You are on page 1of 10

ORIGINAL ARTICLE: REPRODUCTIVE ENDOCRINOLOGY

1 60
2
3
Randomized controlled trial of 61
62
4
5
6
letrozole, berberine, or a 63
64
65
7
8
9
combination for infertility in the 66
67
68
10
11
12
polycystic ovary syndrome 69
70
71
13 a,b
Yong-Yan Wang, M.D.,b Jian-Ping Liu, M.D., Ph.D.,c Rui-Ning Liang, M.D.,d 72
Q4 Xiao-Ke Wu, M.D., Ph.D.,
14 Hui-Ying Xue, M.D.,e Hong-Xia Ma, M.D.,f Xiao-Guang Shao, M.D.,g and Ernest H. Y. Ng, M.D.,h for the 73
15 Reproductive and Developmental Network in Chinese Medicine 74
16 a 75
Department of Obstetrics and Gynecology, First Afliated Hospital, Heilongjiang University of Chinese Medicine, Harbin;
17 b
Institute of Basic Clinical Medicine, China Academy of Chinese Medical Science, Beijing; c Center for Evidence-Based 76
18 Chinese Medicine, Beijing University of Chinese Medicine, Beijing; d Department of Gynecology, First Hospital, Jiangxi 77
19 University of Chinese Medicine, Nanchang; e Center for Reproductive Medicine, Huaian Maternal and Child Health 78
Hospital, Huaian; f Department of Chinese Medicine, First Afliated Hospital, Guangzhou Medical University,
20 Guangzhou; g Center for Reproductive Medicine, Dalian Maternal and Children's Health Center, Dalian; and 79
21 h
Department of Obstetrics and Gynecology, University of Hong Kong, Hong Kong, People's Republic of China 80
22 81
23 82
24 83
25 84
Objective: To study whether a combination of berberine and letrozole results in higher live births than letrozole alone in infertile
26 women with polycystic ovary syndrome (PCOS). 85
27 Design: Not applicable. 86
28 Setting: Nineteen sites in Mainland China within national reproductive and developmental network in Chinese medicine. 87
29 Patient(s): Eligible women had PCOS as dened by the Rotterdam criteria. We enrolled 644 participants randomized 1:1:1 among le- 88
30 trozole, berberine, and combination groups. 89
Interventions(s): Berberine or berberine placebo were administrated orally at a daily dose of 1.5 g for up to 6 months. Patients received
31 90
an initial dose of 2.5 mg letrozole or placebo on days 37 of the rst three treatment cycles. This dose was increased to 5 mg on the last
32 three cycles if not pregnant. 91
33 Main Outcomes Measure(s): Cumulative live births. 92
34 Results: The cumulative live births were similar between the letrozole and combination groups after treatment (36% and 34%), and 93
35 were superior to those in the berberine group (22%). Likely, conception, pregnancy, and ovulation rates were similar between the 94
36 letrozole and combination groups, and these were signicantly higher than in the berberine group. There was one twin birth in the 95
37 letrozole group, three twin births in the combination group, and none in the berberine group. 96
Conclusion(s): Berberine did not add fecundity in PCOS when used in combination with the new ovulation agent letrozole.
38 97
Clinical Trial Registration Number: ChiCTR-TRC-09000376 (http://apps.who.int/trialsearch/). (Fertil Steril 2016;-:--. 2016
39 by American Society for Reproductive Medicine.) 98
40 Key Words: PCOS, infertility, letrozole, berberine, live birth, RCT 99
41 100
42 Discuss: You can discuss this article with its authors and with other ASRM members at http://fertstertforum.com/wux-letrozole- 101
berberine-pcos/
43 102
44 103
45 104
46 105
47 Received November 1, 2015; revised May 21, 2016; accepted May 23, 2016. 106
48 X.-K.W. has nothing to disclose. Y.-Y.W. has nothing to disclose. J.-P.L. has nothing to disclose. R.-N.L. has nothing to disclose. H.-Y.X. has nothing to disclose. 107
H.-X.M. has nothing to disclose. X.-G.S. has nothing to disclose. E.H.Y.N. has nothing to disclose.
49 Supported by National Public Welfare Projects for Chinese Medicine (200807021) of China, National Key Discipline of Chinese Medicine in Gynecolog, 2009 108
50 14, Heilongjiang Province Foundation for Outstanding Youths (JC200804), Intervention for Polycystic Ovary Syndrome Based on Traditional Chinese 109
Medicine TheoryTianGui Shi Xu (2011TD006), and National Clinical Research Base in Chinese Medicine, 200914, at First Afliated Hospital, Heilong-
51 jiang University of Chinese Medicine. The funding sources had no involvement in the study design, the collection, analysis, and interpretation of data, 110
52 the writing of the report, or in the decision to submit the article for publication. 111
This work is the postdoctoral dissertation of Xiao-Ke Wu, M.D., Ph.D., in the specialty of methodology in traditional Chinese medicine, supervised by Acade-
53 mician Professor Yong-Yang Wang, M.D., at the Institute of Basic Clinical Medicine, China Academy of Chinese Medical Science, Beijing, China. 112
54 This work was published as an abstract in The LancetChinese Academy of Medical Sciences Health Summit in The Lancet (2015; 386:S70, http://dx.doi.org/ Q3 113
10.1016/S0140-6736(15)00651-0).
55 114
Reprint requests: Professor Xiao-Ke Wu, M.D., Ph.D., Department of Obstetrics and Gynecology, First Afliated Hospital, Heilongjiang University of Chinese
56 Medicine, Heping Road 26, Dongli District, Harbin 150040, People's Republic of China (E-mail: xiaokewu2002@vip.sina.com). 115
57 116
Fertility and Sterility Vol. -, No. -, - 2016 0015-0282/$36.00
58 Copyright 2016 American Society for Reproductive Medicine, Published by Elsevier Inc. 117
59 http://dx.doi.org/10.1016/j.fertnstert.2016.05.022 118

VOL. - NO. - / - 2016 1


FLA 5.4.0 DTD  FNS30250_proof  18 June 2016  9:00 pm  ce E
ORIGINAL ARTICLE: REPRODUCTIVE ENDOCRINOLOGY

P
119 olycystic ovary syndrome (PCOS) affects 5.6% of were allocated randomly into one of the three groups in a ratio 178
120 Chinese women 1945 years of age, and the main of 1:1:1. We previously reported details of the trial protocol 179
121 clinical manifestations are oligo-anovulation and poly- (13), which was designed by the steering committee of the Na- 180
122 cystic ovaries (1). It is the most common cause of anovulatory tional Clinical Research Base and approved before initiation 181
123 infertility. Although to a less pronounced degree than in Cau- by the State Administration of Traditional Chinese Medicine 182
124 casians, Chinese women with PCOS also suffer from hyperan- of the People's Republic of China, the appointed scientic 183
125 drogenism and hyperinsulinemia together with insulin advisory board. The Institutional Review Boards at partici- 184
126 resistance (2, 3). Given the huge population and a lack of pating hospitals approved the protocol, and every participant 185
127 coverage for infertility treatment by the public health care gave written informed consent. 186
128 system and insurance companies in mainland China, PCOS 187
129 constitutes a considerable economic burden and source of 188
Participants
130 emotional distress (4). 189
131 The rst-line medical treatment for anovulatory infertility A total of 644 women were enrolled in 19 participating sites. 190
132 in women with PCOS is ovulation induction by clomiphene, Chinese women with PCOS attempting to get pregnant were 191
133 an antiestrogen. The drawbacks for clomiphene include a eligible if they fullled the following criteria: 1) age 20 192
134 relatively low cumulative live birth rate together with higher 40 years; 2) diagnosis of PCOS according to two of the three 193
135 multiple pregnancy rates than unassisted conception (5). Rotterdam 2003 criteria (14), including oligo-ovulation or an- 194
136 Insulin-sensitizing agents such as metformin are commonly ovulation, clinical and/or biochemical signs of hyperandro- 195
137 used as adjunct medication for women with PCOS. However, genism, and/or polycystic ovaries; 3) at least one open 196
138 metformin alone is not superior to clomiphene or a combination fallopian tube and normal uterine cavity documented by hys- 197
139 of metformin and clomiphene (5). Recently letrozole, an aroma- terosalpingography, sonohysterography, or diagnostic lapa- 198
140 tase inhibitor, has been shown to be superior to clomiphene for roscopy within the past 3 years; 4) a male partner with 199
141 ovulation and live birth rates in infertile women with PCOS (6). sperm concentration of 15 million/mL and motility of 200
142 Berberine, a major active component of the Chinese herbal R40% in at least one ejaculate; and 5) at least 1 year of infer- 201
143 medicines Rhizoma Coptidis, Cortex Phellodendri, and Cortex tility. Subjects were excluded if they used hormonal drugs or 202
144 Berberidis, has been prescribed empirically for the treatment other medications, including Chinese herbal prescriptions, in 203
145 of diarrhea, metabolic disorders, and infertility (4, 7, 8). the past 3 months; had known severe organ dysfunction or 204
146 Berberine is commonly used in China. A total of 0.8 billion mental illness; were pregnant, post-miscarriage, postpartum, 205
147 0.1 mg tablets were consumed at the end of 2000 and 5.9 or breastfeeding within the past 6 weeks; or had congenital 206
148 billion n 2013, and this is projected to reach 12 billion in adrenal hyperplasia, clinically suspected Cushing syndrome, 207
149 2015 (9). It has been used for thousands of years in its herbal or an androgen-secreting neoplasm. 208
150 form to enhance fertility and recently as an extract combined 209
151 with ovulation induction agents, such as clomiphene or letro- Randomization and Blinding 210
152 zole, to enhance their effectiveness (4). 211
The randomization was performed through a web-based com-
153 We previously demonstrated that berberine could directly 212
puter program (http://210.76.97.192: 8080/cjbyj) operated by
154 alleviate the insulin resistance and androgen synthesis within 213
an independent data coordinating center, the Institute of
155 insulin-resistant ovaries cultured in vitro and when we assessed 214
Basic Clinical Medicine of the China Academy of Chinese
156 its function in vivo (10, 11). Compared with metformin, 215
Medical Sciences. The randomization was stratied by the
157 berberine showed similar metabolic effects on improving 216
participating sites. Participants, investigators, physicians tak-
158 insulin sensitivity and reducing hyperandrogenemia, and 217
ing care of the participants, laboratory technicians, and data
159 berberine had additional effects on body composition and 218
analyzers were blinded to the assignments.
160 dyslipidemia in women with PCOS (12). Because of its favorable 219
161 effects on these metabolic factors, berberine has the potential to 220
162 complement letrozole in improving live birth rates among infer- Study Intervention 221
163 tile women with PCOS. To our knowledge, this is the rst study to After spontaneous menses or withdrawal bleeding induced by 222
164 investigate ovulation and live birth rates following the use of progestin administration (medroxyprogesterone acetate 223
165 berberine in women with PCOS. [Provera; Pzer Italia], 5 mg/d for 7 days), eligible patients 224
166 We sought to determine the effectiveness of letrozole were randomized into one of three interventions: 1) letrozole 225
167 alone, berberine alone, and the combination of the two in and berberine placebo (letrozole group); 2) berberine and le- 226
168 achieving live births among infertile women with PCOS. Our trozole placebo (berberine group); and 3) letrozole and 227
169 primary hypothesis was that the combination of berberine berberine (combination group). Each participant received a 228
170 and letrozole would result in signicantly higher live birth medication package on a monthly visit basis that consisted 229
171 rates than treatment with letrozole or berberine alone (13). of a monthly supply of berberine capsules or placebo capsules 230
172 and one or two packages of pills (letrozole or letrozole pla- 231
173 cebo, one package per month for the rst 3 months, and 232
174
MATERIALS AND METHODS two packages per month for the next 3 months). Berberine 233
175 Study Oversight or berberine placebo was administrated orally at a daily 234
176 This was a multicenter randomized double-blind placebo- dose of 1.5 g for 6 months. Patients received an initial dose 235
177 controlled trial in mainland China. Recruited participants of 2.5 mg (one tablet) of letrozole or one tablet of letrozole 236

2 VOL. - NO. - / - 2016


FLA 5.4.0 DTD  FNS30250_proof  18 June 2016  9:00 pm  ce E
Fertility and Sterility

237 placebo on days 37 of the rst three treatment cycles. This prolonged inpatient hospitalization; overdoses (intentional 296
238 dose was increased to 5 mg letrozole (two tablets) or two tab- or accidental); congenital anomalies; or any event deemed 297
239 lets of letrozole placebo on days 37 of the last three treat- to be serious by the principal investigator at the study site. 298
240 ment cycles if not pregnant. Induction of withdrawal 299
241 bleeding with progestin was scheduled at the discretion of 300
242 the principal investigator at each site. All subjects returned Statistical Analysis 301
243 monthly on day 22 of induced or spontaneous menstrual cy- The sample size calculation was based on anticipated live 302
244 cles for measurement of serum progesterone levels and urine birth rate. A previous meta-analysis suggested that the live 303
245 hCG test in local laboratories to document ovulation and birth rate with the use of letrozole in women with PCOS 304
246 pregnancy, received medication packages, and reported was 22% during a 6-month intervention (13), and our study 305
247 concomitant medication and adverse events. Couples were in- was designed and completed before a more recently published 306
248 structed to have regular intercourse two to three times a week large randomized trial (6). We hypothesized that a combina- 307
249 until achieving pregnancy. Once participants conceived, they tion of letrozole and berberine would increase the live birth 308
250 were followed until a viable intrauterine pregnancy sac was rate from 22% to 30%. Accordingly, we estimated that a sam- 309
251 observed (fetal heart motion and gestational sac visualized ple size of 220 participants per group was required consid- 310
252 on ultrasonography). They were then referred for antenatal ering a 20% dropout, 90% power, and an alpha error of 311
253 care. Outcomes were tracked through regular interviews 0.05. On the basis of these assumptions, we needed to enroll 312
254 with midwives and abstractions of obstetrical records. Ovula- 660 subjects for the study. 313
255 tion detection kits and intrauterine insemination were not Either chi-square test or Fisher exact test was used at a 314
256 used. Berberine and berberine placebo were produced by Re- two-sided signicance level of 0.05 for testing differences 315
257 nhetang Pharmaceutical Co. Letrozole and letrozole placebo among the three study groups for categoric variables. The 316
258 were produced by Jiangsu Hengrui Medicine Co. Neither Kruskal-Wallis was used to test differences among the three 317
259 manufacturer had a nancial role in the study. groups for continuous variables. If signicant, a Mann- 318
260 Fasting blood samples for assessment of metabolic and Whitney U test was used to test differences between the 319
261 hormonal proles were drawn at the baseline visit and at groups. Kaplan-Meier analysis was used to compare time to 320
262 the end of the treatment visit at menstrual cycle days 37 live birth according to treatment groups (5, 6) body mass 321
263 and analyzed at the core laboratory in Harbin. The intra- index (BMI), hirsutism scores, menstrual patterns, age, and 322
264 assay and interassay coefcients of variation of each assay previous infertility duration. Adverse events were 323
265 were <10%. All baseline measures, including assessment of categorized, and the percentage of patients experiencing 324
266 liver and renal function, were repeated at the end of the visits. adverse events and serious adverse events in each treatment 325
267 Normal values for the steroids and proteins were 0.6 arm were compared with the use of chi-square tests. All ana- 326
268 4.7 nmol/L for P, 0.291.67 nmol/L for total T, 46.0607 lyses were performed with the use of SAS software, version 327
269 pmol/L for E2, 510 for free androgen index, 2.412.6 mIU/ 9.2 (SAS Institute). Data were analyzed according to the 328
270 mL for LH, 3.512.5 mIU/mL for FSH, 0.354.94 mIU/L for intention-to-treat principle. 329
271 TSH, 102496 mIU/L for PRL, and 18114 nmol/L for SHBG 330
272 at early follicular stage; and 2.724.9 IU/L for fasting insulin 331
273 and 0.371.47 nmol/L for C-peptide. We did not measure
RESULTS 332
274 serum glucose levels because of inaccuracy with long storage Study Oversight 333
275 of samples in the core laboratory, resulting in missing It was approved by the Ethics Committee on April 10, 2009, 334
276 homeostasis-model assessment index data. and registered in China with identier ChiCTR-TRC- 335
277 09000376 on October 8, 2009 (http://apps.who.int/trial- 336
278 search/). The trial was started on October 2009. Owing to 337
279 Outcome Measures the expiration of the study drug (berberine and matching pla- 338
280 The primary outcome was cumulative live births during inter- cebo), the data safety and monitoring board decided to stop 339
281 vention period. Secondary outcomes included: 1) ovulation: enrollment in November 2013, after 644 patients were 340
282 serum P level >5 ng/mL on day 22 of each treatment cycle enrolled. The rst enrollment was on November 11, 2009, 341
283 in local sites; 2) conception: a positive serum or urinary test and the last enrollment was February 28, 2013. The rst birth 342
284 of hCG; 3) pregnancy: an intrauterine pregnancy sac with occurred on October 5, 2010, in a patient who became preg- 343
285 fetal heart motion as determined by means of ultrasonogra- nant after she was enrolled on November 25, 2009. The last 344
286 phy at 810 weeks of gestation; 4) multiple pregnancy; 5) live birth occurred on November 12, 2013, in a woman 345
287 pregnancy loss: loss before 20 completed weeks of intrauter- enrolled on December 25th, 2012. 346
288 ine gestation; 6) other pregnancy complications, such as still- The owchart of the study is shown in Figure 1. The num- 347
289 birth, gestational diabetes mellitus, pregnancy-induced ber of subjects who withdrew from the study was 16 out of 348
290 hypertension, and small-for-gestational-age fetus; and 7) 215 (7.4%) in the letrozole group, 25 out of 214 (11.7%) in 349
291 adverse events from the study medications. Patients were the berberine group, and 15 out of 215 (7.0%) in the combina- 350
292 asked to record adverse events and to report them to the coor- tion group (P .16; Fig. 1). Reasons for withdrawal were 351
293 dinator at each visit. Serious adverse events were dened as similar among the three groups (P .16 for the three groups; 352
294 events that were fatal or immediately life threatening, that P .19 for lost to follow-up; P .88 for drop-out; P1.0 for 353
295 were severely or permanently disabling, or that required protocol violations; and P .33 for adverse events). 354

VOL. - NO. - / - 2016 3


FLA 5.4.0 DTD  FNS30250_proof  18 June 2016  9:00 pm  ce E
ORIGINAL ARTICLE: REPRODUCTIVE ENDOCRINOLOGY

355 414
356
FIGURE 1 415
357 416
358 417
359 418
360 419
361 420
362 421
363 422
364 423
365 424
366 425
367 426
368 427
369 428
370 429
371 430
372 431
373 432
374 433
375 434
376 435
377 436
378 437
379 Enrollment owchart and reproductive outcomes. 438
380 Wu. Berberine, letrozole, or both for PCOS. Fertil Steril 2016. 439
381 440
382 441
383 442
There were no signicant differences in the ages of the signicantly higher in those groups than in the berberine
384 443
women, duration of infertility, BMI, waist/hip circumference, group. Regarding fecundity among subjects who ovulated,
385 444
presence of hirsutism, menstrual pattern, ultrasound features the letrozole and combination groups were superior to the
386 445
of polycystic ovaries, or baseline hormonal prole among the berberine group in terms of conception rates and pregnancy
387 three groups (Table 1). rates. However, there were no differences among the three 446
388 groups in rates of conception, pregnancies, or live births 447
389 among ovulatory cycles during treatment. The three groups 448
390 Primary Outcomes 449
had similar rates of pregnancy loss after conception. The
391 A total of 199 live births occurred, including 195 singletons within-group changes in BMI and waist circumference be- 450
392 and four sets of twins. The rate of cumulative live births tween last visit and baseline were signicant or marginal in 451
393 was similar between the letrozole and combination groups the berberine group (0.31  4.34 kg/m2 [P .0017]; 0.45 452
394 453
(36.3% vs. 34.4%; odds ratio [OR] 0.95, 95% condence inter-  4.31 cm [P .0553]) and not signicant in the letrozole
395 val [CI] 0.731.23; P .687), and signicantly higher in those 454
group (0.65  10.73 kg/m2 [P .0942]; 0.82  13.13 cm
396 455
groups together than in the berberine group (22%; OR 1.68, [P .0715]) or the combination group (0.08  2.23 kg/m2
397 456
95% CI 1.232.28 [P .001]; OR 1.57, 95% CI 1.152.14 [P .241]; 0.08  4.97 cm [P .2703]), although the
398 [P .004]; respectively; Table 2; Supplemental Fig. 1A [avail- between-group comparisons were not signicant among 457
399 able online at www.fertstert.org). Birth weight among live three groups (P .45). 458
400 births was similar among the three groups. There were three 459
401 twin live births in the combination group, one in the letrozole 460
402 Adverse Events and Pregnancy Complication 461
group, and none in the berberine group.
403 Independently from treatment, subjects with age <33 years No serious adverse events occurred during the intervention 462
404 had signicantly higher rates of live births than did women period in any of the three groups. Berberine was associated 463
405 whose age was >33 years (P .018). There were no signicant with a signicantly higher incidence of constipation and 464
406 differences in live birth rate stratied by BMI (P .782), men- nausea, and letrozole was associated with a signicantly 465
407 strual cycle pattern (P .689), hirsutism ,or duration of infer- higher incidence of fatigue and hot ashes. There were no sig- 466
408 tility (data not shown; Supplemental Fig. 1B1D). nicant differences among the three groups regarding rates of 467
409 total cases of adverse events (37.0%, 48.0%, and 45.5% for the 468
410 letrozole, berberine, and combination groups, respectively) or 469
411 Secondary Outcomes cases of serious adverse events, including ectopic pregnan- 470
412 The rates of ovulation, conception, and pregnancy also were cies, pregnancy loss during the second trimester, or preterm 471
413 similar between the combination and letrozole groups, and labor (21.4%, 20.8%, and 23.5%, respectively; Table 3). 472

4 VOL. - NO. - / - 2016


FLA 5.4.0 DTD  FNS30250_proof  18 June 2016  9:00 pm  ce E
Fertility and Sterility

473 532
474
TABLE 1 533
475 534
Baseline characteristics.
476 535
Letrozole group Berberine group Combination group
477 536
Biometric features (n [ 215) (n [ 214) (n [ 215)
478 537
479 Age of women (y) 27.8  3.6 27.8  3.7 27.8  3.6 538
Body mass index (kg/m2) 24.8  4.5 24.5  4.1 25.1  5.0
480 Waist circumference (cm) 83.5  10.9 82.7  11.8 83.1  11.8 539
481 Hip circumference (cm)a 98.1  11.4 97.7  11.8 97.9  10.9 540
482 WHR 0.85  0.08 0.85  0.08 0.85  0.07 541
Hirsutism (Ferriman-Gallwey R5) 78/202 (38.6) 76/201 (37.8) 60/209 (28.7)
483 542
Menstrual patterna
484 Oligomenorrhea 126/201 (62.7) 116/193 (60.1) 121/198 (61.1) 543
485 Regular menses 75/201 (37.3) 77/193 (39.9) 77/198 (38.9) 544
486 Duration of infertility (mo) 32.7  24.0 28.5  21.6 29.8  21.3 545
Previous infertility therapy 178 (82.8) 180 (84.1) 185 (86.0)
487 Traditional Chinese medicine 107/178 (60.1) 112/180 (62.2) 110/185 (59.5) 546
488 Ovulation drugs 121/178 (68.0) 109/180 (60.6) 107/185 (57.8) 547
489 Assisted reproductive technology 9/178 (5.1) 11/180 (6.1) 12/185 (6.5) 548
Other therapies 8/178 (4.5) 8/180 (4.4) 6/185 (3.2)
490 b 549
Previous pregnancy
491 Conception 67 (31.2) 62 (29.0) 80 (37.2) 550
492 Live birth 9 (4.2) 7 (3.3) 7 (3.3) 551
493 Miscarriage 13 (6.1) 16 (7.5) 26 (12.1) 552
Termination of pregnancy 48 (22.3) 43 (20.1) 64 (29.8)
494 Ultrasonographic ndings 553
495 Polycystic ovary morphology 104/151 (68.9) 113/153 (73.9) 98/155 (63.2) 554
496 Ovarian volume (cm3) 555
497 Left ovary 10.3  6.8 9.5  7.9 11.0  6.8 556
Right ovary 11.0  6.3 10.2  6.4 11.6  7.4
498 Fasting serum levels 557
499 TSH (mIU/L) 2.6  1.9 2.6  1.5 2.7  1.8 558
500 PRL (mIU/L) 318.0  160.9 311.3  168.4 296.7  155.8 559
LH (mIU/L) 10.9  6.7 10.0  6.5 10.4  6.1
501 560
FSH (mIU/L) 5.7  2.1 5.3  1.8 5.5  1.9
502 LH/FSH 2.0  1.1 1.9  1.2 1.9  1.1 561
503 Q2 P 6.2  13.1 5.7  14.9 6.6  15.6 562
504 E2 (pmol/L) 213.9  206.3 244.0  252.4 228.8  233.0 563
T (nmol/L) 1.6  0.8 1.4  0.7 1.4  0.7
505 SHBG (nmol/L) 48.1  31.1 47.6  32.9 42.6  28.3 564
506 Free androgen index b
5.0  4.8 5.3  6.3 5.1  5.8 565
507 Insulin (mIU/L) 11.9  7.7 11.1  8.3 12.3  8.5 566
Peptide C (nmol/L) 0.62  0.45 0.55  0.44 0.59  0.35
508 567
Note: Data are presents as mean  SD or n (%).
509 a
Missing information in some subjects. 568
510
b
The free androgen index was calculated according to the following formula: (total T [nmol/L]/SHBG [nmol/L])  100. 569
511 Wu. Berberine, letrozole, or both for PCOS. Fertil Steril 2016. 570
512 571
513 572
514 During pregnancy, the most common complication was rates of ovulation, conception, pregnancy, and live birth 573
515 pregnancy-induced hypertension, followed by gestational were similar between the letrozole and combination groups. 574
516 diabetes, threatened abortion, and premature rupture of mem- These end points were signicantly higher in both groups 575
517 branes. There were no signicant differences among the three with letrozole than in the berberine alone group. We found 576
518 treatment groups for these events. Two major congenital that the higher ovulation rate per cycle accounted for the su- 577
519 anomalies were reported. One fetal abnormality in the letro- periority of this cumulative live birth rate for letrozole or 578
520 zole group was detected as hydrocephalus by ultrasound ex- combination treatment compared with berberine alone. 579
521 amination at gestational week 16, resulting in termination of The use of insulin-sensitizing agents such as metformin 580
522 the pregnancy without further autopsy. Another abnormality in women with PCOS undergoing ovulation induction has 581
523 in the combination group came to an infant death on day 31 been widely studied. A Cochrane review (15) showed that 582
524 after birth by parent's refusal of further treatment for a major there was no evidence that metformin improved live birth 583
525 ventricular septal defect and pulmonary stenosis. rates, whether used alone or in combination with clomiphene. 584
526 Therefore, the role of metformin in improving reproductive 585
527 outcomes in women with PCOS appears to be limited. 586
528 DISCUSSION Berberine, an active ingredient from Chinese medicinal herbs, 587
529 Our ndings did not support the hypothesis that a combina- has multiple biologic activities and pharmacologic effects in 588
530 tion of letrozole and berberine is superior to letrozole alone several metabolic diseases, such as type 2 diabetes mellitus, 589
531 for achieving live birth in infertile women with PCOS. The hyperlipidemia, and nonalcoholic fatty liver disease 590

VOL. - NO. - / - 2016 5


FLA 5.4.0 DTD  FNS30250_proof  18 June 2016  9:00 pm  ce E
644

600
649
648

646
645

643
642

640

606

604
639
638

636

634

632

630
629
628

626
625

623
622

620

609
608

605

603
602

599
598

596
595
594
593
592
635

633

624

607
627

597
647

637

601

591
641

631

621

612
619

616
615
614
613

610
618
617

611
6

ORIGINAL ARTICLE: REPRODUCTIVE ENDOCRINOLOGY


TABLE 2

Live birth and other fecundity outcomes, n (%).


Berberine Combination OR (95% CI) OR (95% CI) OR (95% CI)
FLA 5.4.0 DTD  FNS30250_proof  18 June 2016  9:00 pm  ce E

Letrozole group group group between combination between combination between letrozole
Outcome (n [ 215) (n [ 214) (n [ 215) and letrozole P value and berberine P value and berberine P value
Primary outcomes
Live birth 78/215 (36.3) 47/214 (22.0) 74/215 (34.4) 0.95 (0.731.23) .687 1.57 (1.152.14) .004 1.68 (1.232.28) .001
Singleton live birth 77/78 (98.7) 47/47 (100) 71/74 (95.9) 0.97 (0.921.03) .357 0.96 (0.921.01) .28 0.99 (0.961.01) 1.000
Twin live birth 1/78 (0.1) 0/47 (0) 3/74 (0.4) 3.16 (0.3429.72) .357 4.48 (0.2484.82) .28 1.82 (0.0843.85) 1.000
Birth weight (g), 3,463  575 3,542  399 3,484  504 21.20 (177.16219.57) .845 58.23 (248.84132.38) .246 79.43 (282.07123.20) .216
mean  SD
Secondary outcomes
Ovulation 473/796 (59.4) (59.4) 302/831 (36.3) 486/797 (61.0) 1.03 (0.951.11) .526 1.68 (1.511.87) < .0001 1.64 (1.471.82) < .0001
Conception 98/215 (45.6) 61/214 (28.5) 105/215 (48.8) 1.07 (0.881.31) .499 1.71 (1.332.21) < .0001 1.60 (1.242.07) .0003
Immediate loss to 3/98 (3.1) 0 4/105 (3.8) 1.24 (0.295.42) 1.000
follow-up
Pregnancy 84/215 (39.1) 48/214 (22.4) 81/215 (37.7) 0.96 (0.761.23) .766 1.68 (1.242.27) .0006 1.741 (1.292.35) .0002
Singleton 83/84 (98.8) 48/48 (100.0) 78/81 (96.3) 0.97 (0.931.02) .361 0.96 (0.921.01) .294 0.99 (0.971.01) 1.000
Twins 1/84 (1.2) 0 3/81 (3.7) 3.11 (0.3329.30) .361
Pregnancy loss 17/98 (17.4) 14/61 (23.0) 27/105 (25.7) 1.48 (0.862.55) .148 1.12 (0.641.97) .691 0.76 (0.401.42) .386
In the 1st trimester 13/98 (13.2) 14/61 (23.0) 24/105 (22.9) 1.72 (0.933.19) .077 0.996 (0.561.78) .989 0.58 (0.291.15) .114
In the 2nd trimester 4/98 (4.1) 0 3/105 (2.9) 0.70 (0.163.05) .714
Fecundity among ovulated cycles
Conception 98/473 (20.7) 61/302 (20.2) 105/486 (21.6) 1.05 (0.771.44) .737 1.09 (0.761.55) .638 1.03 (0.721.48) .861
Pregnancy 84/473 (17.5) 48/302 (15.9) 81/486 (16.1) 0.94 (0.711.24) .654 1.05 (0.761.45) .776 1.13 (0.761.66) .549
Live birth 78/473 (16.3) 47/302 (15.6) 74/486 (14.6) 0.92 (0.691.24) .592 0.98 (0.701.37) .899 1.06 (0.711.57) .791
Fecundity among subjects who ovulated
Conception 98/188 (52.1) 61/147 (41.5) 105/184 (57.1) 1.09 (0.911.32) .339 1.38 (1.091.73) .005 1.26 (0.991.59) .053
Pregnancy 84/188 (44.2) 48/147 (32.7) 81/184 (42.4) 0.99 (0.781.24) .898 1.35 (1.021.79) .035 1.37 (1.031.81) .025
Live birth 78/188 (41.0) 47/147 (32.0) 74/184 (38.6) 0.97 (0.761.24) .803 1.26 (0.941.69) .122 1.30 (0.971.74) .074
Note: Ovulation was dened as a serum P level according to the standard of the local laboratory (minimum value of luteal phase) or >5 ng/mL. Conception was dened as any positive serum level of hCG. Pregnancy was dened as an intrauterine pregnancy sac with fetal
heart motion as determined by ultrasonography. Live birth was dened as the delivery of a viable infant.
Wu. Berberine, letrozole, or both for PCOS. Fertil Steril 2016.
VOL. - NO. - / - 2016

700

668

666

664
689
688
687
686
685
684
683
682
681
680

669

665

663
662

660
706

704

699
698

696
695
694
693
692

690

667

659
658

656
655
654
653
652

650
708

705

703
702

679
678

675
707

697

677

670

661
691

673
672

657
701

671
676

651
674
Fertility and Sterility

709 768
710
TABLE 3 769
711 770
Adverse events during treatment, pregnancy, and neonatal periods, n (%).
712 771
Letrozole group Berberine group Combination group
713 772
Adverse event (n [ 215) (n [ 214) (n [ 215)
714 773
715 Serious adverse event from 0/215 (0) 0/214 (0) 0/215 (0) 774
study medication
716 Other adverse events 775
717 Constipation 10/215 (4.7) 26/214 (12.1)a 12/215 (5.6) 776
718 Nausea 13/215 (6.0) 34/214 (15.9)a 26/215 (12.1) 777
Diarrhea 13/215 (6.0) 5/214 (2.3) 5/215 (2.3)
719 778
Hot ashes 21/215 (9.8) 6/214 (2.8)a 19/215 (8.8)
720 Fatigue 17/215 (7.9) 5/214 (2.3)a 18/215 (8.4) 779
721 Serious events during pregnancy 780
722 First trimester 781
Ectopic pregnancy 2/84 (2.4) 2/48 (4.2) 2/81 (2.5)
723 Second and third trimesters 782
724 Pregnancy loss after 4/84 (4.8) 0/48 (0) 3/81 (3.7) 783
725 12 wk 784
Preeclampsia 4/84 (4.8) 6/48 (12.5) 7/81 (8.6)
726 785
Gestational diabetes 3/84 (3.6) 3/48 (6.3) 7/81 (8.6)
727 Preterm labor 2/84 (2.4) 0/48 (0) 2/81 (2.5) 786
728 Premature rupture of 5/84 (6.0) 1/48 (2.1) 2/81 (2.5) 787
729 membranes 788
Serious events in fetus and infant
730 Fetal abnormality 1/84 (1.2) 0/48 (0) 0/48 (0) 789
731 Neonatal death 0/78 (0) 0/47 (0) 1/74 (1.4) 790
732 a
P<.05 versus other two groups. 791
733 Wu. Berberine, letrozole, or both for PCOS. Fertil Steril 2016. 792
734 793
735 794
736 (7, 8, 16). A systemic review and meta-analysis for berberine receiving clomiphene. In the population-based Northern 795
737 in the treatment of type 2 diabetes mellitus demonstrated gly- Finland Birth Cohort 1966 study (18), previous oligo- 796
738 cemic control similar to other oral hypoglycemic agents. amenorrhea and/or hirsutism and obesity were both found 797
739 However, berberine also additionally showed an antidyslipi- to be independently associated with decreased fecundity. 798
740 demic effect (7). The relevant targets of berberine might link These differences might be related to the distinct phenotypes 799
741 to the insulin pathway, adenosine monophosphateactivated of PCOS, in that a Chinese cohort is more likely to have less 800
742 protein kinase signaling, the gut environment, and hepatic hyperandrogenism and to be more lean than a European 801
743 lipid transportation (16). cohort (1, 2). 802
744 In a study comparing berberine and metformin (12), 89 Letrozole has a shorter half-life than clomiphene, leading 803
745 Chinese women with PCOS and insulin resistance were ran- to a shorter exposure during implantation and early fetal 804
746 domized into one of three treatment groups: berberine and development and, therefore, a low theoretical risk of congen- 805
747 cyproterone acetate, metformin and cyproterone acetate, or ital anomalies in the offspring (6, 19). There has been 806
748 placebo and cyproterone acetate for three months. Berberine signicant concern for congenital anomalies since the 807
749 showed similar restoration of insulin sensitivity and reduc- introduction of letrozole for use in inducing ovulation. In 808
750 tion of hyperandrogenemia compared with metformin. the present trial, we reported one fetal abnormality in the 809
751 Berberine also appeared to have a greater effect on changes letrozole group, one neonatal death in the combination 810
752 in body composition and dyslipidemia in PCOS patients. group, and no abnormalities in the berberine group. 811
753 Our results showed that ovulation and live birth rates for a However, our study was underpowered to detect a 812
754 combination of letrozole and berberine were similar to letro- signicant difference for rare but potentially serious adverse 813
755 zole alone. However, there were no differences among the events. In the PCOS II trial (6), Legro et al. found four major 814
756 three groups in rates of conception, pregnancies, or live births congenital anomalies in the letrozole group and one in the 815
757 when only ovulatory cycles were considered. This implied that clomiphene group. There was no pattern to the four major 816
758 improvement in the metabolic prole by berberine did not anomalies with letrozole, implying that they were random 817
759 affect the ovulation or live birth rates achieved with letrozole. events. The rate of congenital anomalies in the present study 818
760 We did not measure insulin sensitivity before and after expo- is below what has been reported in other studies (20). 819
761 sure to the study medication in the present study. We did not In the present study, the use of berberine alone achieved a 820
762 nd signicant differences in live birth rates stratied by 36% ovulation rate per cycle, similar to metformin, and a 22% 821
763 BMI, hirsutism score, menstrual patterns of regularity or oli- cumulative live birth rate, similar to clomiphene, after 822
764 gomenorrhea, and duration of infertility. Our results contrast 6 months of use (5). To the best of our knowledge, this is 823
765 with those from a study conducted in the Netherlands (17) the rst study to show the effectiveness of berberine alone 824
766 where BMI, age, free androgen index, and cycle history on the ovulation and live birth rates in women with PCOS. 825
767 were all associated with live birth in women with PCOS However, we can not make conclusions regarding efcacy 826

VOL. - NO. - / - 2016 7


FLA 5.4.0 DTD  FNS30250_proof  18 June 2016  9:00 pm  ce E
ORIGINAL ARTICLE: REPRODUCTIVE ENDOCRINOLOGY

827 because berberine was not compared with a placebo or no Mudanjiang, China; Mu-Er An, M.D., Department of Obstet- 886
828 treatment. A study directly comparing metformin and rics and Gynecology, First Afliated Hospital, Harbin Medical 887
829 berberine for ovulation induction is worth performing in the University, Harbin, China; Guang-Zhu Yu, M.D., Department 888
830 future. of Gynecology, Suqian Maternal and Children's Health Hospi- 889
831 One of the limitations of the present study was its early tal, Suqian, China; Robert M. Silver, M.D., Department of Ob- 890
832 termination because of the expiration of the study drug stetrics and Gynecology, University of Utah Health Sciences 891
833 (berberine and its placebo). However, the absolute difference Center, Salt Lake City, Utah; and Elisabet Stener-Victorin, 892
834 (12%14%) in live birth rates for the original two primary Ph.D., Department of Physiology and Pharmacology, Karolin- 893
835 comparisons of letrozole and berberine in this trial was ska Institutet, Stockholm, Sweden. 894
836 greater than the projected 8%; therefore, the unexpected The members of the Steering Committee for this network 895
837 reduction to 644 participants still provided adequate power were Xiao-Ke Wu, Jian-Ping Liu, Elisabet Stener-Victorin, 896
838 (R90%) for the study. Other limitations included not all sub- Ernest H. Y. Ng, Zhaoling You, and Chengzong Xiao. The 897
839 jects having pelvic sonograms to assess for polycystic ovary members of the Scientic Advisory Board were Drs. Sulun 898
840 morphology and the inclusion of subjects with regular cycles Sun, Weiliang Weng, Yanming Xie, Yixun Liu, Yongyan 899
841 for ovulation. The major strength of this study is that it was a Wang for the National Clinical Research Base of Chinese Med- 900
842 large multicenter double-blind trial with close monitoring of icine in this network at the First Afliated Hospital, Heilong- 901
843 adverse and serious adverse events and tracking of live births jiang University of Chinese Medicine, Harbin, People's 902
844 in line with our recent Harbin Consensus (21). Also, it demon- Republic of China. 903
845 strates the high live birth rate achieved with letrozole in a 904
846 population with a relatively lower BMI range. Acknowledgment: The authors thank all of the site partic- 905
847 In summary, berberine did not add fecundity in infertility ipants and the staff in the Harbin Administrative ofce for 906
848 conditions with PCOS when used in combination with the their contributions, including Drs. Hongying Kuang, Yan Li, 907
849 new ovulation agent letrozole. Hongli Ma, Wenjuan Shen, and Jingshu Gao during their do- 908
850 mestic and international training for the trial. 909
851 910
852 PARTICIPANT LIST 911
853
REFERENCES 912
National Reproductive and Developmental Network in Chi-
854 1. Li R, Zhang Q, Yang D, Li S, Lu S, Wu X, et al. Prevalence of polycystic ovary 913
nese Medicine for this trial included Li-Hui Hou, M.D.,
syndrome in women in China: a large community-based study. Hum Reprod
855 Department of Obstetrics and Gynecology, First Afliated 914
2013;28:25629.
856 Hospital, Heilongjiang University of Chinese Medicine, Har- 915
2. Li R, Qiao J, Yang D, Li S, Lu S, Wu X, et al. Epidemiology of hirsutism among
857 bin, China; Yong-Yan Wang, M.D., Feng Tian, Ph.D., and women of reproductive age in the community: a simplied scoring system. 916
858 Yan-Ming Xie, M.D., Institute of Basic Clinical Medicine, Eur J Obstet Gynecol Reprod Biol 2012;163:1659. 917
859 China Academy of Chinese Medical Science, Beijing, China; 3. Li R, Yu G, Yang D, Li S, Lu S, Wu X, et al. Prevalence and predictors of meta- 918
860 Jin-Feng Zhang, M.D., Department of Obstetrics and Gyne- bolic abnormalities in Chinese women with PCOS: a cross-sectional study. 919
861 BMC Endocr Disord 2014;14:76. 920
cology, Shanxi Province Hospital of Chinese Medicine,
4. Wu XK. Polycystic ovary syndrome. In: Hou LH, Wang YT, Wu XK, editors.
862 Taiyuan, China; Ya-Qin Gao, M.D., Center for Reproductive 921
Today gynecology in traditional Chinese medicine. Beijing: Peoples Medical
863 Medicine, Daqing Oileld General Hospital, Daqing, China; Publishing House; 2010:11845.
922
864 Shao-Min Du, M.D., Department of Obstetrics and Gynecol- 5. Legro RS, Barnhart HX, Schlaff WD, Carr BR, Diamond MP, Carson SA, et al. 923
865 ogy, Daqing Longnan Hospital, Daqing, China; Ying Yan, Clomiphene, metformin, or both for infertility in the polycystic ovary syn- 924
866 M.D., Department of Gynecology, First Afliated Hospital, drome. N Engl J Med 2007;356:55166. 925
867 Tianjin University of Chinese Medicine, Tianjin, China; Pei- 6. Legro RS, Brzyski RG, Diamond MP, Coutifaris C, Schlaff WD, Casson P, et al. 926
Letrozole versus clomiphene for infertility in the polycystic ovary syndrome.
868 Lin Li, M.D., Department of Gynecology, Second Afliated 927
N Engl J Med 2014;371:11929.
869 Hospital, Tianjin University of Chinese Medicine, Tianjin, 928
7. Dong H, Wang N, Zhao L, Lu F. Berberine in the treatment of type 2 diabetes
870 China; Jin-Ying Fu, M.D., Department of Obstetrics and Gy- mellitus: a systemic review and meta-analysis. Evid Based Complement Al- 929
871 necology, Second Afliated Hospital, Henan College of Chi- ternat Med 2012;2012:591654. 930
872 nese Medicine, Zhengzhou, China; Wei-Li Li, M.D., 8. Dong H, Zhao Y, Zhao L, Lu F. The effects of berberine on blood lipids: a sys- 931
873 Department of Obstetrics and Gynecology, Afliated Hospi- temic review and meta-analysis of randomized controlled trials. Planta Med 932
874 2013;79:43746. 933
tal, Anhui University of Chinese Medicine, Hefei, China;
9. Xu H. Analysis of market outlook in 2014: Cortex Phellodendri high-quality
875 Zhen-Yu Tan, M.D., Department of Obstetrics and Gynecol- 934
stocks. Available at: http://www.kmzyw.com.cn/pages/channel_467/1140
876 ogy, First Afliated Hospital, Hunan University of Chinese 935
320 467.1395293714000.2579.shtml; 2014. Q1
877 Medicine, Changsha, China; Feng-Jie He, M.D., Department 10. Zhao L, Li W, Han F, Hou L, Baillargeon JP, Kuang H, et al. Berberine reduces 936
878 of Obstetrics and Gynecology, Afliated Hospital, Shanxi insulin resistance induced by dexamethasone in theca cells in vitro. Fertil 937
879 College of Chinese Medicine, Xianyang, China; Cai-Fei Steril 2011;95:4613. 938
880 Ding, M.D., Center for Reproductive Medicine, Zhejiang Prov- 11. Li Y, Ma H, Zhang Y, Kuang H, Ng EH, Hou L, et al. Effect of berberine on 939
881 insulin resistance in women with polycystic ovary syndrome: study protocol 940
ince Hospital of Integrative Medicine, Hangzhou, China;
for a randomized multicenter controlled trial. Trials 2013;14:226.
882 Xiao-Bin Li, M.D., Department of Obstetrics and Gynecology, 941
12. Wei W, Zhao H, Wang A, Sui M, Liang K, Deng H, et al. A clinical study on the
883 Guangdong Province Hospital of Chinese Medicine, Guangz- short-term effect of berberine in comparison to metformin on the metabolic
942
884 hou, China; Xian-Ji Shen, M.D., Outpatient Department, Mu- characteristics of women with polycystic ovary syndrome. Eur J Endocrinol 943
885 danjiang Maternal and Children's Health Hospital, 2012;166:99105. 944

8 VOL. - NO. - / - 2016


FLA 5.4.0 DTD  FNS30250_proof  18 June 2016  9:00 pm  ce E
Fertility and Sterility

945 13. Li Y, Kuang H, Shen W, Ma H, Zhang Y, Stener-Victorin E, et al. Letrozole, ovulation in normogonadotropic oligoamenorrheic infertility. Fertil Steril 1004
946 berberine, or their combination for anovulatory infertility in women with 2002;77:917. 1005
polycystic ovary syndrome: study design of a double-blind randomised 18. Koivunen R, Pouta A, Franks S, Martikainen H, Sovio U, Hartikainen AL, et al.
947 1006
controlled trial. BMJ Open 2013;3:e003934. Fecundability and spontaneous abortions in women with self-reported
948 1007
14. Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group. oligo-amenorrhea and/or hirsutism: Northern Finland Birth Cohort 1966
949 Revised 2003 consensus on diagnostic criteria and long-term health risks Study. Hum Reprod 2008;23:21349. 1008
950 related to polycystic ovary syndrome. Fertil Steril 2004;81:1925. 19. Franik S, Kremer JA, Nelen WL, Farquhar C. Aromatase inhibitors for subfer- 1009
951 15. Tang T, Lord JM, Norman RJ, Yasmin E, Balen AH. Insulin-sensitising drugs tile women with polycystic ovary syndrome. Cochrane Database Syst Rev 1010
952 (metformin, rosiglitazone, pioglitazone, d-chiro-inositol) for women with 2014:CD010287. 1011
953 polycystic ovary syndrome, oligo amenorrhoea and subfertility. Cochrane 20. Davies MJ, Moore VM, Willson KJ, van Essen P, Priest K, Scott H, et al. Repro- 1012
Database Syst Rev 2012:CD003053. ductive technologies and the risk of birth defects. N Engl J Med 2012;366:
954 1013
16. Liu Y, Zhang L, Song H, Ji G. Update on berberine in nonalcoholic fatty liver 180313.
955 1014
disease. Evid Based Complement Alternat Med 2013;2013:308134. 21. Harbin Consensus Conference Workshop Group. Improving the Reporting
956 17. Imani B, Eijkemans MJ, te Velde ER, Habbema JD, Fauser BC. A nomogram to of Clinical Trials of Infertility Treatments (IMPRINT): modifying the CONSORT 1015
957 predict the probability of live birth after clomiphene citrate induction of statement. Hum Reprod 2014;29:207582. 1016
958 1017
959 1018
960 1019
961 1020
962 1021
963 1022
964 1023
965 1024
966 1025
967 1026
968 1027
969 1028
970 1029
971 1030
972 1031
973 1032
974 1033
975 1034
976 1035
977 1036
978 1037
979 1038
980 1039
981 1040
982 1041
983 1042
984 1043
985 1044
986 1045
987 1046
988 1047
989 1048
990 1049
991 1050
992 1051
993 1052
994 1053
995 1054
996 1055
997 1056
998 1057
999 1058
1000 1059
1001 1060
1002 1061
1003 1062

VOL. - NO. - / - 2016 9


FLA 5.4.0 DTD  FNS30250_proof  18 June 2016  9:00 pm  ce E
ORIGINAL ARTICLE: REPRODUCTIVE ENDOCRINOLOGY

1063 1122
1064
SUPPLEMENTAL FIGURE 1 1123
1065 1124
1066 1125
1067 1126
1068 1127
1069 1128
1070 1129
1071 1130
1072 1131
1073 1132
1074 1133
1075 1134
1076 1135
1077 1136
1078 1137
1079 1138
1080 1139
1081 1140
1082 1141
1083 1142
1084 1143
1085 1144
1086 1145
1087 1146
web 4C=FPO

1088 1147
1089 1148
1090 1149
1091 1150
1092 Kaplan-Meier curves for live births according to (A) treatment group, (B) body-mass index (BMI), (C) menstrual pattern, and (D) age. The rate of 1151
1093 cumulative live births was similar between the letrozole and combination groups, and signicantly higher in both groups than in the berberine 1152
group. Independently from treatment, subjects with age <33 years had signicantly higher rates of live births than did women whose age was
1094 >33 years. There were no signicant differences in live birth rate stratied by BMI or menstrual cycle pattern. 1153
1095 Wu. Berberine, letrozole, or both for PCOS. Fertil Steril 2016. 1154
1096 1155
1097 1156
1098 1157
1099 1158
1100 1159
1101 1160
1102 1161
1103 1162
1104 1163
1105 1164
1106 1165
1107 1166
1108 1167
1109 1168
1110 1169
1111 1170
1112 1171
1113 1172
1114 1173
1115 1174
1116 1175
1117 1176
1118 1177
1119 1178
1120 1179
1121 1180

9.e1 VOL. - NO. - / - 2016


FLA 5.4.0 DTD  FNS30250_proof  18 June 2016  9:00 pm  ce E