Study Indicates acupuncture may increase sperm quantity, quality and increase testosterone

A new study from China shows that acupuncture may improve sperm quality in idiopathic male infertility.

http://www.nycacupuncture.com/

In the July issue of Fertility and Sterility (Fertil Steril. 2005;84:141-147), the chief investigator, Jian Pei, PhD, from Shanghai University of Traditional Chinese Medicine in the People’s Republic of China, noted that acupuncture had a positive effect on sperm concentration and motility, an increase in testosterone, and some improvement in luteinizing hormone (LH) level, as well as an increase of normally shaped sperm and a significant decrease in the percentage of morphologically abnormal sperm.

The study consisted of 40 men with unexplained sperm abnormalities (oligospermia, asthenospermia, or teratozoospermia). 28 of the men (70%) received acupuncture twice weekly for five weeks. The semen samples from both groups were quantitatively analysed by transmission electron microscopy.

After acupuncture, there was a statistically significant increase in the percentage and number of sperm, and improvement in overall morphology of the sperm without defects seen in the total ejaculates. Specific sperm pathologies in the form of apoptosis, immaturity, and necrosis did not change significantly between the control and treatment groups before and after treatment.

“The treatment of idiopathic male infertility could benefit from employing acupuncture,” the authors write. “A general improvement of sperm quality, specifically in the ultrastructural integrity of spermatozoa, was seen after acupuncture, although we did not identify specific sperm pathologies that could be particularly sensitive to this therapy.”

Relationship Between Blood Radioimmunoreactive Beta-Endorphin and Hand Skin Temperature During The Electro-Acupuncture Induction of Ovulation

ByChen Bo Ying M.D. Lecturer of Neurobiology Institute of Acupuncture Research, and Yu Jin, MD., Prof of Gynecology Obstetricus and Gynecology Hospital Shanghai Medical University Shanghai, People’s Republic of China.
Published: February 24, 2010 , New York Times

ABSTRACT
Thirteen cycles of anovulation menstruation in 11 cases were treated with Electro-Acupuncture (EA) ovulation induction. In 6 of these cycles which showed ovulation, the hand skin temperature (HST) of these patients was increased after EA treatment. In the other 7 cycles ovulation was not induced. There were no regular changes in HST of 5 normal subjects. The level of radioimmunoreactive beta-endorphin (rß-E) fluctuated, and returned to the preacupunctural level in 30 min. after withdrawal of needles in normal subjects. After EA, the level of blood rß-E in cycles with ovulation declined or maintained the range of normal subjects. But the level of blood rß-E and increase of HST after EA (r=-0.677, P <0.01). EA is able to regulate the function of the hypothalamic pituitary-ovarian axis. Since a good response is usually accompanied with the increase of HST, monitoring HST may provide a rough but simple method for prediciting the curative effect of EA. The role of rß-E in the mechanism of EA ovulation induction was discussed.

KEY WORDS: Electro-Acupuncture (EA), Hand Skin Temperature (HST), radioimmunoreactive beta-endorphin (rß-E), ovulation, radioimmunoassay (RIA)

INTRODUCTION
In our previous work, it has been demonstrated that EA is an effectual method of ovulation induction (1). The present work studied the relationship between the curative effect of EA and the changes of the HST and the level of blood beta-endorphin.

MATERIALS AND METHODS
Selection and Treatment of Cases
Eleven cases of chronically anovulatory patients including 9 cases of polycystic ovarian disease (PCO), 1 case of hypogonadotropic amenorrhea and case of oligomenorrhea were treated with EA in 13 menstruation cycles. They were 22 to 35 years of age and their courses of disease were 3 to 12 years. The basic body temperature (BBT) of these patients was monophase for at least 3 months. Each patient accepted the vaginal dropping cell examination twice or more a week. The results showed that the eosinocyte index (EI) of 10 cases was less than 30% and the EI of 1 case was more than 70%.

On the 10th day of each menstruation cycle, the patients were treated with EA. “Guanyuan” “Zhongji,” “Sanyinjiao” and both sides of “Zigong” points were stimulated for 30 min. at 8:00 AM, OD for 3 days. The stimulation parameters were 7-10mA and 4-5HZ with G6805 model generator. The electric current of EA was bearable for every patient. Before and after the EA, HST was measured by a semiconduct thermometer and blood samples were collected from the forearm vien of patients for ß-E RIA. Five healthy woman voluteers with normal menstruation cycle were selected as controls. They were 31 to 35 years old and the menstruation cycle was 28 days. BBT showed change of biphase. All of them were healthy in premenorrhea and did not take any drug one month before EA. The stimulation points and parameters of EA were the same as above mentioned.

Plasma ß-Enorphin Radioimmunoassay
The blood samples were added to 100ug/ml bacitracin for inhibiting blood aminopeptidase and centrifuged at 3,000g for 15 min. The plasma was stored at -40°C.

The sensitive radioimmunoassay was performed as a routine in our lab (2,3), to determine the concentration of ß-E in the samples of plasma. Each estimative tube was added 0.1ml 1:8000 rabbit ß-E antiserum, 0.1ml[125]I-ß-E . That is 0.03ml sheep antiserum to rabbit gamma-globulin diluted 20-fold with RIA buffer was added to each tube, than shaken and incubated at 0-4°C for 24 hours, and centrifuged at 3,000g for 15 min. The supernatant was poured out and the precipitate was counted for radioactivity in Model FH 408 gamma counter. ß-E contents were quantitated according to the standard curve which was performed at the same time with the sample tubes. The least detected quantity of RIA was 10pg/tube.

RESULTS
Clinical Observation

It was adopted standards of ovulation that BBT showed biphase and EI became cyclic variation. Six of 13 menstruation cycles treated with EA showed ovulation, while the other 7 cycles failed to do so. No EA effect was found in normal control subjects.

In the 13 anovulatary cycles, increased HST occurred in 6 cycles, of which 5 cycles showed ovulation after EA treatment. 7 cycles manifested decreased HST and only one of them produced ovulation (Table 1). No regular change was seen in HST in normal subjects.

Table l. Effect of EA Induction of Ovulation in 13 Cycles

Change of Plasrna rß-E

In normal menstruation cycles the level of plasma rß-E before and after EA fluctuated and returned to the preacupural level after 30 minutes.

In the 13 anovulatory cycles the level of plasma rß-E on the 10th day of the cycles was higher but not statistically significant from that of normal subjects.

After EA the plasma rß-E contents of 6 cycles with ovulation either declined or maintained within the range of normal. And the plasma level of 7 cycles that failed to show ovulation after EA were significantly higher than those of normal subjects and 6 ovulatory cases as estimated by t test (P<0.05), (Table 2).

Table 2. Changes of blood ß-E level before and after EA* (pg/ml)

Cycles which showed increase of HST after EA were associated with a declination of plasma rß-E Ievel but in cycles where HST decreased, the plasma rß-E level elevated after EA. There was a negative correlation between changes of plasma rß-E and HST as measured by rank correlation (r=0.677, P<0.01).

DISCUSSION
According to our clinical practice of using EA to cure barreness, the curative effect was related to the changes of patients’ HST. In general, provided that the 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.

From present results, it seems that the successful rate of EA ovulation induction was higher in patients with the depression of sympathetic activity. In normal subject whether HST increased or declined, no influence in ovulation was found. These results suggest that the relationship of ovulation and HST in normal women is different from that in anovulatory patients. Yen and his colleagues (4) first reported that enogenous opioid peptides can inhibit pituitary pulse secreting LH. Fumiko, Akio and Michael reported in succession that morphine, ß-E and dynorphin can also depress LH pulse secretion (5,6,7). These substances may exert their action via regulating the secretion of LH-RH in hypothalmus. EA can affect the central opioid peptide level (2,8,9) thus it may regulate the function of hypothalamic-pituitary-ovarian axis via brain endogenous opiod peptides, such as ß-E and dynorphin etc.

In this study 11 cycles were PCO and the blood LH level in these cycles was marked higher than that of normal subjects. EA may promote the release of ß-E in the brain and reduce LH-RH secretion from hypothalamus. Therefore, the blood LH content released from the pituitary was decreased. This might be one of the mechanisms of EA ovulation induction.

The injection of ß-E into rat cerebellomedullary cisterm resulted in the increase of blood epinephrine (E), norepinephrine (NE) and dopamine (DA) levels, and there was a positive correlation in the dose of ß-E and the levels of blood E, NE, and DA (10). The result suggests that control ß-E may influence the activity of the sympathetic system. Our study showed that the sympathetic activity in normal subjects was not affected and the level of blood ß-E was relatively stable. Thus EA was not able to influence the normal ovulatory cycles. In anovulatory patients, especially in PCO cases, EA can depress sympathetic activity resulting in the increase of HST and the lowering the level of blood ß-E.

These results suggest that in anovulatory cases the hyperactive sympathetic system can be depressed by EA and the function of the hypothalamus-pituitary-ovarian axis can be regulated by EA via central sympathetic system. This might be another possible mechanism of EA ovulation induction.

Our study also suggest that measuring HST my provide a rough but simple method for predicting the effect of EA ovulation induction.

ACKNOWLEDGEMENT
This report has been directed by Prof. He Lian Fang.

REFERENCES
1.Yu Jin, Zheng Hua-Mei, Chen Bo-Yeng, Relationship of hand temperature and blood ß-endorphinelike immunoreactive substance with electroacupuncture induction of ovulation, Acupuncture Research vol. 11 (2), pp. 86-90, 1986.
2.Chen Bo-Ying, Pan Xiao-Ping, Jiang Cheng-Chuan, Chen Shang-Qun, Correlation of pain threshold and level of ß-endoprphin like immuno-reactive substance in human CST during electroacupuncture analgesia, Acta Physiologica Sinica vol. 36 (2), pp. 193-197, 1984.
3.He Xiao-Ping, Chen Bo-Ying, Zhu Jin-Ming, Cao Xiao-Ding, Change of Leu-enkephalin and ß-endorphin-like immunoreactivity in the Hippocampus after electroconvulsive shock and electroacupuncture, Acupuncture & Electro-Therapeutics Res., Int. J., vol. 14 (1), pp.131-139, 1989.
4.Quigley, M.E., Sheeham, K.L., Casper, R.F. and Yen, S.S.C., Evidence for an increased opioid inhibition of luteinizing hormone secretion in hyperprolactinemic patients with pituitary microadenoma, J. Clin. Endocrinol, Metabol, vol.50 (3), pp. 427-436, 1980.
5.Fumiki Kinoshita, Yoshikatsu Nakai, Hideki Katakami, Hiroo Imura, Suppressive effect of dynorphin (1-13) on luteinizing hormone release in conscious rat, Life Sci. vol. 30 (22), pp. 1915-1919, 1982.
6.Akio Adabori, Charles A. Barraclough, Effect of morphine on luteinizing hormone secretion and catecholmine turnover in the hypothalamus of estrogen-treated rats, Brain Res. vol. 362 (2) pp. 221-226, 1986.
7.K. Michael Orstead, Harold G. Spics, Inhibition of hypothalamic gonadotropin releasing hormone release by endogenous opioid peptides in the female rabbit, Neuroendocrinology, vol. 46 (1), pp. 14-23, 1987.
8.Richard S.S., Cheng, S, Pomeranz, B., Electroacupuncture analgesia could be mediated by least two pain relieving endorphin and non-endorphin systems, Life Sci., vol. 25 (22), pp. 1951-1968, 1979.
9.Chen Bo-ying, Wang De-Ling, Pan Xiao-Ping, Changes of opiate likesubstances (OLS) level in perfusate of periaqueductal gray (PAG) after electroacupuncture and brain stimulation, Acta Physiologica Sinica vol. 34 (4), pp.385-391, 1982.
10.Glen R. Van Loon, Nathan M. Appel, Doris Ho, ß-endorphin-induced stimulation of central sympathetic outflow: ß-endorphin increases plasma concentration of epinephrine, norepinephrine, and dopamine in rats, Endocrinology, vol. 109 (1), pp. 46-53, 1981.

Substitution of Acupuncture for HCG in Ovulation Induction

Cai Xuefen, Obstetrical & Gynecological Hospital, Zhejiang Medical University, Zhejiang Province 310006
Source: Journal of Traditional Chinese Medicine 17 (2):119-121,1997

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

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

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

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

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

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

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

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Effects of Electro-Acupuncture on Nerve Growth Factor and Ovarian Morphology in Rats with Experimentally Induced Polycystic Ovaries

Elisabet Stener-Victorin,[2,3] Thomas Lundeberg,[4] Urban Waldenström,[3] Luigi Manni,[5] Luigi Aloe,[5] Stefan Gunnarsson,[6] and Per Olof Janson[3]
Department of Obstetrics and Gynecology,[3] Göteborg University, SE-413 45 Goteborg, Sweden. Department of Physiology and Pharmacology,[4] Karolinska Institutet, SE-164 01 Stockholm, Sweden, Institute of Neurobiology (CNR),[5] Rome, Italy Department of Evolutionary Biology,[6] SE-752 36 Uppsala, Sweden

ABSTRACT
Despite extensive research on the pathogenesis of polycystic ovary syndrome (PCOS), there is still disagreement on the underlying mechanisms. The rat model for experimentally induced polycystic ovaries (PCO)–produced by a single injection of estradiol valerate–has similarities with human PCOS, and both are associated with hyperactivity in the sympathetic nervous system. Nerve growth factor (NGF) is known to serve as a neurotrophin for both the sympathetic and the sensory nervous systems and to enhance the activity of catecholaminergic and possibly other neuron types. Electro-acupuncture (EA) is known to reduce hyperactivity in the sympathetic nervous system. For these reasons, the model was used in the present study to investigate the effects of EA (12 treatments, approximately 25 min each, over 30 days) by analyzing NGF in the central nervous system and the endocrine organs, including the ovaries. The main findings in the present study were first, that significantly higher concentrations of NGF were found in the ovaries and the adrenal glands in the rats in the PCO model than in the control rats that were only injected with the vehicle (oil or NaCI). Second, that repeated EA treatments in PCO rats resulted in concentrations of NGF in the ovaries that were significantly lower than those in non-EA-treated PCO rats but were within a normal range that did not differ from those in the untreated oil and NaCI control groups. The results in the present study provide support for the theory that EA inhibits hyperactivity in the sympathetic nervous system.

adrenal, central nervous system, follicular development, hypothalamus, ovary, ovulation, pituitary, stress

INTRODUCTION
Polycystic ovary syndrome (PCOS), one of the most common causes of anovulation in women of reproductive age. is a complex endocrine and metabolic disorder [1]. Despite extensive research seeking the pathogenesis of PCOS, there is still disagreement on the underlying mechanisms. Different hypotheses of its pathophysiology have emerged, which indicates that the etiology is multifactorial and poorly understood.

Women with PCOS have an increased risk of endometrial cancer, hypertension, and type II diabetes, and they need some kind of long-standing treatment [2]. Traditional pharmacological treatment for ovulation induction is effective, but side effects such as superovulation are quite common. A previous clinical study on anovulatory women with PCOS showed that sensory stimulation (i.e., electro-acupuncture [EA]) affects endocrinological and neuroendocrinological parameters [3]. In addition, regular ovulations were induced in more than one-third of the women without negative side effects. These findings accord with previous reports [4-6] but do not enlighten underlying mechanisms. The mechanisms behind the beneficial effect of EA on PCOS in the human are difficult to study because tissue samples from the ovaries and the central nervous system (CNS) are for obvious reasons unobtainable. Studies on, for instance, neuropeptides in the gonads and the CNS would be possible to conduct in an animal model, provided that such a model exists.

Experiments on normal cycling rats have shown that exogenous estradiol valerate (EV), a long-acting estrogen, causes acyclicity and the formation of polycystic ovaries (PCO) [7, 8]. The changes include atretic antral follicles, follicular cysts with a well-developed theca cell layer, a diminished granulosa cell compartment, and luteinized cysts [7, 8]. Furthermore, the rats exhibited alterations in basal and pulsatile LH and FSH concentrations, changes in the pituitary response to GnRH, degenerative changes in the hypothalamus, altered opioid inhibitory tone on GnRH release, and high estradiol levels with a persistent pattern of constant estrus as assessed by vaginal smear [9, 10]. In addition, EV-induced PCO is associated with an increase in peripheral sympathetic outflow, evidenced by an increase in the release of norepinephrine (NE), an increase in ovarian NE content, and a decrease in the number of ß-adrenergic receptors in the ovarian compartments receiving catecholaminergic innervation [9-11]. Even if it is not possible to reproduce human PCOS using a rat model, it may provide important leads because a single injection of EV induces an anovulatory state that shares many endocrinological and morphological characteristics of human PCOS [7-13]. Thus, comparisons between the rat PCO model and human PCOS must be interpreted with caution because rat PCO ovaries contain multiple follicular cysts, the structure of which does not replicate the follicular growth arrest found in human PCOS. Contrary to previously held notions, the granulosa cells in the follicles accumulating in the human ovary are not atretic. However, both human PCOS and EV-induced PCO in rats may be associated with hyperactivity in the sympathetic nervous system.

According to one theory, elevated concentrations of neurotransmitters found in women with PCOS and anovulation may be associated with psychological stress and with hyperactivity in the sympathetic nervous system [3, 12, 13]. That superior ovarian nerve transection restores estrus cyclicity and ovulatory capacity in rats with EV-induced PCO further supports the theories of sympathetic hyperactivity [9]. Other evidence of neuronal involvement is that ovarian sympathetic innervation is under trophic control by nerve growth factor (NGF) [14]. This is also supported by the fact that the expression of the genes that encode NGF and one of its receptors, the low-affinity NGF-receptor, was dramatically increased in the ovary 30 days after EV injection [11]. Ovarian NGF is principally synthesized in the cells of the follicular wall [15], which is the site where the sympathetic neurons project to the ovaries [14]. The increase in the synthesis of NGF and its receptor that precedes the formation of cysts suggests that after PCO has been induced by EV injection, the neurons innervating the ovary are subjected to an enhanced neurotrophic influence that contributes to their hyperactivation and to the maintenance of an abnormally elevated catecholaminergic tone in ovarian steroid secretions [9-11].

Aim of the Study: Because NGF is known to serve as a neurotrophin for both the sympathetic and the sensory nervous systems and to enhance the activity of catecholaminergic and possibly other neuron types [9, 11, 14, 16-22], and because EA is known to reduce hyperactivity in the sympathetic nervous system [23-25], the experimentally induced PCO model was used to study the effects of EA by analyzing NGF in the CNS and the endocrine organs, including the ovaries.

The first part of the present study investigated dose-response–the discovery of the exact dose of EV needed to produce fully developed polycystic ovaries. The second part of this study investigated treatment with EA–what contribution NGF made to the etiology and maintenance of EV-induced PCO in rats and if and to what extent EA has an effect on NGF and ovarian morphology in experimentally induced PCO.

MATERIALS AND METHODS
Fifty-nine virgin adult cycling Sprague-Dawley rats (Möllegaard, Denmark) weighing 190-210 g and with regular 4-day estrous cycles were used. The rats were housed at 22°C, four to a cage, with free access to pelleted food and tap water and with a 12L:12D cycle for at least 1 wk before and throughout the experimental period. All rats received a single i.m. injection of either EV (Riedeldehaen, Germany), oil, or 0.15 M NaCI (Kabi Pharmacia AB, Sweden) and were anesthetized with enfluran (EFRANE, Abbott Scandinavia, Kista, Sweden) and killed by decapitation. The local Animal Ethics Committee at Göteborg University, Sweden approved the study.

Dose-Response: Twenty-seven rats were injected with one of two different doses of EV in an oil solution or with oil alone to ascertain the optimal dose for induction of PCO [8]. They were decapitated on three different occasions (15, 30, or 60 days after i.m. injection) to elucidate precisely when the ovaries display characteristic features of well-defined PCO [7, 8]. Nine rats received 2 mg EV in 0.2 ml oil/rat, nine rats 4 mg EV in 0.2 ml oil/rat, and nine rats 0.2 ml oil alone. Three rats per dose were killed on Day 15, three on Day 30, and three on Day 60.

FIG. 1. Schematic drawing of the dorsal side of a rat and the placement of acupuncture needles. Two needles were placed bilaterally in the erector spinae muscle at the level of Th12 and two were placed in the quadriceps muscle bilaterally. The needles were then attached to an electrical stimulator for EA treatment.

Treatment with EA: The optimal dose (4 mg EV in 0.2 ml oil/rat) and timing (30 days after injection) were chosen for the experiments. In total, 32 rats took part. Eight rats in the EV control group and eight in the EA-treated EV group were injected i.m. with 4 mg EV in 0.2 ml oil/rat, eight rats in the oil control group with 0.2 ml oil, and eight rats in the NaCI control group with 0.2 ml 0.15 M NaCI. All 32 were decapitated on Day 30 after injection, that is, 1-2 days after the last EA treatment. All groups were anesthetized 12 times for about 25 min each time. Anesthesia was induced by inhalation of enfluran at 5.5-6.5 ml/h with an O2 and air flow of 0.25 L/min. The EA-treated EV group was subjected to 12 EA treatments every second or third day, beginning 2 days after the i.m. injection of EV. The stimulation points were bilateral in the quadriceps and erector spinae muscles at the level of thoracic (Th) 12 in the somatic segments according to the innervation of the ovaries (Th 12-lumbar [L]2, sacral [S]2-S4) (Fig. 1). The needles (Hegu; Hegu AB, Landsbro, Sweden) were inserted to depths of 0.5-0.8 cm and then bilaterally attached to an electrical stimulator (CEFAR ACU II, Cefar, Lund, Sweden) with a low burst frequency of 2 Hz. Individual pulses within the frequency were square wave pulses with alternating polarities and with a pulse duration of 0.2 msec, 80 pulses/sec. The intensity was adjusted so that local muscle contractions were seen to reflect the activation of muscle-nerve afferents (A delta fibers and possibly C fibers) [26, 27]. The location and type of stimulation were the same in all rats.

Nerve Growth Factor Measurements by Enzyme Immunoassay: In the second part of the study, after the rats were decapitated, the pituitary gland, the hypothalamus, the hippocampus, one ovary, and the adrenal glands were quickly removed and dissected on dry ice, weighed, and stored at -80°C until extraction. The samples were sonicated in extraction buffer (0.1% Triton X-100, 100 mM Tris-HCI, pH 7.2, 400 mM NaCI, 4 mM EDTA, 0.2 mM PMSF, 0.2 mM benzethonium chloride, 2 mM benzamidine, 40 U/ml aprotinin, 0.05% sodium azide, 2% BSA, and 0.5% gelatin; 1 ml/100 mg of tissue), followed by centrifugation at 10,000 x g for 30 min. The supernatants were used for the assay. The bioactive form of 2.5S NGF purified from mouse sub-maxillary glands and prepared in the laboratory at the Institute of Neurobiology (CNR) in Rome, Italy, according to the method of Bocchini and Angeletti [28] was used as a standard. The NGF was dissolved in extraction buffer and the standard curve was in a range of 31.25 pg ml (-1) and 1 ng ml (-1). An ELISA was performed as described by Weskamp and Otten [29] with a minor modification [30]. Specific NGF binding was assessed by use of monoclonal mouse anti-ß-2.5S NGF (Boehringer Mannheim GmbH, Mannheim, Germany) that reacts with both the 2.5S and the 7S biologically active forms of NGF. The absorbency of samples and standards was corrected for nonspecific binding (i.e., the absorbency in a well coated with purified mouse IgG). The NGF content in the samples was determined in relation to the NGF standard curve. Data were not corrected for recovery of NGF from samples, which was routinely 70-90%, and was accepted only when the values were >2 SD above the blank. With these criteria, the limit of sensitivity of NGF ELISA averaged 0.5 pg per assay.

Morphology: One ovary per rat was removed, cleaned of adherent connective fat tissue, and fixed in 4% formaldehyde buffer; sections were stained with hematoxylin-eosin, and a trained pathologist performed a quantitative analysis of the follicle population. If ovum degeneration or at least one pyknotic granulosa cell was seen, the follicle populations were classified as atretic, otherwise they were classified as healthy. Morphological characteristics of follicular atresia were, for instance, scattered pyknotic nuclei in the granulosa cell layer [31], detachment of the granulosa cell layer from the basement membrane [32], fragmentation of the basal lamina [33], and the presence of cell debris in the antrum of the follicle [34].

STATISTICAL ANALYSIS
Statistical analyses were carried out using the SPSS 8.0 software. The NGF concentrations in the pituitary gland, the hypothalamus, the hippocampus, the ovary, and the adrenal glands were analyzed and the groups compared using ANOVA followed by multiple comparison procedures (Bonferroni test). All results are presented as mean ± SEM. A P value less than 0.05 was considered significant. The 95% confidence interval (Cl) was given when P < 0.05.

RESULTS
Ovarian Morphology–Dose-Response: In the first part of the present study, dose-response, injection of 0.2 ml oil alone (control) was associated with a normal appearance of the ovaries and no differences were seen between rats sacrificed on Day 15, 30, or 60 (Fig. 2, a and b). No changes were seen in the ovaries of rats injected with 2 mg EV in 0.2 ml oil/rat and killed on Day 15. The ovaries of rats injected with the same dose of EV in oil exhibited small morphological changes resembling PCO when killed on Day 30 and 60 (Fig. 3, a and b). The ovaries of rats injected with a higher dose of EV (4 mg EV in 0.2 ml oil/rat) exhibited only small morphological changes on Day 15. Rats injected with the same dose of EV in oil and killed on Day 30 (Fig- 4, a-c) showed a progressive decrease in the number of primary and secondary follicles but it was on Day 60 (Fig- 5, a and b) that the true cystic follicles appeared and the well-defined PCO was fully developed in accordance with previous reports by Brawer et al. [8].

Ovarian Morphology–Treatment with EA: In the second part of the present study, treatment with EA, all rats were killed at Day 30 after EV injection, i.e., before the appearance of cystic follicles. The ovaries in the EV control group (4 mg EV in 0-2 ml oil/rat) displayed the same morphological changes as previously shown in the dose-response section (see Fig- 4, a-c). The ovaries in the oil control group and the NaCI control group exhibited a typically normal appearance (see Fig- 2, a and b). No substantial morphological differences were found between the EA-treated, EV group, and the EV control group.

Nerve Growth Factor–Treatment with EA: In the second part of the present study, treatment with EA, NGF measurements were made at Day 30 after EV injection. Means ± SEM for NGF (pg/g wet weight) in the hypothalamus, the pituitary gland, the hippocampus, the ovary, and the adrenal gland in all groups are presented in Table 1. Ovarian NGF concentrations were significantly higher in the EV control group compared to the oil control group (P < 0.001, CI = 178.7, 821.6) and the NaCl control group (P < 0.01, CI = 144.6, 787.5). The NGF concentrations in the ovary were significantly lower in the EA-treated, EV group compared to the EV control group (P < 0.05 Cl = 6.2, 614.9) and did not differ from the (Jil and the NaCI control groups) The NGF concentrations in the adrenal glands were significantly higher in the EV control group and the EA-treated. EV group compared to both the oil control group (P < 0.001, CI = 45.7, 169.3 and P < 0.01, CI = 38.5, 166.5) and the NaCI control group (P < 0.001, Cl = 21.9, 162.9 and P < 0.01, Cl = 15.0, 159.8).

Weights of Ovaries and Adrenal Gland–Treatment with EA: Means ± SEM for weights (mg) of the ovaries and the adrenal glands in all groups are presented in Table 2. Ovarian weights in the control EV group and in the EV-treated EV group were significantly lower compared to the oil control group (both P < 0.001) and the NaCI control group (both P < 0.001).

FIG. 2. a) Section of an ovary from a rat injected with 0.2 ml in oil and sacrificed on Day 30. In total, 11 corpora lutea (CL) marked with CL and three secondary follicles (SF) marked with SF are seen. One secondary follicle is framed (b). Magnification x2.5. Section stained with hematoxylin-eosin. b) Normal secondary follicle. Magnification x20. FIG. 2. a) Section of an ovary from a rat injected with 0.2 ml in oil and sacrificed on Day 30. In total, 11 corpora lutea (CL) marked with CL and three secondary follicles (SF) marked with SF are seen. One secondary follicle is framed (b). Magnification x2.5. Section stained with hematoxylin-eosin. b) Normal secondary follicle. Magnification x20.

 

FIG. 5. a) Section of an ovary from a rat injected with 4 mg EV in 0.2 ml oil and sacrificed on Day 60. In total, two corpora lutea marked with CL, five cystic follicles marked with CF and one secondary follicle marked with SF are seen. One cystic follicle is framed (b). Magnification x2.5 Section stained with hematoxylin-eosin. b) A cystic degenerating follicle showing a thin granulosa layer and debris in follicular fluid. Magnification x20.
FIG. 4. a) Section of an ovary from a rat injected with 4 mg EV in 0.2 ml oil and sacrificed on Day 30. In total, seven corpora lutea marked with CL three cystic follicles (CF) marked with CF, and two atretic secondary follicles marked with ASF are seen. One cystic follicle (b) and one atretic secondary follicle are framed (c). Magnification x2 5: Section stained with hematoxylin-eosin. b) Cystic degenerating follicle showing a thin granulosa layer and debris in follicular fluid. Magnification x20. c) An atretic secondary follicle with detachment of the oocyte from the cumulus mass of pyknotic granulosa cells. Magnification x20.

 

DISCUSSION
The main findings in the present study are as, follows: First, PCO induced in rats by a single injection or EV results in significantly higher concentrations of NGF in the ovaries and the adrenal glands without any changes in the brain tissue when measured 30 days after EV injection.

Second, repeated EA treatments with low frequency (2 Hz) significantly decrease the elevated NGF concentrations in the ovaries, to within a normal range, without affecting NGF concentrations in the adrenal glands or brain tissue when measured 30 days after EV injection.

The histological examination of the ovaries in the first part of the present study, dose-response, revealed that the optimal dose of EV that caused typical PCO-like morphological changes was 4 mg and that PCO was fully developed at Day 60. This dose was twice that used by Brawer and coworkers [7, 8] to achieve full development of a well defined PCO in rats. The reason might be differences in the strain of rat and/or the estrogen preparation that was used. In addition, the ovarian weight in the two EV-injected groups was significantly lower compared to that in the vehicle-injected (oil and NaCI) control rats. The reduction in ovarian weight and size, as well, are in accordance with the findings of Brawer et al. [8]. The reduction in weight and size of the ovaries might be explained by a reduction in the number of corpora lutea. In the second part, treatment with EA, no substantial influence in ovarian morphology was seen at Day 30, after EV injection with the number and duration of the EA treatments used in this study. However, the main reason for beginning EA treatment as early as 2-3 days after EV injection and to decapitate at Day 30 after EV injection was to estimate whether EA could influence the increased ovarian NGF concentrations that have been shown to precede the development of morphological changes in rats with PCO [11]. It remains to be shown whether EA influences the ovarian morphology 60 days after EV injection. It would therefore be of interest to study the effects of EA after extended treatment periods. Such a study would provide a unique opportunity to collect experimental evidence of the effectiveness of EA in humans. In fact, we have observed that the multifollicular pattern characteristic of the ovarian morphology of women with PCOS and anovulation, as assessed by ultrasonography, began to disappear after they had received repeated EA treatments [3].

An involvement of the nervous system in the etiology and/or maintenance of PCOS is suggested by both clinical and experimental findings [9-13]. Clinical studies show that women with PCOS temporarily recover normal ovarian function after bilateral wedge resection or ovarian drilling that partially denervates the ovary [35, 36]. There is thus a possibility that the ovarian nerves are involved in the successful outcome of bilateral wedge resection and ovarian drilling.

Experimental observations in rats reveal that superior ovarian nerve transection in EV-induced PCO reduces the steroid response, increases ß-adrenoreceptor concentrations to more normal levels, and restores estrus cyclicity and ovulation [9]. These effects were linked to reduced activity in the ovarian sympathetic nerve fibers, indicating a peripheral neurogenic effect [9].

Sensory stimulation, i.e., EA, activates muscle-nerve afferents, mainly A-delta and possibly C fibers [23, 26, 27], that initiate a number of peripheral reactions at the spinal level and centrally in the brain. That EA may reduce hyperactivity in the ovarian peripheral sympathetic nerve fibers is in accordance with the theory that EA could modulate sensory, motor, and autonomic outflow at the segmental level [24]. In parallel, higher control systems are activated, resulting in the release of a number of neuropeptides, important in the modulation of central and segmental autonomic outflow, of the hypothalamic-pituitary-ovarian axis (HPO axis), and of the descending pain-inhibiting systems [23-25].

TABLE 1. Treatment with EA.

TABLE 1. Treatment with EA.

For obvious reasons it is not possible to subject control animals to true sham needle insertion. As soon as a needle penetrates the skin, it may be seen as a form of sensory stimulation that activates afferent nerve fibers. If a sham needle insertion without electrical stimulation is performed, then different acupuncture methods/stimulation techniques are being compared, and this does not provide proper information on the effect of EA versus no EA. We chose EA because the stimulation intensity is easy to standardize and it has been shown to be superior to manual needle stimulation [37]. In addition, to show a difference between two or more stimulation techniques would require a very large number of study subjects. In the present study, the control rats received the same enfluran anesthesia protocol as the rats treated with EA, which, in our opinion, is the best way to control completely environmental and/or emotional factors and the EA effect. The acupuncture needles in the present study were placed in the somatic segments that correspond to ovarian innervation. The needles were stimulated with low frequency EA for optimal activation of muscle nerve afferents to inhibit the autonomic outflow at the segmental level and at the central level and to modulate the HPO axis. The choice of acupuncture points and the aim of stimulation has been the same as in our other EA studies on the female reproductive tract that dealt with blood flow in the uterine arteries prior to in vitro fertilization (IVF) [38], pain-relief during oocyte aspiration in connection with IVF treatment [39], and induction of ovulation in women with PCOS [3].

We have shown that repeated EA treatments restore regular ovulations in more than one-third of the anovulatory women with PCOS. In addition, EA-influenced neuroendocrine and endocrine parameters indicative of PCOS, such as LH/FSH ratios, mean testosterone concentrations, and ß-endorphin concentrations, decreased significantiy [3]. The effects of repeated EA on anovulation were then attributed to an inhibition of hyperactivity in the sympathetic nervous system [3, 5, 6].

The findings of the present study support recent reports that ovarian NGF concentrations in rats with experimentally induced PCO [11] are elevated and that this increase can be related to a hyperactivity in the ovarian sympathetic nerves. Lara et al. [11] also suggests that activation of this neurotrophic-neurogenic regulatory loop is a component of the pathological process by which EV induces cyst formation and anovulation. They also stated that there is evidence that the alteration in neurotrophic input to the ovary contributes to the etiology and/or maintenance of human PCOS [11].

Furthermore, the present study shows that repeated EA treatments reduce peripheral sympathetic nerve hyperactivity, as revealed by the reduction in increased NGF concentrations in the ovaries into a normal range 30 days after EV injection, that did not differ from that of the untreated oil and NaCI control groups.

It remains to be shown whether EA directly affects sympathetic nerve activity. Measurements of the nervous output by analyses of the catecholamine release can resolve this. In addition, because receptors for NGF are expressed on the endocrine cells of the ovary, activities of ovarian NGF may mediate and/or be mediated by alterations in endocrine factors, for example, by corticotropin-releasing hormone, prolactin, oxytocin, and/or adrenal corticosteroid secretion. To resolve this, the same experimental protocol regarding EA and controls used here must be supplemented with measurements of serum levels of these hormones.

Whether this condition can be reversed with EA treatment at higher stimulation intensities, in higher numbers, and/or over longer periods remains to be shown.

The conclusion of this study is that repeated EA treatments reduce ovarian NGF concentrations to within normal ranges. This suggests that EA inhibits the hyperactivity in the ovarian sympathetic nerves, which may be of importance for the development and maintenance of experimentally induced PCO.

ACKNOWLEDGMENTS
The authors thank Professor Owe Lundgren and laboratory assistant Britt-Marie Fin, Department of Physiology, Goteborg University, for providing excellent working facilities and for invaluable laboratory help at their Department. We also thank Associate Professor Folke Knutsson for his invaluable assistance in the morphological analyses of the ovaries. Carl Lofman, M.D., Stockholm is acknowledged for skillful preparation of morphological specimens.

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[1]Supported by grants from the Hjalmar Svensson Foundation, Wilhelm och Martina Lundgrens Vetenskapsfond Wilhelm and Martina Lundgren’s Science Fund, and the Foundation for Acupuncture and Alternative Biological Treatment Methods.
[2]Correspondence: Elisabet Stener-Victorin, Department of Obstetrics and Gynecology, Kvinnokliniken, Sahlgrenska University Hospital, SE-413 45 Göteborg, Sweden. FAX: 46 31829248;
e-mail: elisabet.stenervictorin@medstud.gu.se

Received: 29 February 2000.
First decision: 30 March 2000.
Accepted: 11 July 2000.
© 2000 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org

Acupuncture Normalizes Dysfunction of Hypothalamic-Pituitary-Ovarian Axis

By Bo-Ying Chen M.D. Professor of Neurobiology. Institute of Acupuncture and Department of Neurobiology, Shanghai Medical University, Shanghai 200032, P.R. China
(Received June 3, 1997; Accepted with revisions June 30,1997)

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

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

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

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

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

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

Pushpull perfusion in hypothalamic preoptic area (POA) and elution of pituitary and LH and ß-endorphin (ß-EP)
The technique of brain pushpull perfusion was processed as previously described by our laboratory [1]. The perfusate from hypothalamic POA was kept at -70°C for GnRX and ß-EP RIA.

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

Radioimmunoassay (RIA) of hormones

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

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

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

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

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

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

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

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

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

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

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

(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

Table 4. Weight of adrenal

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

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

 

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

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

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

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

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

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

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

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

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

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

REFERENCES
1. Chen, BY et al, Correlation of pain threshold and level of beta-endorphin like immunoreactive substance in human CSF during electroacupuncture analgesia. Acta Physiologica Sinica (in Chinese), 34(4), 385-391, 1984
2. Riahard, ss et al, Electroacupuncture analgesia could be mediated by least two pain-releasing endorphins and one endorphin. Life Science, 25, 1957-1968, 1980
3. Yu, J et al, Relationship of hand temperature and blood ß-endorphin immunoreactive substance with electroacupuncture induction of ovulation. Acupuncture Research (in Chinese), 11(2), 86-90, 1986
4. Liu, WC et al, The influence of acupuncture on serum hormones of dysfunction uterine bleeding. Chinese Acupuncture and Moxibustion (in Chinese), 11(5), 37-38, 1991
5. Zhou, CH et al, Experimental study of the mechanism of acupuncture inducing ovulation. J Combining Chinese and Westen Medicine (in Chinese), 6(12), 764, 1986
6. Shatina, GV et al, Corretive effect of reflextherapy on the hypophyseal-ovarian and sympathetic-adrenal system after ovariectomy. Akush Ginekol MOsk (in Russian), 10, 58-61,1991
7. Zheng, W et al, Electroacupuncture-induced acceleration of proopiomelanocortin mRNA in the pituitary and proenkephalin mRNA in the adrenal in rat. Chinese J Physiological Sciences, 3, 106-108, 1986
8. YU, YH et al, Time course of alteration of proopiomelanocorting mRNA level in rat hypothlamic arcuate nucleus following electroacupuncture. Acta Academiae Medicinae Shanghai, 21(Suppl.), 59-62, 1994
9. Nett, TM et al, Aradioimmunoassay for gonadotropin-releasing hoemone (GnRH) in serum. J Clinical Endocrine and Metabolism, 36, 880-883, 1973
10. Howell, WM and Black, DA, Controlled silver-staining organizer reginos with protective coppoidal developer: A 1-step method. Experiment, 36, 1014-1016, 1980
11. Wu, ZT et al, The change of c-fos expression in ovariectomized rats following electroacupuncture treatment-An immunohistochemistry study. Acupuncture & Electro-Therapeutics Research The International Journal, 18, 117-124, 1993
12. Stallcup, MR and Washington, LD, Region-specific initiation of mouse mammary tumor virus RNA synthesis by endogenous RNA polymerase II in preparations of cell nuclei. J Biologic Chemisty 258, 2802-2904, 1083
13. Sambrook, J et al, Molecular Cloning-A Laboratory Manual. 343-355, 2nd edition, Cold Spring Harbor Laboratory Press, 1989
14 Chen, BY et al, Relationship between blood radioimmunoreactive beta endorphin and hand skin temperature during the electro-acupuncture induction of ovulation. Acupuncture & Elctro-Therapeutics Research The International Journal 16(1), 1-5, 1991
15. Crocker, C and Paramyit, NAR, Nucleolar organizer regions in lymphomas. J Pathology, 155, 111-118, 1987
16. Omura, Y et al, Simple non-invasive mapping of pain pathway in living humans, and the effect of acute non-invasively induced pain on substance P, oncogen C-FOS Ab1, oncogen C-fos Ab2, dopamine and acetycholine. Acupuncture & Electro-Therapeutics Research The International Journal, 17(4), 291-300, 1992
17. Morgan, TI et al, Mapping patterns of C-fos expression in the central nervous system after seizure. Science, 237, 192-199, 1984
18. Sagar, S et al, Expression of C-fos protein in brain: Metabolic rnapping at cellular level. Science, 240, 1326-1331, 1988
19. Dragunow, M and Full, R, The use of C-fos as a metabolic marker in neuronal pathway tracing. J Neuroscience Method, 29, 251-265, 1989
20. Micheal, KS and Harold, GS, Inhibition of hypothalamic-gonadotropin-releasing hormone release of endogenous opioid peptides in the female rabbit. Neuroendocrinology, 46, 14-21, 1987
21. Lauber, AH, et al, Estrogen receptor mRNA expression in rat hypothlamus as a function of genetic sex and estrogen dose. Endocrinology, 129, 3180-3186, 1990
22. Medhabanada, S et al, immunohistochemical localization of estrogen receptor in rat brain, pituitary and uterus with monoclonal antibody. Steroid Biochemistry, 24, 497-503, 1986
23. Simerly, RB, Distribution of androgen and estrogen receptor mRNA containing cell in rat brain an in situ hybridization study. J Comparative Neurology, 294, 76-95, 1990
24. Shughrue, PJ et al, Estrogen receptor mRNA in female rat brain during estrous cycle, a comparision with ovariectomized female and intact male rat. Endocrinoloy, 131, 3180-3186, 1992

Acupuncture Treatment For Infertile Women Undergoing Intracytoplasmic Sperm injection

Sandra L. Emmons, MD, Phillip Patton, MD
Source: Medical Acupuncture, A Journal For Physicians By Physicians, Spring / Summer 2000- Volume 12 / Number 2

Up to a quarter of all women suffer from depression during pregnancy, and many are reluctant to take antidepressants. Now a new study suggests that acupuncture may provide some relief during pregnancy, even though it has not been found to be effective against depression in general.

The Stanford University study recruited 150 depressed women who were 12 to 30 weeks pregnant, and randomly assigned 52 to receive acupuncture specifically designed for depressive symptoms, 49 to regular acupuncture and 49 to Swedish massage.

Each woman received 12 sessions of 25 minutes each; those given acupuncture did not know which type they were getting. (In the depression-specific treatment, needles are inserted at body points that are said to correspond to symptoms like anxiety, withdrawal and apathy.)

After eight weeks, almost two-thirds of the women who had depression-specific acupuncture experienced a reduction in at least 50 percent of their symptoms, compared with just under half of the women treated with either massage or regular acupuncture.

The findings appear in the March issue of Obstetrics & Gynecology. The lead author, Rachel Manber, a professor of psychiatry and behavioral sciences at Stanford, said the results suggested that some symptoms of depression during pregnancy might be related to physical discomfort that is alleviated by acupuncture. Still, the results were striking, she said.

Reduction of blood flow impedance in the uterine arteries of infertile women with electro-acupuncture

Elisabet Stener-Victorin[1.4], Urban Waldenström[2], Sven A. Andersson[3] and Matts Wikland[2] [1]Department of Obstetrics and Gynaecology [2]Fertility Centre Scandinavia. Department of Obstetrics and Gynaecology and [3]Department of Physiology University of Gothenburg. S-413 45 Gothenburg, Sweden [4]To whom correspondence should be addressed: Department of Obstetrics and Gynecology. Kvinnokliniken Sahlgrensh sjukhuset, S-413 45 Golhenburg, Sweden
Source: European Society for Human Reproduction and Embryology

In order to assess whether electro-acupuncture (EA) can reduce a high uterine artery blood flow inpedance, 10 infertile but otherwise healthy women with a pulsatility index (PI) ≥3.0 in the uterine arteries were treated with EA in a prospective, non-randomized study. Before inclusion in the study and throughout the entire study period, the women were down-regulated with a gonadotrophin-releasing hormone analogue (GnRHa) in order to exclude any fluctuating endogenous hormone effects on the PI. The baseline PI was measured when the serum oestradiol was ≤0.1 nmol/l, and thereafter the women were given EA eight times, twice a week for 4 weeks. The PI was measured again closely after the eighth EA treatment, and once more 10-14 days after the EA period. Skin temperature on the forehead (STFH) and in the lumbosacral area (STLS) was measured during the flrst, fifth and eighth EA treatments. Compared to the mean baseline PI, the mean PI was significantly reduced both shortly after the eighth EA treatment (P < 0.0001) and 10-14 days after the EA period (P < 0.0001). STFH increased significantly during the EA treatments. It is suggested that both ot these effects are due to a central inhibition of the sympathetic activity.

Key words: electro acupuncture/pulsatilily index (PI)/trans-vaginal colour Doppler curve/uterine artery blood flow

INTRODUCTION
Successful in-vitro fertilization (IVF) and embryo transfer demand optimal endometrial receptivity at the time of implantation. Blood flow impedance in the uterine arteries, measured as the pulsatility index (PI) using transvaginal ultrasonography with pulsed Doppler curves, has been considered valuable in assessing endometrial receptivity (Goswamy and Steptoe, 1988; Sterzik et al., 1989; Steer et al., 1992, 1995a,b; Coulam et al., 1995; Tekay et al., 1995). Steer et al. (1992) found that a PI ≥3.0 at the time of embryo transfer could predict 35% of the failures to become pregnant. Coulam et al. (1995) did not observe any significant differences between PI measurements done on the day of oocyte retrieval compared with PI measurements on the day of embryo transfer. This would allow prediction of non-receptive endometria earlier in the cycle.

Previous studies on rats have shown a decreased blood pressure after electro-acupuncture (EA) with low frequency (2 Hz) stimulation of muscle afferents (A-d fibres). The decreased blood pressure was related to reduced sympathetic activity (Yao et al., 1982; Hoffman and Thoren, 1986; Hoffman et al.. 1987, 1990a,b), and was paralleled by an increase in the ß-endorphin concentration in the cerebrospinal fluid (CSF), suggesting a causal relationship to central sympathetic inhibition (Cao et al., 1983; Moriyama 1987; Reid and Rubin, 1987). The cardiovascular effects of acupuncture treatment are probably mediated by central opioid activity via the ß-endorphin system from the hypothalamus.

The aim of this study was to evaluate whether EA can reduce a high impedance in the uterine arteries. There are several conceivable mechanisms which may give this effect.

In addition to central sympathetic inhibition via the endorphin system, vasodilatation may be caused by stimulation of sensory nerve fibres which inhibit the sympathetic outflow at the spinal level, or by antidromic nerve impulses which release substance-P and calcitonin gene-related peptide from peripheral nerve terminals (Jansen et al., 1989; Andersson, 1993; Andersson and Lundeberg, 1995).

It has been assumed that various disorders in the autonomic nervous system, such as hormonal disturbances, may be normalized during auricular acupuncture (Gerhard and Postneck, 1992). It has also been suggested that the concentrations of central opioids may regulate the function of the hypothalamic-pituitary-ovarian axis via the central sympathetic system, and that a hyperactive sympathetic system in anovulatory patients could be normalized by EA (Chen and Yin, 1991).

MATERIALS AND METHODS
Subjects, design and Pl measurements: The study was approved by the ethics committee of the University of Gothenburg and was conducted at the Fertility Centre Scandinavia, Gothenburg, Sweden, a tertiary private IVF unit. All women attending the clinic for information about the IVF/embryo transfer procedure, had the PI of their uterine arteries measured by transvaginal ultrasonography and pulsed Doppler curves (Aloka SSD 680: Berner Medecinteknik, Stockholm, Sweden). The PI value for each artery was calculated electronically from a smooth curve fitted to the average waveform over three cardiac cycles, according to the formula: Pl = (A – B)/mean, where A is the peak systolic Doppler shift, B is the end diastolic shift frequency and mean is the mean maximum Doppler shifted frequency over the cardiac cycle. A reduction in the value of PI is thought to indicate a reduction in impedance distal to the point of sampling (Steer et al., 1990).

In the routine preparation for their IVF/embryo transfer treatment, all women were down-regulated with a gonadotrophin-releasing hormone analogue (GnRHa) (Suprecur: Hoechst. Germany). When their oestradiol concentration in serum was <0.1 nmol/1, the women were considered down-regulated and the PI of their uterine arteries was again measured in those women showing a mean Pl ≥3.0 before down-regulation. The measurements were done by two of the authors (M.W. and U.W.) between 08.30 h and 14.30 h. These hours were chosen for practical reasons, and also to reduce the risk that the PI measurements would be affected by the circadian rhythm in blood flow, recently reported by Zaidi et al. (1995). Three measurements were made on the right and three on the left uterine artery of each patient. Before the study was conducted, the observers were well trained in PI measurements with the equipment used. Steer et al. (1995) has shown that in trained hands, the inter-, and intra-observer variations in vaginal colour Doppler ultrasound are sufficiently small to provide a basis for clinically reliable work.

PI measurements were done on all women attending the unit for an IVF/embryo transfer treatment between November 1992 and February 1993. Of these, all infertile but otherwise healthy women, with a mean PI ≥3.0 in the uterine arteries both before and after down-regulation, were invited to be included in the study.

In all, 10 women accepted after informed consent and they had a mean age of 32.3 years (range 25-40 years). The infertility diagnoses were unexplained infertility (n = 6), tubal factor (n = 3) and polycystic ovarian syndrome (n = 1).

From their inclusion and onwards, the women were kept on the GnRHa and were given no other pharmacological treatment. Consequently, their gonadotrophins and ovarian steroids were kept at a constantly low concentration, both at their inclusion in the study and throughout the whole study period. Thus, PI changes due to hormonal fluctuations were avoided.

EA was then given eight times, twice a week for 4 weeks. The mean PI of the uterine arteries was measured (mean of three PI on each side) directly after the eighth EA treatment and again 10-14 days after the EA period.

Of the 10 women included, two were later excluded. One of them, with tubal factor infertility, was excluded because she started taking medications for her migraine, which could have affected her PI. The other excluded woman, with unexplained infertility, stopped her GnRHa treatment because she preferred IVF/embryo transfer in a natural cycle.

Acupuncture Treatment
The sympathetic outflow may be inhibited at the segmental level and, for this reason, acupuncture points were selected in somatic segments according to the innervation of the uterus (Thl2-L2, S2-S3) (Bonica, 1990).

The needles were inserted i.m. to a depth of 10-20 mm. The aim of the stimulation was to activate group III muscle-nerve afferents. The needles were twirled to evoke `needle sensation,’ often described as tension, numbness, tingling and soreness, sometimes radiating from the point of insertion. The needles were then attached to an electrical stimulator (WQ-6F: Wilkris & Co. AB, Stockholm, Sweden) for 30 min. The location of the needles was the same in all women (Table I).

Table 1. Acupuncture points, their anatomical position and their innervation

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.

 

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.

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16. 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.
17. Reid, J.L. and Rubin, P.C. (1987) Peptides and central neural regulation of circulation. Physiol. Rev., 67, 725-749.
18. 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.
19. 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.
20. 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.
21. 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.
22. 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.
23. 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.
24. Tekay, A., Martikainen, H. and Jouppila, P. (1995) Blood flow changes in uterine and ovorian vasculature, and predictive value of transvaginal pulsed colour Doppler ultrasonography in an in-vitro fertilization programme. Hum. Reprod., 10, 688-693.
25. 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.
26. 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

Influence of acupuncture on the pregnancy rate in patients who undergo assisted reproduction therapy

Influence of acupuncture on the pregnancy rate in patients who undergo assisted reproduction therapy
Wolfgang E. Paulus, M.D.,[a] Mingmin Zhang, M.D.,[b] Erwin Strehler, M.D.,[a]
Imam El-Danasouri, Ph.D.,[a] and Karl Sterzik, M.D.[a] Christian-Lauritzen-Institut, Ulm, Germany
FERTILITY AND STERILITY® VOL. 77, NO. 4, APRIL 2002
Copyright ©2002 American Society for Reproductive Medicine
Published by Elsevier Science Inc.

Objective: To evaluate the effect of acupuncture on the pregnancy rate in assisted reproduction therapy (ART) by comparing a group of patients receiving acupuncture treatment shortly before and after embryo transfer with a control group receiving no acupuncture.

Design: Prospective randomized study.

Setting: Fertility center.

Patient(s): After giving informed consent, 160 patients who were undergoing ART and who had good quality embryos were divided into the following two groups through random selection: embryo transfer with acupuncture (n = 80) and embryo transfer without acupuncture (n = 80).

Intervention(s): Acupuncture was performed in 80 patients 25 minutes before and after embryo transfer. In the control group, embryos were transferred without any supportive therapy.

Main Outcome Measure(s): Clinical pregnancy was defined as the presence of a fetal sac during an ultrasound examination 6 weeks after embryo transfer.

Result(s): Clinical pregnancies were documented in 34 of 80 patients (42.5%) in the acupuncture group, whereas pregnancy rate was only 26.3% (21 out of 80 patients) in the control group.

Conclusion(s): Acupuncture seems to be a useful tool for improving pregnancy rate after ART. (Fertil Steril®2002;77:721- 4. ©2002 by American Society for Reproductive Medicine.)

Key Words: Acupuncture, assisted reproduction, embryo transfer, pregnancy rate

Acupuncture is an important element of traditional Chinese medicine (TCM), which can be traced back for at least 4,000 years. Acupuncture has been shown to alleviate nausea and vomiting, dental pain, addiction, headache, menstrual cramps, tennis elbow, fibromyalgia, myofascial pain, osteoarthritis, carpal tunnel syndrome, and asthma. Both physiologic and psychological benefits of acupuncture have been scientifically demonstrated in recent years.

However, so far there have been only a few serious trials concerning the use of acupuncture in reproductive medicine. Publications focus primarily on acupuncture therapy for male infertility (1, 2). Electroacupuncture may reduce blood flow impedance in the uterine arteries of infertile women (3). A positive impact of electroacupuncture on endocrinologic parameters and ovulation in women with polycystic ovary syndrome has been demonstrated (4). In addition, auricular acupuncture was successfully used in the treatment of female infertility (5). In the present study, we chose acupuncture points that relax the uterus according to the principles of TCM. Because acupuncture influences the autonomic nervous system, such treatment should optimize endometrial receptivity (6). Our main objective was to evaluate whether acupuncture accompanying embryo transfer increases clinical pregnancy rate.

Materials and Methods
This study was a prospective randomized trial at the Christian-Lauritzen-Institut in Ulm, Germany. It was approved by the ethics committee of the University of Ulm. A total of 160 healthy women undergoing treatment with in vitro fertilization (IVF; n = 101) or intracytoplasmic sperm injection (ICSI; n = 59) were recruited into the study. The age of the patients ranged from 21 to 43 (mean age: 32.5 = 4.0 years). The cause of infertility was the same for both groups (Table 1). Only patients with good embryo quality were included in the study. Using a computerized randomization method, patients were assigned into either the acupuncture group or the control group.

Table 1
Descriptive data on acupuncture and control group (mean ± SD or total number).
NS = not significant (P>.05).
Paulus. Acupuncture in ART. Fertil Steril 2002.

Ovarian stimulation, oocyte retrieval, and in vitro culture were performed as previously described (7). Transvaginal ultrasound-guided needle aspiration of follicular fluid was performed 36 to 38 hours after hCG administration. Immediately after follicle puncture, the oocytes were retrieved, assessed, and fertilized in vitro. Sperm preparation and culture conditions did not differ for either group.

In cases of severe male subfertility, ICSI was preferred, as described in the literature (8). Forty-eight hours after the IVF or ICSI procedure, embryos were evaluated according to their appearance as type 1 or 2 (good), type 3 or 4 (poor), as described in literature (9).

Just before and after embryo transfer, all patients underwent ultrasound scans of the uterus using a 7-MHz transvaginal probe (LOGIQ 400 Pro, GE Medical Systems Ultra-sound Europe, Solingen, Germany). Pulsed Doppler curves of both uterine arteries were measured by one observer. The pulsatility index (PI) for each artery was calculated electronically from a smooth curve fitted to the average waveform over three cardiac cycles.

A maximum of three embryos, in accordance with German law, were transferred into the uterine cavity on day 2 or 3 after oocyte retrieval. For embryo replacement, the patient was placed in a dorsal lithotomy position, with an empty bladder. The cervix was exposed with a bivalved speculum, then washed with culture media prior to embryo transfer. Labotect Embryo Transfer Catheter Set (Labotect GmbH, Go&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 2000;50:225-30

Quantitative evaluation of spermatozoa ultrastructure after acupuncture treatment for idiopathic male infertility

Jian Pei, Ph.D.ab, Erwin Strehler, M.D.b, Ulrich Noss, M.D.c, Markus Abt, Ph.D.d, Paola Piomboni, Ph.D.e, Baccio Baccetti, Ph.D.e, Karl Sterzik, M.D.b
Received 19 February 2004; accepted 23 December 2004.

Objective

To evaluate the ultramorphologic sperm features of idiopathic infertile men after acupuncture therapy.

 

Design

Prospective controlled study.

 

Setting

Christian-Lauritzen-Institut, Ulm, IVF center Munich, Germany, and Department of General Biology, University of Siena, Siena, Italy.

 

Patient(s)

Forty men with idiopathic oligospermia, asthenospermia, or teratozoospermia.

 

Intervention(s)

Twenty eight of the patients received acupuncture twice a week over a period of 5 weeks. The samples from the treatment group were randomized with semen samples from the 12 men in the untreated control group.

 

Main Outcome Measure(s)

Quantitative analysis by transmission electron microscopy (TEM) was used to evaluate the samples, using the mathematical formula based on submicroscopic characteristics.

 

Result(s)

Statistical evaluation of the TEM data showed a statistically significant increase after acupuncture in the percentage and number of sperm without ultrastructural defects in the total ejaculates. A statistically significant improvement was detected in acrosome position and shape, nuclear shape, axonemal pattern and shape, and accessory fibers of sperm organelles. However, specific sperm pathologies in the form of apoptosis, immaturity, and necrosis showed no statistically significant changes between the control and treatment groups before and after treatment.

 

Conclusion(s)

The treatment of idiopathic male infertility could benefit from employing acupuncture. A general improvement of sperm quality, specifically in the ultrastructural integrity of spermatozoa, was seen after acupuncture, although we did not identify specific sperm pathologies that could be particularly sensitive to this therapy.

 

Key Words: Idiopathic male infertility , sperm ultrastructure , transmission electron microscopy , acupuncture

 

The use of traditional or complementary/alternative medicine (CAM) for health care has been increasing (1, 2), including the use of acupuncture for the treatment of infertility. In 2002, the World Health Organization (WHO) released a global policy to assist countries in regulating traditional medicine to increase safety and effectiveness, improve standardization, protect cultural heritage, and preserve traditional knowledge.

It is estimated that about 10% of men are infertile, and that the male partner is responsible for up to 50% of infertility among couples. In 40% to 50% of males with infertility, the etiology is unknown. Research has shown that acupuncture can result in endocrine changes and relief for women with menstrual disorders (3, 4, 5). Numerous studies of acupuncture treatment on infertile men have also been conducted. Reports from uncontrolled trials using acupuncture on infertile men have shown a positive effect on sperm concentration and motility (6, 7, 8), an increase in testosterone, and some improvement in luteinizing hormone (LH) level (7, 8). These studies have also shown an increase of normally shaped sperm and a significant decrease in the percentage of morphologically abnormal sperm (7, 9). Some studies also have shown that acupuncture did not trigger subjective behavior alterations (6, 7) or influence sexual behavior (7).

Several controlled acupuncture studies have shown a positive effect on sperm production in males with low sperm quality (10, 11). A controlled trial on men with idiopathic, normal gonadotropic oligospermia revealed that pregnancy rate or normalization of semen parameters increased significantly in 74% of patients receiving acupuncture plus clomiphene compared with 52% of those receiving clomiphene alone (12). Another controlled trial on infertile men showed a positive effect on the 279 cases of male infertility treated by the combination of acupuncture and Chinese herbal medicine (13). In our previous studies, the results showed that acupuncture could improve sperm quality, fertilization rate (14), and pregnancy rate (15) in assisted reproductive technology (ART).

Encouraging results prompted us to analyze the possible effect of acupuncture treatment on sperm structure. Standard semen analysis is a relatively blunt instrument for the diagnosis of male infertility, and sperm morphology at light microscopic level has been insufficiently evaluated. Thus, we considered transmission electron microscopy (TEM) to be the appropriate tool for estimating ultrastructural changes occurring in spermatozoa or in fine structure of organelles involved in fertilization after acupuncture treatment (16, 17, 18, 19).

Materials and methods

Patients

Male patients were recruited from the couples visiting the Christian-Lauritzen-Institut, Ulm, Germany, who had been unable to initiate a pregnancy during a period of at least 2 years of unprotected sexual intercourse. All participants had thorough clinical workups that included a clinical history, physical examination, endocrinologic studies, and laboratory testing of ejaculates. The results of combined gynecologic and andrologic examinations pointed to an idiopathic male factor responsible for the infertility of all these couples. The female partners also were required to complete infertility workups, including laparoscopy and chromopertubation to verify the patency of the fallopian tubes.

 

The inclusion criteria were [1] male partner with idiopathic infertility for at least 2 years whose female partner had had at least two failed intrauterine insemination treatment cycles; [2] a minimum of two pathological spermiograms at an interval of 6 weeks showing oligospermia, asthenospermia, and/or teratozoospermia according to WHO criteria (20); [3] normal values in a baseline endocrine evaluation that measured follicle-stimulating hormone (FSH), LH, prolactin (PRL), estradiol (E2), and testosterone levels; [4] a minimum of 12 months without having received andrologically effective treatment; and [5] availability of the female partner’s clinical fertility data.

Exclusion criteria were [1] thyroid dysfunction, adrenal disorders, hyperprolactinemia, or any pathologic hormone parameter; [2] any cause of infertility detectable after systematic physical examination and laboratory testing, including genetic testing; [3] azoospermia; [4] infectious disease or immunologic-associated disease, or presence of any major systemic disease; or [5] abnormal psychological stresses.

Study Design and Treatment Protocol

This study was a prospective, controlled trial, approved by the ethics committee of the University of Ulm, Germany. Forty patients who fulfilled the inclusion criteria were selected for the study. All of them were willing to use acupuncture, and each patient gave his written informed consent before the start of treatment in the study. The median age of patients was 33 years (range: 25 to 46 years). The experimental group consisted of 28 men who received acupuncture treatments twice a week over a period of 5 weeks. Semen samples of 12 patients with untreated idiopathic infertility, examined as the control group, were randomized with the treated idiopathic infertile men by an independent researcher using computer software. No further treatment was allowed in the study.

 

We used the following acupoints as main points: Guan Yuan (Ren 4), Shen Shu (UB 23, bilateral), Ci Liao (UB32, bilateral), Tai Cong (LR 3, bilateral), and Tai Xi (KI 3, bilateral). The secondary points were Zhu San Li (St 36, bilateral), Xue Hai (Sp 10, bilateral), San Yin Jiao (Sp6, bilateral), Gui Lai (St 29, bilateral), and Bai Hui (DU 20). The location of acupoints followed the international standardized location of acupoints (21).

The needles (Viva, 0.25 × 25 mm, or 0.25 × 40 mm; Helio Medical Supplies, Inc. San Jose, CA) were made of sterile disposable stainless steel and were inserted in acupuncture point locations to a depth of 15–25 mm, depending on the region of the body undergoing treatment. To evoke the needle sensation, or De Qi, often described as variable feelings of soreness, numbness, tingling, warmness, and/or tension, the needles were rotated to activate the muscle-nerve afferents, the A delta and possibly C fibers (22, 23). When puncturing Shen Shu (UB 23, bilateral) and Ci Liao (UB32, bilateral), the needling sensation should be transmitted to the sacral or perineum area and anterior hypogastric zone. After 10 minutes the needles were manipulated to maintain needle sensation. The needles were left in acupuncture points for 25 minutes and then removed.

Semen Collection and Analysis

Semen samples were collected by masturbation under hygienic conditions, after a period of sexual abstinence of 3 days. Two samples from each patient, one obtained the day before treatment and one after acupuncture treatment, were analyzed following standard protocols of the WHO laboratory manual (20). Semen samples were liquefied at 37°C, then the sperm count and the different motility grades were subjectively assessed using a Makler counting chamber (El-OP, Rehovoth, Israel). An aliquot of each sample was processed for examination by TEM.

 

Transmission Electron Microscopy

Ultramorphologic analysis of spermatozoa was assessed by TEM, performed at the Biology Section, University of Siena, Italy. Spermatozoa were fixed in cold Karnovsky fluid and maintained at 4°C for 2 hours. The fixed semen was then centrifuged at 3000 × g for 15 minutes. The pellet was washed in 0.1 M cacodylate buffer (pH 7.2) for 12 hours, postfixed in 1% buffered osmium tetroxide for 1 hour at 4°C, dehydrated, embedded in Epon Araldite, and cut with the LKB ultramicrotome. The sections were collected on copper grids, and stained by uranyl acetate and lead citrate.

 

The observation and photography were made using Philips EM 301 and CM 10 TEM (Philips Scientifics, Eindhoven, The Netherlands) at magnifications of ×15,000 to ×75,000.

One hundred sections of each sperm sample were selected randomly for observation. Ultramorphologic features of sperm were selected to evaluate the acrosome, nucleus, chromatin, axoneme, accessory fibers, and fibrous sheath according to submicroscopic characteristics and our previous experience (17, 18, 19, 24, 25). The samples from the treatment and control groups were randomized before being examined by two skilled investigators, blinded to the groups to exclude any bias.

The quantitative evaluation of the TEM data was performed by applying the mathematical formula based on the Bayesian technique proposed by Baccetti et al. (24). This formula can evaluate, by considering all statistical possibilities for defects of examined sperm, the total number of affected spermatozoa and, consequently, the sperm devoid of defects (“healthy” sperm).

Statistical Analysis

Statistical analysis was performed at the Institute of Mathematics, the University of Augsburg. After suitable transformation, one-way analysis of variance was used for data analysis. The analysis was adjusted for values obtained before treatment by including these as covariates in the model. Number of sperm, volume, and number of healthy sperm can be assumed to follow a log-normal distribution and were thus analyzed on a logarithmic scale. For percentages, the logit transformation was used before analysis, as to correspond to the variance stabilizing transformation for the binomial distribution. P <.05 was considered a statistically significant difference between the acupuncture group and the control group after 5 weeks of treatment. All P values reported correspond to two-sided tests for differences.

 

Results

Semen Analysis on a Light Microscopic Level

Semen analysis of the treatment and control groups by light microscopy showed no statistically significant changes of the median number of sperm/mL (P = .657) and the median volume of the ejaculate (P = .731). The median percentage of total motility in ejaculate increased from 32% to 37% in the control group and from 44.5% to 50% in acupuncture group, a statistically significant difference between the two groups (P = .017).

 

The semen samples from the 40 patients selected for this study showed the usual structural defects observed by TEM in infertile men. The results of ultrastructural analysis were available for all 40 patients (Fig. 1).

FIGURE 1. Median percentage of sperm submicroscopic characteristics in the control group (n = 12) and acupuncture group (n = 28) before and after treatment. The bars represent the median of the data; approximate 95% confidence intervals for the upper, lower quartiles and ranges.

Pei. Acupuncture and male factor infertility. Fertil Steril 2005.

Number and Percentage of Healthy Spermatozoa in the Total Ejaculate

The TEM data of 40 sperm samples were analyzed using the formula of Baccetti et al. (24); according to the formula, the threshold of natural fertility is 2 × 106 healthy spermatozoon in the total ejaculate. The median percentage of “healthy” spermatozoa was very low, 0.16% in the control group and 0.06% in acupuncture group, confirming the presence of male factor infertility. The median number of healthy spermatozoa, calculated in the total ejaculate, was 0.14 × 106 in the control group and 0.04 × 106 in acupuncture group.

 

After 10 sessions of acupuncture treatment, TEM evaluation was performed again in both groups. A statistically significant improvement was found of the percentage (P = .012) and the number (P = .002) of healthy sperm after 5 weeks of therapy. The median of percentage of healthy sperm was increased to 0.26%, and the median number of healthy sperm reached 0.2 × 106.

Response of Organelles to Acupuncture Therapy

We used TEM and mathematical statistical analysis (24) to assess the reaction of individual organelles responsible for sperm integrity to acupuncture therapy. The ultrastructural characteristics of organelles that indicate perfect sperm functionality were analyzed before (Fig. 2a) and after (Fig. 2b) acupuncture therapy.

FIGURE 2. (a) Structural characteristics of semen before acupuncture therapy. Spermatozoa generally showed misshapen nuclei (N) and acrosomes (A) with uncondensed, necrotic, or marginated chromatin, cytoplasmic residues (CR), and coiled axonemes (arrow). Magnification ×8000. (b) Structural characteristics of semen after acupuncture therapy. Semen contained spermatozoa characterized by regularly shaped acrosome (A) and nuclei (N) with well-condensed chromatin, regularly assembled mitochondria (M), and normal cytoskeletal structures (AX). Some sperm showed altered acrosomes and nuclei, with uncondensed chromatin. Magnification ×13,500.

 

Pei. Acupuncture and male factor infertility. Fertil Steril 2005.

 

 

Acrosome in Normal Position

In the control group, 65% of sperm showed the acrosome in a normal position, compared with 69.5% in the pretreatment acupuncture group. In the other cases, the acrosome was displaced and localized far from the nucleus. After the therapy, this value reached 71.5% in the control group and 77.5% in acupuncture group. The increase was statistically significant in the acupuncture group after 5 weeks of therapy (P = .013).

 

Acrosome of Normal Shape

Only about 26% of spermatozoa in the control group and 22.5% in the acupuncture group had a normal acrosomal shape before treatment. After the therapy, the median percentage of normal acrosomal shapes in the acupuncture group showed a statistically significant improvement up to 38.5% (P <.001).

 

Normal Nuclear Shape

Approximately 29% of the sperm population had a normal nuclear shape in the control group and the acupuncture group before treatment. The acupuncture treatment group had a statistically significant improvement the population of normal nuclear shape, from 30% to 42.5% (P<.001).

 

Condensed Chromatin

About 36% to 39% of sperm population had condensed chromatin in the control and acupuncture groups before treatment. No statistically significant change was found between the two groups after 5 weeks of treatment (P = .506).

 

Normal Axoneme Pattern

Infertile men frequently show a disturbed spermatogenesis that results in axonemal patterns different from “9 + 2” configuration. The 9 + 2 pattern was present in 52% of sperm in the control group and 46.06% in the acupuncture group before treatment. After acupuncture therapy, the median percentage showed a statistically significant increase, from 46.1% to 52.19% (P = .005). This value had decreased to 38.18% in the control group after 5 weeks.

 

Normal Axoneme Shape

Before treatment, normal axoneme shape was 67.44% in the control group and 63.64% in the acupuncture group. After acupuncture therapy, the median percentage of the normal axonemal shape showed a statistically significant increase, from 63.64% to 67.71% (P = .022). In control group, this value decreased to 55.85% after 5 weeks.

 

Normal Accessory Fibers

After acupuncture treatment, the median percentage of normal accessory fibers statistically significantly increased from 34.06% to 48.53%. In the control group, this value decreased from 48.68% to 34.06% of spermatozoa after 5 weeks. The groups showed a statistically significant difference (P = .005).

 

Normal Fibrous Sheath

The normal fibrous sheath was 44.41% in the control group and 33.33% in the acupuncture group before therapy. After acupuncture treatment, the median percentage of normal fibrous sheath increased to 40.59%. No statistically significant improvement could be demonstrated between the two groups, although the acupuncture group showed a tendency toward an increase after 5 weeks of treatment.

 

Typical Pathologies Affecting Spermatozoa in Infertile Men

The mathematical formula of Baccetti et al. (24) was applied to the TEM data of the 40 sperm samples to evaluate the probability percentage of the presence of the most common sperm pathologies.

 

Apoptosis

Before treatment, the median percentage of apoptosis in ejaculated spermatozoa was 8.18% in the control group and 7.80% in the acupuncture group. After 5 weeks, the median percentage of apoptosis decreased to 6.43% in the control group and 7.15% in the acupuncture group. No statistically significant difference between the two groups was observed (P = .863).

 

Immaturity

Before treatment, the percentage of immature spermatozoa was 68.23% in the control group and 71.29% in the acupuncture group. After 5 weeks, no statistically significant changes between the two groups were observed (P = 0.146); the percentage of immaturity in ejaculated spermatozoa was 74.11% in the control group and 68.43% in the acupuncture group.

 

Necrosis

Before treatment, the median percentage of necrosis in ejaculated spermatozoa was 37.28% in the control group and 36.70% in the acupuncture group. After 5 weeks, the median percentage of necrosis in ejaculated spermatozoa was 44.03% in the control group and 34.3% in the acupuncture group. There was no statistically significant difference between the two groups (P = .072), although there was a trend toward a decrease in the acupuncture group after 5 weeks of treatment.

 

Discussion

Traditional Chinese medicine and acupuncture are based on ancient medical theories, but modern, scientific neurobiological perspectives have begun to evolve over the past 40 years. These new perspectives can help us to understand acupuncture effects and mechanisms, such as how the “acupuncture signal” transfers from a mechanical signal to an electric signal to a biological signal, which produces biological response. In infertility treatment, a controlled study by Siterman et al. (11) analyzed sperm density to define the most appropriate responders to acupuncture treatment. The results showed that acupuncture might be a useful, nontraumatic treatment for individuals with very poor sperm density, especially those with a history of genital tract inflammation.

Sperm morphology assessment is a valuable and stable method for predicting the in vivo and in vitro fertilizing ability of sperm. Conventional light microscopy tests cannot identify the entire variety of morphologic defects that can occur in sperm organelles, head structures (26, 27), and tail organization. Electron microscopy is currently the only tool able to analyze the ultramorphologic status of sperm cells (detecting organelles’ shape, structure, and function) to determine specific sperm quality; TEM enables viewing of sperm sections and provides two-dimensional, detailed anatomic information of all subcellular structures.

Using scanning electron microscopy (SEM) and TEM, Bartoov et al. (26) evaluated the advantages of quantitative ultramorphologic sperm analysis in the diagnosis and treatment of male infertility. This methodology can successfully predict a patient’s natural fertility potential by identifying the cause of infertility, and thus enable directing the patient to specific therapeutic options (10, 26).

Nevertheless, the use of TEM in andrology has been limited due to its inability to analyze data collected by observation of ultrathin sections of elongated and tortuous cells, such as spermatozoa that could appear several times in the same field. Another problem is the interdependence of submicroscopic sperm defects; for example, the probability of a spermatozoon to be morphologically normal is related to the degree of interdependence of each defect with the others. Probability analysis using a Bayesian technique solves the difficulties mentioned; the Baccetti formula (24) is a very sensitive and useful tool for assessing the relationship between sperm ultrastructure, the success of different ART techniques (17, 18, 19), and the effect of FSH therapy on sperm ultrastructure to test the improvement of sperm quality (16, 25).

In the present study, submicroscopic and mathematical analysis performed before and after 5 weeks of acupuncture treatment showed a general improvement in the ultrastructural characteristics of sperm in the 28 treated patients. The median percentage and number of healthy sperm in the total ejaculate had increased. As far as the responsiveness of organelles to the therapy is concerned, the characteristics of the acrosome were shown to be sensitive to acupuncture therapy. Statistically significant improvements were seen in acrosome position and shape after 5 weeks. The nucleus was also sensitive to acupuncture therapy: nuclear shape showed statistically significant improvement, although chromatin condensation remained at the same level after therapy. The evaluation of the main structures of sperm head, acrosome, and nucleus allowed a prospective assessment of sperm penetration and fertilization ability.

Motility is a sperm function of highest relevance for reproduction, as each of the flagellar elements plays a key role in allowing spermatozoa to move effectively in a forward direction. The axoneme responded quite well to acupuncture therapy. The two characteristics of the axoneme, the classic 9 + 2 pattern and the shape, showed statistically significant improvement. The accessory fibers were also sensitive to the therapy, although the fibrous sheath was less affected by acupuncture treatment.

Combined with semen analysis at light microscopy level, the median percentage of progressive motility in ejaculate increased from 44.5% to 50% after acupuncture therapy. This statistically significant increase in motility was correlated with the improvement of axonemal pattern, axonemal shape, and accessory fibers. It was in agreement with the data of Siterman et al. (10), who found that the positive response to acupuncture therapy, related to improvement of total motility in ejaculate, was highly correlated with the axonemal integrity.

Our mathematical formula is able to detect the probability of the presence of pathologies affecting an ejaculate—specifically, apoptosis, immaturity, and necrosis. In spite of the statistically significant improvement of sperm quality, no statistical significance was found when the probability percentage of the presence of the main three sperm pathologies was compared before and after the therapy.

In conjunction with ART or even for reaching natural fertility potential, acupuncture treatment is a simple, noninvasive method that can improve sperm quality. Further research is needed to demonstrate what stages and times in spermatogenesis are affected by acupuncture, and how acupuncture causes the physiologic changes in spermatogenesis. Our future aim is strengthen our findings by enlarging the study group for more investigations.

Acknowledgments

The authors thank Corinne Axelrod, M.P.H., L.Ac., Dipl.Ac. for her review.

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a Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, People’s Republic of China

b Christian-Lauritzen-Institut, Ulm, Germany

c In Vitro Fertilization Center Munich, Munich, Germany

d Institute for Mathematics, University of Augsburg, Augsburg, Germany

e Department of Paediatrics, Obstetrics and Reproductive Medicine, Section of Biology, Siena University, Siena, Italy

Reprint requests: Jian Pei, Ph.D., Christian-Lauritzen-Institut, Frauenstrasse 56, 89073 Ulm, Germany (FAX: 0049-731-9665130

Supported in part by grants from the National Natural Science Foundation of China (No. 399 00196) and the Key Projects Foundation, State Administration of Traditional Chinese Medicine, People’s Republic of China.

Pregnancy: Some Depression Relief, Without Drugs

By RONI CARYN RABIN
Published: February 24, 2010 , New York Times

Up to a quarter of all women suffer from depression during pregnancy, and many are reluctant to take antidepressants. Now a new study suggests that acupuncture may provide some relief during pregnancy, even though it has not been found to be effective against depression in general.

The Stanford University study recruited 150 depressed women who were 12 to 30 weeks pregnant, and randomly assigned 52 to receive acupuncture specifically designed for depressive symptoms, 49 to regular acupuncture and 49 to Swedish massage.

Each woman received 12 sessions of 25 minutes each; those given acupuncture did not know which type they were getting. (In the depression-specific treatment, needles are inserted at body points that are said to correspond to symptoms like anxiety, withdrawal and apathy.)

After eight weeks, almost two-thirds of the women who had depression-specific acupuncture experienced a reduction in at least 50 percent of their symptoms, compared with just under half of the women treated with either massage or regular acupuncture.

The findings appear in the March issue of Obstetrics & Gynecology. The lead author, Rachel Manber, a professor of psychiatry and behavioral sciences at Stanford, said the results suggested that some symptoms of depression during pregnancy might be related to physical discomfort that is alleviated by acupuncture. Still, the results were striking, she said.