.24 | 2.07 | 2.37 | 2.57 | 2.59 | 2.80 | 2.54 | 3.34 | 2.68 |
| 10-14 days after eight EA | 2.25 | 2.01 | 2.40 | 2.60 | 2.40 | 3.84 | 2.54 | 3.20 | 2.65 |
The right and left uterine arteries responded similarly to EA. The diffcrence in mean PI between the two arteries was =0.3 (not significant), both before down-regulation, during down-regulation and throughout the whole study period. There was no significant difference in the mean PI for patients with different causes of infertility.
Skin temperature
The pooled results from all skin temperature measurements are presented in Figure 3. Compared with the starting point, mean STFH increased significantly after 21 min of EA (P = 0.02), and directly after the EA treatments (P = 0.002). STLS did not change significantly.
Figure 3. Pooled mean values (n = 8) of skin temperature on forehead (STFH) and skin temperature in the lumbosacral area (STLS) during the first, fifth and eighth electro-acupuncture (EA) treatments. * = significant changes (P = 0.02) after 21 min and ** = significant changes (P = 0.002) immediately after EA compared to the time just before needles were inserted. 0 = `baseline'.
Discussion It has been shown in previous studies that a high PI in the uterine arteries is associated with a decreased pregnancy rate following IVF-embryo transfer (Goswamy et al., 1988; Sterzik et al., 1989; Steer et al., 1992, 1995a.b; Coulam et al., 1995). The results reported by Tekay et al. (1995) support the hypothesis postulated by Steer et al. (1992) that uterine receptivity is improved when the PI value is between 2.0 and 2.99 on the day of embryo transfer. When a high PI is found before embryo transfer in a stimulated cycle, treatment options are few. Goswamy et al. (1988) successfully tried pre-treatment with exogenous oestrogens in the next cycle, but their results have not been verified by others. It has been proposed that the embryos should be frozen, thawed and transferred in an unstimulated cycle (Goswamy et al., 1988; Steer et al., 1992, 1994), but there is little support for the hypothesis that the PI would be lower under these contitions.
In experiments on spontaneously hypertensive rats, EA at low frequency (2-3 Hz) induced a long-lasting, significant fall in blood pressure which was associated with decreased activity in sympathetic fibres (Yao et al., 1982; Hoffman and Thoren, 1986; Hoffman et al., 1987, 1990a,b). A decrease in sympathetic activity appears to be generalized. In microneurographic studies on humans, EA in the upper limbs resulted in an initial increase and then a decrease in activity of sympathetic efferents in the tibial nerve, with a parallel increase in the temperature of the skin (Moriyama, 1987). Kaada (1982) reported that transcutaneous stimulation of acupuncture points in the hand increased the skin temperature, giving pain relief in limbs suffering from Reynaud's phenomenon. Kaada (1982) also found that electrical stimulation of accupuncture hand points in patients with ischaemic conditions of the lower limbs, increased the skin temperature in the lower limbs and possibly enhanced the healing of long-standing ulcers. It has been noted in both animals and humms that EA has greater effects on pathological conditions, e.g. hypertension or hypotension, whereas normal blood pressure is only slightly changed (Yao et al., 1982: Hoffman and Thoren, 1986: Hoffman et al., 1987, 1990a,b).
The mechanisms of sympathetic inhibition following EA are poorly understood. Based on animal experiments, Hoffmann and Thoren (1986) and Hoffman et al. (1987, 1990a,b) suggested that electrical slimulation of muscle efferents innervating ergoreceptors increases the eoncentration of ß-endorphin in the CSF. They found support for the hypothesis that the hypothalamic ß-endorphinergic system has inhibitory effects on the vasomotor centre, and thereby a central inhibition of sympathetic activity. It has been suggested that this central mechanism, involving hypothalamic and brain stem systems, is important in changing the descending control of many different organ systems, including the vasomotor system (Andersson. 1993; Andersson and Lundeberg, 1995).
In this study, the PI of the uterine arteries was signifieantly decreased soon after the eighth EA treatment and remained significantly decreased 10-14 days after the EA period. These findings suggest that a series of EA treatments increases the uterine artery blood flow. Another effect observed in this study was the signifieantly inereased STFH during the EA treatments.
The most likely cause of these effects is a decreased tonic activity in the sympathetic vasoconstrictor fibres to the uterus and an involvement of the central mechanisms with general inhibition of the sympathetic outflow, in accordance with previously observed EA effects (Kaada. 1982; Yao et al., 1982; Cao et al., 1983: Hoffman and Thoren, 1986; Hoffman et al., 1987, 1990a,b; Moriyama, 1987; Reid and Rubin, 1987; Jansen et al., 1989).
In conclusion. the present study showed a decrease of the PI in the uterine arteries following EA treatment. Randomized studies on a greater number of patients are needed to verify these results and to exclude non-specific effects.
References
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Andersson, S.A. and Lundeberg. T. (1995) Acupuncture - from empiricism to science: functional background to acupuncture effects in pain and disease. Med. Hypoth., 45, 271-281.
Bonica, J. (1990) The Management of Pain, vol. 1, 2nd edn, revised. Lea & Febiger, Philadelphia, London, 156 pp.
Cao, :X.D., Xu. S.F. and Lu. W.X. (1983) Inhibition of sympathetic nervous system by acupuncture. Acupuncturc Electro-Ther. Res. Int. J., 8, 25-35.
Chen, B.Y. and Jin. Y. (1991) Relationship between blood radioimmunoreactive beta-endorphin and hand skin temperature during the electro-acupuncture induction of ovulation. Acupuncture Electro-Ther. Res. Int. J., 16, 1-5.
Coulam, C.B., Stem. IJ.. Soenksen D.M., Britten, S. and Bustillo, M. (1995) Companson of pulsatility indices on the day of oocyte retrieval and embryo transfer. Hum. Reprod., 10, 82-84.
Goswamy, R.K. and Steptoe, P.C. (1988) Doppler ultrasound studies of the uterine atery in spontaneous ovarian cycles. Hum. Reprod., 3, 721-726.
Goswamy, R.K., Williams, G. and Steptoe, P.C. (1988) Decreased uterine pertusion - cause of infentlity. Hum. Reprod., 3, 955-959.
Gerhard, I. and Posteck, F. (1992) Auricular acupuncture in the treatment of female infertility. Gynecol, Endocinol., 6, 171-181.
Hoffmann, P. and Thoren, P. (1986) Long-lasting cardiovascular depression induced by acupuncture-like stimulation of the sciatic nerve in unanaesthetized rats. Effects of arousal and type of hypertension. Acta Physiol., Scand., 127, 119-112.
Hoffman, P., Friberge, P., Ely, D. and Thoren, P. (1987) Effect of spontaneous running on blood pressure, heart rate and cardiac dimension in developing and established spontaneous hypertension in rats. Acta Physiol., Scand., 129, 535-542.
Hoffman, P., Skarphedinsson, J.O., Delle, M. and Thoren, P. (1990a) Electrical stimulation of the gastrocnemius muscle in spontaneously hypertensive rat increases the pain threshold: role of different serotonergic receptors. Acta Physiol., Scand., 138, 125-131.
Hoffman, P., Terenius, L. and Thoren, P. (1990b) Cerebrospinal fluid immunoreactive beta-endorphin concentration is increased by long-lasting voluntary exercise in the spontaneously hypertensive rat. Regul. Pept., 28, 233-239.
Jansen, G., Lundeberg, T., Kjartansson, J. and Samuelsson, U.E. (1989) Acupuncture and sensory neuropeptides increase cutaneous blood flow in rats. Neurosci. Lett., 97, 305-309.
Kaada, B. (1982) Vosodilatation induced by transcutaneous nerve stimulation in peripheral ischemia (Raynaud's phenomenon and diabetic polyneuropathy), Eur. Heart J., 3, 303-314.
Moriyama, T. (1987) Microneurographic analysis of the effects of acupuncture stimulation on sympathetic muscle nerve activity in humans: excitation followed by inhibition. Nippon Seirigaku Zasshi., 49, 711-721.
Reid, J.L. and Rubin, P.C. (1987) Peptides and central neural regulation of circulation. Physiol. Rev., 67, 725-749.
Steer, C.V., Campbell, S., Pampiglione. J.S. et al. (1990) Transvaginal colour flow imaging of uterine arteries during the ovarian and menstrual cycles. Hum. Reprod., 5, 391-395.
Steer. C.V., Campbell, S., Tan, S.L. et al. (1992) The use of transvaginal colour flow imaging after in vitro fertilization to identify optimum uterine conditions before embryo transfer. Fertil. Steril., 57, 372-376.
Steer, C.V., Tan. S.L., Mason, B.A. and Campbell, S. (1994) Midluteal-phase vaginal color Doppler assessment of uterine artery impedance in a subfertile population. Fertil. Steril., 61, 53-58.
Steer, C.V., Williams, J., Zaidi, J., Campbell, S. and Tan, S.L. (1995a) Intra-observer, interobserver, interultrasound transducer and intercycle variation in colour Doppler assessment of uterine artery impedance. Hum. Reprod., 10, 479-481.
Steer, C.V., Tan, S.L., Mason, B.A. and Campbell, S. (1995b) Vaginal color Doppler assessment of uterine artery impedance correlates with immunohistochemical markers of endometrial receptivity required for the implantation of an embryo. Fertil., Steril., 61, 101-108.
Sterzik, K., Hütter, W., Grab, D. et al. (1989) Doppler sonographic findings and their correlation with implantation in an in vitro fertilizalion program. Fertil. Steril., 52, 825-828.
Tekay, A., Martikainen, H. and Jouppila, P. (1995) Blood flow changes in uterine and ovorian vasculature, and predictive value of transvaginal pulsed colour Dooppler ultrasonography in an in-vitro fertilization programme. Hum. Reprod., 10, 688-693.
Yao. T., Andersson, S. and Thoren, P. (1982) Long-lasting cardiovascular depressor response following sciatic stimulation in SHR. Evidence for the involvement of central endorphin and serotonin systems. Brain Res., 244, 295-303.
Zaidi, J., Jurkovic. D., Campbell, S. et al. (1995) Description of circadian rhythm in artery blood flow during the peri-ovulatory period. Hum. Reprod., 10, 1642-l646.
Received on June 27. 1995; accepted on March 20, 1996
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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)
| Group of cases | N | Before EA | After EA |
| Ovulation | 6 | 65.59 ± 24.15 | *38.86 ± 10.11 |
| No ovulation | 7 | 65.59 ± 24.15 | 80.09 ± 22.16 |
| Control | 5 | 38.84 ± 10.13 | 41.52 ± 6.40 |
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)
| Group | N | Before EA | After EA |
| OVX | 10 | *5.47 ± 0.63 | **11.58 ± 0.98 |
| INT | 10 | 18.00 ± 3.26 | 18.34 ± 8.77 |
*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
Group N |
INT 4 |
INI+EA 3 |
OVX 4 |
OVX+EA 7 |
F value |
Number
of AgNORs
(mean/100 cells) |
1.55 1.82 1.24 1.30 |
1.19 1.28 1.16 |
1.25 1.61 1.66 1.96 |
2.53 2.05 1.82 2.86 2.86 2.93 3.92 |
9.614* |
*P < 0.01 tested with ANOVA
Table 4. Weight of adrenal
Group N |
INT 5 |
INI+EA 3 |
OVX 5 |
OVX+EA 8 |
F value |
Weight
(mg) |
57 56 57 43 57 |
54 57 58 |
45 68 56 50 58 |
67 72 66 71 57 74 74 68 |
5.825* |
*P < 0.01 tested with ANOVA
Table 5. The levels of blood corticosterone in OVX and lNT (mean ± SE, ng/ml)
Group |
N |
Before EA |
After EA |
OVX |
12 |
4.78 ± 0.42 |
*6.06 ± 0.73 |
INT |
12 |
3.64 ± 0.15 |
4.76 ± 1.25 |
*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).
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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
Nett, TM et al, Aradioimmunoassay for gonadotropin-releasing hoemone (GnRH) in serum. J Clinical Endocrine and Metabolism, 36, 880-883, 1973
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Wu, ZT et al, The change of c-fos expression in ovariectomized rats following electroacupuncture treatment-An immunohistochemistry study. Acupuncture & Electro-Therapeutics Research The International Journal, 18, 117-124, 1993
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
Sambrook, J et al, Molecular Cloning-A Laboratory Manual. 343-355, 2nd edition, Cold Spring Harbor Laboratory Press, 1989
Chen, BY et al, Relationship between blood radioimmunoreactive beta endorphin and hand skin temperature during the electro-acupuncture induction of ovulation. Acupuncture & Elctro-Therapeutics Research The International Journal 16(1), 1-5, 1991
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Acupuncture Treatment For Infertile Women Undergoing Intracytoplasmic Sperm injection
Sandra L. Emmons, MD
Phillip Patton, MD
Source: Medical Acupuncture, A Journal For Physicians By Physicians
Spring / Summer 2000- Volume 12 / Number 2
"Aurum Nostrum Non Est Aurum Vulgi"
ABSTRACT
Background Little information exists regarding the use of acupuncture in combination with allopathic treatment of infertility.
Objective To describe the use of acupuncture to stimulate follicle development in women undergoing in vitro fertilization.
Design, Setting, and Patients Prospective case series of 6 women receiving intracytoplasmic sperm injection and acupuncture along with agents for ovarian stimulation.
Main Outcome Measures Number of follicles retrieved, conception, and pregnancy past the 1st trimester before and after acupuncture treatment.
Results No pregnancies occurred in the non-acupuncture cycles. Three women produced more follicles with acupuncture treatment (mean, 11.3 vs 3.9 prior to acupuncture; P=.005). All 3 women conceived, but only 1 pregnancy lasted past the 1st trimester.
Conclusion Acupuncture may be a useful adjunct to gonadotropin therapy to produce follicles in women undergoing in vitro fertilization.
KEY WORDS
Female Infertility, Intracytoplasmic Sperm Injection, In Vitro Fertilization, Acupuncture
INTRODUCTION
Infertility is an area of women's health that has sparked much consumer interest in acupuncture. However, there is little published information concerning the combination of acupuncture with allopathic infertility technology.
We present results from 6 women treated with acupuncture to enhance follicle development during in vitro fertilization with intracytoplasmic sperm injection (ICSI) cycles. Our patients all had difficulty with follicle production despite maximum gonadotropin therapy. They were referred for acupuncture as a last resort. We compare results for the acupuncture cycle with results previous to acupuncture.
MATERIALS AND METHODS
The methods used for ovarian hyperstimulation have been described.1 Briefly, ovarian hyperstimulation was achieved using a long-acting gonadotropin-releasing hormone agonist (Lupron, TAP Pharmaceuticals Inc, Deerfield, Ill) administered either in the mid-luteal phase or following a minimum of 2 weeks of oral contraceptive treatment. After biochemical evidence of pituitary suppression (serum estradiol <40 pg/mL), subcutaneous follicle-stimulating hormone was given twice daily (3-6 amps/d). Follicular response was monitored with serial pelvic ultrasonography and serum estradiol measurements. When at least 2 follicles were >17 mm, 7500 IU of human chorionic gonadotropin was given intramuscularly, and transvaginal ultrasound-directed oocyte retrieval was scheduled 36 hours later. Oocytes were identified and then rinsed free of follicular fluid, blood, and debris in TALP-Hepes plus 10% serum substitute supplement (SSS) before being placed in 0.9 mL of bicarbonate-buffered human tubal fluid (HTF) medium plus 10% SSS.2 Spermatozoa were prepared using a discontinuous Percoll gradient. Oocytes for injection were denuded of cumulus cells using hyaluronidase followed by mechanical removal and then assessment for maturity. Metaphase II oocytes were injected with a single immobilized sperm.
Following ICSI, oocytes were cultured in 0.9 mL of HTF plus 10% SSS in organ culture dishes and housed in individually gassed chambers at 37ºC with 5% CO2, 5% O2, and 90% N2. At 15-18 hours following insemination, oocytes were assessed for pronuclei as evidence of fertilization. On the morning of day 3, cleaving embryos were transferred to 50-µL drops of S2 (Scandinavian IVF Sciences, Gothenburg, Sweden) under oil. Embryos of similar quality were grouped together. Embryos cultured beyond day 5 were transferred to fresh medium.
Luteal support consisted of intravaginal progesterone (300 mg/d) beginning on the day following embryo transfer in combination with 1500 IU of hCG intramuscularly given 5 days after oocyte retrieval. Embryo transfer was performed on day 5 or 6 of extended culture using a Soft-Pass catheter (Cook Ob-Gyn, Bloomington, Ind).
The women began acupuncture treatment at the same time that they began follicle-stimulating hormone injections. They had 3 or 4 twice-weekly treatments, on days 1-3, 4-6, 7-9 and in some cases 9-11, with the final treatment on the day of or prior to egg retrieval.
Acupuncture treatments were aimed at stimulating Ming Men (BL 23, GV 4), Chong Mo, and Ren Mo. Points BL 23 and GV 4 were used at all treatments, whereas the Chong Mo (SP 4, MH 6) and Jenn Mo (KI 6, LU 7) Master and Couple points were alternated. Additional points were added on an individual basis, including LR 3, CV 4, 6, SP 30, BL 18, 20, 60, and 62.
Main outcome measures included the number of follicles retrieved, the incidence of pregnancy, and pregnancy lasting past the 1st trimester. Statistical analyses were calculated using SPSS version 10 (SPSS Inc, Chicago, Ill).
RESULTS Results are shown in Table 1. None of the women achieved pregnancy during the non-acupuncture cycles. Three of the women (patients 1-3) clearly recruited more follicles with acupuncture than prior to acupuncture. For the 3 who responded, the mean number of follicles with acupuncture was 11.3 vs 3.9 prior to acupuncture (P=.005). All 3 achieved chemical pregnancy, but only 1 continued the pregnancy past the 1st trimester.
Patient 4 recruited fewer follicles during the acupuncture cycle than during previous cycles. Patients 5 and 6 recruited more follicles with acupuncture, but still recruited few follicles (P=.13). Patient 6 did achieve a chemical pregnancy, whereas patient 5 had the retrieval cancelled due to too few follicles.
On average, significantly more follicles were recruited with acupuncture than without (P=.02). Data on estrogen levels and endometrial lining thickness were not routinely collected in all cycles. For the 4 women (patients 1, 3, 4, and 5) who had estradiol levels measured during both acupuncture and non-acupuncture cycles, mean estradiol levels were higher during the acupuncture cycles than the non-acupuncture cycles (mean [SD], 1471 [480] pg/mL for acupuncture vs 731 [505] pg/mL for non-acupuncture), but this finding did not reach statistical significance (P=.08). Three women (patients 1, 3, and 6) had endometrial lining measurements recorded for both acupuncture and non-acupuncture cycles. The difference in average endometrial lining thickness, measured on the day of follicle retrieval, did not approach statistical significance (acupuncture, 10.4 [2.2] mm vs non-acupuncture, 12.1 [1.1] mm, P=.33).
None of the 6 women reported any adverse reaction to the acupuncture treatments. There were no adverse reactions from the follicle retrievals or embryo transfers during either acupuncture or non-acupuncture cycles.
Table 1. Outcomes for Acupuncture vs Non-Acupuncture Cycles Among 6 Women Undergoing ICSI* |
||||||
Patient No. |
Age, y |
Non-Acupuncture Cycles |
AcupunctureCycles |
|||
Follicles |
Cycles |
Follicles |
Cycles |
|||
Mean No. |
No. |
Mean No. |
No. |
Outcome |
||
1 |
29 |
4.7 |
3 |
8 |
1 |
IUP |
2 |
34 |
5 |
1 |
10 |
2 |
SAB twice |
3 |
36 |
3 |
2 |
14 |
1 |
SAB |
4 |
37 |
8 |
1 |
6 |
1 |
No pregnancy |
5 |
38 |
4 |
1 |
4 |
1 |
Cycle canceled |
6 |
41 |
2 |
1 |
6 |
1 |
SAB |
Mean (SD) |
3.7(1.0) |
8.4(1.3) |
||||
*ICSI indicates intracytoplasmic sperm injection; IUP, intrauterine pregnancy; and SAB, early spontaneous abortion. P=.02 for overall acupuncture follicles vs non-acupuncture follicles.
DISCUSSION
Our findings suggest that acupuncture may be a useful adjuvant to gonadotropin therapy among women undergoing ICSI. In this context, acupuncture increased the number of follicles produced and appeared to also increase the estradiol level, but did not appear to affect endometrial lining thickness. However, none of the women in this report had difficulty with achieving adequate endometrial linin
Although there is significant consumer interest in using alternative and complementary therapies for infertility, there is little research that addresses the combination of techniques. Stener-Victorin et al3 published a report of using acupuncture to decrease the uterine pulsatility index among women with a history of poor uterine lining response to in vitro fertilization. They demonstrated a significant decrease in uterine pulsatility index, which was maintained for 2 weeks, by using 4 set acupuncture points with electric stimulation. Gerhard and Postneek4 published results of infertile women treated with acupuncture vs similar women treated hormonally, and showed a similar pregnancy rate among the 2 groups. Siterman et al5 showed improvement in sperm quality among subfertile men treated with acupuncture.
The mechanisms responsible for the systemic actions of acupuncture have been debated but not yet clearly defined. Traditional Chinese Medicine (TCM) speaks to increasing and harmonizing Qi within the reproductive organs.6 Scientific analysis of acupuncture used in the context of pain syndromes has shown acupuncture to raise the level of endogenous opiates7 and to decrease the level of sympathetic nerve stimulation8 at the painful area. The decrease in sympathetic stimulation may be 1 of the factors that results in an increased level of blood flow to the area.7,8 In the context of infertility, acupuncture may be helpful by increasing blood supply to the reproductive organs, or may simply increase relaxation or reduce subjective stress surrounding the infertility diagnosis and treatment.
Study Limitations
These cases have an obvious bias. The group was selected from those who responded poorly to gonadotropin therapy. The patients served as their own historical controls, but there was no similar group that simply had another ICSI attempt without acupuncture to compare before and after results. The acupuncture treatments were not standardized. Even though similar points were chosen for all women, points based on the individual TCM diagnosis were also used.
CONCLUSION
The cases do present evidence that a structured clinical trial of acupuncture to assist in follicle development for women undergoing in vitro fertilization and/or ICSI would be of interest. Many women undergoing infertility treatment seek alternative care; knowing the interaction of these 2 systems would be most useful.
REFERENCES
1. Patton PE, Eaton D, Burry KA, Wolf DP. The use of gonadotropin-releasing hormone agonist to regulate oocyte retrieval time. Fertil Steril. 1990; 54:652-655.
2. Bavister BD, Boatman DE, Leibfried L, Loose M, Vernon MW. Fertilization and cleavage of rhesus monkey oocytes in vitro. Biol Reprod. 1983;28: 983-999.
3. Stener-Victorin E, Waldenstrom U, Andersson SA, Wikland M. Reduction of blood flow impedance in the uterine arteries of infertile women with electro-acupuncture. Hum Reprod. 1996;11:1314-1317.
4. Gerhard I, Postneek F. Auricular acupuncture in the treatment of female infertility. Gynecol Endocrinol. 1992;6:171-181.
5. Siterman S, Eltes F, Wolfson V, Zabludovsky N, Bartoov B. Effect of acupuncture on sperm parameters of males suffering from subfertility related to low sperm quality. Arch Androl. 1997;39:155-161.
6. Vincent CA, Richardson PH. The evaluation of therapeutic acupuncture: concepts and methods. Pain. 1986;24:1-13.
7. Andersson S, Lundeberg T. Acupuncture: from empiricism to science: functional background to acupuncture effects in pain and disease. Med Hypotheses. 1995;45:271-281.
8. Cai W. Acupuncture and the nervous system. Am J Chin Med. 1992; 20: 331-337.
AUTHORS' INFORMATION
Dr Sandra Emmons is an Assistant Professor of Obstetrics and Gynecology at Oregon Health Sciences University. Dr Emmons practices Obstetrics and Gynecology, and incorporates Medical Acupuncture in her practice. She is a Fellow of the American Academy of Obstetrics and Gynecology.
Sandra L. Emmons, MD
Assistant Professor, Obstetrics and Gynecology
OHSU, L466
3181 SW Sam Jackson Park Rd
Portland, OR 97201
Phone: 503-494-3102
Fax: 503-494-3111
E-mail: emmonss@ohsu.edu
Dr Phillip Patton is an Associate Professor of Obstetrics and Gynecology at Oregon Health Sciences University with specialty boards in Reproductive Endocrinology. Dr Patton's practice at OHSU emphasizes infertility and assisted reproductive technology, and he is a Fellow of the American Academy of Obstetrics and Gynecology.
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Effects of Electro-Acupuncture on Nerve Growth Factor and Ovarian Morphology in Rats with Experimentally Induced Polycystic Ovaries
Elisabet Stener-Victorin,[2,3] Thomas Lundeberg,[4] Urban Waldenström,[3] Luigi Manni,[5] Luigi Aloe,[5] Stefan Gunnarsson,[6] and Per Olof Janson[3]
Department of Obstetrics and Gynecology,[3] Göteborg University, SE-413 45 Goteborg, Sweden
Department of Physiology and Pharmacology,[4] Karolinska Institutet, SE-164 01 Stockholm, Sweden,
Institute of Neurobiology (CNR),[5] Rome, Italy
Department of Evolutionary Biology,[6] SE-752 36 Uppsala, Sweden
ABSTRACT
Despite extensive research on the pathogenesis of polycystic ovary syndrome (PCOS), there is still disagreement on the underlying mechanisms. The rat model for experimentally induced polycystic ovaries (PCO)--produced by a single injection of estradiol valerate--has similarities with human PCOS, and both are associated with hyperactivity in the sympathetic nervous system. Nerve growth factor (NGF) is known to serve as a neurotrophin for both the sympathetic and the sensory nervous systems and to enhance the activity of catecholaminergic and possibly other neuron types. Electro-acupuncture (EA) is known to reduce hyperactivity in the sympathetic nervous system. For these reasons, the model was used in the present study to investigate the effects of EA (12 treatments, approximately 25 min each, over 30 days) by analyzing NGF in the central nervous system and the endocrine organs, including the ovaries. The main findings in the present study were first, that significantly higher concentrations of NGF were found in the ovaries and the adrenal glands in the rats in the PCO model than in the control rats that were only injected with the vehicle (oil or NaCI). Second, that repeated EA treatments in PCO rats resulted in concentrations of NGF in the ovaries that were significantly lower than those in non-EA-treated PCO rats but were within a normal range that did not differ from those in the untreated oil and NaCI control groups. The results in the present study provide support for the theory that EA inhibits hyperactivity in the sympathetic nervous system.
adrenal, central nervous system, follicular development, hypothalamus, ovary, ovulation, pituitary, stress
INTRODUCTION
Polycystic ovary syndrome (PCOS), one of the most common causes of anovulation in women of reproductive age. is a complex endocrine and metabolic disorder [1]. Despite extensive research seeking the pathogenesis of PCOS, there is still disagreement on the underlying mechanisms. Different hypotheses of its pathophysiology have emerged, which indicates that the etiology is multifactorial and poorly understood.
Women with PCOS have an increased risk of endometrial cancer, hypertension, and type II diabetes, and they need some kind of long-standing treatment [2]. Traditional pharmacological treatment for ovulation induction is effective, but side effects such as superovulation are quite common. A previous clinical study on anovulatory women with PCOS showed that sensory stimulation (i.e., electro-acupuncture [EA]) affects endocrinological and neuroendocrinological parameters [3]. In addition, regular ovulations were induced in more than one-third of the women without negative side effects. These findings accord with previous reports [4-6] but do not enlighten underlying mechanisms. The mechanisms behind the beneficial effect of EA on PCOS in the human are difficult to study because tissue samples from the ovaries and the central nervous system (CNS) are for obvious reasons unobtainable. Studies on, for instance, neuropeptides in the gonads and the CNS would be possible to conduct in an animal model, provided that such a model exists.
Experiments on normal cycling rats have shown that exogenous estradiol valerate (EV), a long-acting estrogen, causes acyclicity and the formation of polycystic ovaries (PCO) [7, 8]. The changes include atretic antral follicles, follicular cysts with a well-developed theca cell layer, a diminished granulosa cell compartment, and luteinized cysts [7, 8]. Furthermore, the rats exhibited alterations in basal and pulsatile LH and FSH concentrations, changes in the pituitary response to GnRH, degenerative changes in the hypothalamus, altered opioid inhibitory tone on GnRH release, and high estradiol levels with a persistent pattern of constant estrus as assessed by vaginal smear [9, 10]. In addition, EV-induced PCO is associated with an increase in peripheral sympathetic outflow, evidenced by an increase in the release of norepinephrine (NE), an increase in ovarian NE content, and a decrease in the number of ß-adrenergic receptors in the ovarian compartments receiving catecholaminergic innervation [9-11]. Even if it is not possible to reproduce human PCOS using a rat model, it may provide important leads because a single injection of EV induces an anovulatory state that shares many endocrinological and morphological characteristics of human PCOS [7-13]. Thus, comparisons between the rat PCO model and human PCOS must be interpreted with caution because rat PCO ovaries contain multiple follicular cysts, the structure of which does not replicate the follicular growth arrest found in human PCOS. Contrary to previously held notions, the granulosa cells in the follicles accumulating in the human ovary are not atretic. However, both human PCOS and EV-induced PCO in rats may be associated with hyperactivity in the sympathetic nervous system.
According to one theory, elevated concentrations of neurotransmitters found in women with PCOS and anovulation may be associated with psychological stress and with hyperactivity in the sympathetic nervous system [3, 12, 13]. That superior ovarian nerve transection restores estrus cyclicity and ovulatory capacity in rats with EV-induced PCO further supports the theories of sympathetic hyperactivity [9]. Other evidence of neuronal involvement is that ovarian sympathetic innervation is under trophic control by nerve growth factor (NGF) [14]. This is also supported by the fact that the expression of the genes that encode NGF and one of its receptors, the low-affinity NGF-receptor, was dramatically increased in the ovary 30 days after EV injection [11]. Ovarian NGF is principally synthesized in the cells of the follicular wall [15], which is the site where the sympathetic neurons project to the ovaries [14]. The increase in the synthesis of NGF and its receptor that precedes the formation of cysts suggests that after PCO has been induced by EV injection, the neurons innervating the ovary are subjected to an enhanced neurotrophic influence that contributes to their hyperactivation and to the maintenance of an abnormally elevated catecholaminergic tone in ovarian steroid secretions [9-11].
Aim of the Study
Because NGF is known to serve as a neurotrophin for both the sympathetic and the sensory nervous systems and to enhance the activity of catecholaminergic and possibly other neuron types [9, 11, 14, 16-22], and because EA is known to reduce hyperactivity in the sympathetic nervous system [23-25], the experimentally induced PCO model was used to study the effects of EA by analyzing NGF in the CNS and the endocrine organs, including the ovaries.
The first part of the present study investigated dose-response--the discovery of the exact dose of EV needed to produce fully developed polycystic ovaries. The second part of this study investigated treatment with EA--what contribution NGF made to the etiology and maintenance of EV-induced PCO in rats and if and to what extent EA has an effect on NGF and ovarian morphology in experimentally induced PCO.
MATERIALS AND METHODS
Fifty-nine virgin adult cycling Sprague-Dawley rats (Möllegaard, Denmark) weighing 190-210 g and with regular 4-day estrous cycles were used. The rats were housed at 22°C, four to a cage, with free access to pelleted food and tap water and with a 12L:12D cycle for at least 1 wk before and throughout the experimental period. All rats received a single i.m. injection of either EV (Riedeldehaen, Germany), oil, or 0.15 M NaCI (Kabi Pharmacia AB, Sweden) and were anesthetized with enfluran (EFRANE, Abbott Scandinavia, Kista, Sweden) and killed by decapitation. The local Animal Ethics Committee at Göteborg University, Sweden approved the study.
Dose-Response
Twenty-seven rats were injected with one of two different doses of EV in an oil solution or with oil alone to ascertain the optimal dose for induction of PCO [8]. They were decapitated on three different occasions (15, 30, or 60 days after i.m. injection) to elucidate precisely when the ovaries display characteristic features of well-defined PCO [7, 8]. Nine rats received 2 mg EV in 0.2 ml oil/rat, nine rats 4 mg EV in 0.2 ml oil/rat, and nine rats 0.2 ml oil alone. Three rats per dose were killed on Day 15, three on Day 30, and three on Day 60.
FIG. 1. Schematic drawing of the dorsal side of a rat and the placement of acupuncture needles. Two needles were placed bilaterally in the erector spinae muscle at the level of Th12 and two were placed in the quadriceps muscle bilaterally. The needles were then attached to an electrical stimulator for EA treatment.
Treatment with EA
The optimal dose (4 mg EV in 0.2 ml oil/rat) and timing (30 days after injection) were chosen for the experiments. In total, 32 rats took part. Eight rats in the EV control group and eight in the EA-treated EV group were injected i.m. with 4 mg EV in 0.2 ml oil/rat, eight rats in the oil control group with 0.2 ml oil, and eight rats in the NaCI control group with 0.2 ml 0.15 M NaCI. All 32 were decapitated on Day 30 after injection, that is, 1-2 days after the last EA treatment. All groups were anesthetized 12 times for about 25 min each time. Anesthesia was induced by inhalation of enfluran at 5.5-6.5 ml/h with an O2 and air flow of 0.25 L/min. The EA-treated EV group was subjected to 12 EA treatments every second or third day, beginning 2 days after the i.m. injection of EV. The stimulation points were bilateral in the quadriceps and erector spinae muscles at the level of thoracic (Th) 12 in the somatic segments according to the innervation of the ovaries (Th 12-lumbar [L]2, sacral [S]2-S4) (Fig. 1). The needles (Hegu; Hegu AB, Landsbro, Sweden) were inserted to depths of 0.5-0.8 cm and then bilaterally attached to an electrical stimulator (CEFAR ACU II, Cefar, Lund, Sweden) with a low burst frequency of 2 Hz. Individual pulses within the frequency were square wave pulses with alternating polarities and with a pulse duration of 0.2 msec, 80 pulses/sec. The intensity was adjusted so that local muscle contractions were seen to reflect the activation of muscle-nerve afferents (A delta fibers and possibly C fibers) [26, 27]. The location and type of stimulation were the same in all rats.
Nerve Growth Factor Measurements by Enzyme Immunoassay
In the second part of the study, after the rats were decapitated, the pituitary gland, the hypothalamus, the hippocampus, one ovary, and the adrenal glands were quickly removed and dissected on dry ice, weighed, and stored at -80°C until extraction. The samples were sonicated in extraction buffer (0.1% Triton X-100, 100 mM Tris-HCI, pH 7.2, 400 mM NaCI, 4 mM EDTA, 0.2 mM PMSF, 0.2 mM benzethonium chloride, 2 mM benzamidine, 40 U/ml aprotinin, 0.05% sodium azide, 2% BSA, and 0.5% gelatin; 1 ml/100 mg of tissue), followed by centrifugation at 10,000 x g for 30 min. The supernatants were used for the assay. The bioactive form of 2.5S NGF purified from mouse sub-maxillary glands and prepared in the laboratory at the Institute of Neurobiology (CNR) in Rome, Italy, according to the method of Bocchini and Angeletti [28] was used as a standard. The NGF was dissolved in extraction buffer and the standard curve was in a range of 31.25 pg ml (-1) and 1 ng ml (-1). An ELISA was performed as described by Weskamp and Otten [29] with a minor modification [30]. Specific NGF binding was assessed by use of monoclonal mouse anti-ß-2.5S NGF (Boehringer Mannheim GmbH, Mannheim, Germany) that reacts with both the 2.5S and the 7S biologically active forms of NGF. The absorbency of samples and standards was corrected for nonspecific binding (i.e., the absorbency in a well coated with purified mouse IgG). The NGF content in the samples was determined in relation to the NGF standard curve. Data were not corrected for recovery of NGF from samples, which was routinely 70-90%, and was accepted only when the values were >2 SD above the blank. With these criteria, the limit of sensitivity of NGF ELISA averaged 0.5 pg per assay.
Morphology
One ovary per rat was removed, cleaned of adherent connective fat tissue, and fixed in 4% formaldehyde buffer; sections were stained with hematoxylin-eosin, and a trained pathologist performed a quantitative analysis of the follicle population. If ovum degeneration or at least one pyknotic granulosa cell was seen, the follicle populations were classified as atretic, otherwise they were classified as healthy. Morphological characteristics of follicular atresia were, for instance, scattered pyknotic nuclei in the granulosa cell layer [31], detachment of the granulosa cell layer from the basement membrane [32], fragmentation of the basal lamina [33], and the presence of cell debris in the antrum of the follicle [34].
Statistical Analyses
Statistical analyses were carried out using the SPSS 8.0 software. The NGF concentrations in the pituitary gland, the hypothalamus, the hippocampus, the ovary, and the adrenal glands were analyzed and the groups compared using ANOVA followed by multiple comparison procedures (Bonferroni test). All results are presented as mean ± SEM. A P value less than 0.05 was considered significant. The 95% confidence interval (Cl) was given when P < 0.05.
RESULTS
Ovarian Morphology--Dose-Response
In the first part of the present study, dose-response, injection of 0.2 ml oil alone (control) was associated with a normal appearance of the ovaries and no differences were seen between rats sacrificed on Day 15, 30, or 60 (Fig. 2, a and b). No changes were seen in the ovaries of rats injected with 2 mg EV in 0.2 ml oil/rat and killed on Day 15. The ovaries of rats injected with the same dose of EV in oil exhibited small morphological changes resembling PCO when killed on Day 30 and 60 (Fig. 3, a and b). The ovaries of rats injected with a higher dose of EV (4 mg EV in 0.2 ml oil/rat) exhibited only small morphological changes on Day 15. Rats injected with the same dose of EV in oil and killed on Day 30 (Fig- 4, a-c) showed a progressive decrease in the number of primary and secondary follicles but it was on Day 60 (Fig- 5, a and b) that the true cystic follicles appeared and the well-defined PCO was fully developed in accordance with previous reports by Brawer et al. [8].
Ovarian Morphology--Treatment with EA
In the second part of the present study, treatment with EA, all rats were killed at Day 30 after EV injection, i.e., before the appearance of cystic follicles. The ovaries in the EV control group (4 mg EV in 0-2 ml oil/rat) displayed the same morphological changes as previously shown in the dose-response section (see Fig- 4, a-c). The ovaries in the oil control group and the NaCI control group exhibited a typically normal appearance (see Fig- 2, a and b). No substantial morphological differences were found between the EA-treated, EV group, and the EV control group.
Nerve Growth Factor--Treatment with EA
In the second part of the present study, treatment with EA, NGF measurements were made at Day 30 after EV injection. Means ± SEM for NGF (pg/g wet weight) in the hypothalamus, the pituitary gland, the hippocampus, the ovary, and the adrenal gland in all groups are presented in Table 1. Ovarian NGF concentrations were significantly higher in the EV control group compared to the oil control group (P < 0.001, CI = 178.7, 821.6) and the NaCl control group (P < 0.01, CI = 144.6, 787.5). The NGF concentrations in the ovary were significantly lower in the EA-treated, EV group compared to the EV control group (P < 0.05 Cl = 6.2, 614.9) and did not differ from the (Jil and the NaCI control groups) The NGF concentrations in the adrenal glands were significantly higher in the EV control group and the EA-treated. EV group compared to both the oil control group (P < 0.001, CI = 45.7, 169.3 and P < 0.01, CI = 38.5, 166.5) and the NaCI control group (P < 0.001, Cl = 21.9, 162.9 and P < 0.01, Cl = 15.0, 159.8).
Weights of Ovaries and Adrenal Gland--Treatment with EA
Means ± SEM for weights (mg) of the ovaries and the adrenal glands in all groups are presented in Table 2. Ovarian weights in the control EV group and in the EV-treated EV group were significantly lower compared to the oil control group (both P < 0.001) and the NaCI control group (both P < 0.001).
| FIG. 2. a) Section of an ovary from a rat injected with 0.2 ml in oil and sacrificed on Day 30. In total, 11 corpora lutea (CL) marked with CL and three secondary follicles (SF) marked with SF are seen. One secondary follicle is framed (b). Magnification x2.5. Section stained with hematoxylin-eosin. b) Normal secondary follicle. Magnification x20. | FIG. 3. a) Section of an ovary from a rat injected with 2 mg EV in 0.2 ml oil and killed on Day 30. In total, six corpora lutea marked with CL and three atretic secondary follicles (ASF) marked with ASF are seen. The atretic secondary follicle is framed (b): Magnification x2.5: Section stained with hematoxylin-eosin. b) An atretic secondary follicle with granulosa cells showing signs of atresia and intact theca cells. Magnification x20. |
| FIG. 4. a) Section of an ovary from a rat injected with 4 mg EV in 0.2 ml oil and sacrificed on Day 30. In total, seven corpora lutea marked with CL three cystic follicles (CF) marked with CF, and two atretic secondary follicles marked with ASF are seen. One cystic follicle (b) and one atretic secondary follicle are framed (c). Magnification x2 5: Section stained with hematoxylin-eosin. b) Cystic degenerating follicle showing a thin granulosa layer and debris in follicular fluid. Magnification x20. c) An atretic secondary follicle with detachment of the oocyte from the cumulus mass of pyknotic granulosa cells. Magnification x20. | FIG. 5. a) Section of an ovary from a rat injected with 4 mg EV in 0.2 ml oil and sacrificed on Day 60. In total, two corpora lutea marked with CL, five cystic follicles marked with CF and one secondary follicle marked with SF are seen. One cystic follicle is framed (b). Magnification x2.5 Section stained with hematoxylin-eosin. b) A cystic degenerating follicle showing a thin granulosa layer and debris in follicular fluid. Magnification x20. |
DISCUSSION
The main findings in the present study are as, follows: First, PCO induced in rats by a single injection or EV results in significantly higher concentrations of NGF in the ovaries and the adrenal glands without any changes in the brain tissue when measured 30 days after EV injection.
Second, repeated EA treatments with low frequency (2 Hz) significantly decrease the elevated NGF concentrations in the ovaries, to within a normal range, without affecting NGF concentrations in the adrenal glands or brain tissue when measured 30 days after EV injection.
The histological examination of the ovaries in the first part of the present study, dose-response, revealed that the optimal dose of EV that caused typical PCO-like morphological changes was 4 mg and that PCO was fully developed at Day 60. This dose was twice that used by Brawer and coworkers [7, 8] to achieve full development of a well defined PCO in rats. The reason might be differences in the strain of rat and/or the estrogen preparation that was used. In addition, the ovarian weight in the two EV-injected groups was significantly lower compared to that in the vehicle-injected (oil and NaCI) control rats. The reduction in ovarian weight and size, as well, are in accordance with the findings of Brawer et al. [8]. The reduction in weight and size of the ovaries might be explained by a reduction in the number of corpora lutea. In the second part, treatment with EA, no substantial influence in ovarian morphology was seen at Day 30, after EV injection with the number and duration of the EA treatments used in this study. However, the main reason for beginning EA treatment as early as 2-3 days after EV injection and to decapitate at Day 30 after EV injection was to estimate whether EA could influence the increased ovarian NGF concentrations that have been shown to precede the development of morphological changes in rats with PCO [11]. It remains to be shown whether EA influences the ovarian morphology 60 days after EV injection. It would therefore be of interest to study the effects of EA after extended treatment periods. Such a study would provide a unique opportunity to collect experimental evidence of the effectiveness of EA in humans. In fact, we have observed that the multifollicular pattern characteristic of the ovarian morphology of women with PCOS and anovulation, as assessed by ultrasonography, began to disappear after they had received repeated EA treatments [3].
An involvement of the nervous system in the etiology and/or maintenance of PCOS is suggested by both clinical and experimental findings [9-13]. Clinical studies show that women with PCOS temporarily recover normal ovarian function after bilateral wedge resection or ovarian drilling that partially denervates the ovary [35, 36]. There is thus a possibility that the ovarian nerves are involved in the successful outcome of bilateral wedge resection and ovarian drilling.
Experimental observations in rats reveal that superior ovarian nerve transection in EV-induced PCO reduces the steroid response, increases ß-adrenoreceptor concentrations to more normal levels, and restores estrus cyclicity and ovulation [9]. These effects were linked to reduced activity in the ovarian sympathetic nerve fibers, indicating a peripheral neurogenic effect [9].
Sensory stimulation, i.e., EA, activates muscle-nerve afferents, mainly A-delta and possibly C fibers [23, 26, 27], that initiate a number of peripheral reactions at the spinal level and centrally in the brain. That EA may reduce hyperactivity in the ovarian peripheral sympathetic nerve fibers is in accordance with the theory that EA could modulate sensory, motor, and autonomic outflow at the segmental level [24]. In parallel, higher control systems are activated, resulting in the release of a number of neuropeptides, important in the modulation of central and segmental autonomic outflow, of the hypothalamic-pituitary-ovarian axis (HPO axis), and of the descending pain-inhibiting systems [23-25].
TABLE 1. Treatment with EA.
| NGF concentration (pg/g)[a] | ||||
| EA-treated, EV (n = 8) |
EV control (n=8) |
Oil control (n=8) |
NaCI control (n=8) | |
| Ovary | 647.8 ± 69.9[b] | 952.2 ± 95.1[d] | 452 ± 42.4 | 486 ± 90.0 |
| Adrenal gland | 157.4 ± 11.8[c] | 162.4 ± 22.1[e] | 54.9 ± 9.7 | 70 ± 14.4 |
| Pituitary gland | 63.3 ± 9.6 | 95.5 ± 15.5 | 122.6 ± 36.2 | 125.8 ± 26.0 |
| Hypothalamus | 293.1 ± 26.8 | 293 ± 64.7 | 531.2 ± 155.7 | 315.6 ± 21.3 |
| Hippocampus | 3412.4 ± 210.2 | 3589.6 ± 292.2 | 2837.9 ± 122.7 | 3166.0 ± 164.8 |
[a] NGF (pg/g wet weight) in the ovary, the adrenal gland, the pituitary gland, the hypothalamus, and the hippocampus in the different groups: EA-treated, EV; EV control; oil control (0.2 ml); and NaCI control (0.2 ml 0.15 M). All EV iniections were 4 m8 EV In 0.2 ml oil/rat. All data values are mean ± SEM. [b] P < 0.05, EA EV versus EV control.
[c] P < 0.001, EA EV versus oil control; and P < 0.01, EA EV versus NaCI control.
[d] P < 0.001, EV control versus oil control; and P < 0.01, EV control versus NaCI control.
[e] < 0.01, EV control versus oil control; and P < 0.001, EV control versus NaCI control.
TABLE 2. Treatment with EA.
| Weight (mg)[a] | ||||
| EA-treated, EV (n = 8) |
EV control (n=8) |
Oil control (n=8) |
NaCI control (n=8) |
|
| Ovary | 0.011 ± 0.0007[b] | 0.011 ± 0.0007[c] | 0.021 ± 0.001 | 0.021 ± 0.001 |
| Adrenal gland | 0.018 ± 0.0008 | 0.016 ± 0.006 | 0.017 ± 0.001 | 0.018 ± 0.0011 |
[a] Weights of the ovary and the adrenal gland shown as mean ± SEM in the different groups: EA-treated, EV; EV control; oil control (0.2 ml); and NaCI control (0.2 ml 0.15 M). All EV injections were 4 mg EV in 0.2 ml oil/rat.
[b] P < 0.001, EA EV versus oil control and NaCI control.
[c] P < 0.001, EV control versus oil control and NaCI control
For obvious reasons it is not possible to subject control animals to true sham needle insertion. As soon as a needle penetrates the skin, it may be seen as a form of sensory stimulation that activates afferent nerve fibers. If a sham needle insertion without electrical stimulation is performed, then different acupuncture methods/stimulation techniques are being compared, and this does not provide proper information on the effect of EA versus no EA. We chose EA because the stimulation intensity is easy to standardize and it has been shown to be superior to manual needle stimulation [37]. In addition, to show a difference between two or more stimulation techniques would require a very large number of study subjects. In the present study, the control rats received the same enfluran anesthesia protocol as the rats treated with EA, which, in our opinion, is the best way to control completely environmental and/or emotional factors and the EA effect. The acupuncture needles in the present study were placed in the somatic segments that correspond to ovarian innervation. The needles were stimulated with low frequency EA for optimal activation of muscle nerve afferents to inhibit the autonomic outflow at the segmental level and at the central level and to modulate the HPO axis. The choice of acupuncture points and the aim of stimulation has been the same as in our other EA studies on the female reproductive tract that dealt with blood flow in the uterine arteries prior to in vitro fertilization (IVF) [38], pain-relief during oocyte aspiration in connection with IVF treatment [39], and induction of ovulation in women with PCOS [3].
We have shown that repeated EA treatments restore regular ovulations in more than one-third of the anovulatory women with PCOS. In addition, EA-influenced neuroendocrine and endocrine parameters indicative of PCOS, such as LH/FSH ratios, mean testosterone concentrations, and ß-endorphin concentrations, decreased significantiy [3]. The effects of repeated EA on anovulation were then attributed to an inhibition of hyperactivity in the sympathetic nervous system [3, 5, 6].
The findings of the present study support recent reports that ovarian NGF concentrations in rats with experimentally induced PCO [11] are elevated and that this increase can be related to a hyperactivity in the ovarian sympathetic nerves. Lara et al. [11] also suggests that activation of this neurotrophic-neurogenic regulatory loop is a component of the pathological process by which EV induces cyst formation and anovulation. They also stated that there is evidence that the alteration in neurotrophic input to the ovary contributes to the etiology and/or maintenance of human PCOS [11].
Furthermore, the present study shows that repeated EA treatments reduce peripheral sympathetic nerve hyperactivity, as revealed by the reduction in increased NGF concentrations in the ovaries into a normal range 30 days after EV injection, that did not differ from that of the untreated oil and NaCI control groups.
It remains to be shown whether EA directly affects sympathetic nerve activity. Measurements of the nervous output by analyses of the catecholamine release can resolve this. In addition, because receptors for NGF are expressed on the endocrine cells of the ovary, activities of ovarian NGF may mediate and/or be mediated by alterations in endocrine factors, for example, by corticotropin-releasing hormone, prolactin, oxytocin, and/or adrenal corticosteroid secretion. To resolve this, the same experimental protocol regarding EA and controls used here must be supplemented with measurements of serum levels of these hormones.
Whether this condition can be reversed with EA treatment at higher stimulation intensities, in higher numbers, and/or over longer periods remains to be shown.
The conclusion of this study is that repeated EA treatments reduce ovarian NGF concentrations to within normal ranges. This suggests that EA inhibits the hyperactivity in the ovarian sympathetic nerves, which may be of importance for the development and maintenance of experimentally induced PCO.
ACKNOWLEDGMENTS
The authors thank Professor Owe Lundgren and laboratory assistant Britt-Marie Fin, Department of Physiology, Goteborg University, for providing excellent working facilities and for invaluable laboratory help at their Department. We also thank Associate Professor Folke Knutsson for his invaluable assistance in the morphological analyses of the ovaries. Carl Lofman, M.D., Stockholm is acknowledged for skillful preparation of morphological specimens.
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3. Stener-Victorin E, Waldenstrom U, Tagnfors U, Lundeberg T, Lindstedt G, Janson PO. Effects of electro-acupuncture on anovulation in women with polycystic ovary syndrome. Acta Obstet Gynecol Scand 2000; 79:180-188.
4. Gerhard I, Postneek F. Auricular acupuncture in the treatment of female infertility. Gynecol Endocrinol 1992; 6:171-181.
5. Chen BY, Yu J. Relationship between blood radioimmunoreactive beta-endorphin and hand skin temperature during the electro-acupuncture induction of ovulation. Acupunct Electro-Ther Res 1991; 16:1-5.
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7. Brawer JR, Naftolin F, Martin J, Sonnenschein C. Effects of a single injection of estradiol valerate on the hypothalamic arcuate nucleus and on reproductive function in the female rat. Endocrinology 1978; 103:501-512.
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11. Lara HE, Dissen GA, Leylon V, Paredes A, Fuenzalida H, Fedler JL, Ojeda SR. An increased intraovarian synthesis of nerve growth factor and its low affinity receptor is a principal component of steroid-induced polycystic ovary in the rat. Endocrinology 2000; 141:1059-1072.
12. Lobo RA, Granger LR, Paul WL, Goebelsmann U, Mishell DR Jr. Psychological stress and increases in urinay norepinephrine metabolites, platelet serotonin. and adrenal androgens in women with polycystic ovary syndrome. Am J Obstet Gynecol 1983; 115:496-503.
13. Lobo RA. The role of neurotransmitters and opioids in polycystic ovarian syndrome. Endocrinol Metab Clin North Arn 1988; 17:667-683.
14. Lara HE, McDonald JK, Ojeda SR. Involvement of nerve growth factor in female sexual development. Endocrinology 1990; 126:364-375.
15. Dissen GA, Hill DF, Costa ME, Les Dees CW, Lara HE, Ojeda SR, A role for trkA nerve growth factor receptors in mammalian ovulation. Endocrinology 1996: 137:198-209.
16. Levi-Montalcini R, Angeleni PU. Immunosympathectomy. Pharmacol Rev 1966; 18:619-628.
17. Johnson EMJ, Osbom PA, Rydel RE, Schmidt RE, Pearson J. Characterization of the effects of autoimmune nerve growth factor deprivation in the developing guinea pig. Neuroscience 1983; 8:631-642.
18. Aloe L. The effect of nerve growth factor and its antibody on mast cells in vivo. J Neuroimmunol 1988; 18:1-12.
19. Lockhart ST, Turrigiano GG, Birren SJ. Nerve growth factor modulates synaptic transmission between sympathetic neurons and cardiac myocytes. J Neurosci 1997; 17:9573-9582.
20. Cowen T, Gavazzi I. Plasticity in adult and ageing sympathetic neurons. Prog Neurobiol 1998; 54:249-288.
21. Heath BM, Xia J, Dong E, An RH, Brooks A, Liang C, Federoff HG, Kass RS. Overexpression of nerve growth factor in the heart alters ion channel activity and beta-adrenergic signalling in an adult transgenic mouse. J Physiol (Lond) 1998; 512:779-791.
22. Lockhart ST, Mead JN, Pisano JM, Slonimsky JD, Birren SJ. Nerve growth factor collaborates with myocyte-derived factors to promote development of presynaptic sites in cultured sympathetic neurons. J Neurobiol 2000; 42:460-476.
23. Andersson S. The functional background in acupuncture effects. Scand J Rehabil Med Suppl 1993; 29:31-60.
24. Andersson S, Lundeberg T. Acupuncture--from empiricism to science: functional background to acupuncture effects in pain and disease. Med Hypotheses 1995; 45:271-281.
25. Sato A, Sato Y, Schmidt RF. The Impact of Somatosensory Input on Autonomic Functions. Heidelberg: Springer-Verlag; 1997.
26. Lundeberg T, Hurtig T, Lundeberg S, Thomas M. Long-term results of acupuncture in chronic head and neck pain. Pain Clinic 1988; 2:161-164.
27. Haker E, Lundeberg T. Acupuncture treatment in epicondylalgia: a comparative study of two acupuncture techniques. Clin J Pain 1990; 6:221-226.
28. Bocchini V, Angeletti PU. The nerve growth factor: purification as a 30,000-molecular-weight protein. Proc Natl Acad Sci U S A 1969; 64:787-794.
29. Wescamp G, Otten U. An enzyme-linked immunoassay for nerve growth factor (NGF): a tool for studying regulatory mechanisms involved in NGF production in brain and peripheral tissues. J Neurochem 1987; 48:1779-1786.
30. Tirassa P, Stenfors C, Lundeberg T, Aloe L. Cholecystokinin-8 regulation of NGF concentrations in adult mouse brain through a mechanism involving CCKA and CCKB receptors. Br J Pharmacol 1998; 123(6):1230-1236.
31. Hirshfield AN. Rescue of atretic follicles in vitro and in vivo. Biol Reprod 1989; 40:181-190.
32. Junquiera LC, Cameiro J, Kelly RO. Basic Histology. Norwalk: Appleton and Lange; 1989.
33. Bagavandoss P, Midgley ARJ, Wicha M. Developmental changes in the follicular basal lamina detected by immunofluorescence and electron microscopy. J Histochem Cytochem 1983; 31:633-640.
34. Hay MF, Cran DG, Moor RM. Structural changes occurring during atresia in sheep ovarian follicles. Cell Tissue Res 1976; 169:515-529.
35. Nakamura Y. Treatment of polycystic ovary syndrome: an overview. Horm Res 1990; 33:(suppl 2):31.
36. Vaitukaitis JL. Polycystic-ovary syndrome-what is it? N Engl J Med 1983; 309:1245-1246.
37. Litscher G, Wang L, Yang NH, Schwarz G. Ultrasound-monitored effects of acupuncture on brain and eye. Neurol Res 1999; 21:373-377.
38. Stener Victorin E, Waldenström U, Andersson SA, Wikland M. Reduction of blood flow impedance in the uterine arteries of infertile women with electro-acupuncture. Hum Reprod 1996; 11:1314-1317.
39. Stener-Victorin E, Waldenström U, Nilsson L, Wikland M, Janson PO. A prospective randomized study of electro-acupuncture versus alfentanil as anaesthesia during oocyte aspiration in in-vitro fertilization. Hum Reprod 1999; 14:2480-2484.
[1]Supported by grants from the Hjalmar Svensson Foundation, Wilhelm och Martina Lundgrens Vetenskapsfond Wilhelm and Martina Lundgren's Science Fund, and the Foundation for Acupuncture and Alternative Biological Treatment Methods.
[2]Correspondence: Elisabet Stener-Victorin, Department of Obstetrics and Gynecology, Kvinnokliniken, Sahlgrenska University Hospital, SE-413 45 Göteborg, Sweden. FAX: 46 31829248;
e-mail: elisabet.stenervictorin@medstud.gu.se
Received: 29 February 2000. First decision: 30 March 2000. Accepted: 11 July 2000. © 2000 by the Society for the Study of Reproduction, Inc. ISSN: 0006-3363. http://www.biolreprod.org
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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.
A Brief Introduction to the Training Center of China Academy of Traditional Chinese Medicine
The Training Center of China Academy of Traditional Chinese Medicine is an educational institution of traditional Chinese medicine, and has excellent teachers and good bases for clinical practice and provides proper board and lodging.
The Center regularly conducts three-month advanced and general courses of traditional Chinese medicine, acupuncture, Tuina (massage), Qigong (breathing exercises) and Taiji (shadow boxing). It also runs short-term training courses on some special topics, and preparatory guidance courses for licensure examination of tradi tional Chinese medicine, acupuncture and moxibustion. In addition, various courses based on the participant's requirements may be arranged in the center. All those who complete the required courses will receive relevant certificates.
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FERTILITY AND STERILITY® VOL. 77, NO. 4, APRIL 2002
Copyright ©2002 American Society for Reproductive Medicine
Published by Elsevier Science Inc., Printed on acid-free paper in U.S.A.
Influence of acupuncture on the pregnancy rate in patients who undergo assisted reproduction therapy
Wolfgang E. Paulus, M.D.,[a] Mingmin Zhang, M.D.,[b] Erwin Strehler, M.D.,[a]
Imam El-Danasouri, Ph.D.,[a] and Karl Sterzik, M.D.[a]
Christian-Lauritzen-Institut, Ulm, Germany
Received June 5, 2001; revised and accepted October 16, 2001. Reprint requests: Wolfgang E. Paulus, M.D., Christian-Lauritzen-Institut, Frauenstr. 51, D-89073, Ulm, Germany (FAX: ++49-731-9665130; E-mail: paulus@reprotox.de).
[a] Department of Reproductive Medicine, Christian-Lauritzen-Institut.
[b] Department of Traditional Chinese Medicine, Tongji Hospital, Tongji Medical University, Wuhan, People's Republic of China.
0015-0282/02/$22.00
PII S0015-0282(01)03273-3
Objective: To evaluate the effect of acupuncture on the pregnancy rate in assisted reproduction therapy (ART) by comparing a group of patients receiving acupuncture treatment shortly before and after embryo transfer with a control group receiving no acupuncture.
Design: Prospective randomized study.
Setting: Fertility center.
Patient(s): After giving informed consent, 160 patients who were undergoing ART and who had good quality embryos were divided into the following two groups through random selection: embryo transfer with acupuncture (n = 80) and embryo transfer without acupuncture (n = 80).
Intervention(s): Acupuncture was performed in 80 patients 25 minutes before and after embryo transfer. In the control group, embryos were transferred without any supportive therapy.
Main Outcome Measure(s): Clinical pregnancy was defined as the presence of a fetal sac during an ultrasound examination 6 weeks after embryo transfer.
Result(s): Clinical pregnancies were documented in 34 of 80 patients (42.5%) in the acupuncture group, whereas pregnancy rate was only 26.3% (21 out of 80 patients) in the control group.
Conclusion(s): Acupuncture seems to be a useful tool for improving pregnancy rate after ART. (Fertil Steril®2002;77:721- 4. ©2002 by American Society for Reproductive Medicine.)
Key Words: Acupuncture, assisted reproduction, embryo transfer, pregnancy rate
Acupuncture is an important element of traditional Chinese medicine (TCM), which can be traced back for at least 4,000 years. Acupuncture has been shown to alleviate nausea and vomiting, dental pain, addiction, headache, menstrual cramps, tennis elbow, fibromyalgia, myofascial pain, osteoarthritis, carpal tunnel syndrome, and asthma. Both physiologic and psychological benefits of acupuncture have been scientifically demonstrated in recent years.
However, so far there have been only a few serious trials concerning the use of acupuncture in reproductive medicine. Publications focus primarily on acupuncture therapy for male infertility (1, 2). Electroacupuncture may reduce blood flow impedance in the uterine arteries of infertile women (3). A positive impact of electroacupuncture on endocrinologic parameters and ovulation in women with polycystic ovary syndrome has been demonstrated (4). In addition, auricular acupuncture was successfully used in the treatment of female infertility (5). In the present study, we chose acupuncture points that relax the uterus according to the principles of TCM. Because acupuncture influences the autonomic nervous system, such treatment should optimize endometrial receptivity (6). Our main objective was to evaluate whether acupuncture accompanying embryo transfer increases clinical pregnancy rate.
Materials and Methods
This study was a prospective randomized trial at the Christian-Lauritzen-Institut in Ulm, Germany. It was approved by the ethics committee of the University of Ulm. A total of 160 healthy women undergoing treatment with in vitro fertilization (IVF; n = 101) or intracytoplasmic sperm injection (ICSI; n = 59) were recruited into the study. The age of the patients ranged from 21 to 43 (mean age: 32.5 = 4.0 years). The cause of infertility was the same for both groups (Table 1). Only patients with good embryo quality were included in the study. Using a computerized randomization method, patients were assigned into either the acupuncture group or the control group.
Table 1
| Descriptive data on acupuncture and control group (mean ± SD or total number). | |||
| Control group (n = 80) |
Acupuncture group (n = 80) |
Statistics Statistics |
|
| Age of patients (years) | 32.1 ± 3.9 | 32.8 ± 4.1 | NS |
| No. of previous cycles | 2.0 ± 2.0 | 2.1 ± 2.1 | NS |
| No. of transferred embryos | 2.1 ± 0.5 | 2.2 ± 0.5 | NS |
| VF (n) | 54 | 47 | NS |
| ICSI (n) | 26 | 33 | NS |
| No. of cycles with male factor infertility | 46 | 47 | NS |
| No. of cycles with tubal disease | 21 | 22 | NS |
| No. of cycles with polycystic ovaries | 2 | 2 | NS |
| No. of cycles with unknown cause of infertility | 11 | 9 | NS |
| Endometrial thickness (mm) | 9.9 ± 2.7 | 9.1 ± 2.4 | NS |
| Plasma estradiol on day of embryo transfer (pg/mL) | 1001 -± 635 | 971 ± 832 | NS |
| Pulsatility index of uterine arteries (PI) before embryo transfer | 2.00 ± 0.56 | 2.02 ± 0,45 | NS |
| Pulsatility index of uterine arteries (PI) after embryo transfer | 2.19 ± 0.52 | 2.22 ± 0,44 | NS |
| Pregnant | 21/80 26.3%) | 34/80 (42.5%) | P=.03 |
NS = not significant (P>.05).
Paulus. Acupuncture in ART. Fertil Steril 2002.
Ovarian stimulation, oocyte retrieval, and in vitro culture were performed as previously described (7). Transvaginal ultrasound-guided needle aspiration of follicular fluid was performed 36 to 38 hours after hCG administration. Immediately after follicle puncture, the oocytes were retrieved, assessed, and fertilized in vitro. Sperm preparation and culture conditions did not differ for either group.
In cases of severe male subfertility, ICSI was preferred, as described in the literature (8). Forty-eight hours after the IVF or ICSI procedure, embryos were evaluated according to their appearance as type 1 or 2 (good), type 3 or 4 (poor), as described in literature (9).
Just before and after embryo transfer, all patients underwent ultrasound scans of the uterus using a 7-MHz transvaginal probe (LOGIQ 400 Pro, GE Medical Systems Ultra-sound Europe, Solingen, Germany). Pulsed Doppler curves of both uterine arteries were measured by one observer. The pulsatility index (PI) for each artery was calculated electronically from a smooth curve fitted to the average waveform over three cardiac cycles.
A maximum of three embryos, in accordance with German law, were transferred into the uterine cavity on day 2 or 3 after oocyte retrieval. For embryo replacement, the patient was placed in a dorsal lithotomy position, with an empty bladder. The cervix was exposed with a bivalved speculum, then washed with culture media prior to embryo transfer. Labotect Embryo Transfer Catheter Set (Labotect GmbH, Go¨ ttingen, Germany) was used for atraumatic replacement owing to the curved guiding cannula with a ball end, allowing the set to be used reliably even with difficult anatomic conditions. The metallic reinforced inner catheter shaft al lowed safe passage through the cervical canal. When the catheter tip lay close to the fundus, the medium containing the embryos was expelled and the catheter withdrawn gently. After this procedure, the patient was placed at bed rest for 25 minutes. All oocyte retrievals and embryo transfers were performed by one examiner using the same method. The examiner was not aware of the patient's treatment group (control or acupuncture).
At the time of the embryo transfer, blood samples (10 mL) were obtained from the cubital vein. Plasma estrogen was determined by an immunometric method using the IMMULITE 2000 Immunoassay System (DPC Diagnostic Product Corporation, Los Angeles, CA).
Luteal phase support was given by transvaginal progesterone administration (Utrogest®, 200 mg, three times per day; Kade, Berlin, Germany). Progesterone administration was initiated on the day after oocyte retrieval and was continued until the serum ß-hCG measurement 14 to 16 days after transfer and, in cases of pregnancy, until gestation week 8.
Each patient in the experimental group received an acupuncture treatment 25 minutes before and after embryo transfer. Sterile disposable stainless steel needles (0.25 X 25 mm) were inserted in acupuncture point locations. Needle reaction (soreness, numbness, or distention around the point = Deqi sensation) occurred during the initial insertion. After 10 minutes, the needles were rotated in order to maintain Deqi sensation. The needles were left in position for 25 minutes and then removed. The depth of needle insertion was about 10 to 20 mm, depending on the region of the body undergoing treatment. Before embryo transfer, we used the following locations: Cx6 (Neiguan), Sp8 (Diji), Liv3 (Taichong), Gv20 (Baihui), and S29 (Guilai).
After embryo transfer, the needles were inserted at the following points: S36 (Zusanli), Sp6 (Sanyinjiao), Sp10 (Xuehai), and Li4 (Hegu).
In addition, we used small stainless needles (0.2 X 13 mm) for auricular acupuncture at the following points, without rotation: ear point 55 (Shenmen), ear point 58 (Zhigong), ear point 22 (Neifenmi), and ear point 34 (Naodian). Two needles were inserted in the right ear, the other two needles in the left ear. The four needles remained in the ears for 25 minutes. The side of the auricular acupuncture was changed after embryo transfer. The patients in the control group also remained lying still for 25 minutes after embryo transfer. All treatments were performed by the same well-trained examiner, in the same way.
The primary point of the study was to determine whether acupuncture improves the clinical pregnancy rate after IVF or ICSI treatment. Student's t-test was used as a corrective against any possible imbalance between the two groups regarding the following variables: age of patient, number of previous cycles, number of transferred embryos, endometrial thickness, plasma estradiol on day of transfer, method of treatment (IVF or ICSI), and blood flow impedance in the uterine arteries (pulsatility index). Chi-square test was used to compare the two groups. All statistical analyses were carried out using the software package Statgraphics (Manugistics, Inc., Rockville, MD).
Results
A total of 160 patients was recruited for the study. Patients who failed to conceive during the first treatment cycle were not reentered into the study. According to the randomization, 80 patients were treated with acupuncture, and 80 patients underwent the usual therapy without acupuncture.
As Table 1 shows, there were no statistically significant differences between the two groups with respect to the following covariants: age of patient, number of previous cycles, number of transferred embryos, endometrial thickness, plasma estradiol on day of transfer, or method of treatment (IVF or ICSI). Clinical indications for ART were the same for patients of both groups. The blood flow impedance in the uterine arteries (pulsatility index) did not differ between the groups before and after embryo transfer.
The analysis shows that the pregnancy rate for the acupuncture group is considerably higher than for the control group (42.5% vs 26.3%; P=.03).
Discussion
The acupuncture points used in this study were chosen according to the principles of TCM (10): Stimulation of Taiying meridians (spleen) and Yangming meridians (stomach, colon) would result in better blood perfusion and more energy in the uterus. Stimulation of the body points Cx6, Liv3, and Gv20, as well as stimulation of the ear points 34 and 55, would sedate the patient. Ear point 58 would influence the uterus, whereas ear point 22 would stabilize the endocrine system.
The anesthesia-like effects of acupuncture have been studied extensively. Acupuncture needles stimulate muscle afferents innervating ergoreceptors, which leads to increased ß-endorphin concentration in the cerebrospinal fluid (11). The hypothalamic ß-endorphinergic system has inhibitory effects on the vasomotor center, thereby reducing sympathetic activity. This central mechanism, which involves the hypothalamic and brainstem systems, controls many major organ systems in the body (12). In addition to central sympathetic inhibition by the endorphin system, acupuncture stimulation of the sensory nerve fibers may inhibit the sympathetic outflow at the spinal level. By changing the concentration of central opioids, acupuncture may also regulate the function of the hypothalamic-pituitary-ovarian axis via the central sympathetic system (13).
Kim et al. (14) suggested that Li4 acupuncture treatment could be useful in inhibiting the uterus motility. In their rat experiments, treatment on the Li4 acupoint suppressed the expression of COX-2 enzyme in the endometrium and myometrium of pregnant and nonpregnant uteri.
Stener-Victorin et al. (3) reduced high uterine artery blood flow impedance by a series of eight electroacupuncture treatments, twice a week for 4 weeks. They suggest that a decreased tonic activity in the sympathetic vasoconstrictor fibers to the uterus and an involvement of central mechanisms with general inhibition of the sympathetic outflow may be responsible for this effect. In our study, we could not see any differences in the pulsatility index between the acupuncture and control group before or after embryo transfer. This may be due to a different acupuncture protocol and the selected sample of patients with high blood flow impedance of the uterine arteries (PI = 3.0) in the Stener-Victorin et al. study.
As we could not observe any significant differences in covariants between the acupuncture and control groups, the results demonstrate that acupuncture therapy improves pregnancy rate.
Further research is needed to demonstrate precisely how acupuncture causes physiologic changes in the uterus and the reproductive system. To rule out the possibility that acupuncture produces only psychological or psychosomatic effects, we plan to use a placebo needle set as a control in a future study.
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