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). 

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 

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 

Andersson, S.A. (1993) The functional background in acupuncture effects. Scand J. Rehab, Med. Suppl., 29. 31-60. 

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

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. 

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&die; 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. 

References 

1. Siterman S, Eltes F, Wolfson V, Lederman H, Bartoov B. Does acupuncture treatment affect sperm density in males with very low sperm count? A pilot study. Andrologia 2000;32:31-9. 

2. Bartoov B, Eltes F, Reichart M, Langzam J, Lederman H, Zabludovsky N. Quantitative ultramorphological analysis of human sperm: fifteen years of experience in the diagnosis and management of male factor infertility. Arch Androl 1999;43:13-25. 

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 -7. 

4. Stener-Victorin E, Waldenstrom U, Tagnfors U, Lundeberg T, Lindst-edt G, Janson PO. Effects of electro-acupuncture on anovulation in women with polycystic ovary syndrome. Acta Obstet Gynecol Scand 2000;79:180 -8. 

5. Gerhard I, Postneek F. Auricular acupuncture in the treatment of female infertility. Gynecol Endocrinol 1992;6:171-81. 

6. Stener-Victorin E, Lundeberg T, Waldenstrom U, Manni L, Aloe L, Gunnarsson S, Janson PO: Effects of electro-acupuncture on nerve growth factor and ovarian morphology in rats with experimentally induced polycystic ovaries. Biol Reprod 2000;63:1497-503. 

7. Strehler E, Abt M, El-Danasouri I, De Santo M, Sterzik K. Impact of recombinant follicle-stimulating hormone and human menopausal gonadotropins on in vitro fertilization outcome. Fertil Steril 2001;75: 332-6. 

8. Palermo GD, Schlegel PN, Colombero LT, Zaninovic N, Moy F, Rosenwaks Z. Aggressive sperm immobilization prior to intracytoplasmic sperm injection with immature spermatozoa improves fertilization and pregnancy rates. Hum Reprod 1996;11:1023-9. 

9. Plachot M, Mandelbaum J: Oocyte maturation, fertilization and embryonic growth in vitro. Br Med Bull 1990;46:675-94. 

10. Maciocia G. Obstetrics and gynecology in Chinese medicine. New York: Churchill Livingstone, 1998. 

11. Hoffmann P, Terenius L, Thoren P. Cerebrospinal fluid immunoreactive beta-endorphin concentration is increased by voluntary exercise in the spontaneously hypertensive rat. Regul Pept 1990;28:233-9. 

12. Andersson SA, Lundeberg T. Acupuncture-from empiricism to science: functional background to acupuncture effects in pain and disease. Med Hypotheses 1995;45:271-81. 

13. Chen BY, Yu J. Relationship between blood radioimmunoreactive beta-endorphin and hand skin temperature during the electro-acupuncture induction of ovulation. Acupunct Electrother Res 1991;16:1-5. 

14. Kim J, Shin KH, Na CS. Effect of acupuncture treatment on uterine motility and cyclooxygenase-2 expression in pregnant rats. Gynecol Obstet Invest 20

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. 

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 

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 

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. 

REFERENCES 

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

2. Ku Y, Chang Y. Beta-endorphin- and GABA-mediated depressor effect of specific electroacupuncture surpasses pressor response of emotional circuit. Peptides 2001;22:1465–70.

3. NIH Consensus Development Panel of Acupuncture. Acupuncture. JAMA 1998;280:1518 –24.

4. Streitberger K, Diefenbacher M, Bauer A, Conradi R, Bardenheuer H, Martin E, et al. Acupuncture compared to placebo-acupuncture for postoperative nausea and vomiting prophylaxis: a randomized placebocontrolled patient and observer blind trial. Anaesthesia 2004;59:142–9.

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

6. Chien LW, Au HK, Chen PL, Xiao J, Tzen CR. Assessment of uterine receptivity by the endometrial-subendometrial blood flow distribution pattern in women undergoing in vitro fertilization-embryo transfer. Fertil Steril 2002;78:245–51.

7. Ayoubi JM, Epiney M, Brioschi PA, Fanchin R, Chardonnens D, Ziegler D. Comparison of changes in uterine contraction frequency after ovulation in the menstrual cycle and in in vitro fertilization cycles. Fertil Steril 2003;79:1101–5.

8. 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.

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

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

11. Cho ZH, Chung SC, Jones JP, Park JB, Park HJ, Lee HJ, et al. New findings of the correlation between acupoints and corresponding brain cortices using functional MRI. Proc Natl Acad Sci U S A 1998;3: 2670–3.

12. Zhang WT, Jin Z, Cui GH, Zhang KL, Zhang L, Zeng YW, et al. Relations between brain network activation and analgesic effect induced by low vs. high frequency electrical acupoint stimulation in different subjects: a functional magnetic resonance imaging study. Brain Res 2003;29:168 –78.

13. Wu MT, Hsieh JC, Xiong J, Yang CF, Pan HB, Iris Chen YC, et al. Central nervous pathway for acupuncture stimulation: localization of processing with functional MR imaging of the brain—preliminary experience. Radiology 1999;212:133– 41.

14. Andersson S, Lunderberg T. Acupuncture from empiricism to science: functional background to acupuncture effects in pain and disease. Med Hypotheses 1995;45:271– 81.

15. Luo H, Meng F, Jia Y, Zhao X. Clinical research on the therapeutic effect of the electroacupuncture treatment in patients with depression. Psychiatry Clin Neurosi 1998;52:338–40.

16. Sato A, Sato Y, Schmidt RF. The impact of somatosensory input on autonomic functions. Heidelberg: Springer-Verlag, 1997:325.

17. Chen BY, Yu J. Relationship between blood radioimmunoreactive betaendorphin and hard skin temperature during the electro-acupuncture of ovulation. Acupunct Electrother Res 1991;16:1–5.

18. Kim J, Shin KH, Na CS. Effect of acupuncture treatment on uterine motility and cyclooxygenase-2 expression in pregnant rats. Gynecol Obstet Invest 2000;50:225–30.

19. Jinno M, Ozaki T, Iwashita M, Nakamura Y, Kudo A, Hirano H. Measurement of endometrial tissue blood flow: a novel way to assess uterine receptivity for implantation. Fertil Steril 2001;76: 1168 –74.

20. 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 –7.

21. Fanchin R, Righini C, Olivennes F, Taylor S, de Ziegler D, Frydman R. Uterine contractions at the time of embryo transfer alter pregnancy rates after in-vitro fertilization. Hum Reprod 1998;13:1968 –74.

22. Stener-Victorin E, Waldenstrom 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;14:2480–4.

23. Domar AS, Siebel MM, Benson H. The mind/body program for infertility: a new behavioral treatment approach for women with infertility. Fertil Steril 1990;53:246 –9.

24. Verhaak AM, Smeenk MJ, Eugster A, Minnen A, Kremer JA, Kraaimaat FW. Stress and marital satisfaction among women before and after their first cycle of in vitro fertilization and intracytoplasmic sperm injection. Fertil Steril 2001;76:525–31.

25. Dong JT. Research on the reduction of anxiety and depression with acupuncture. Am J Acupunct 1993;21:327–30.

26. Middlekauff HR. Acupuncture in the treatment of heart failure. Cardiol Rev 2004;12:171–3.

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 

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). 

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 

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. 

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. 

*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. 

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

ABSTRACT 

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

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

INTRODUCTON 

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

MATERIALS AND METHODS 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

RESULTS 

Effect of EA on ovulatary induction and curing sterility in woman 

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

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

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

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

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

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

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

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

Table 3. AgNORs number in OVX and INT 

(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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