黄素化未破裂卵泡综合征的发生及相关治疗

黄素化未破裂卵泡综合征(luteinized unruptured follicle syndrome, LUFS)是一种无法排出卵子的特殊临床症候群,占不孕症妇女的25%～43%。其发生可能与卵巢周期各种激素水平调控和卵巢内各种因子调控异常有关。子宫内膜异位症、盆腔环境、各种药物的使用以及患者本身精神因素亦可导致LUFS的发生。由于机制尚未完全阐明,目前多根据各种可能病因进行相关治疗。     

Sequential clomiphene citrate and human menopausal gonadotrophin for ovulation induction: comparison to clomiphene citrate alone and human menopausal gonadotrophin alone

The need for frequent injections and monitoring, the possibilityof multiple gestations, and the higher cost compared to clomiphenecitrate, prevents many clinicians from using human menopausalgonadotrophin (HMG) for ovulation induction. A sequential medicationregimen, in which HMG is taken after clomiphene, overcomes theseproblems. We retrospectively compared per cycle fecundity andbirth rates in 119 cycles of clomiphene—HMG, 524 cyclesof clomiphene alone, 57 cycles of HMG alone, and 79 cycles ofconcurrent HMG and clomiphene in patients receiving intra-uterineinsemination (IUI), who were free of endometriosis or tubaldisease. Per cycle fecundity for clomiphene—HMG was 22%[95% confidence interval (CI) 12–34%], double that ofclomiphene alone (11%) (95% CI 8–14%) (P < 0.01), andequal to HMG alone (18%) (95% CI 7–29%) or HMG and clomiphenetogether (19%) (95% CI 10–28%). The multiple birth ratefor clomiphene—HMG (7/21) equalled that for HMG alone(3/12) and HMG and clomiphene together (3/8). The average numberof ampoules of HMG required [follicle stimulating hormone (FSH)75 mIU, luteinizing hormone (LH) 75 mIU] was decreased by 65%from 24.5 ± 1.0 for HMG or HMG and clomiphene togetherto 8.6 ± 0.3 for clomiphene—HMG (P < 0.001).Per cycle fecundity was identical when one, two or three ampoulesof HMG per day were administered after clomiphene. We concludethat ovulation induction with sequential clomiphene—HMGresults in fecundity double that of clomiphene alone and equalto HMG alone or concurrent with clomiphene, thereby reducingthe requirement for HMG.     

Gonadotrophin-releasing hormone agonist compared with human chorionic gonadotrophin for ovulation induction after clomiphene citrate treatment

The objective of this study was to compare hormonal response,luteal phase adequacy and pregnancy and abortion rates in patientsrandomized to receive human chorionic gonadotrophin (HCG) orgonadotrophinreleasing hormone agonist (GnRHa) during ovulationcycles stimulated by clomiphene citrate. Anovulatory patientsreceived either one s.c. dose of tryptorelin (0.1 mg; n = 104)or one i.m. dose of HCG (10 000 IU; n = 106) after clomiphenecitrate stimulation had induced enlarged ovarian follicles (>17mm in diameter). A short-lived, transitory increase in serumluteinizing hormone (98 ± 9 IU/1) and follicle-stimulatinghormone (30 ± 5 IU/1) concentrations was measured at12 h following the injection of GnRHa, and these concentrationsreturned to baseline levels by 36 h post-injection. Midlutealprogesterone concentrations were similar in both groups (>10ng/ml), and the mean luteal phase duration was also not significantlydifferent (13 days). There were no significant differences inthe mean number of pregnancies (12.0 versus 12.6% per cycle)and the abortion rate (18.2 versus 12.5%) between the GnRHa-and HCG-treated groups respectively. There were no complicationsrelated to treatment in either group. The results show thata relatively low dose of GnRHa can be used in place of HCG toinduce ovulation in clomiphene citrate-treated patients.     

三种方法治疗未破裂卵泡黄素化综合征的疗效分析

目的 比较3种方法治疗未破裂卵泡黄素化综合征（LUFS）疗效。方法 将34个病例56个治疗周期随机分成3个治疗组：A组（HCG组），B组（单次GnRH—a组），C组（GnRH—a＋HCG组），观察3组患者的妊娠率，排卵率，并分别于HCG／达必佳注射日及排卵后7日抽取外周血测LH，P，观察各组有无差异。结果 3种治疗方法的排卵率有统计学差异（P〈0．05），C组的排卵率显著高于B组，A组。而3组的妊娠率则无明显差异（P〉0．05）。HCG／达必佳注射日3组血LH有统计学差异（P〈0．05），C组低于A组与B组，而血P值则3组之间无统计学差异（P〉0．05）。排卵后7日血P值3组之间亦有统计学差异（P〈0．05），A组高于C组，C组高于B组。结论 GnRH—a＋HMG方案能有效的降低HCG注射日LH值。提高LUFS患者的排卵率，但妊娠率无明显改善。     

Luteinized unruptured follicle syndrome: incidence and recurrence rate in infertile women with unexplained infertility undergoing intrauterine insemination

BACKGROUND: The objective of this study was to determine the incidence and recurrence rate of luteinized unruptured follicle (LUF) syndrome in women with unexplained infertility undergoing intrauterine insemination (IUI). METHODS: A total of 167 women with unexplained infertility who underwent 292 cycles of IUI were enrolled in the study. All patients were treated with clomiphene citrate, 50-150 mg/daily from day 5 to 9 of their menstrual cycle. Ultrasound examination to confirm ovulation was performed on the day of IUI (day 0) and every day thereafter for another 3 days (days 1, 2 and 3). A total of 69 women who failed to conceive in the first cycle and 56 women who failed to conceive in the second cycle underwent second and third cycles, respectively. RESULTS: Of the total 167 patients who underwent first cycle, 42 (25%) had LUF. The incidence of LUF was 56.5% in 69 patients who underwent a second cycle of IUI treatment, of whom 33 patients had LUF in the first cycle with recurrence rate of 78.6%. In 56 patients who underwent 3 consecutive cycles, the incidence of LUF was 58.9% and recurrence rate of 90%. No pregnancies were recorded in patients with LUF during the study period. CONCLUSION: The incidence and recurrence rate of LUF are significantly increased in subsequent cycles of IUI. In these patients, other options of infertility treatment might be justified.     

Consecutive versus alternating cycles of ovarian stimulation using human menopausal gonadotrophin.

Ovarian stimulation is an effective treatment for patients with ovulatory dysfunction and unexplained infertility. An initial report has suggested that consecutive cycles of ovarian stimulation can be employed without causing a diminished response in the second cycle. However, this observation has neither been confirmed nor has a regimen of consecutive stimulation cycles been compared to one of alternating stimulation cycles. Accordingly, 44 consecutive and 54 alternating cycles of stimulation were evaluated in patients (n = 42) who were treated with human menopausal gonadotrophin (HMG) alone. Human chorionic gonadotrophin (HCG) 10,000 IU was administered i.m. when at least one follicle exceeded 16 mm in mean diameter, and this was followed by either intercourse or intrauterine insemination. Using each patient as her own control, we were unable to demonstrate any differences in mean HMG dose requirements, endocrine parameters or follicular development on the day of HCG administration, or ovulation rates in the second consecutive cycle compared to the second alternating cycle. Clinical pregnancies resulted significantly more often in a consecutive cycle (8/22) than in an alternating cycle (2/27, P = 0.029). We conclude that consecutive cycles of ovarian stimulation with HMG are not detrimental and may, in fact, result in increased cycle fecundity compared to alternating stimulation cycles.     

Establishing pregnancies after follicular stimulation for IVF with clomiphene citrate and human menopausal gonadotrophin only

Data are presented on establishing pregnancies by IVF during 1987 using only clomiphene citrate and human menopausal gonadotrophin for follicular stimulation. Of the 562 patients undergoing follicular stimulation, 80% reached oocyte recovery and 70% had at least one conceptus replaced. Patients having one or more (up to a maximum of four) conceptuses replaced demonstrated a significant increase in the establishment of pregnancies from one to two (14-29%: P = 0.035) and from two to three conceptuses (29-42%: P = 0.037). There was a significant decline in pregnancies when four conceptuses were replaced compared with three (P = 0.004). The data were also analysed according to the cause of infertility, specifically tubal, endometriosis, unexplained infertility and male factors only. After the replacement of conceptuses, the incidence of implantation and abortion was not significantly different. The incidence of pregnancy declined significantly after 35 years (26%) compared with women under 31 years (43%; P = 0.043). Of 129 women having three conceptuses replaced, in those greater than 35 years (63 patients) 23 (37%) became pregnant whereas in those less than 31 years (65 patients), 34 (52%; P = 0.05) became pregnant. Twenty-two per cent of stimulated cycles resulted in an endogenous LH surge and the incidence of patients having three conceptuses replaced in this group was lower than those in the HCG group (P = 0.007). Fertilization per oocyte was also significantly reduced (P less than 0.001) in patients with an LH surge. In total, 2824 oocytes were recovered and 57% fertilized with 54% of patients having three conceptuses replaced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recurrent empty follicle syndrome successfully treated with recombinant human chorionic gonadotrophin.

We report a case of a patient with polycystic ovary syndrome and primary infertility who was admitted to our in-vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) programme because of her partner's severe oligozoospermia and asthenozoospermia. Ovarian stimulation was accomplished in the three treatment cycles using gonadotrophin therapy after a dual approach with ovarian suppression using oral contraceptive pills followed by gonadotrophin-releasing hormone agonist therapy. Oocyte retrieval was unsuccessful in the first two treated cycles despite the fact that human chorionic gonadotrophin (HCG) from three different batches was used. In the third treatment cycle, recombinant HCG was used and five oocytes were retrieved. This is the first report of recurrent empty follicle syndrome despite the use of different batches of commercially available urinary HCG, and of its successful treatment using recombinant HCG.     

Folliculogenesis and ovulation in infertile women with mild endometriosis

Twenty-one infertile women with laparoscopically documented minimal-mild endometriosis (AFS score 2-10) were studied during 27 cycles (six women had two cycles each) to investigate follicular development and ovulation. Of the 27 cycles studies, 24 (89%) appeared to be endocrinologically normal and ovulatory. Luteinized unruptured follicle (LUF) occurred in only one cycle (4%). One further patient exhibited abnormal endocrinology with evidence of premature ovulation over two (8%) consecutive cycles. This study indicates that the majority of women with minimal-mild endometriosis have endocrinologically normal menstrual cycles and that luteinized unruptured follicles occur infrequently.     

Response to clomiphene citrate in the polycystic ovarian syndrome according to different LH/FSH ratios

Twenty-nine clomiphene tests were carried out, 19 on subjectswith polycystic ovarian syndrome (PCO) who did not wish to conceiveand 10 on women with correct ovarian function (control women).The intention was to determine if the response of the hypothalamic—pituitary—ovaryaxis varies according to different LH/FSH ratios. The resultsindicate that with repeated LH/FSH ratios of <3, the positiveand negative feedback systems are preserved in most subjects,but when the ratio is higher than 3, both systems are significntlyaltered (P < 0.01). In the control group, both systems werepreserved in all subjects. In another group composed of 26 infertilepatients with PCO, the rate of ovulation and pregnancy afteradministration of clomiphene citrate (CC) was correlated accordingto different LH/FSH ratios. A significant (P < 0.0001) redudonin the rate of ovulation and a lesser reduction in pregnancywas found with increasing LH/FSH ratios. In conclusion we suggestthat the LH/FSH ratio is a good marker of this syndrome, andcan be used to give a prognosis on the severity of the syndrome,as well as the ovulatory capacity of the subject after administrationof CC.     

Endocrinology: The influence of human chorionic gonadotrophin administration upon the next ovarian cycle

We examined the influence of human chorionic gonadotrophin (HCG),used as an ovulation inducer and/or for supporting the lutealphase, on the next ovarian cycle. Four women received 10 000IU of HCG at mid-cycle and another four received the same doseplus 1500 IU on the 17th, 19th and 21th days of the cycle. Inthe menstrual cycle prior to our experiments, venous blood sampleswere collected and vaginal ultrasound of the ovaries was performedevery other day from day 21–28; the same data were alsocollected on days 1–10 of the experiment cycle. In sucha way, control values were obtained. After the administrationof HCG, venous blood samples were collected and ultrasound wasperformed in the same way and on the same days as in the controls.Follicle stimulating hormone (FSH) and luteinizing hormone weredetermined by radioimmunoassay in all blood samples, and HCGonly in samples collected after the experiment. The resultsshowed that only FSH was lower in the late luteal phase afterthe administration of 10 000 IU of HCG. Follicular diameterswere higher during the follicular phase than during the previouscycle only in women who received the low dose of HCG. In addition,one woman presented with detectable HCG in the following ovariancycle. The use of HCG in the preceding cycle may reduce FSHand develop persistent follicles in the subsequent cycle. Wesuggest that an ultrasound of the ovaries should be performedbefore starting a new ovulation induction cycle in a woman whohas received HCG in the previous cycle. Our results also implythat the ‘biochemical pregnancy’ could be a falsepregnancy.     

Endocrinology: Nuclear maturity and oocyte morphology after stimulation with highly purified follicle stimulating hormone compared to human menopausal gonadotrophin

Several studies have shown that high concentrations of luteinizinghormone (LH) in the follicular phase of stimulation can havea negative effect on oocyte quality, pregnancy rate and incidenceof miscarriage. The aim of the present study was to examinethe effects of highly purified follicle stimulating hormone(FSH HP) on ovarian stimulation and particularly on nuclearmaturity and morphological appearance of the oocyte in intracytoplasmicsperm injection (ICSI) therapy and to compare the results withhuman menopausal gonadotrophin (HMG) stimulation. For this purpose,50 patients for ICSI (HMG: 30; FSH HP: 20) and 26 patients forin-vitro fertilization (TVF; HMG: 14, FSH HP: 12) were stimulatedwith either HMG of FSH HP using a short-term protocol. Patientswere divided into the two groups according to the first letterof their family name. No differences were observed among thegroups in relation to patient age, duration of stimulation,number of aspirated oocytes or maturity of the oocyte-cumuluscomplex. After removal of the cumulus-corona cells in the ICSIoocytes, a significantly higher proportion of oocytes in theFSH HP group were nuclear mature (metaphase II) than in theHMG group (FSH HP: 88.8%, HMG: 80.6%; P = 0.009). Furthermore,in the FSH HP group, significantly fewer oocytes with dark cytoplasmwere observed (FSH HP: 14.4%, HMG: 22.4%; P = 0.02). Fertilization,cleavage and pregnancy rates (FSH HP 38%, HMG: 34% per retrieval)were comparable in both groups. Based on the results obtained,it can be concluded that the short-term FSH HP treatment protocolsynchronizes oocyte maturation better than comparable stimulationwith HMG.     

Laparoscopic removal of an abdominal pregnancy adherent to the appendix after ovulation induction with human menopausal gonadotrophin

The laparoscopic management of tubal pregnancies has becomea standard form of treatment. We present, for the first time,a successful laparoscopic approach in the management of earlyabdominal pregnancy. Although it is difficult to reach conclusionsfrom such limited published experience, it nevertheless appearsthat early abdominal pregnancies can be treated via operativelaparoscopy. The possible advantages of such a therapeutic approachinclude lower morbidity and mortality, and a better fertilityprognosis.     

Oocyte maturation,follicle rupture and luteinization in human cryopreserved ovarian tissue following xenografting

BACKGROUND: Previous studies have demonstrated development of antral follicles in cryopreserved human ovarian tissue after autografting and xenografting, thus indicating successful preservation of follicular function. The study aim was to assess whether these follicles could also undergo periovulatory changes in response to hCG. METHODS: Ovarian tissue from three patients were dehydrated in propanediol (PROH)/sucrose and cryopreserved using the slow cooling/rapid thaw procedure. Thawed tissue was placed under the kidney capsule in immunodeficient mice. Following growth (>20 weeks) in the presence of gonadotrophin, hCG was administered and ovarian tissue examined histologically. RESULTS: Thirty-two antral follicles (diameter range 0.6 to 5 mm) were examined. Histological evidence of a response to hCG was evident in all follicles. Disruption of the concentric layers of mural granulosa and theca cells was apparent in all antral cavities. In 17 (53%) follicles the exterior follicular wall had reduced to a few cells thick, and in eight (25%) the wall had ruptured. Mucified oocyte-cumulus cell complexes were present in 32 follicles, 17 of which had begun to detach from the pedicle. Resumption of meiosis had occurred in over half the oocytes (five metaphase II and seven metaphase I oocytes, eight germinal vesicle breakdown). Two corpora lutea were also detected. CONCLUSIONS: Follicles cryopreserved within human ovarian tissue using the PROH procedure, can develop to the antral stage and undergo periovulatory changes following xenografting and exposure to a luteinizing stimulus.     

Predictors for treatment failure after laparoscopic electrocautery of the ovaries in women with clomiphene citrate resistant polycystic ovary syndrome

BACKGROUND: Laparoscopic electrocautery has been put forward as the treatment of choice in women with clomiphene citrate (CC)-resistant polycystic ovary syndrome (PCOS). In order to make an informed treatment decision it would be helpful if we could identify women with PCOS with a high probability of treatment failure following electrocautery of the ovaries. METHODS: Eighty-three women with CC-resistant PCOS were allocated to receive laparoscopic electrocautery followed by CC when anovulation persisted as part of a randomized controlled trial. Multivariable logistic regression analyses using clinical, ultrasonographic and endocrinological parameters were performed to predict (i) failure to ovulate within 8 weeks after electrocautery, and (ii) failure to reach an ongoing pregnancy after electrocautery with or without CC. RESULTS: Of the 83 women, 56 (67%) ovulated within 8 weeks after electrocautery. The model for predicting anovulation after electrocautery included LH/FSH rate, year of menarche and glucose level. Women who were younger at menarche, had a lower LH/FSH ratio and a lower glucose level were more likely to have persistent anovulation. The area under the curve was 0.74. After electrocautery and CC, 41 women reached an ongoing pregnancy. No prognostic parameters could be identified to predict failure to reach an ongoing pregnancy after electrocautery followed by CC. CONCLUSIONS: Persistence of anovulation after electrocautery could be predicted and women with a high risk of persisting anovulation could be distinguished. We were, however, not able to predict treatment failure after electrocautery followed by CC.     

Ovarian stimulation in women undergoing in-vitro fertilization and embryo transfer using recombinant human follicle stimulating hormone (Gonal-F) in non-down-regulated cycles

In order to assess the efficacy and safety of recombinant humanfollicle stimulating hormone (FSH) in routine clinical use,ovarian stimulation with recombinant human FSH was performedin 71 patients prior to in-vitro fertilization (IVF) withoutgonadotrophin-releasing hormone (GnRH) analogues in a multicentre,non-comparative study. Human chorionic gonadotrophin (HCG) wasadministered to 58 patients (81.7%), 15 of whom underwent 19cycles with an initial dosage of three ampoules daily of recombinantFSH and 43 of whom underwent 152 cycles with four ampoules dailyfrom day 3 onwards. No significant differences were detectedbetween these two groups in all test parameters. The mean durationof treatment was 9.06 and 8.86 days respectively with a meannumber of 24.06 and 23.25 vials of recombinant human FSH administered.A mean number of 6.26 and 5.88 oocytes respectively was collected.The number of transferred embryos was 2.4 and 2.2. A clinicalpregnancy rate of 23.8% (10 out of 42) per transfer was achieved(30.9 and 20.6% respectively). Local tolerance of s.c. administrationwas excellent. Mild pain at the injection site was the dominantfinding in <20% of patients. Two cases of ovarian hyperstimulationsyndrome were noted. Recombinant human FSH is very attractiveto patients because it can be self-administered s.c. and thepreparation does not come from a human source. In conclusion,these data support the safety and efficacy of recombinant humanFSH in routine use for IVF.     

Pure FSH for ovulation induction in patients with polycystic ovary syndrome and resistant to clomiphene citrate therapy

Twenty-nine infertile women with polycystic ovary disease whichwas resistant to therapy with clomiphene citrate underwent acombined treatment for follicle recruitment consisting of pureFSH during the first days of the cycle and HMG during the lastdays of the follicular phase. Sixty cycles were stimulated ofwhich 83% were ovulatory. Eighteen pregnancies were achieved(36% of cycles, 62% of patients). The multiple pregnancy ratewas 39%. Twelve cycles (20%) showed the ovarian hyperstimulationsyndrome (OHS) although seven of these resulted in full termdeliveries. There were no miscarriages among the patients studied.     

Programming of ovarian stimulation with norethindrone acetate in IVF/GIFT cycles

Twenty patients were given norethindrone acetate (NET) to program the initiation of controlled ovarian hyperstimulation and to coordinate follicular aspiration with surgery to obtain spermatozoa from the husband. Patients received NET, 10 mg/day orally, starting between days 2 and 4 of the cycle. The duration of NET therapy varied from 9 to 37 days. The mean time of onset of vaginal bleeding, after cessation of NET, was 2.9 +/- 0.7 days. Ovarian stimulation was carried out with a combination of a luteinizing hormone releasing hormone analog, follicle-stimulating hormone and human menopausal gonadotrophin. The day of human chorionic gonadotrophin (HCG) administration ranged from day 8 to day 15 of the cycle (10.1 +/- 1.7). On the day of HCG injection, the mean E2 level was 2188 +/- 1126. The mean number of follicles aspirated was 18.4 +/- 9.9 per cycle. The mean number of oocytes collected per cycle was 15.5 +/- 8.5. There was no correlation between duration of NET suppression and the number of days of gonadotrophin therapy needed to reach HCG administration. The large number of oocytes retrieved is probably related more with the fact that the patients represented a group with a purely male factor of infertility, than by the specific drug protocol utilized. Our results demonstrate that the ovarian response to gonadotrophin stimulation was not affected by NET administration. The main advantages of the use of this drug for cycle control are that its administration is oral, simple and inexpensive.     

Endocrinology: Comparison of gonadotrophin-releasing hormone analogues and human chorionic gonadotrophin for the induction of ovulation and prevention of ovarian hyperstimulation syndrome: a case-control study

Gonadotrophin-releasing hormone analogue (GnRHa) has been suggestedas an alternative to human chorionic gonadotrophin (HCG) fortriggering ovulation, while preventing ovarian hyperstimulationsyndrome (OHSS). Since a prospective, controlled study wouldbe unethical at this point, we used a retrospective, case-selfcontrol approach to compare GnRHa with HCG in that context.A group of 16 in-vitro fertilization (IVF) patients who hadsevere OHSS in previous cycles, in which HCG was given to triggerovulation, were studied in subsequent cycles in which GnRHawas used. Each GnRHa cycle (case) was compared to a previousHCG cycle that resulted in OHSS (self control). None of thesesubsequent cydes resulted in severe OHSS. The use of GnRHa didnot affect the number of oocytes retrieved or their quality.Serum oestradiol concentrations on the day of ovulation triggeringwere signilicantly (P<0.01) higher in the GnRHa cycles comparedto HCG cycles. Exogenous progesterone and oestra diol were effectivein maintaining relatively constant serum oestradiol and progesteroneserum concentrations during the luteal phase. Pregnancy rateper cycle was similar in the two groups. In conclusion, theuse of GnRHa to induce ovulation in IVF patients, who are athigh risk for developing OHSS, effectively eliminates this riskwithout affecting other parameters of the stimulation cycle.     

Randomized controlled trial comparing laparoscopic ovarian diathermy with clomiphene citrate as a first-line method of ovulation induction in women with polycystic ovary syndrome

BACKGROUND: Laparoscopic ovarian diathermy (LOD) is currently accepted asa successful second-line treatment for ovulation induction (OI)in clomiphene citrate (CC)-resistant women with polycystic ovarysyndrome (PCOS). The aim of this study was to test the hypothesisthat LOD may be superior to CC as a first-line treatment. METHODS: The study included 72 anovulatory women with PCOS who were randomizedto LOD (n = 36) or CC (n = 36). Women who remained anovulatoryafter LOD were offered CC. Similarly, women receiving CC whofailed to ovulate or conceive were offered LOD. Pregnancy rateswere compared between the two groups using ² and odds ratiowith 95% confidence interval (OR, 95% CI). RESULTS: After randomization, six women conceived before starting treatmentand another patient postponed treatment. The remaining 65 womenreceived the treatment (33 underwent LOD and 32 received CC).After the primary treatment, more pregnancies (44%) occurredin women receiving CC than in those undergoing LOD (27%), althoughthe difference did not reach statistical significance [P = 0.13,OR 2.1 (0.7 – 5.8)]. After adding the second treatment,the pregnancy rate was still higher, but to a less extent, inthe CC group [63% versus 52%, P = 0.2, OR 1.6 (0.6 – 4.2)]. CONCLUSIONS: LOD is not superior to CC as a first-line method of OI in womenwith PCOS. The trial is registered with ClinicalTrials.gov withan identifier number NCT00220545 [ClinicalTrials.gov] .