Analysis of the risk factors and treatment for repeated implantation failure: OPtimization of Thyroid function, IMmunity, and Uterine Milieu (OPTIMUM) treatment strategy.

PROBLEM: What are the pregnancy outcomes after the OPtimization of Thyroid function, Immunity, and Uterine Milieu (OPTIMUM) treatment strategy in patients with repeated implantation failure (RIF)? METHOD OF STUDY: Infertile women with a history of RIF after more than three embryo transfer (ET) cycles underwent implantation testing, including a hysteroscopy, endometrial biopsy for CD138 immunostaining and bacterial culture, and serum 25-hydroxyvitamin D3 , interferon-γ-producing helper T (Th1) cell, IL-4-producing helper T (Th2) cell, thyroid-stimulating hormone, thyroid peroxidase antibody, and thrombophilia screening between April 2017 and August 2018. We treated chronic endometritis with antibiotics, aberrant high Th1/Th2 cell ratios with vitamin D and/or tacrolimus intake, overt/subclinical hypothyroidism with levothyroxine, and thrombophilia with low-dose aspirin. Of the 116 RIF women, 88 women with 133 ET cycles were recruited from a questionnaire-based survey regarding pregnancy outcomes. Fifty-nine consecutive RIF patients without the OPTIMUM treatment strategy were also recruited as a control. RESULTS: The 116 women with RIF after the OPTIMUM treatment strategy were 38.3 ± 3.8 years old and had an implantation failure history over 5 (3-19) ET cycles. Implantation testing identified impaired intrauterine circumstances in 75 women (64.7%), an aberrant elevated Th1/Th2 cell ratio in 56 women (48.3%), and thyroid abnormalities in 33 women (28.4%). Cumulative ongoing pregnancy rates including spontaneous pregnancy in the patients aged < 40 and ≥ 40 years were 72.7% and 45.5% within two ET cycles, respectively. The pregnancy outcomes in the OPTIMUM group were significantly higher than those in the control. CONCLUSIONS: The OPTIMUM treatment strategy improved pregnancy outcomes in patients with RIF.

A Novel Treatment for Cervical and Cesarean Section Scar Pregnancies by Transvaginal Injection of Absolute Ethanol to Trophoblasts: Efficacy in 19 Cases.

STUDY OBJECTIVE: To evaluate the efficacy of a nonsurgical treatment for cervical pregnancy (CP) and cesarean section scar pregnancy (CSP). DESIGN: Retrospective clinical study (Canadian Task Force classification III). SETTING: Private assisted reproductive technology practice. PATIENTS: Nineteen women with CP (n = 16) or CSP (n = 3), including 6 patients with positive fetal heartbeat. INTERVENTION: Transvaginal local injection of absolute ethanol (AE) into the hyperechoic ring (lacunar space) around the gestational sac under ultrasound guidance. MEASUREMENTS AND MAIN RESULTS: Serum beta-human chorionic gonadotropin (ß-hCG) was measured at frequent intervals, and ultrasound and/or magnetic resonance imaging was used to observe the gestational sac. In 9 patients, the serum ß-hCG level was effectively reduced with a single AE injection at 2 hours. In the remaining 10 patients, the level decreased but then increased in 4 and slowly decreased in the other 6; all of these 10 patients required 2 to 5 repeat AE injections. In all patients, serum ß-hCG level was reduced by 50% within 3 days and decreased to <10% of the initial level within 14 days. In 18 patients (95%), the level was decreased to 1.0 mIU/mL within 40 days. Seven patients were treated on an outpatient basis. Twelve patients received no anesthesia. Five patients subsequently became pregnant, and each had a live birth. There was no recurrent CP or CSP. The procedure was successful in all 19 patients. CONCLUSION: This procedure is an effective treatment for CP or CSP that could be used in place of conventional surgical interventions and medical treatment using MTX.

High serum progesterone associated with infertility in a woman with nonclassic congenital adrenal hyperplasia.

Nonclassic congenital adrenal hyperplasia (NCAH) is an autosomal-recessive disorder caused by 21-hydroxylase deficiency and manifests as hirsutism and oligomenorrhea due to excess adrenal androgen and progesterone. We report a case of a woman with NCAH who showed continuous high serum progesterone levels in the follicular phase associated with impaired folliculogenesis. NCAH was diagnosed based on high 17-hydroxyprogesterone levels after rapid adrenocorticotropic hormone loading, and three heterozygous missense mutations in CYP21A2, encoding 21-hydroxylase, were identified. Recurrent failure of in vitro fertilization programs occurred because of empty follicles and preterm rupture of leading follicles, and vaginal stenosis with a hypoplastic cervix. Glucocorticoid administration normalized serum progesterone levels, restored folliculogenesis, and stretched the vaginal wall, which contributed to the success of the in vitro fertilization program. The patient delivered at term following blastocyst transfer. Correction of hyperandrogenism and high progesterone levels using glucocorticoids may improve fertility in women with NCAH.

Cytogenetic analysis of the retained products of conception after missed abortion following blastocyst transfer: a retrospective, large-scale, single-centre study.

Cytogenetic analysis of the retained products of conception (POC) is the most effective test for identifying miscarriage causes. However, there has been no large-scale study limited to blastocyst transfer. This study retrospectively reports the findings of 1030 cases in which POC analysis was performed after missed abortion following single blastocyst transfer performed at the Shinbashi Yume Clinic. We identified 19.4% as normal karyotypes and 80.6% as aneuploid. These cases broke down into: 62.3% trisomy; 7.8% double trisomy; 0.5% triple or quadruple trisomy; 1.3% monosomy 21; 3.2% monosomy X; 0.1% 47,XXY; 1.0% polyploidy; 1.0% mixed; 1.1% embryonic mosaicism; and 2.4% structural anomalies. In samples with normal karyotypes, 49.5% were female while 50.5% were male. The occurrence of trisomy and double trisomy were both significantly more frequent in the ≥38 years group than in the ≤37 years group (P < 0.01). Trisomy was significantly more frequently associated with fetal heartbeat (P < 0.01); double trisomy, polyploidy and normal karyotype were significantly more frequent with no fetal heartbeat (P < 0.01). There was no significant difference in the frequency of chromosomal abnormalities between the number of miscarriages or blastocyst quality. Thus, POC cytogenetic testing is highly valuable for ascertaining the cause of miscarriage.

Changes in estrone and estradiol levels during follicle development: a retrospective large-scale study.

BACKGROUND: The improved reagent for measuring estradiol (E2), the ST AIA-PACK iE2 reagent, has a higher specificity for the measurement of E2 levels than the original ST AIA-PACK E2 reagent, because of its lower cross-reactivity with estrone (E1). As we had E2 data obtained with either of the reagents, we analyzed changes in E1 and E2 levels during follicle development. METHODS: The study included 14371 serum hormone measurements from 4412 patients who underwent oocyte retrieval or frozen/thawed embryo transfer in natural cycle in vitro fertilization in Shinbashi YUME clinic, Tokyo, between June 2011 and May 2014. The age of the patients ranged from 24 to 48 year (mean and standard deviation, 39.8 ± 4.0 year). Patients were categorized into three age groups (<38 year, 38-40 year, and >40 year) and into 10 groups of largest follicle diameter from 11 to 20 mm, with 1-mm intervals. Serum E2 levels were measured in the follicular phase with either the ST AIA-PACK E2 reagent or the ST AIA-PACK iE2 reagent, and the data were compared. Also, for 26 randomly selected samples, E2 was measured using both reagents, together with E1 and E3, and the E1/E2 ratios were compared. RESULTS: E2 concentrations measured with the ST AIA-PACK iE2 reagent were significantly lower than those measured with the ST AIA-PACK E2 reagent in the largest follicle diameter category of 11-17 mm in the <38 year group, in the largest follicle diameter category of 11-18 mm in the 38-40 year group, and in the largest follicle diameter category of 11-15 mm in the >40 year group. The serum E1/E2 ratio in the 26 samples was 3.4 ± 1.1 and 0.7 ± 0.1 in the early follicular phase and in the ovulatory phase, respectively. CONCLUSIONS: The difference between the E2 concentrations measured with the ST AIA-PACK E2 reagent and the ST AIA-PACK iE2 reagent tended to decrease as the follicle diameter increased, particularly in the older patients, which suggests E1 secretion is more abundant in the early follicular phase and in younger patients than in the ovulatory phase and in older patients.

The efficacy and safety of managing ectopic pregnancies with transvaginal ultrasound-guided local injections of absolute ethanol.

PURPOSE: To describe the efficacy and safety of managing ectopic pregnancies (EP) with ultrasound-guided local injections of absolute ethanol (AE). METHODS: 69 cases of EP following IVF performed in our clinic were treated with a local injection of 0.3 ml AE with a 23-gauge needle under transvaginal ultrasonic guidance. The efficacy was evaluated comparing serum beta-human chorionic gonadotropin (beta-hCG) levels before and after the injection. RESULTS: In the 60 successful cases (87%), the serum beta-hCG level decreased by 10-30% in two hours postinjection. Of these, 46 were effective with a single injection and the half-life of beta-hCG was achieved within 4 days in 45 cases. In 56 cases (including repetitive administration) serum beta-hCG levels decreased to 20 mIU/mL within 20 days. The treatment showed no side effects and could be given on an outpatient basis without anesthesia. CONCLUSIONS: This method was shown to be a safe, effective new approach to treating EP.

Aromatase expression in stromal cells of endometrioid endometrial cancer correlates with poor survival.

PURPOSE AND EXPERIMENTAL DESIGN: To assess the prognostic significance of intratumoral aromatase in endometrioid endometrial cancer, sections from 55 patients with endometrial cancer were evaluated for expression of aromatase using immunohistochemistry, and the correlation between aromatase expression and clinicopathologic parameters were analyzed. RESULTS: Immunohistochemical staining for aromatase was positive for 32 (58%), 20 (36%), and 19 (34%) patients in cancer epithelial cells, stromal cells, and myometrial cells around the flank invasion, respectively. In situ hybridization also detected aromatase mRNA in all three types of cells. RT-PCR analysis revealed that aromatase mRNA was 2.5 +/- 1.0 amol/mug total RNA (mean +/- SE; n = 7) in tumor tissue. Western blot analysis detected the expected aromatase protein size of 58 kDa in cancer tissues more abundantly than in cancer-free endometrium (n = 3). The immunoreactivity in stromal cells correlated positively with advanced surgical stage and poor survival. Survival analysis revealed that the immunoreactivity of stromal cells was a significant prognostic factor, independent of histologic grade, muscular invasion, and lymph node metastasis, but dependent on surgical stage. By contrast, the immunoreactivity of aromatase both in cancer epithelial cells and myometrial cells did not correlate with prognosis. CONCLUSIONS: To the best of our knowledge, this is the first evidence associating intratumoral aromatase expression in stromal cells and poor survival in endometrioid endometrial cancer. This positive linkage indicates that local expression of aromatase plays a role in tumor progression through the formation of in situ estrogens. In situ expression of aromatase may offer a potential target for management of endometrial cancers.

Increased expression of type I 17beta-hydroxysteroid dehydrogenase enhances in situ production of estradiol in uterine leiomyoma.

Expression of 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) was compared between leiomyoma and myometrium. Cytosolic fractions from leiomyoma homogenate displayed 5-fold higher activity (estrone to estradiol), compared with surrounding myometrium (n = 6, P < 0.05), whereas microsomal fractions showed no difference. Oxidative activity (estradiol to estrone) did not differ between leiomyoma and myometrium. Levels of mRNA for 17beta-HSDs were then measured using real-time PCR techniques. Among the eight different types of 17beta-HSDs (types 1-5, 7, 8, and 10), type 1 was the only enzyme displaying differential expression between leiomyoma and myometrium. Mean concentration of type 1 17beta-HSD mRNA was 4-fold higher in leiomyoma than in surrounding myometrium (n = 20, P < 0.05). Type 1 transcript levels correlated significantly with reductive activity in individual samples (n = 6, P < 0.05). Northern blot analysis of leiomyoma and myometrium tissues detected 2.3- and 1.0-kb transcripts of type 1 enzyme, whereas the major 1.3-kb transcript for 17beta-HSD in placenta-derived JEG-3 cells was not detected. None of the factors increasing mRNA levels for type 1 enzyme in placenta increased mRNA levels in leiomyoma. These results indicate that leiomyoma tissues overexpress type 1 17beta-HSD, resulting in high conversion of estrone to estradiol. In situ expression of type 1 17beta-HSD may play a role in self-supported growth of leiomyoma cells.

Decreased expression of early growth response-1 and its role in uterine leiomyoma growth.

Expression of early growth response (Egr)-1, a transcriptional factor implicated in growth regulation, is suppressed in several malignant tumors. The present study investigated the expression of Egr-1 and related genes in uterine leiomyoma and normal myometrium to determine possible contributions of Egr-1 to neoplastic growth in leiomyoma cells. Levels of Egr-1 transcripts were decreased in all leiomyomas (n = 20) to approximately 10% of levels in corresponding myometrium, where basal expression was high. Preoperative leuprorelin acetate therapy increased levels of Egr-1 mRNA in normal myometrium only. Northern blot analysis using additional sample sets (n = 5) revealed the full-length Egr-1 transcript. Western blot analysis (n = 5) confirmed decreased expression of Egr-1 protein. Southern blot analysis of the Egr-1 gene and microsatellite analysis of the chromosomal location at 5q31 (D5S414, D5S500, and D5S476) revealed neither DNA recombination nor loss of heterozygosity in leiomyomas. Moreover, Egr-1 retained identical responsiveness to phorbol 12-myristate 13-acetate in primary cultures derived from both leiomyoma and normal tissues. Electrophoretic mobility shift analysis revealed that phorbol 12-myristate 13-acetate-induced Egr-1 in leiomyoma cells retained DNA binding ability. Egr-1 thus appears functionally intact in leiomyoma cells. Finally, consistent with the role of Egr-1 in growth inhibition, transfection of Egr-1 expression vector into a myometrial cell line (KW) that expresses low levels of Egr-1 and displays rapid growth inhibited thymidine uptake in these cells. Egr-1 may display tumor-suppressing activity and offers a potential target for leiomyoma management.

Successful treatment of a symptomatic uterine leiomyoma in a perimenopausal woman with a nonsteroidal aromatase inhibitor.

OBJECTIVE: To assess the management of symptomatic leiomyomas using a nonsteroidal aromatase inhibitor in perimenopausal women. DESIGN: Case report. SETTING: Academic clinical practice. PATIENT(S): A 53-year-old woman suffering from recurrent urinary retention secondary to a uterine leiomyoma. INTERVENTION(S): Fadrozole, orally, 2 mg daily for 8 weeks and then 1 mg daily for 4 weeks. MAIN OUTCOME MEASURE(S): Measurements of leiomyoma volume, and levels of serum E(2), LH, and FSH. RESULT(S): Urinary retention resolved after 2 weeks of treatment and did not recur. Leiomyoma volume estimated by ultrasonography revealed a 71% reduction after 8 weeks of treatment. CONCLUSION(S): Fadrozole was useful for the management of a symptomatic leiomyoma without transient deterioration of symptoms. Clinical trials are warranted.

Spatially heterogeneous expression of aromatase P450 through promoter II is closely correlated with the level of steroidogenic factor-1 transcript in endometrioma tissues.

Endometriosis is an estrogen-dependent disease of women of reproductive age. Recent studies demonstrate that endometriosis per se express high levels of estrogen synthetase (aromatase P450). The resulting estrogen synthesized in situ may play a role in the development and exacerbation of the disease. For ovarian endometrioma, previous studies have been conducted ex vivo using cells obtained from endometrioma and have demonstrated that steroidogenic factor-1 is involved in the expression of aromatase. The aim of the present study was to provide in vivo evidence that steroidogenic factor-1 plays an important role in the regulation and overexpression of aromatase P450 in situ. First, promoter use of aromatase P450 in endometrioma tissue was determined using quantitative methods. Ovarian endometrioma tissue was chopped into small pieces, and two exon 1-specific transcripts of aromatase P450 (PII-specific and I.4-specific transcripts) were quantified using competitive RT-PCR. PII-specific transcript was more abundant than the I.4-specific transcript in 13 of the 15 endometriomas and less abundant in the remaining two. Spatial distribution of aromatase P450 transcripts in these endometrioma tissues revealed heterogeneous expression in the cyst wall, demonstrating wide variability even in the same endometrioma. Two possible regulators of aromatase expression (steroidogenic factor-1 and IL-1 beta) were then measured in all endometrioma samples and the correlation between aromatase P450 transcripts and these possible regulators in the endometrioma samples were tested using Spearman's rank order correlation test. Levels of steroidogenic factor-1 transcript were found to correlate closely with levels of PII-specific transcript in eight of nine endometriomas examined. On the other hand, the level of IL-1 beta weakly correlated with I.4-specific transcripts in three of the nine endometriomas. We next histologically examined samples of four endometriomas in which complete sets of tissue samples corresponded to the RNA samples. We could not identify any specific pathology to explain the heterogeneous expression of PII-specific transcripts of aromatase P450, although the number of CD-68 positive macrophages in the tissue sections weakly correlated with the level of I.4-specific transcript in two of four endometriomas. These results provide strong evidence that promoter II is the predominant promoter of aromatase P450 in endometrioma tissues in vivo and that steroidogenic factor-1 in situ is a major determinant of aromatase P450 overexpression in endometrioma tissues in vivo.

Overexpression of aromatase P450 in leiomyoma tissue is driven primarily through promoter I.4 of the aromatase P450 gene (CYP19).

The CYP19 gene encoding aromatase P450 (estrogen synthetase) is expressed in several extragonadal sites and regulated in a tissue-specific fashion, which is achieved by alternative use of the seven different promoters (and corresponding exons 1) of the CYP19 gene. Previously, we demonstrated that aromatase P450 is overexpressed in leiomyoma tissue and that in situ estrogen synthesized in leiomyoma tissues possibly plays a role in leiomyoma growth. To elucidate the mechanism of overexpression of aromatase P450, we determined the promoter use of aromatase P450 in leiomyomas. 5'-Rapid amplification of cDNA ends analysis revealed that of six leiomyoma nodules tested, four nodules contained I.4-specific transcript of aromatase P450 alone, one nodule contained PII-specific transcript alone, and the remaining nodule contained both I.4- and PII-specific transcripts simultaneously. The levels of aromatase transcripts were then quantified by competitive RT-PCR assay. Among 21 leiomyomas, I.4-specific transcript and PII-specific transcript were predominant in 18 and 2 leiomyomas, respectively, whereas the remaining leiomyoma was negative for aromatase P450 expression. We next compared the aromatase activity of leiomyoma cells stimulated by promoter-specific regulatory factors. A combination of IL-1beta and dexamethasone, known as a potent inducer of promoter I.4-driven transcription, effectively increased aromatase activity. A combination of (Bt)(2)cAMP, 3-isobutyl-1-myethylxanthine, and PGE(2), known as inducers of promoter II-driven transcription, also increased aromatase activity, but the increases found were smaller than that induced by dexamethasone and IL-1beta. The transcriptional ability of the promoter I.4 sequence was confirmed by transient transfection assay using primary cells released from leiomyomas and established cells from normal myometrium (KW cells). Luciferase vectors containing promoter I.4 sequence (-340/+14 or longer) showed a significant increase in luciferase activity in response to dexamethasone. Deletion or mutation of a putative glucocorticoid-responsive element in the promoter I.4 sequence eliminated promoter activity. These results indicate that promoter I.4 is the major promoter responsible for overexpression of aromatase P450 in leiomyomas and that a glucocorticoid-responsive element within it plays a substantial role in the expression of aromatase P450.