Objective: to determine the effect of follicular phase acupuncture on anti-mullerian hormone (AMH) levels in PCOS women undergoing IVF/ intracytoplasmic sperm injection (ICSI) and the effect of that on the outcome.
Materials and method: randomized, prospective, controlled clinical study was carried out. A total of 81 PCOS women undergoing IVF/ICSI were invited to participate. The enrolled women underwent follicular phase acupuncture together with medical therapy, the control group was comprised of PCOS women who underwent IVF/ICSI without acupuncture treatment .Follicular fluid and blood samples were collected from both groups at day of oocyte retrieval for AMH measurement using ELISA kit.
Results: A total of 81 PCOS women aged 20-40 years were enrolled in this study. The results of this study show no significant role of acupuncture on serum and follicular fluid AMH levels as the p values 0.451, 0.607 respectively. But it show that acupuncture had a significant effect on number of oocyte retrieved as p 0.040 and had a positive effect on implantation rate (15.14%, 9.37% respectively). Clinical pregnancy rate (30.3%, 18.8% respectively) and ongoing pregnancy rate (30.3%, 16.7% respectively) and reducing the most common complication of PCOS patients underwent ART which is OHSS (36.4%, 47.9% respectively) comparing group exposed to acupuncture and group not exposed to acupuncture (control).
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Conclusions: The result of this study support the significance of follicular phase acupuncture for the outcome of IVF/ICSI for PCOS patients and decrease complications lead to an increase the number of embryo transferred patients. But it had no role on AMH hormone concentrations in the serum and follicular fluid. Further randomized controlled trials are needed to further assess the role of TCM on AMH concentrations as a part of treatment management for increasing the number of healthy pregnancies among PCOS women who undergo IVF/ICSI.
Keywords: PCOS, Acupuncture, AMH, IVF or ICSI, infertility.
Polycystic ovary syndrome (PCOS) is heterogeneous collection of sign and symptoms that gathered together form a spectrum of a disorder with a mild presentation in some, while in others a sever disturbance of reproductive, endocrine and metabolic function . The pathophysiology of PCOS appears to be multifactorial and polygenic. Features include menstrual cycle disturbance, hyperandrogenism and obesity. The morphology of polycystic ovary, has been redefined as an ovary with 12 or more follicles measuring 2-9mm in diameter and increased ovarian volume (>10cm3) (1) on trans vaginal ultrasound. . It has been suggested that the disorder occurs in 4% to 7% of women of reproductive age (2) (3) Family studies have revealed that about 50% of 1st degree relatives have PCOS suggesting a dominant mode of inheritance (4)
Although PCOS patients are typically characterized by producing an increased number of oocytes, they are often of poor quality, leading to lower fertilization, cleavage and implantation rates, and a higher miscarriage rate (5) (6) (7) (8) (9) (10).
Therefore a better understanding of how PCOS is related to abnormalities in extra- and intra-ovarian factor and their impact on granulosa cell (GC) - oocyte interactions.
Anti-mullerian hormone (AMH) also known as mullerian-inhibiting substance (MIS) is a member of transforming growth factor -β (TGF-β) family. In men, AMH is produced by sertoli cells of fetal testis and it causes regression of the mullerian ducts, which is a requirement for normal male reproductive tract development (11).
AMH gene is located on the short arm of chromosome 19 (12). AMH was produced from 36 weeks of gestation in human GCs (13)and was expressed until menopause.
The highest level of AMH expression is seen in the granulosa cells of secondary, pre antral, and small antral follicles no more than 4mm in diameter and disappears as follicles develop to the larger antral stage (4-8mm) in diameter. AMH protein is undetectable in granulosa cells from large antral or pre ovular follicles (14)
AMH Concentration is supra physiological in those with excess small antral follicles, classically in the patient with PCOS (15). The main physiological role of AMH in the ovary seems to be limited to the inhibition of the early stages of follicular development (16) (17).The pathogenesis of PCOS remains largely unknown although resent studies have suggested that AMH may have a role to play in the ovarian follicular status in PCOS (18) (19).Also in an ovulatory women with PCOS, granulosa cell function is abnormal (20) and this abnormality may influence oocyte or embryo quality (20) (21), PCOS women are known to have excessive amount of small antral follicles in the ovaries and at the same time increased serum AMH level (18) (19).
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IVF-embryo transfer (IVF-ET) is an effective treatment for various causes of infertility. In 2000, about 200,000 babies were conceived throughout IVF worldwide (22) (23). Acupuncture is a popular treatment choice for infertility (24) and it has been used during IVF treatment. Acupuncture is an ancient traditional Chinese treatment with an empirical basis, originating 2500 years ago. It is one of the most wide spread form of complementary and alternative medicine (CAM) in the USA and Europe (25) (26). The general theory of acupuncture is based on the premise that disruptions of natural balanced energy flow (Qi) are responsible for disease pathogenesis.
More recent randomized controlled studies evaluated the effect of acupuncture on reproductive out come in patients treated with IVF/intra cytoplasmic sperm injection (ICSI) (27) (28).
Some of the studies show that acupuncture improves clinical pregnancy rates (29) (30) , implantation rate and ongoing pregnancy rate (30).
The aim of this study was to investigate the effect of follicular phase acupuncture on AMH hormone in PCOS women undergoing IVF/ICSI and the effect of that on assisted reproduction outcome.
Materials and methods:
A total of 81 infertile PCOS, 20-40 years of age: were prospectively recruited for this study between March 2011 and January 2012. The diagnosis of PCOS was made when at least two out of three of the following criteria existed, as proposed at the Rotterdam Consensus Meeting 1-oligomenorrhoea or amenorrhoea 2- hyperandrogenism3- Polycystic ovaries (Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group, 2004).
The inclusion criteria included: only PCOS patients with patent at least one fallopian tube, normal uterine cavity evaluated by hysterosalpingography, and partners with normal semen parameters. The semen quality was assessed using the World Health Organization guidelines WHO, 1999 (31).
The exclusion criteria: it is important to exclude other disorders with a similar clinical presentation such as congenital adrenal hyperplasia, Cushing's syndrome and androgen-secreting tumors, thyroid dysfunction and hyperprolactinaemia.
Protocol for ovarian stimulation
All patients were down regulated according to long protocol adopted in infertility department. They received a standard gonadotrophin-releasing hormone (GnRH) agonist regimen on day 21 of the preceding menstrual cycle until the day of HCG injection. Pituitary and ovarian suppression is confirmed by a plasma FSH, LH levels should be less than 5mIU/ml and plasma estradiol level should be less than 50pg/ml and/or an endometrial thickness of less than 5 mm on vaginal ultrasound scan. Trans-vaginal ultrasound was performed on the second day of the cycle to count the number of antral follicles as a base line measurement. Then ovarian stimulation was performed with recombinant FSH (rFSH) Gonalf 75IU(5.5µg) or Serono 75IU(Follitropin alpha recombinant) was initiated on the third day of subsequent withdrawal bleeding and at that time the dose determined according to age. Ovulation was triggered with a single dose of HCG (10,000 IU) intra muscularly when at least three follicles had a diameter of more than or equal to 18mm and the endometrial thickness is more than 8 mm with an adequate serum E2 concentration. Oocyte retrieval was performed 36 h later under trans-vaginal ultrasound guidance and vaginal sedation. Oocyte was examined for pro nuclei (PN) score 16-18 h. Following oocyte retrieval, the oocytes are inseminated, fertilization checked for 24h later and the embryo further cultured for another 24 or 48h. The embryos were transferred on day 3 after oocyte retrieval.
The embryos were judged to be the ´best´ are transferred to the patient´s uterus, using an Edwards Wallace's catheter (depending on previous measurement of uterine cavity by touch technique) otherwise all fresh embryos were cryopreserved if patient had developed symptoms suggestive of OHSS.
All patients received luteal-phase support progesterone (60 mg) orally daily starting from the day of oocyte retrieval.
Biochemical pregnancy was established when serum β-HCG concentration was found >2mIU/ml on day 14 after ET. Clinical pregnancy was defined as the presence of at least one gestational sac with fetal heart beat on vaginal ultra sound performed at 4-6 weeks after ET. The implantation rate was calculated as the number of gestational sacs/number of embryos transferred -100.
All patients should inform about sites of acupuncture and time of putting needles. Acupuncture was performed in the follicular phase of the cycle, starting from day three of the cycle, together with IVF/ICSI protocol, and continues every day till day of giving HCG which is the last day of doing acupuncture.
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Acupuncture was performed after iodine wipe of the skin at the specific points. It applied for 30-40 min by using 4cm long disposable stainless steel hair-thin needles manufactured by Suzhou medical appliance factory, China. The needles were inserted into the skin to a depth of 15-30mm, depending on the region of the body. Needle reaction (soreness, numbness, feeling of heaviness, distention around the puncture site or some time propagate along the corresponding meridians which termed the DiQi sensation) was elicited during initial insertion. Some patients feel minimal pain as the needle is inserted. Every 10 min, the needles were stimulated manually by rotating, lifting and thrusting the handle of the needle in order to maintain DeQi sensation. The needle were retained in position for 30-40 min and then removed. The following acupuncture points used in this study:Tai chongå¤ªè¡ (å¤ªå†²)(LIV 3), san yin jiao (ä¸‰°äº¤) (SP 6), diji (°æœº) (SP 8), zu san li (è¶³ä¸‰é‡Œ) (ST 36), xue hai (è¡€æµ·) (SP 10), gui lai æ¸ä¾† (å½’æ¥) (ST 29), hegu (åˆ°) (LI 4), guan yuan (-å…ƒ) (Ren 04).
Follicular fluid and serum collection
To obtain the exact follicular-fluid AMH concentration, only the follicular fluid from the first retrieved follicle of bilateral ovaries was collected. It is important to avoid contamination of follicular fluid with blood or culture medium or mixed follicular fluid during oocyte retrieval. Any sample contaminated with blood or with culture medium should be discarded.
The average AMH concentration of two samples (one from right ovary and one from left ovary) were used from each patient. A total of 162 follicular fluid samples (one from each ovary) that collected from 81 patients were analyzed and the average value of AMH concentration was used. The follicular fluid was collected from follicle >18 mm, clear follicular fluid, not contaminated with blood or culture medium, then all samples processed by centrifugation at 1000g for 20 min at 4C(according to instruction notice of ELISA kit company) and immediately frozen at -80C for biochemical and hormonal analysis. Time elapsed between follicular fluid collection and cryopreservation is between 30 min-1h.
On the same day of oocyte retrieval, blood samples were obtained by venipuncture from all patients in the morning prior to oocyte retrieval and the collection time between 9:00am-11:00am then the samples processed within ½h-1h after withdrawal (centrifugation at 1000g for 20 min. at 4C then serum should be stored immediately at -80C according to instruction notice of ELISA Kit Company).Analysis of both follicular and serum AMH concentration should be within a period of less than two months, otherwise loss of samples bioavailability and give false results.
Serum and follicular AMH concentration were determined using AMH ELISA kit (Uscn Company, Wuhan /China). The minimum detectable dose of human AMH is typically less than 0.058ng/ml with intra-assay: CV<10% and inter-assay: CV<12% .the results were expressed as ng/ml.
Data analysis was performed by using Statistical Package for Social Sciences (SPSS, version 19).We used the student t-test and Chi-square test to assess for differences in independent variables at baseline between intervention and control groups. The data are shown as mean and standard deviation. A P-value less than 0.05 were considered statistically significant.
A total of 81 PCOS eligible patients were entered into the study among all infertility patients. All patients were randomized: 33 patients received acupuncture according to the principle of traditional Chinese medicine (group 1), and 48 patients with no acupuncture (group 2). No statistical significant regarding age, BMI, duration of infertility, GnRH ampules, FSH ampules and ovarian stimulation duration (ds). (Data not mentioned).
Table one shows statistical difference among the number of oocyte retrieved, MII oocyte, II PN as p values (0.040, 0.004, 0.009 respectively). Also show statistical difference among serum E2 (pg/ml), serum P (ng/ml) on dOPU as p values (0.01, 0.02 respectively).and also serum P (ng/ml) on HCG day.
Table 1: Characteristics of ovarian response in women with PCOS (interventional and non-interventional groups)
2-tailed P- value
15.33 ± 7.84
16.89 ± 6.14
23.72 ± 12.34
29.52 ± 12.23
M II oocyte
18.39 ± 9.65
25.18 ± 10.50
12.72 ± 6.84
17.20 ± 7.74
Serum Estradiol (pg/ml) on HCG day
9990.46 ± 2943
Serum Estradiol on dOPU.
7352.33 ± 3681
Serum Progesterone (ng/ml) on HCG day
1.15 ± 0.33
1.51 ± 0.66
Serum Progesterone on dOPU
12.02 ± 8.39
16.89 ± 10.49
Serum AMH (ng/ml)
Follicular fluid AMH (ng/ml)
7.54 ± 2.40
7.82 ± 2.32
Endometrial thickness (mm)
10.48 ± 1.98
10.10 ± 2.29
There is no statistical significance regarding serum and follicular fluid AMH (ng/ml) between two groups as the p values (0.451, 0.607 respectively).
Table two shows the outcome of IVF/ICSI in both groups. The number of ET, implantation rate, clinical pregnancy rate and ongoing pregnancy rate which were higher in interventional group than non-interventional although, it is statistically not significant due to small sample size.
Table2: Outcome of Assisted Reproductive Technique
2-tailed P- value
Biochemical pregnancy rate
Clinical pregnancy rate
Ongoing pregnancy rate
Table three shows the complications of ART, the most common one occurs in PCOS patients underwent ovarian stimulation was OHSS which is lower in interventional than non-interventional group (36.4%, 47.9% respectively).
Table3: Complications of Assisted Reproductive Technique (ART)
2-tailed P- value
High progesterone level
Ectopic pregnancy and abortion
Many women with PCOS require prolonged treatment. Treatment strategies such as acupuncture need to be evaluated in PCOS. Acupuncture, a treatment that dates back 2500 years, is an integral part of traditional Chinese medicine. The physiological mechanism and clinical significance of acupuncture have not been completely revealed and has been the subject of controversy. Recent study shows that intramuscular needle insertion causes a particular pattern of afferent activity in peripheral nerve.Acupuncture affect the hypothalamic-pituitary-adrenal (HPA) axis by decreasing cortisol concentrations (32) and the hypothalamic-pituitary-gonadal (HPG) axis by modulating central and peripheral B-endorphin production and secretion ,thereby influencing the release of hypothalamic GnRH and pituitary secretion of gonado-trophin (33) (34) (35).On the other hand, the positive effect of acupuncture during IVF treatment may be related to the changes in uterine blood flow and uterine contractility and relaxation of stress (36)acupuncture affects PCOS symptoms via modulation of endogenous regulatory systems, including the sympathetic nervous system, the endocrine system and the neuroendocrine system. The changes are most likely mediated via the endogenous opioid system (37). The result of this comparative study demonstrated that traditional Chinese medicine may be effective in enhancing fertility of PCOS women undergoing ART and decrease complication.
The BMI for studying and control group 23.06±SD, 22.49±SD respectively (data not mentioned) i.e. this values were low for PCOS patients as it should be more than 25 kg/m2, this belong to racial group (Asian population) as the study was done in China. Also some cases involved in this study decided to do IVF but after oocyte retrieval it changed to ICSI because of poor fertilization in spite of normal semen count.
In interventional group, values of serum AMH hormone concentration taken as mean± SD was 3.50±1.47 and for non-interventional group was 3.77±1.58 with p (0.451) .And for follicular AMH hormone concentrations were 7.54±2.40 and 7.82±2.32 respectively with p (0.607).
This study shows that there is no effect of acupuncture on AMH hormone levels both in serum and follicular fluid as there is no significance difference in AMH concentration between studying and control group.
The granulosa cells of small antral follicles secrete AMH into both follicular fluid and the circulation. Regarding serum AMH levels, it's shown than the AMH concentrations lower than that for follicular fluid, This may be due to that serum AMH concentration declined progressively during ovarian stimulation when using GnRH agonist protocol as same as some studies published before ( La Marca et al., 2004a, Fanchin R et al., 2003a ) this reduction of AMH levels could be due to a negative direct or indirect effect of FSH on ovarian AMH secretion or may be due to that the circulating AMH concentrations reflect the growing follicular pool on day of oocyte retrieval and are less effective to discriminate per-follicle AMH production mainly if blood samples collected during follicular phase or may be due to that AMH has a paracrine effect on the process of oocyte development in women with PCOS undergoing IVF or ICSI this may explain why follicular fluid AMH concentrations were higher than serum concentrations . As in this study there is no significant difference in AMH concentrations between interventional and non-interventional group but it's shown that the group exposed to acupuncture has high Implantation rate and clinical pregnancy rate than patients not exposed to acupuncture as in one of the studies published before (Stefan Dieterle, et al., 2006) although its statistically not significant, may be due to small sample size.
The number of oocytes retrieved in interventional group is 23.72±12.34 while in control group 29.52±12.23 with p value 0.04. That means that there is statistical significance as the p value <0.05 .Also regarding MII oocyte and IIPN, the values are statistically lower in experimental group than control group with p values 0.004, 0.009 respectively. This may explain the positive effect of acupuncture for PCOS women undergoing IVF as the number of oocytes retrieved was less in experimental group than in control group therefore, decrease complication of ovarian hyper stimulation syndrome. The effect of acupuncture in reducing the number of oocytes retrieved unknown but may be due to that acupuncture has an effect on ovaries by modifying the PCOS pathogenesis or modulate the hormonal effect on PCOS ovaries, such result need further study. Acupuncture has been shown to affect hormone levels as same as one study ( Stener-Victorin E et al., 2010 ) by promoting the release of B-endorphin in the brain, which affect the release of gonadotrophin releasing hormone by the hypothalamus, follicle stimulating hormone from the pituitary gland and estrogen and progesterone levels from the ovary (38).
Embryo transfer in group exposed to acupuncture 57.6% higher than that of control group 45.8%, although it's statistically not significant because of small sample size. Implantation rate and clinical pregnancy rate both were affected by acupuncture; Implantation rate in study group is higher than control group as it was 15.14%, 9.37% respectively also it´s statistically not significant (p 0.283).Clinical pregnancy rate in study group was also higher about 30.3% comparing with 18.8% in control group; as in (Zheng CH. et al., 2012).
Also ongoing pregnancy rate is higher in group exposed to acupuncture 30.3%, 16.7%respectively in group 1 and 2. This can be explained by the effect of acupuncture in increasing blood flow to the reproductive organs, increase blood flow to the uterus (39), improving the thickness of endometrial lining and increasing the chance of embryo implantation. OHSS refers to an exaggerated ovarian response to gonadotrophin treatment. The specific risk factors include: young age, low BMI, signs of PCOS and high estradiol on the day of HCG (40).percentage of OHSS is higher in control group as its 47.9% compared with 36.4%in studying group. This may explain the role of acupuncture in reducing the complication of hyper stimulation protocol that delay implantation of embryo mainly OHSS. Relating to AMH hormone concentration which was higher in both groups, it seems that hyper response and OHSS may be associated with significantly higher mean basal AMH levels (41) (42) (43).
The result of this study support the significance of follicular phase acupuncture for the outcome of IVF/ICSI for PCOS patients by increase implantation rate, clinical pregnancy rate and ongoing pregnancy rate, at the same time, it decrease the number of oocyte retrieved leading to low percentage of ovarian hyper response and OHSS lead to an increase the number of embryo transferred patients but it shows no role on AMH hormone concentrations in the serum and follicular fluid. Further randomized controlled trials are needed to further assess the role of TCM on AMH concentrations as a part of treatment management for increasing the number of healthy pregnancies among PCOS women who undergo IVF/ICSI.
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