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Dr H. Zeisler, Department of Obstetrics and Gynecology, University of Vienna, General Hospital Vienna, Währinger Gürtel 18–20, A–1090 Vienna, Austria (e-mail: harald.zeisler@akh-wien.ac.at) :12, 1419–1419
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Caroline Smith, Ph.D.,a Meaghan Coyle, B.Hlth.Sc. (Acup.),b and Robert J. Norman, M.D.c,d Fertility & Sterility |
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a randomized, prospective, controlled clinical study
Stefan Dieterle, M.D.,a Gao Ying, M.D.,a,b Wolfgang Hatzmann, M.D.,a and Andreas Neuer, M.D.a Fertility & Sterility |
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A prospective, randomized trial
Lars G. Westergaard, M.D., Ph.D.,a Qunhui Mao, M.D.,b Marianne Krogslund,a Steen Sandrini,c Suzan Lenz, M.D., Ph.D.,a and Jørgen Grinsted, M.D., Ph.D.a |
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With more patients asking for it, some clinics are offering the ancient technique.By Elena Conis, Special to The Times
Jackie Apuzzo is 16 weeks pregnant - something she was beginning to think would never happen.
I was a little apprehensive about the needles at first," said Apuzzo. But in April, Apuzzo's acupuncturist in Santa Monica looked at her tongue, checked her pulse and declared the Long Beach resident pregnant. Apuzzo later confirmed the diagnosis with a blood test. |
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Traditional Chinese Medicine has been used for thousands of years to help couples become pregnant. With the technology of Modern Medicine couples that have been struggling to become parents now have a good chance of fulfilling their dreams. In Vitro Fertilization (IVF) has become a popular choice for many couples. Now, high tech joins with the ancient wisdom of acupuncture. Many studies have shown that Acupuncture can enhance the pregnancy rates of women going through IVF. Two studies, which have gotten the most attention on the use of Acupuncture combined with IVF, are 1) Influence of Acupuncture on the Pregnancy rate on patients who undergo assisted reproduction therapy. Paulus et al. Fertility & Sterility Vol 77:No.4, April 2002 2) Reduction In blood flow impedance in the uterine arteries of infertile women with electro-acupuncture. Stener-Victorin et al. European Society for Human Reproduction and Embryology. The Paulus protocol uses two acupuncture treatments. One is given just before Embryo transfer and one just after Embryo transfer. The first treatment prepares the woman's body to open and accept the Embryo and the second (post transfer) uses acupoints for holding and keeping the Embryo. The Stener-Victorin study used a series of acupuncture treatments using electro acupuncture. This incorporates using gentle electro stimulation on needles places in the back and legs. The protocol requires at least eight acupuncture treatments before retrieval of the eggs. This specifically increases the blood flow to the uterus which in tern helps the fertility medications reach the ovaries. This provides a better stimulation to the ovaries. The other benefit is that the uterine wall lining develops a nice thick environment and makes a better place for the embryo to imbed. Without this plush lining the embryo has no place to imbed. These studies demonstrated a 10 to 15% improvement in pregnancy rates in those patients who augmented their traditional IVF treatments with acupuncture. It took Western Reproductive Techniques over 8 years for a 10% improvement in IVF statistics and 4 more years to improve it by another 10%. These studies would suggest providing patients with acupuncture treatments is a very positive practice. Dr. Paul Magarelli of RMFC (Reproductive Medicine & Fertility Center) Board Certified Reproductive Endocrinologist and Diane K. Cridennda Licensed Acupuncturist of East Winds Acupuncture have just completed a 4-year study showing the efficacy of Acupuncture being used in conjunction with IVF. Both electro acupuncture and Pre and Post embryo transfer acupuncture protocols were used. The study (which has been submitted for presentation and publication) studied reproductive outcomes in over 160 patients receiving IVF, or IVF & ICSI (50% pregnancy rates per cycle in the acupuncture patients). It is reported that the "good prognosis" patients (those patients who have a good chance of success) could be made to have better outcomes (16% more pregnancy) by using acupuncture during their IVF cycle. The most remarkable discovery was that the acupuncture group had a significantly higher live birth rate. Since the goal here is to have a baby not just to achieve pregnancy, this is a remarkable finding? Another remarkable finding was that no ectopic pregnancies were reported in the acupuncture group versus 9% in the non- acupuncture group. More research needs to be done to support this finding. There are secondary benefits from acupuncture that our patients report:
Acupuncture is the placement of fine needles into specific acupoints along the pathways of energy in the body. This works by sending messages to the brain almost like programming a computer. The basis of all TCM is energy or "Qi". Energy flows in patterns of electricity through our bodies, cells and tissues. This Qi is conducted through protein molecules giving us energy and life. This energy is carried through our bodies via pathways of energy called meridians. These meridians make up a web or network of channels, which sets up communication between every organ in your body. Oriental doctors have mapped these channels out over the course of thousands of years of experimentation and observation. The insertion of fine needles into the acupoints along the meridians produces measurable effects on different parts of the body. This in how acupuncture can affect abdominal organs, such as the kidneys, liver and uterus by placing needles in your arms and legs. Some of the acupuncture points used in the studies mentioned above make absolute sense to a TCM practitioner. These acupoints achieve balance by holding the Qi up, moving the blood (a natural heparin affect) regulates the uterus by building a nice plush uterine wall lining. |
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Medical experts are finding potential in the use of acupuncture as adjunctive therapy for women undergoing infertility treatment. In a new study testing the feasibility of this alternative approach, doctors compared outcomes between two groups of patients: those who underwent acupuncture combined with IVF, and those who underwent IVF alone.(1) A Medical Approach with a Long History Acupuncture is one of the most ancient medical treatments in the world, dating back several millennia, originating in China, but finally becoming popular in more modern times in the United States in the early 1970s. The practice is used not just as an adjunctive infertility treatment, but also a wide variety of medical conditions. It involves applying a range of procedures that stimulate anatomical points on the body. The technique that has been studied the most scientifically involves penetrating the skin with thin, solid, metallic needles that are manipulated by the hands or by electrical stimulation. Traditional Chinese medicine holds that there are more than 2,000 acupuncture points on the body, and that these connect with more than a dozen main and secondary pathways known as meridians. Chinese medicine practitioners believe these meridians conduct energy, or qi (chee), throughout the body. Essentially, acupuncture helps maintain the balance between qi and the opposing forces of yin and yang, which in turn regulates spiritual, emotional, mental and physical balance.(2) "Western medicine takes a very different approach," explained lead study investigator Paul Magarelli, MD, PhD, a practicing reproductive endocrinologist and medical director of the Reproductive Medicine & Fertility Center in Colorado Springs. "We use medications. We override [biological] systems." Yet while treatment for infertility, such as in-vitro fertilization (IVF), has taken major strides in the last two decades, odds are that couples won't achieve a pregnancy 60% of the time by solely using the Western approach, Magarelli explained. "A lot of the time, the patient gets one try at becoming pregnant, mainly because of the cost," he said. Considering the Alternative Thus, patients have begun looking at alternative options, mainly integrative medicine, in hopes of boosting those odds, Magarelli said. That was one motivation for this study. But Magarelli says he didn't always believe in the potential of complementary approaches like acupuncture. "I just did not feel that the data existed that supported acupuncture," he recalled. Over time, studying the medical literature about the topic intrigued him enough to launch a study of his own. In a previous analysis, Magarelli recruited patients with the poorest prognoses (over age 35, several male factor, elevated FSH level, or those who failed IVF in the past) in his clinic, who were given acupuncture in combination with IVF, and compared them with the same types of patients who received IVF alone. Two years later, he and his colleagues retrospectively evaluated the data. "Those patients who were treated with the acupuncture [and] who were poor prognosis had equivalent pregnancies to those [considered] good prognosis patients," Magarelli explained, an outcome he found "amazing". This led to the latest study involving "good prognosis" patients. Specifically, the researchers wanted to determine the efficacy of electro stimulation or traditional acupuncture combined with auricular (or ear) acupuncture. Electro stimulation acupuncture is the term used to describe the approach that involves electrical stimulation. These approaches are used to either improve uterine bloodflow or help relax the uterus prior to embryo transfer in IVF. One hundred fourteen patients undergoing in-vitro fertilization after controlled ovarian hyperstimulation were included in the study. Only those women described as having a good response to ovarian hyperstimulation and whose partners' sperm morphology (quality) was also good were analyzed for the research. Each patient underwent ovarian hyperstimulation using a standard protocol, including the use of gonadotropins and a gonadotropin releasing-hormone (GnRH) agonist or antagonist. Each woman who then responded well to ovulation induction underwent either electro stimulation acupuncture or traditional combined with auricular acupuncture in conjunction with IVF. For this study, 53 underwent acupuncture combined with IVF, and 61 underwent IVF alone. The investigators then analyzed successful pregnancies in the group of women, as well as the miscarriage rate. Acupuncture Patients Had Improved Outcomes Of those in the acupuncture group, 51 percent achieved a successful pregnancy, the research team noted. That compares to 36 percent of those who underwent IVF alone. Comparatively, the miscarriage rate was 8% and 20% in the acupuncture versus non-acupuncture group, respectively. There were no ectopic (Tubal) pregnancies in the group of women who underwent acupuncture, but 9% of those in the group without acupuncture had a Tubal pregnancy. The investigators also reported 23% more births per pregnancy among those who had acupuncture as part of their infertility treatment. "In previously published data, acupuncture was reserved for poorer prognosis patients, and enhanced outcomes were observed," wrote Magarelli and his colleagues. "In this study, we demonstrated that good prognosis patients would also benefit from inclusion of published acupuncture protocols." They say this is the first published study to include birth-related IVF outcome in patients also treated with acupuncture. Why does acupuncture theoretically work for infertility? Medical experts hypothesize that the ancient approach positively impacts opioid production in the central nervous system, which in turn, positively influences gonadotropin secretion.(3) These naturally-produced opioids, like endorphins, are similar to the actions of opiate drugs. Gonadotropins are the hormones that help promote normal reproductive function in the body. They include follicle-stimulating hormone (FSH), which helps promote follicle maturity prior to the release of an egg in women, and sperm production in men.(4) Experts also suggest acupuncture has a positive effect on uterine bloodflow. For the Future The study will likely be expanded early next year, Magarelli study. In the next phase, data will be collected on all patients, regardless of prognosis. "I would suspect that ... we're going to see that, across the board, we can improve anywhere between 5 percent and 15 percent the number of babies successfully created through the process of IVF and acupuncture," he said. "What blows me away, personally, is how I have absolutely seen things that I didn't think were possible by placing a needle somewhere."
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by Catherine Donaldson-Evans NEW YORK - At 36, Lucy Appert has suffered through two miscarriages, a stillbirth at 8 1/2 months and, because of a rare pregnancy-related liver dysfunction, intensive illness and surgery. Yet after enduring five painful years of trying to have their own baby, Appert and her husband Edward finally saw their dream come true last month when their son Henry was born - premature, but healthy. For all the fertility treatments, technologies and prenatal care available to women today, Appert credits the success of her pregnancy to an ancient Chinese secret. "I recommend acupuncture to everyone," Appert said. "It does work. I did everything possible for years to have a baby. I almost lost hope." The millennias-old Asian medical practice - in which the acupuncturist places tiny needles in various pressure points, or "Qi" (Chee), in the body to improve circulation and reduce stress - has been around in the United States for years as an "alternative" treatment for numerous ailments. But recently, acupuncture has been picking up steam as a possible remedy for female infertility, with a handful of American and European studies showing that it enhances the success rate of in vitro fertilization (IVF). "Do I believe in it? Absolutely," said Dr. Paul C. Magarelli, an infertility doctor at the Reproductive Medicine & Fertility Center in Colorado Springs, Colo., and co-author of an ongoing study into the use of acupuncture with IVF with Dr. Diane K. Cridennda. Cridennda is a licensed acupuncturist with a master's degree in Oriental medicine from the International Institute of Chinese Medicine who owns East Winds Acupuncture, also in Colorado Springs. Magarelli said he joined the study at the urging of Cridennda, who had approached him about using acupuncture with IVF based on her knowledge of its history as an Eastern fertility treatment. A skeptic at first, Magarelli said he dismissed the idea for a while before signing on. "I thought, this is rubbish - it can't be true," Magarelli said. "But no matter how I look at this data, I see an improvement. ... I'm pretty much a convert." In general, studies seem to indicate that doing acupuncture about 30 minutes before and after in vitro fertilization can increase the chance that the embryo will be implanted successfully and reduce the chance of miscarriage. There are also indications that the effectiveness of the IVF drugs and procedure may improve if acupuncture is done about once a week in the month or two leading up to the start of IVF and then continued regularly - once or twice a week - during the whole cycle. And, as in Appert's case, there is anecdotal evidence that acupuncture can help with other fertility and pregnancy problems. Appert didn't need IVF to conceive, but she was told she probably couldn't carry a healthy baby to term because of her liver disorder. But some doctors caution that there is no "magic pill" for fertility, pregnancy and IVF troubles - whether it's acupuncture or something else. "The jury is still out on that," said Dr. Eric Surrey, president of the Society for Assisted Reproductive Technology (SART), who has a practice at the Colorado Center for Reproductive Medicine. "I don't think we have good data to show that acupuncture before and after the embryo transfer is truly beneficial." And they warn against making too much of claims that acupuncture can help with having babies. "It's impossible to say at this point," said Dr. Robert Schenken, president of the American Society for Reproductive Medicine (ASRM), who has a practice at the University of Texas Health Science Center. "In the absence of any controlled data, I don't think we can come to a firm conclusion." Promising Research Acupuncture seems to help some women because it improves circulation to the ovaries - which makes for healthier eggs - and to the uterus, which increases the chances that the lining will be strong enough to hold those eggs to full-term. "Acupuncture provides better circulation and better blood flow to the womb," said Dr. Raymond Chang, director of New York's Meridian Medical Group, who has been incorporating acupuncture into fertility treatments for the past decade. "It will give a better chance for the eggs to be nourished and therefore carried." There's also the fact that acupuncture can be a stress-reliever during an emotional time. "Trying to get pregnant is incredibly stressful," said Victoria Koos, the acupuncturist who treated Appert at Yin and Tonic Acupuncture in New York. "They're crossing their fingers. The longer they're trying to get pregnant, the worse it gets ... Part of [acupuncture's success] is simply relaxation. When the body is relaxed, all systems function better." The Colorado study Magarelli and Cridennda presented at a conference this fall is one of a series the pair have done with acupuncture and in vitro. That one looked at 114 patients who had a good chance of IVF being effective, some who did acupuncture and some who didn't. It found, among other things, that there were fewer miscarriages, more pregnancies and a 7 percent higher birth rate among those who got acupuncture treatment over those who didn't, according to Magarelli. It piggybacked off other research the team did on 147 "poor responders" to IVF, which found that the pregnancy rate was 40 percent, with 11 percent more babies born, among those who did acupuncture with in vitro fertilization compared to those who didn't. In March, Magarelli and Cridennda released findings in Italy involving patients with an average prognosis for IVF success. Those yielded clear numbers that the pregnancy rate increased with acupuncture by 24 percent, according to Magarelli. "What got us was that now we were seeing a firm trend toward getting more people pregnant," he said. The Colorado research seems to support some findings of two earlier studies, one in Germany by lead researcher Dr. Wolfgang E. Paulus - published in ASRM's "Fertility and Sterility" in April 2002 - and one in Sweden by lead researcher Elisabet Stener-Victorin in the 1990s. Of course, even those who believe in acupuncture concede that while the existing studies have yielded good information, there still isn't sufficient evidence, or a broad enough sample of patients tested, to call acupuncture a proven remedy. "We are convinced, but scientifically you need proof - or so-called proof," Chang said. "There is a whole set of proof from lab experiments and animal studies to human studies, but it's very difficult to do human studies." Schenken noted that even though there might be one set of data showing positive results, "it really needs to be corroborated, preferably with several different studies and different patient populations." For example, there can be bias when the entire study sample comes from the same clinic, or when patients know they're doing something different from usual. Schenken said he doesn't get asked about acupuncture often, but when patients do, "we don't recommend it, but we do not discourage it." Surrey takes a similar approach. In his opinion, the data "is not bad" on the theory that acupuncture can help when administered before IVF, but as far as acupuncture generally improving fertility or helping after the embryo transfer in IVF, "there really isn't a whole lot of data on that." But at the very least, "there is absolutely nothing to show that it's harmful if it's done with a trained and appropriately skilled acupuncturist," he said. It's a notion that nearly everyone in the medical field - whether they believe in needles and Qi or not - seems to agree upon. Some Eastern medicine-Western medicine rivalry may come into play with how to treat reproductive problems, but Chang said he sees more resistance with the use of Chinese herbs - which are ingested - than he does with acupuncture. Often, it's the in vitro specialists themselves who refer their patients to him for acupuncture after a couple of failed IVF attempts. As for the couples trying to bring a child into the world - particularly through a complicated, invasive procedure like IVF- anything that helps the process along is welcome. "IVF is so technical that patients feel like they're being pushed and pulled ... with acupuncture, they're in a sense taking some control," Magarelli said. "Acupuncture isn't a needle, it's an environment." Added Koos: "They're on these incredibly strong drugs that make the poor women crazy. They're running around like Catwoman. This is to help them stay sane while they're going through the process." The emotional cost of infertility comes with a hefty financial price tag as well - in vitro fertilization can cost anywhere from $10,000 to $20,000 a cycle and generally isn't covered by insurance; acupuncture ranges from about $30 to over $200 per treatment - Koos and Chang charge about $90 a pop - and certain health plans do cover at least a portion of it. Meanwhile, researchers and experts in the field are excited at what they're seeing in the studies. Chang said he's currently working with NYU Medical Center on a trial that looks at IVF with and without acupuncture. Appert, for her part, was at the end of her rope and felt she had nothing to lose. She started acupuncture with Koos about two months before she began trying to conceive - with needles in her toes and a couple of liver points - and continued with the treatments throughout the pregnancy. "The first time I went, I was completely terrified. My husband went with me and held my hand," she said. "I could feel the muscles in my liver jump and an electric current going through my body. It was very strange but also felt right." She said being monitored by both her obstetrician and Koos helped reassure her about what was going on during her high-risk pregnancy. "She would tell me things about how I was doing physically and then I would go to the doctor and he would tell me the same thing," remembers Appert, who works as a professor. When she got sick late in the pregnancy, both Koos and Appert's OB/GYN were able to detect when her liver went dangerously haywire and get her to the hospital for delivery six weeks early, before the problem harmed the fetus and caused another stillbirth. Regardless of the skeptics, Appert said she's relieved that she was finally able to have a nearly full-term baby of her own. At 4 pounds, 6 ounces, Henry has been in intensive care but otherwise is doing "fine." "It really was a miracle," the new mom gushed. "It's one of these weird things that Western medicine can't explain." |
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New Mexico Woman
The sunrise in the east affirms the balance and harmony within the universe. As the golden glow of the western sky sets, it shimmers with the promise of hope for tomorrow. The endless facets of nature complement one another; light and dark, cold and warmth, life and death. Having a baby is no different; the integration of mind and body, male and female, must work together to achieve the balance needed. For many couples facing infertility, maintaining this equilibrium seems elusive and out of reach.
In the Southwest, the symmetry of East and West are working in concert to bring options and hope to couples experiencing infertility. In March of 2004, Reproductive Medicine & Fertility Center (RMFC) joined with the University of New Mexico, Department of Obstetrics & Gynecology to form RMFC@UNM™, whose private practice specialization is in vitro fertilization (IVF) treatments. Medical director Paul Magarelli, M.D., Ph.D., a board certified reproductive endocrinologist and infertility specialist, says RMFC@UNM™ seeks to provide New Mexico’s infertile couples with compassionate, exceptional, ad-vanced IVF services. Nationwide, the Centers for Disease Control and Prevention reports 99,629 procedures were performed in 2000 by in vitro fertilization (IVF) — fertility treatments in which the egg and sperm are handled in the laboratory — resulting in 25,228 live births and 35,025 infants. In New Mexico limited access to fertility specialists has resulted in declining IVF procedures. With the opening of RMFC@UNM™, Magarelli estimates that within two years, 150 to 200 procedures will be performed annually in Albuquerque. In 2002, in an effort to enhance reproductive services in New Mexico, Magarelli conducted a seminar in Santa Fe. Three people attended. At a subsequent seminar, a few months later, the room was filled. “We have the capability and people want this information. New Mexico as a whole is interested in integrative care.” RMFC@UNM™ is an outgrowth of Dr. Magarelli’s fertility practice in Colorado Springs and a response to genuine desire for expanded services in New Mexico. Working with local professionals in Albuquerque and Colorado Springs, Magarelli offers comprehensive services to his patients at both centers. He hopes the new center in Albuquerque and his affiliation with the University of New Mexico will enhance the one other program operating in New Mexico.
The Problem Ask your doctor to check the following: Are there sperm? A semen analysis at an Andrology Lab; Are there eggs? Day 2, 3, or 4 after your menstrual cycle starts have your doctor check your blood for Estradiol, FSH, and LH hormones; Can the sperm and egg meet? Have your MD order an HSG or X-ray dye test on Day 10 of your cycle — this will tell him if your fallopian tubes are open or not; Does the baby have a safe place to grow? The same HSG above will also tell your MD that you can safely carry a child.
A Partnership Diane Cridennda is a doctor of oriental medicine (D.O.M.) practicing in Colorado Springs. Magarelli began referring some of his more challenging patients, those with a limited chance of getting pregnant, to Cridennda for acupuncture treatments. Two articles have appeared in the scientific literature that thrust acupuncture into the spotlight as a viable complementary modality for the treatment of infertility. The protocols outlined in the articles are widely accepted and used successfully in conjunction with IVF to treat infertility. Cridennda has been following the protocol for 10 years. “Participating in the acupuncture treatment gives women a sense of control; it’s empowering to be able to contribute and actively participate in their care,” she says. Cridennda adds, “The rewards are heartwarming.” The protocol includes:
The acupuncture treatments before and after egg transfer are to bolster the heart energy and connect the heart with the head. The other treatments promote increased blood flow to the uterine arteries, preparing to accept the embryo. The final treatment facilitates closure and enables the woman to hold the embryo and bring the baby to term. Dr. Cridennda treats prospective fathers as well as mothers. The process for men is a little different. It takes three months of treatments to affect the sperm. The treatments consist of vitamins, antioxidants, and specific herbs. Because of their collaboration, Doctors Magarelli and Cridennda have given birth to a three-year retrospective study, assessing the efficacy of combining IVF and acupuncture to treat infertility. Their results have not been duplicated.
The Bottom Line Despite the focus on babies, cost is an enormous factor. In the past, IVF treatments were “up front, pay as you go” procedures. In New Mexico and Colorado, things have changed, thanks to an innovative funding program called Shared Risk, implemented by Dr. Magarelli. Using in vitro, it takes approximately 2.2 attempts to get pregnant. Multiple attempts are costly and may not result in pregnancy. Through the Shared Risk program, patients receive four complete cycles of in vitro for the price of one. By making a deposit and affordable monthly payments, patients and doctors share the financial and fertility risk. This lowers the cost to families, increases the chances for pregnancy, and broadens access to these specialized services. Magarelli maintains, “We want to provide New Mexico women with options.” For couples facing infertility, having a baby can be an arduous journey. The time and energy spent identifying resources and agonizing over how to pay for it all, coupled with the spiritual and emotional toll, can be daunting. Through it all, couples wrap themselves in hope and persevere. East and West have converged in the high desert plains of the Southwest with a message of hope, anchored in traditional Chinese medicine and the promise of innovation that characterizes Western medicine. This balanced approach emanates from the very core of human existence and will move into the future helping couples, one baby at a time.Jahaan Martin is a freelance writer living in Albuquerque. |
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by Bill Radford
The newest technology has been joined by an ancient form of Chinese medicine at the Reproductive Medicine & Fertility Center in Colorado Springs. |
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OB/Gyn News, Copyright © 2005 by International Medical News Group, an Elsevier company.
PHILADELPHIA - Women who receive acupuncture during the stimulation phase of an in vitro fertilization cycle and again immediately after embryo transfer have a higher live-birth rate than do controls, according to the first acupuncture study with this end point. "Other studies have looked at pregnancy rates, but what is really important is whether or not there is a baby," said Paul C. Magarelli, M.D., who reported his findings at the annual meeting of the American Society for Reproductive Medicine. The retrospective study included 131 women who were undergoing standard in vitro fertilization (IVF) or Intracytoplasmic sperm injection (ICSI). All of these women were considered good prognosis candidates for IVF/ICSI and were given the choice of having acupuncture. A total of 83 women declined (controls) and 48 accepted. There were no significant differences between the two groups in terms of infertility diagnoses, demographics, and treatment protocols, except that sperm morphology was slightly better in the partners of women receiving acupuncture (7.3% vs. 5.9 % normal forms with strict criteria evaluation), and the average uterine artery pulsatility index was lower in the acupuncture group (1.57 vs. 1.72), said Dr. Magarelli of the department of ob.gyn. at the University of New Mexico, Albuquerque. The study found that pregnancy rates per embryo transfer were not significantly different between the two groups (50% in the acupuncture group and 45% in controls). The miscarriage rate was almost halved in the acupuncture group (8% vs. 14%). In addition, the rate of ectopic pregnancies was significantly lower in the acupuncture group-0 of 24 pregnancies (0%) vs. 2 of 37 pregnancies (9%), said Dr. Magarelli, who is also in private practice in Colorado Springs and Albuquerque. Thus, the live-birth rate per IVF/ICSI cycle was significantly higher in the acupuncture group than in controls (21% vs. 16%). "The live-birth rate per pregnancy is an even more telling number, since some cycles get cancelled. There was a 42% live-birth rate per pregnancy in the acupuncture group, compared to a 35% rate in the nonacupuncture group," Dr. Magarelli said in an interview with this newspaper. "We believe that what we are doing is improving the uterine environment such that implantation is improved," he added. The study used two acupuncture protocols. The Stener-Victorin electrostimulation protocol-which has been shown to reduce high uterine artery blood flow impedance, or pulsatility index (Hum. Reprod. 1996;11:1314-7)-was used for eight treatments during ovarian stimulation. The second acupuncture technique-the Paulus protocol, which has been associated with improved pregnancy rates (Fertil. Steril. 2002;77:721-4)-was used within 24 hours before the embryo transfer and 1 hour after. "This protocol has demonstrated reductions in uterine contractility, so by relaxing the uterus before the embryo transfer and immediately after, we felt we were setting up a better environment for implantation," Dr. Magarelli said. |
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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]
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. 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.
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. 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.
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Yasuhiro Yoshimoto, M.D., Akira Miyake. M.D., Keiichi Tasaka, M.D., Toshihiro Aono, M.D.
Department Of Obstetrics and Gynecology, Osaka University Medical School, Osaka and "School of Medicine, The University of Tokushima, Tokushima 770, Japan (Accepted for publication August 15, 1988)
Abstract: The effect of combined therapy with Wen-Jing-Tang and clomiphene citrate on anovulation was studied in 16 infertile patients who did not respond to clomiphene citrate alone. Wen-Jing-Tang was given at a dose of 5 g a day every day from day 2 of the menstrual cycles and clomiphene citrate was given at 150 mg day for 5 days from day 5 of the cycle. Ovulation occurred in 43.8% of the patients and 48.6% of the cycles. No case of ovarian hyperstimulation syndrome or pregnancy were observed. These results suggest that the combined therapy with Wen-Jing-Tang and clomiphene citrate should be used before therapy with human menopausal gonadotropin . Addition of Wen-Jing-Tang (Japanese) name, Unkeito) to the perfusion medium of rat mediobasal hypothalamus in vitro is reported to increase LH-RH release. Clomiphene citrate (clomiphene) is also known to increase LH-RH release from the hypothalamus, resulting in induction of ovulation in patients with anovulation due to hypothalamic dysfunction. Since the rate of ovulation on clomiphene citrate treatment ranges from 60.5% to 80.5%. some patients do not respond to clomiphene citrate, Therefore, we evaluated the effect of combination treatment with Wen-Jing-Tang and clomiphene in patients who did not ovulate after clomiphene treatment.
Materials and Methods
Results
Discussion No case of ovarian hyperstimulation syndrome was observed in the present study. The rates of ovarian hyperstimulation syndrome by clomiphene alone and Wen-Jing-Tang alone were both less than, 5%. These findings suggest that the ovarian hyperstimulation syndrome is rarely induced by this combined therapy. Therefore, this combined therapy should be tried before treatment with human menopausal gonadotropin-human chorionic gonadotropin, which has side effects, such as inductions of multiple pregnancy and the ovarian hyperstimulation syndrome.
Acknowledgment
Reference |
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Lynn M. Westphal, M.D., Mary Lake Polan, M.D., Ph.D., M.P.H., Aileen Sontag Trant, Ph.D., and Stephen B. Mooney, M.D. |
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Raymond Chang, M.D.[a,b] Pak H. Chung, M.D.[b] and Zev Rosenwaks, M.D.[c]
BackgroundAcupuncture is the manipulation of thin metallic needles inserted into anatomically defined locations on the body to affect bodily function. The US Food and Drug Administration has recently removed acupuncture needles from the category of experimental medical devices and now regulates them just like it does other devices, such as surgical scalpels and hypodermic needles, under good manufacturing practices and single-use standard of sterility (1). The general theory of acupuncture is based on the premise that there are patterns of energy flow (Qi) through the body, which are essential for health. Disruption of this flow is believed to be responsible for disease. Acupuncture can correct imbalances of flow at identifiable points close to the skin. According to the proposed international acupuncture nomenclature by The World Health Organization in 1991 (17), the meridian system consists of 20 meridians interconnecting about 400 acupoints. These acupoints correspond to specific areas on the surface of the body, which demonstrate higher electrical conductance because of the presence of higher density of gap junctions along cell borders. They act as converging points (or sinks) for electromagnetic fields. A higher metabolic rate, temperature, and calcium ion concentration, are also observed at these points. In principle, positive (anode) pulse stimulation of a point inhibits the organ function, whereas negative (cathode) pulse stimulation enhances that function (18). This forms the basis of electroacupuncture, which applies small electrical needles inserted in specific acupoints. Effects of acupuncture on the hypothalamic-pituitary-ovarian axis and menstrual cycleAlthough traditional Chinese medicine understanding of acupuncture is based on ancient medical theory, a modern and scientific neuroendocrine perspective has begun to evolve in the past two decades. Mayer et al. (19) first reported that acupuncture analgesia was induced through endorphin production and antagonized by the narcotic antagonist naloxone. Other studies similarly suggested that certain effects of acupuncture are mediated through the nervous system, within which ß-endorphin and other neuropeptides have been implicated (20-22).Acupuncture was shown by Petti et al. (20) to cause a significant increase in ß-endorphin levels during treatment, which lasted for up to 24 hours. ß-endorphin is derived from its precursor protein pro-opiomelanocortin, which is present in abundant amounts in neuronal cells of the arcuate nucleus of the hypothalamus, pituitary, medulla, and in peripheral tissues including intestines and ovaries (23-25). Pro-opiomelanocortin cleaves to form adrenocorticotropic hormone and ß-lipoprotein. Further cleavage of ß-lipoprotein yields neuropeptides including ß-endorphin. Aleem et al. (26, 27) demonstrated the presence of immunoreactive ß-endorphin in follicular fluids of both normal and polycystic ovaries. The influence on gonadotropin secretion and the menstrual cycle by endogenous opioid peptides is believed to be mediated by their action on GnRH secretion (28). The hypothalamic ß-endorphin center and the GnRH pulse generator, in fact, are both situated within the arcuate nucleus. Quigley et al. (29) first reported an increased opioid inhibition of LH secretion in hyperprolactinemic patients with pituitary microadenomas. Ching (30) and Orstead and Spics (31), respectively, showed that opioid peptides suppress GnRH release in rats and rabbits. The role of these neuropeptides, including ß-endorphin, in the regulation of GnRH secretion in humans has recently been reviewed by Kalra et al. (32) and Pau and Spies (33). Rossmanith et al. (34) demonstrated the role of opioid peptides in the initiation of the mid-cycle LH surge in normal cycling women. Meanwhile, measurement of ß-endorphin in ovarian follicular fluid of healthy ovulatory women revealed much higher levels than that in circulating plasma (35). The highest level of ß-endorphin was noted to be in the preovulatory follicle. Because acupuncture treatment impacts on ß-endorphin levels, which in turn affect GnRH secretion and the menstrual cycle, it is logical to hypothesize that acupuncture may influence ovulation and fertility. Animal studies have revealed that acupuncture treatment normalized GnRH secretion and affected peripheral gonadotropin levels (36, 37). Various investigators have shown that in normally ovulatory or anovulatory women, acupuncture also influenced plasma levels of FSH, LH, E2, and P (38-40). Acupuncture as a surrogate for hCG in ovulation induction was successfully used by Cai (41). Chen and Yu (42) showed that electroacupuncture normalized they hypothalamic-pituitary-ovarian axis, and in another study Chen (43) reported that 6 of 13 anovulatory cycles responded to acupuncture treatment. A series published from the University of Heidelberg in Germany (44) used auricular acupuncture on 45 infertile women suffering from ovulatory dysfunction such as oligomenorrhea and luteal phase defect. The control group received medical treatment including bromocriptine, dexamethasone, levothyroxine, clomiphene citrate (CC), and gonadotropin. Although the investigators concluded that resumption of ovulatory cycles occurred significantly more often in the acupuncture group compared to the control group, pregnancy rates were not different between the two groups. However, interpretation of study data was very difficult due to the heterogeneity of the patient population and treatment modalities. Moreover, seven pregnancies in the acupuncture group were actually achieved with hormone treatment 6 months after acupuncture was stopped. Another study by Stenver-Victorin et al. (45) evaluated the use of electroacupuncture for ovulation induction on 24 oligo/amenorrheic women with polycycstic ovarian syndrome (PCOS). The percentage of ovulatory cycles in all subjects was shown to improve from 15% (in a total of 3 months before treatment) to 66% up to 3 months after treatment. Responsive patients were noted to have significantly lower body mass index (BMI), waist-to-hip circumference ratio, serum T concentration, serum T/sex hormone-binding globulin ratio, and serum basal insulin level. They suggested that, in these selected patients with PCOS, acupuncture could be considered as an alternative or adjunct to pharmacological ovulation induction. A recent prospective randomized controlled study by Paulus et al. (46) compared pregnancy rates in a total of 160 patients undergoing IVG. Acupuncture was performed in 80 patients 25 minutes before and after ET. After controlling confounding variables, clinical pregnancy rate for the acupuncture group (42.5%) was significantly higher than the control group (26.3%). Peripheral effects of acupunctureIn addition to the central modulation of the hypothalamic-pituitary-ovarian axis, the effects of acupuncture on the autonomic nervous system have been well documented (47). In the early 1980s, Yao et al. (48) reported long-lasting cardiovascular depression induced by acupuncture stimulation of the sciatic nerve in unanesthetized hypertensive rats. In the human, acupuncture was also shown to be sympathoinhibitory. After acupuncture, sympathetic nerve activity as measured by norepinephrine level, skin temperature, blood pressure, and pain tolerance threshold was shown to be decreased (49).Endometrial thickness, morphology, and uterine artery blood flow have been implicated as important parameters for success of implantation of human embryos (50-57). Despite conflicting results in the utilization of these parameters during various stages of treatment to predict outcome in IVF, it is generally believed that adequate endometrial thickness is required to optimize pregnancy rate. Because endometrial thickness is a function of uterine artery blood flow, Sher and Fisch (58) reported a novel method of using vaginal sildenafil in an attempt to improve uterine artery blood flow and endometrial development in patients undergoing IVF. With its central sympathoinhibitory effect, acupuncture may contribute to reduce uterine artery impedance and therefore, increase blood flow to the uterus. In fact, Sterner-Victorin et al. (59) demonstrated this when they performed acupuncture in 10 infertile women who were down-regulated by GnRH analog to avoid the effect of endogenous hormone on the uterine artery blood flow. Pulsatility index in the uterine artery and skin temperature (on the forehead and lumbosacral area) were evaluated in three time periods-before, right after, and 2 weeks after acupuncture treatment (twice a week for 4 weeks). Pulsatility index and skin temperatures were found to be significantly decreased and increased, respectively, both right after and 14 days after acupuncture treatment. This effect was hypothesized to be caused by central inhibition of sympathetic activity. Acupuncture and stress reductionIt has been well documented that infertility causes stress (60-65), and stress reduction may, in turn, improve fertility (66). However, the relationship between stress and infertility is that of a vicious cycle. Social stigmatization, decreased self-esteem, unmet reproductive potential of sexual relationship, physical and mental burden of treatment, and the lack of control on treatment outcome are just some of the factors that can lead to psychological stress in any couple pursuing infertility treatment. In turn, stress may lead to the release of stress hormones and influence mechanisms responsible for a normal ovulatory menstrual cycle through its impact on the hypothalamic-pituitary-ovarian axis.The use of acupuncture for reducing anxiety and stress possibly through its sympathoinhibitory property and impact on ß-endorphin levels has been reviewed (67, 68), and the efficacy of acupuncture in depression has also been studied (69). Because the pharmacological side effects of anxiolytic and antidepressant drugs on infertility treatment outcome are largely unknown, acupuncture may provide an excellent alternative for stress reduction in women undergoing infertility treatment. DiscussionThe practice of acupuncture to treat identifiable patho-physiological conditions has been a subject of intense research. The underlying physiologic mechanisms of acupuncture such as the release of opioids and other peptides in the central peripheral nervous system, and its inhibition of the sympathetic nervous system have been increasingly established. Promising results from credible trials have emerged for the use of acupuncture in treating various pain syndromes, substance abuse, and chemotherapy-induced nausea and vomiting.Although the definitive role of acupuncture in the treatment of female infertility is yet to be established, its neuroendocrine effect on the hypothalamic-pituitary-ovarian axis and the preliminary clinical data reviewed here justifies further clinical trials to systematically examine the efficacy of acupuncture in treating various conditions related to female infertility such as ovulatory dysfunction associated with PCOS. The peripheral impact of acupuncture in improving uterine artery blood flow and hence endometrial thickness also provides encouraging data regarding its potential positive effect on implantation. Whether these potential beneficial effects of acupuncture on the reproductive system can be translated into improving infertility treatment outcomes will eventually mandate randomized controlled studies of adequate design. Because acupuncture is nontoxic and relatively affordable, its indications as an adjunct in assisted reproduction or as an alternative for women who are intolerant, ineligible, or contraindicated for conventional hormone induction of ovulation deserves serious research and exploration. Appropriate training, credentialing, and certification of acupuncture practitioners by state agencies can facilitate the integration of acupuncture into the treatment of female infertility, and healthcare in general. The NIH Consensus Conference (1) agreed that this is necessary to allow the public and other health practitioners to identify qualified acupuncture practitioners. With the help of the US Department of Education, issues of training and licensure of non-physician and physician practitioners have been addressed. There is sufficient evidence to acupuncture's value to expand its use into conventional medicine and treatment of female infertility, and to encourage further studies of its underlying mechanisms as well as to establish its clinical value. References
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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., Printed on acid-free paper in U.S.A. Received June 5, 2001; revised and accepted October 16, 2001. Reprint requests: Wolfgang E. Paulus, M.D., Christian-Lauritzen-Institut, Frauenstr. 51, D-89073, Ulm, Germany (FAX: ++49-731-9665130; E-mail: paulus@reprotox.de). [a] Department of Reproductive Medicine, Christian-Lauritzen-Institut. [b] Department of Traditional Chinese Medicine, Tongji Hospital, Tongji Medical University, Wuhan, People's Republic of China. 0015-0282/02/$22.00 PII S0015-0282(01)03273-3 Objective: To evaluate the effect of acupuncture on the pregnancy rate in assisted reproduction therapy (ART) by comparing a group of patients receiving acupuncture treatment shortly before and after embryo transfer with a control group receiving no acupuncture. Design: Prospective randomized study. Setting: Fertility center. Patient(s): After giving informed consent, 160 patients who were undergoing ART and who had good quality embryos were divided into the following two groups through random selection: embryo transfer with acupuncture (n = 80) and embryo transfer without acupuncture (n = 80). Intervention(s): Acupuncture was performed in 80 patients 25 minutes before and after embryo transfer. In the control group, embryos were transferred without any supportive therapy. Main Outcome Measure(s): Clinical pregnancy was defined as the presence of a fetal sac during an ultrasound examination 6 weeks after embryo transfer. Result(s): Clinical pregnancies were documented in 34 of 80 patients (42.5%) in the acupuncture group, whereas pregnancy rate was only 26.3% (21 out of 80 patients) in the control group. Conclusion(s): Acupuncture seems to be a useful tool for improving pregnancy rate after ART. (Fertil Steril®2002;77:721- 4. ©2002 by American Society for Reproductive Medicine.) Key Words: Acupuncture, assisted reproduction, embryo transfer, pregnancy rate Acupuncture is an important element of traditional Chinese medicine (TCM), which can be traced back for at least 4,000 years. Acupuncture has been shown to alleviate nausea and vomiting, dental pain, addiction, headache, menstrual cramps, tennis elbow, fibromyalgia, myofascial pain, osteoarthritis, carpal tunnel syndrome, and asthma. Both physiologic and psychological benefits of acupuncture have been scientifically demonstrated in recent years. However, so far there have been only a few serious trials concerning the use of acupuncture in reproductive medicine. Publications focus primarily on acupuncture therapy for male infertility (1, 2). Electroacupuncture may reduce blood flow impedance in the uterine arteries of infertile women (3). A positive impact of electroacupuncture on endocrinologic parameters and ovulation in women with polycystic ovary syndrome has been demonstrated (4). In addition, auricular acupuncture was successfully used in the treatment of female infertility (5). In the present study, we chose acupuncture points that relax the uterus according to the principles of TCM. Because acupuncture influences the autonomic nervous system, such treatment should optimize endometrial receptivity (6). Our main objective was to evaluate whether acupuncture accompanying embryo transfer increases clinical pregnancy rate. Materials and MethodsThis 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¨ 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). ResultsA 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). DiscussionThe 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
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Chen 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 (Received October 24, 1990; Accepted with revisions, December 8, 1990) ACUPUNCTURE & ELECTRO-THERAPEUTICS RES., Vol. 16, pp. 1-5,1991 Abstract:
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. 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 
* P<0.05 as estimated by X[2] test 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)
*The values in this table are Mean ± SE **P<0.05 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). 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.
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Cai Xuefen
Obstetrical & Gynecological Hospital, Zhejiang Medical University, Zhejiang Province 310006 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. of China Academy of Traditional Chinese Medicine The Center regularly conducts three-month advanced and general courses of traditional Chinese medicine, acupuncture, Tuina (massage), Qigong (breathing exercises) and Taiji (shadow boxing). It also runs short-term training courses on some special topics, and preparatory guidance courses for licensure examination of tradi tional Chinese medicine, acupuncture and moxibustion. In addition, various courses based on the participant's requirements may be arranged in the center. All those who complete the required courses will receive relevant certificates. The Training Center is always ready to establish friendly relations of exchange and cooperation with medical institutions of various countries. It warmly welcomes medical professionals from home and abroad to take training courses. Address: Training Center of China Academy of Traditional Chinese Medicine No 18 Beixincang Dongzhimennei, Beijing 100700 China Dr. Pan Ping Dr. Zhao Jihui Tel: 86-10-64075193 64062096 Fax: 86-10-64061635 64062096 |
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ABSTRACT
Despite extensive research on the pathogenesis of polycystic ovary syndrome (PCOS), there is still disagreement on the underlying mechanisms. The rat model for experimentally induced polycystic ovaries (PCO)–produced by a single injection of estradiol valerate–has similarities with human PCOS, and both are associated with hyperactivity in the sympathetic nervous system. Nerve growth factor (NGF) is known to serve as a neurotrophin for both the sympathetic and the sensory nervous systems and to enhance the activity of catecholaminergic and possibly other neuron types. Electro-acupuncture (EA) is known to reduce hyperactivity in the sympathetic nervous system. For these reasons, the model was used in the present study to investigate the effects of EA (12 treatments, approximately 25 min each, over 30 days) by analyzing NGF in the central nervous system and the endocrine organs, including the ovaries. The main findings in the present study were first, that significantly higher concentrations of NGF were found in the ovaries and the adrenal glands in the rats in the PCO model than in the control rats that were only injected with the vehicle (oil or NaCI). Second, that repeated EA treatments in PCO rats resulted in concentrations of NGF in the ovaries that were significantly lower than those in non-EA-treated PCO rats but were within a normal range that did not differ from those in the untreated oil and NaCI control groups. The results in the present study provide support for the theory that EA inhibits hyperactivity in the sympathetic nervous system. adrenal, central nervous system, follicular development, hypothalamus, ovary, ovulation, pituitary, stress INTRODUCTION Polycystic ovary syndrome (PCOS), one of the most common causes of anovulation in women of reproductive age. is a complex endocrine and metabolic disorder [1]. Despite extensive research seeking the pathogenesis of PCOS, there is still disagreement on the underlying mechanisms. Different hypotheses of its pathophysiology have emerged, which indicates that the etiology is multifactorial and poorly understood. Women with PCOS have an increased risk of endometrial cancer, hypertension, and type II diabetes, and they need some kind of long-standing treatment [2]. Traditional pharmacological treatment for ovulation induction is effective, but side effects such as superovulation are quite common. A previous clinical study on anovulatory women with PCOS showed that sensory stimulation (i.e., electro-acupuncture [EA]) affects endocrinological and neuroendocrinological parameters [3]. In addition, regular ovulations were induced in more than one-third of the women without negative side effects. These findings accord with previous reports [4-6] but do not enlighten underlying mechanisms. The mechanisms behind the beneficial effect of EA on PCOS in the human are difficult to study because tissue samples from the ovaries and the central nervous system (CNS) are for obvious reasons unobtainable. Studies on, for instance, neuropeptides in the gonads and the CNS would be possible to conduct in an animal model, provided that such a model exists. Experiments on normal cycling rats have shown that exogenous estradiol valerate (EV), a long-acting estrogen, causes acyclicity and the formation of polycystic ovaries (PCO) [7, 8]. The changes include atretic antral follicles, follicular cysts with a well-developed theca cell layer, a diminished granulosa cell compartment, and luteinized cysts [7, 8]. Furthermore, the rats exhibited alterations in basal and pulsatile LH and FSH concentrations, changes in the pituitary response to GnRH, degenerative changes in the hypothalamus, altered opioid inhibitory tone on GnRH release, and high estradiol levels with a persistent pattern of constant estrus as assessed by vaginal smear [9, 10]. In addition, EV-induced PCO is associated with an increase in peripheral sympathetic outflow, evidenced by an increase in the release of norepinephrine (NE), an increase in ovarian NE content, and a decrease in the number of ß-adrenergic receptors in the ovarian compartments receiving catecholaminergic innervation [9-11]. Even if it is not possible to reproduce human PCOS using a rat model, it may provide important leads because a single injection of EV induces an anovulatory state that shares many endocrinological and morphological characteristics of human PCOS [7-13]. Thus, comparisons between the rat PCO model and human PCOS must be interpreted with caution because rat PCO ovaries contain multiple follicular cysts, the structure of which does not replicate the follicular growth arrest found in human PCOS. Contrary to previously held notions, the granulosa cells in the follicles accumulating in the human ovary are not atretic. However, both human PCOS and EV-induced PCO in rats may be associated with hyperactivity in the sympathetic nervous system. According to one theory, elevated concentrations of neurotransmitters found in women with PCOS and anovulation may be associated with psychological stress and with hyperactivity in the sympathetic nervous system [3, 12, 13]. That superior ovarian nerve transection restores estrus cyclicity and ovulatory capacity in rats with EV-induced PCO further supports the theories of sympathetic hyperactivity [9]. Other evidence of neuronal involvement is that ovarian sympathetic innervation is under trophic control by nerve growth factor (NGF) [14]. This is also supported by the fact that the expression of the genes that encode NGF and one of its receptors, the low-affinity NGF-receptor, was dramatically increased in the ovary 30 days after EV injection [11]. Ovarian NGF is principally synthesized in the cells of the follicular wall [15], which is the site where the sympathetic neurons project to the ovaries [14]. The increase in the synthesis of NGF and its receptor that precedes the formation of cysts suggests that after PCO has been induced by EV injection, the neurons innervating the ovary are subjected to an enhanced neurotrophic influence that contributes to their hyperactivation and to the maintenance of an abnormally elevated catecholaminergic tone in ovarian steroid secretions [9-11]. Aim of the Study Because NGF is known to serve as a neurotrophin for both the sympathetic and the sensory nervous systems and to enhance the activity of catecholaminergic and possibly other neuron types [9, 11, 14, 16-22], and because EA is known to reduce hyperactivity in the sympathetic nervous system [23-25], the experimentally induced PCO model was used to study the effects of EA by analyzing NGF in the CNS and the endocrine organs, including the ovaries. The first part of the present study investigated dose-response–the discovery of the exact dose of EV needed to produce fully developed polycystic ovaries. The second part of this study investigated treatment with EA–what contribution NGF made to the etiology and maintenance of EV-induced PCO in rats and if and to what extent EA has an effect on NGF and ovarian morphology in experimentally induced PCO. MATERIALS AND METHODS Fifty-nine virgin adult cycling Sprague-Dawley rats (Möllegaard, Denmark) weighing 190-210 g and with regular 4-day estrous cycles were used. The rats were housed at 22°C, four to a cage, with free access to pelleted food and tap water and with a 12L:12D cycle for at least 1 wk before and throughout the experimental period. All rats received a single i.m. injection of either EV (Riedeldehaen, Germany), oil, or 0.15 M NaCI (Kabi Pharmacia AB, Sweden) and were anesthetized with enfluran (EFRANE, Abbott Scandinavia, Kista, Sweden) and killed by decapitation. The local Animal Ethics Committee at Göteborg University, Sweden approved the study. Dose-Response Twenty-seven rats were injected with one of two different doses of EV in an oil solution or with oil alone to ascertain the optimal dose for induction of PCO [8]. They were decapitated on three different occasions (15, 30, or 60 days after i.m. injection) to elucidate precisely when the ovaries display characteristic features of well-defined PCO [7, 8]. Nine rats received 2 mg EV in 0.2 ml oil/rat, nine rats 4 mg EV in 0.2 ml oil/rat, and nine rats 0.2 ml oil alone. Three rats per dose were killed on Day 15, three on Day 30, and three on Day 60.  FIG. 1. Schematic drawing of the dorsal side of a rat and the placement of acupuncture needles. Two needles were placed bilaterally in the erector spinae muscle at the level of Th12 and two were placed in the quadriceps muscle bilaterally. The needles were then attached to an electrical stimulator for EA treatment. Treatment with EA The optimal dose (4 mg EV in 0.2 ml oil/rat) and timing (30 days after injection) were chosen for the experiments. In total, 32 rats took part. Eight rats in the EV control group and eight in the EA-treated EV group were injected i.m. with 4 mg EV in 0.2 ml oil/rat, eight rats in the oil control group with 0.2 ml oil, and eight rats in the NaCI control group with 0.2 ml 0.15 M NaCI. All 32 were decapitated on Day 30 after injection, that is, 1-2 days after the last EA treatment. All groups were anesthetized 12 times for about 25 min each time. Anesthesia was induced by inhalation of enfluran at 5.5-6.5 ml/h with an O2 and air flow of 0.25 L/min. The EA-treated EV group was subjected to 12 EA treatments every second or third day, beginning 2 days after the i.m. injection of EV. The stimulation points were bilateral in the quadriceps and erector spinae muscles at the level of thoracic (Th) 12 in the somatic segments according to the innervation of the ovaries (Th 12-lumbar [L]2, sacral [S]2-S4) (Fig. 1). The needles (Hegu; Hegu AB, Landsbro, Sweden) were inserted to depths of 0.5-0.8 cm and then bilaterally attached to an electrical stimulator (CEFAR ACU II, Cefar, Lund, Sweden) with a low burst frequency of 2 Hz. Individual pulses within the frequency were square wave pulses with alternating polarities and with a pulse duration of 0.2 msec, 80 pulses/sec. The intensity was adjusted so that local muscle contractions were seen to reflect the activation of muscle-nerve afferents (A delta fibers and possibly C fibers) [26, 27]. The location and type of stimulation were the same in all rats. Nerve Growth Factor Measurements by Enzyme Immunoassay In the second part of the study, after the rats were decapitated, the pituitary gland, the hypothalamus, the hippocampus, one ovary, and the adrenal glands were quickly removed and dissected on dry ice, weighed, and stored at -80°C until extraction. The samples were sonicated in extraction buffer (0.1% Triton X-100, 100 mM Tris-HCI, pH 7.2, 400 mM NaCI, 4 mM EDTA, 0.2 mM PMSF, 0.2 mM benzethonium chloride, 2 mM benzamidine, 40 U/ml aprotinin, 0.05% sodium azide, 2% BSA, and 0.5% gelatin; 1 ml/100 mg of tissue), followed by centrifugation at 10,000 x g for 30 min. The supernatants were used for the assay. The bioactive form of 2.5S NGF purified from mouse sub-maxillary glands and prepared in the laboratory at the Institute of Neurobiology (CNR) in Rome, Italy, according to the method of Bocchini and Angeletti [28] was used as a standard. The NGF was dissolved in extraction buffer and the standard curve was in a range of 31.25 pg ml (-1) and 1 ng ml (-1). An ELISA was performed as described by Weskamp and Otten [29] with a minor modification [30]. Specific NGF binding was assessed by use of monoclonal mouse anti-ß-2.5S NGF (Boehringer Mannheim GmbH, Mannheim, Germany) that reacts with both the 2.5S and the 7S biologically active forms of NGF. The absorbency of samples and standards was corrected for nonspecific binding (i.e., the absorbency in a well coated with purified mouse IgG). The NGF content in the samples was determined in relation to the NGF standard curve. Data were not corrected for recovery of NGF from samples, which was routinely 70-90%, and was accepted only when the values were >2 SD above the blank. With these criteria, the limit of sensitivity of NGF ELISA averaged 0.5 pg per assay. Morphology One ovary per rat was removed, cleaned of adherent connective fat tissue, and fixed in 4% formaldehyde buffer; sections were stained with hematoxylin-eosin, and a trained pathologist performed a quantitative analysis of the follicle population. If ovum degeneration or at least one pyknotic granulosa cell was seen, the follicle populations were classified as atretic, otherwise they were classified as healthy. Morphological characteristics of follicular atresia were, for instance, scattered pyknotic nuclei in the granulosa cell layer [31], detachment of the granulosa cell layer from the basement membrane [32], fragmentation of the basal lamina [33], and the presence of cell debris in the antrum of the follicle [34]. Statistical Analyses Statistical analyses were carried out using the SPSS 8.0 software. The NGF concentrations in the pituitary gland, the hypothalamus, the hippocampus, the ovary, and the adrenal glands were analyzed and the groups compared using ANOVA followed by multiple comparison procedures (Bonferroni test). All results are presented as mean ± SEM. A P value less than 0.05 was considered significant. The 95% confidence interval (Cl) was given when P < 0.05. RESULTS Ovarian Morphology–Dose-Response In the first part of the present study, dose-response, injection of 0.2 ml oil alone (control) was associated with a normal appearance of the ovaries and no differences were seen between rats sacrificed on Day 15, 30, or 60 (Fig. 2, a and b). No changes were seen in the ovaries of rats injected with 2 mg EV in 0.2 ml oil/rat and killed on Day 15. The ovaries of rats injected with the same dose of EV in oil exhibited small morphological changes resembling PCO when killed on Day 30 and 60 (Fig. 3, a and b). The ovaries of rats injected with a higher dose of EV (4 mg EV in 0.2 ml oil/rat) exhibited only small morphological changes on Day 15. Rats injected with the same dose of EV in oil and killed on Day 30 (Fig- 4, a-c) showed a progressive decrease in the number of primary and secondary follicles but it was on Day 60 (Fig- 5, a and b) that the true cystic follicles appeared and the well-defined PCO was fully developed in accordance with previous reports by Brawer et al. [8]. Ovarian Morphology–Treatment with EA In the second part of the present study, treatment with EA, all rats were killed at Day 30 after EV injection, i.e., before the appearance of cystic follicles. The ovaries in the EV control group (4 mg EV in 0-2 ml oil/rat) displayed the same morphological changes as previously shown in the dose-response section (see Fig- 4, a-c). The ovaries in the oil control group and the NaCI control group exhibited a typically normal appearance (see Fig- 2, a and b). No substantial morphological differences were found between the EA-treated, EV group, and the EV control group. Nerve Growth Factor–Treatment with EA In the second part of the present study, treatment with EA, NGF measurements were made at Day 30 after EV injection. Means ± SEM for NGF (pg/g wet weight) in the hypothalamus, the pituitary gland, the hippocampus, the ovary, and the adrenal gland in all groups are presented in Table 1. Ovarian NGF concentrations were significantly higher in the EV control group compared to the oil control group (P < 0.001, CI = 178.7, 821.6) and the NaCl control group (P < 0.01, CI = 144.6, 787.5). The NGF concentrations in the ovary were significantly lower in the EA-treated, EV group compared to the EV control group (P < 0.05 Cl = 6.2, 614.9) and did not differ from the (Jil and the NaCI control groups) The NGF concentrations in the adrenal glands were significantly higher in the EV control group and the EA-treated. EV group compared to both the oil control group (P < 0.001, CI = 45.7, 169.3 and P < 0.01, CI = 38.5, 166.5) and the NaCI control group (P < 0.001, Cl = 21.9, 162.9 and P < 0.01, Cl = 15.0, 159.8). Weights of Ovaries and Adrenal Gland–Treatment with EA Means ± SEM for weights (mg) of the ovaries and the adrenal glands in all groups are presented in Table 2. Ovarian weights in the control EV group and in the EV-treated EV group were significantly lower compared to the oil control group (both P < 0.001) and the NaCI control group (both P < 0.001).
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.
[a] NGF (pg/g wet weight) in the ovary, the adrenal gland, the pituitary gland, the hypothalamus, and the hippocampus in the different groups: EA-treated, EV; EV control; oil control (0.2 ml); and NaCI control (0.2 ml 0.15 M). All EV iniections were 4 m8 EV In 0.2 ml oil/rat. All data values are mean ± SEM. [b] P < 0.05, EA EV versus EV control. [c] P < 0.001, EA EV versus oil control; and P < 0.01, EA EV versus NaCI control. [d] P < 0.001, EV control versus oil control; and P < 0.01, EV control versus NaCI control. [e] < 0.01, EV control versus oil control; and P < 0.001, EV control versus NaCI control. TABLE 2. Treatment with EA.
[a] Weights of the ovary and the adrenal gland shown as mean ± SEM in the different groups: EA-treated, EV; EV control; oil control (0.2 ml); and NaCI control (0.2 ml 0.15 M). All EV injections were 4 mg EV in 0.2 ml oil/rat. [b] P < 0.001, EA EV versus oil control and NaCI control. [c] P < 0.001, EV control versus oil control and NaCI control. For obvious reasons it is not possible to subject control animals to true sham needle insertion. As soon as a needle penetrates the skin, it may be seen as a form of sensory stimulation that activates afferent nerve fibers. If a sham needle insertion without electrical stimulation is performed, then different acupuncture methods/stimulation techniques are being compared, and this does not provide proper information on the effect of EA versus no EA. We chose EA because the stimulation intensity is easy to standardize and it has been shown to be superior to manual needle stimulation [37]. In addition, to show a difference between two or more stimulation techniques would require a very large number of study subjects. In the present study, the control rats received the same enfluran anesthesia protocol as the rats treated with EA, which, in our opinion, is the best way to control completely environmental and/or emotional factors and the EA effect. The acupuncture needles in the present study were placed in the somatic segments that correspond to ovarian innervation. The needles were stimulated with low frequency EA for optimal activation of muscle nerve afferents to inhibit the autonomic outflow at the segmental level and at the central level and to modulate the HPO axis. The choice of acupuncture points and the aim of stimulation has been the same as in our other EA studies on the female reproductive tract that dealt with blood flow in the uterine arteries prior to in vitro fertilization (IVF) [38], pain-relief during oocyte aspiration in connection with IVF treatment [39], and induction of ovulation in women with PCOS [3]. We have shown that repeated EA treatments restore regular ovulations in more than one-third of the anovulatory women with PCOS. In addition, EA-influenced neuroendocrine and endocrine parameters indicative of PCOS, such as LH/FSH ratios, mean testosterone concentrations, and ß-endorphin concentrations, decreased significantiy [3]. The effects of repeated EA on anovulation were then attributed to an inhibition of hyperactivity in the sympathetic nervous system [3, 5, 6]. The findings of the present study support recent reports that ovarian NGF concentrations in rats with experimentally induced PCO [11] are elevated and that this increase can be related to a hyperactivity in the ovarian sympathetic nerves. Lara et al. [11] also suggests that activation of this neurotrophic-neurogenic regulatory loop is a component of the pathological process by which EV induces cyst formation and anovulation. They also stated that there is evidence that the alteration in neurotrophic input to the ovary contributes to the etiology and/or maintenance of human PCOS [11]. Furthermore, the present study shows that repeated EA treatments reduce peripheral sympathetic nerve hyperactivity, as revealed by the reduction in increased NGF concentrations in the ovaries into a normal range 30 days after EV injection, that did not differ from that of the untreated oil and NaCI control groups. It remains to be shown whether EA directly affects sympathetic nerve activity. Measurements of the nervous output by analyses of the catecholamine release can resolve this. In addition, because receptors for NGF are expressed on the endocrine cells of the ovary, activities of ovarian NGF may mediate and/or be mediated by alterations in endocrine factors, for example, by corticotropin-releasing hormone, prolactin, oxytocin, and/or adrenal corticosteroid secretion. To resolve this, the same experimental protocol regarding EA and controls used here must be supplemented with measurements of serum levels of these hormones. Whether this condition can be reversed with EA treatment at higher stimulation intensities, in higher numbers, and/or over longer periods remains to be shown. The conclusion of this study is that repeated EA treatments reduce ovarian NGF concentrations to within normal ranges. This suggests that EA inhibits the hyperactivity in the ovarian sympathetic nerves, which may be of importance for the development and maintenance of experimentally induced PCO. ACKNOWLEDGMENTS The authors thank Professor Owe Lundgren and laboratory assistant Britt-Marie Fin, Department of Physiology, Goteborg University, for providing excellent working facilities and for invaluable laboratory help at their Department. We also thank Associate Professor Folke Knutsson for his invaluable assistance in the morphological analyses of the ovaries. Carl Lofman, M.D., Stockholm is acknowledged for skillful preparation of morphological specimens. REFERENCES
[1]Supported by grants from the Hjalmar Svensson Foundation, Wilhelm och Martina Lundgrens Vetenskapsfond Wilhelm and Martina Lundgren's Science Fund, and the Foundation for Acupuncture and Alternative Biological Treatment Methods. [2]Correspondence: Elisabet Stener-Victorin, Department of Obstetrics and Gynecology, Kvinnokliniken, Sahlgrenska University Hospital, SE-413 45 Göteborg, Sweden. FAX: 46 31829248; e-mail: elisabet.stenervictorin@medstud.gu.se Received: 29 February 2000. First decision: 30 March 2000. Accepted: 11 July 2000. © 2000 by the Society for the Study of Reproduction, Inc. ISSN: 0006-3363. http://www.biolreprod.org |
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Sandra L. Emmons, MD
Phillip Patton, MD Medical Acupuncture, A Journal For Physicians By Physicians Spring / Summer 2000- Volume 12 / Number 2 "Aurum Nostrum Non Est Aurum Vulgi"
ABSTRACT
KEY WORDS
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSION
REFERENCES
AUTHORS' INFORMATION
Sandra L. Emmons, MD Dr Phillip Patton is an Associate Professor of Obstetrics and Gynecology at Oregon Health Sciences University with specialty boards in Reproductive Endocrinology. Dr Patton's practice at OHSU emphasizes infertility and assisted reproductive technology, and he is a Fellow of the American Academy of Obstetrics and Gynecology. |
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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) ACUPUNCTURE & ELECTRO-THERAPEUTICS RES., INT. J., Vol. 22, pp. 97-108, 1997 Copyright (c) 1997 Cognizant Communication Corp. Printed in the USA. 0360-1293/95 $10.00 + .00 ABSTRACT
INTRODUCTON 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. 
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. 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].
The values in this table are mean±SE, *P<0.05 Effect of EA on dysfunction of HPOA in ovariectomized rats For a further study of the mechanism of EA effect on HPOA a series of experiments in the animal models was performed. (1). EA induces maturation and exfoliation of vaginal epithelium cell and enhances blood level of E2. After ovariectomy two weeks late, the exfoliated epithelium cell disappeared from the vaginal smears of the rats, but it reappeared in the smears following EA treatment. The blood level of E2 in OVX was increased significantly (table 2). No obvious change was seen in INT after EA treatment and in OVX following EA treatment with the control acupoints. Table 2. The level of blood E2 following EA treatment (pg/ml)
*P < 0.05 compared with INT, **P<0.01 compared with before EA (2). EA promotes enlargement of adrenals and enhances activity of adrenal AgNORs as well as blood level of corticosterone We found the adrenals of OVX+EA were enlarged and the weight of the adrenals was raised significantly. Using histochemical method, the AgNORs of the cells in inner adrenal cortex were examined. The result shows that the activity of AgNORs of OVX was enhanced (table 3, 4), and the level of blood corticosterone in OVX+EA was also increased (table 5). There were no similar effects in INT following EA treatment and in OVX after EA with control acupoints. Table 3. AgNORs number in OVX and INT
Table 4. Weight of adrenal
*P < 0.001 compared with before EA (3). EA decreases the level of hypothalamic GnRH, pituitary LH and increases the contents of hypothalamic and pituitary ß-endorphin After EA treatment the levels of GnRH released from hypothalamus was rnarkedly decreased however, the ß-endorphin (ß-EP) secretion in hypothalamus was raised. The pituitary content of LH was also fallen, but the ß-EP of pituitary was increased, as well as peripheral LH and ß-EP level (Fig.2). 
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). 
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). 
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.
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Elisabet Stener-Victorin[1.4], Urban Waldenström[2], Sven A. Andersson[3] and Matts Wikland[2]
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
BL - bladder channel. SP - spleen channel. Four needles were located bilaterally at the thoracolumbar and lumbosacral levels of the erector spinae, and were stimulated with high frequency (100 Hz) pulses of 0.5 ms duration. The intensity was low, giving non-painful paraesthesia. Four needles were located bilaterally in the calf muscles, and were stimulated with low frequency (2 Hz) pulses of 0.5 ms duration. The intensity was sufficient to cause local muscle contractions. Skin temperature The skin temperature was measured with a digital infrared thermometer (Microscanner D-series: Exergen, Watertown, MA, USA) between the applied acupuncture needles in the lumbosacral region (25 mm from each needle), skin temperature lumbosacral (STLS), and on the forehead, skin temperature forehead (STFH). The measurements were made during the first, fifth and eighth EA treatments. The first measurements were made after 10 min rest, and just before the EA, these being considered as `baseline.' Thereafter, further measurements of STLS and STFH were done every seventh minute during the EA and immediately after the EA. The room temperature was constant during the three EA treatments. Statistics Analysis of variance (ANOVA: Newman-Keul's range test) was used to analyze the data. Results Blood flow impedance Compared to the mean baseline PI, the mean PI was significantly reduced both soon after the eighth EA treatment (P < 0.0001) and 10-14 days after the EA period (P < 0.0001) (Figure 1), at which time six women had a mean PI <2.6 (Table II and Figure 2).  Figure 1. The mean pulsatility index (PI) (n = 8) for all women before the first electro-acupuncture (EA) treatment, immediately after the eighth EA treatment and 10-14 days after the EA period. *** = significant changes (P < 0.0001) compared to the mean PI before the first EA treatment.  Figure 2. The individual mean pulsatility index (PI) before down-regulation, before the first electro-acupuncture (EA) treatment, immediately after the eighth EA treatment and 10-14 days after the EA period. Table II. The individual mean pulsalility index (PI) before down-regulation, before the first electro-acupunclure (EA) treatment, immedialely after the eighth EA trealment, 10-11 days after the EA period, and average mean values
The right and left uterine arteries responded similarly to EA. The diffcrence in mean PI between the two arteries was ≤0.3 (not significant), both before down-regulation, during down-regulation and throughout the whole study period. There was no significant difference in the mean PI for patients with different causes of infertility. Skin temperature The pooled results from all skin temperature measurements are presented in Figure 3. Compared with the starting point, mean STFH increased significantly after 21 min of EA (P = 0.02), and directly after the EA treatments (P = 0.002). STLS did not change significantly.  Figure 3. Pooled mean values (n = 8) of skin temperature on forehead (STFH) and skin temperature in the lumbosacral area (STLS) during the first, fifth and eighth electro-acupuncture (EA) treatments. * = significant changes (P = 0.02) after 21 min and ** = significant changes (P = 0.002) immediately after EA compared to the time just before needles were inserted. 0 = `baseline'. Discussion It has been shown in previous studies that a high PI in the uterine arteries is associated with a decreased pregnancy rate following IVF-embryo transfer (Goswamy et al., 1988; Sterzik et al., 1989; Steer et al., 1992, 1995a.b; Coulam et al., 1995). The results reported by Tekay et al. (1995) support the hypothesis postulated by Steer et al. (1992) that uterine receptivity is improved when the PI value is between 2.0 and 2.99 on the day of embryo transfer. When a high PI is found before embryo transfer in a stimulated cycle, treatment options are few. Goswamy et al. (1988) successfully tried pre-treatment with exogenous oestrogens in the next cycle, but their results have not been verified by others. It has been proposed that the embryos should be frozen, thawed and transferred in an unstimulated cycle (Goswamy et al., 1988; Steer et al., 1992, 1994), but there is little support for the hypothesis that the PI would be lower under these contitions. In experiments on spontaneously hypertensive rats, EA at low frequency (2-3 Hz) induced a long-lasting, significant fall in blood pressure which was associated with decreased activity in sympathetic fibres (Yao et al., 1982; Hoffman and Thoren, 1986; Hoffman et al., 1987, 1990a,b). A decrease in sympathetic activity appears to be generalized. In microneurographic studies on humans, EA in the upper limbs resulted in an initial increase and then a decrease in activity of sympathetic efferents in the tibial nerve, with a parallel increase in the temperature of the skin (Moriyama, 1987). Kaada (1982) reported that transcutaneous stimulation of acupuncture points in the hand increased the skin temperature, giving pain relief in limbs suffering from Reynaud's phenomenon. Kaada (1982) also found that electrical stimulation of accupuncture hand points in patients with ischaemic conditions of the lower limbs, increased the skin temperature in the lower limbs and possibly enhanced the healing of long-standing ulcers. It has been noted in both animals and humms that EA has greater effects on pathological conditions, e.g. hypertension or hypotension, whereas normal blood pressure is only slightly changed (Yao et al., 1982: Hoffman and Thoren, 1986: Hoffman et al., 1987, 1990a,b). The mechanisms of sympathetic inhibition following EA are poorly understood. Based on animal experiments, Hoffmann and Thoren (1986) and Hoffman et al. (1987, 1990a,b) suggested that electrical slimulation of muscle efferents innervating ergoreceptors increases the eoncentration of ß-endorphin in the CSF. They found support for the hypothesis that the hypothalamic ß-endorphinergic system has inhibitory effects on the vasomotor centre, and thereby a central inhibition of sympathetic activity. It has been suggested that this central mechanism, involving hypothalamic and brain stem systems, is important in changing the descending control of many different organ systems, including the vasomotor system (Andersson. 1993; Andersson and Lundeberg, 1995). In this study, the PI of the uterine arteries was signifieantly decreased soon after the eighth EA treatment and remained significantly decreased 10-14 days after the EA period. These findings suggest that a series of EA treatments increases the uterine artery blood flow. Another effect observed in this study was the signifieantly inereased STFH during the EA treatments. The most likely cause of these effects is a decreased tonic activity in the sympathetic vasoconstrictor fibres to the uterus and an involvement of the central mechanisms with general inhibition of the sympathetic outflow, in accordance with previously observed EA effects (Kaada. 1982; Yao et al., 1982; Cao et al., 1983: Hoffman and Thoren, 1986; Hoffman et al., 1987, 1990a,b; Moriyama, 1987; Reid and Rubin, 1987; Jansen et al., 1989). In conclusion. the present study showed a decrease of the PI in the uterine arteries following EA treatment. Randomized studies on a greater number of patients are needed to verify these results and to exclude non-specific effects. References Andersson, S.A. (1993) The functional background in acupuncture effects. Scand J. Rehab, Med. Suppl., 29. 31-60. Andersson, S.A. and Lundeberg. T. (1995) Acupuncture - from empiricism to science: functional background to acupuncture effects in pain and disease. Med. Hypoth., 45, 271-281. Bonica, J. 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