Clinical relevance of a newly developed endometrial receptivity test for patients with recurrent implantation failure in Japan.

Purpose: To assess the clinical efficacy of personalized embryo transfer (pET) guided by a new endometrial receptivity test, ERPeakSM, in patients with recurrent implantation failure (RIF). Methods: Recurrent implantation failure patients of all ages at two private Japanese clinics from April 2019 to June 2020 were retrospectively analyzed. The intervention group (n = 244) received pET in accordance with endometrial receptivity testing results and was compared to control group (n = 306) receiving standardized timing, non-personalized embryo transfer (npET). In propensity score matching analysis, the clinical pregnancy rate (CPR) and live birth rate (LBR) were compared between groups, and a subanalysis of advanced maternal age (AMA) (≥38 years old) versus non-AMA (<38 years old) patients was also conducted. Results: The CPR and LBR of the pET group were significantly higher than those of the npET group (37.7% vs. 20.0%, adjusted OR: 2.64; 95%CI, 1.70-4.11, p < 0.001 and 29.9% vs. 9.7%, adjusted OR: 4.13; 95%CI, 2.40-7.13, p < 0.001, respectively). Furthermore, in the subanalyses, the CPR and LBR of the pET group were significantly higher than those of the npET group in both the AMA non-AMA patients. Conclusions: The new ERPeakSM endometrial receptivity test is a useful alternative diagnostic tool for poor-prognosis patients, regardless of age.

Falloposcopic tuboplasty: an easy, quick, and safe technique.

OBJECTIVE: To describe our simplified technique for falloposcopic tuboplasty (FT) and demonstrate its principle and results. DESIGN: A step-by-step description of the technique and demonstration of its principle using a clay model. SETTING: Private infertility clinics in Osaka and Tokyo operated by 10 physicians. PATIENT(S): A total of 431 infertile women with a diagnosis of unilateral or bilateral proximal tubal occlusion (6 cm from the uterotubal ostia), between October 2013 and February 2019 were included. These patients underwent routine work-ups for infertility, including a semen analysis, hysterosalpingography, antimüllerian hormone, basal luteinizing hormone/follicle-stimulating hormone and prolactin concentrations during menstruation, postcoital test in the periovulatory period, and estradiol and progesterone concentrations in the middle of the luteal phase. Physicians performed hysterosalpingography to evaluate tubal patency and uterine shape. Saline infusion sonography was not conducted because it does not accurately identify regions of tubal occlusion and/or stenosis. INTERVENTION(S): The principle of our simplified technique for FT is that a hole is located at the side of the FT catheter tip. Therefore, the balloon and fiberscope move away from the catheter line (Fig. 1). The uterotubal ostium is located at the tip-end of the triangle of the uterine cavity. When a balloon is inserted while visualizing the uterotubal ostium at the nearest position to the ostium, the balloon hits the uterine wall. When a balloon is inserted 5-10 mm from the uterotubal ostium without visualization, the balloon may be easily placed in the ostium through its convex angle, allowing it to slide into the uterine wall (Figs. 2 and 3). Step 1: Confirm anteflexion or retroflexion of the uterus by ultrasound. Step 2: Confirm the direction of the uterotubal ostia by hysteroscopy. Step 3: Adjust the angle of the FT catheter according to steps 1 and 2, insert the catheter into the end of the uterus, pull it back 5-10 mm (without visualizing the uterotubal ostia), and then fix it to the forceps. Catheter placement away from the tubal ostium is confirmed by the residual length of the moving part of the catheter. An attending instructor should ask the operator about the feeling of rigidity when the catheter does not advance and then suggest whether to proceed or stop. In the latter case, the catheter is not moved, saline is infused for 1 minute for lubrication, the balloon is pulled back using the fiberscope to remove the bunching of the balloon, and balloon pressure is changed as follows: 6→8→6→10→6 mmHg. Our institutional review board stated that approval was not required because the video describes the technique of our routine procedure. MAIN OUTCOME MEASURE(S): A description of the FT technique using a clay model and a demonstration of its application in our clinic. RESULT(S): The average operative time was 15.4 minutes, and the clinical pregnancy rate was 24.4% (natural conception and intrauterine insemination without in vitro fertilization). No significant differences were observed in the operative time or pregnancy rate among physicians. Approximately 17 FT procedures may be performed using one fiberscope. CONCLUSION(S): Our simplified technique, which was described and demonstrated in this video article, is a feasible and practical approach for performing FT. It provides excellent cost performance by saving fiberscopes. The most important point is "Introduce the balloon and fiberscope 5-10 mm away from the uterotubal ostia without visualizing it." To facilitate learning this technique, we recommend watching the video and then practicing FT without searching for the uterotubal ostia. Physicians master FT without any assistance by an attending instructor in ≤3 attempts.

Chromosomal copy number analysis of products of conception by conventional karyotyping and next-generation sequencing.

PURPOSE: Chromosomal abnormalities are a major cause of spontaneous abortion, and conventional G-banded karyotyping (G-banding) is mainly utilized for chromosomal analysis. Recently, next-generation sequencing (NGS) has been introduced for chromosomal analysis. Here, we aimed to investigate the applicability and utility of NGS-based chromosomal analysis of products of conception (POC) on chorionic villus samples from spontaneous abortion. METHODS: The results of chromosomal analysis of 7 chorionic villus samples from spontaneous abortion were compared between conventional G-banding and NGS-based chromosomal copy number analysis. Age dependency and frequency of each chromosomal aneuploidy were evaluated for 279 cases analyzed by NGS. RESULTS: Excluding two cases (culture failure and maternal cell contamination), the results were consistent between G-banding and NGS. For cases analyzed by NGS, the rate of chromosomal abnormality increased in a maternal age-dependent manner. The frequency of each chromosomal aneuploidy detected by NGS was almost the same as that previously reported. Finally, NGS analysis was possible for difficult cases by G-banding analysis, such as culture failure, maternal cell contamination, long-term storage cases, and low cell number. CONCLUSIONS: Chromosome analysis using NGS not only obtains comparable results to conventional G-banding, but also can analyze POC more accurately and efficiently.

Efficacy of the endometrial receptivity array for repeated implantation failure in Japan: A retrospective, two-centers study.

Aim: This study aimed to assess the efficacy of the endometrial receptivity array (ERA) as a diagnostic tool and the impact of personalized embryo transfer (pET) for the treatment of patients with recurrent implantation failure (RIF) in Japan. Methods: Fifty patients with a history of RIF with frozen-thawed blastocyst transfers were recruited from July, 2015 to April, 2016. Endometrial sampling for the ERA and histological dating and a pET according to the ERA were performed. The receptive (R) or non-receptive (NR) status of the endometrium as a result of the first ERA, endometrial dating, and pregnancy rates after the pET were analyzed. Results: Of the patients with RIF, 12 (24%) were NR. Among them, eight (66.7%) were prereceptive. A clinical follow-up was possible in 44 patients who underwent the pET. The pregnancy rates were 58.8% per patient and 35.3% per first pET in the R patients and 50.0% per patient and 50.0% per first pET in the NR patients. Discrepancies between the ERA results and histological dating were seen more in the NR patients than in the R patients. Conclusions: For patients with unexplained RIF, there is a significance in searching for their personal window of implantation (WOI) using the ERA, considering the percentage of those who were NR and the pregnancy rates that resulted from the pET. By transferring euploid embryos in a personal WOI, much better pregnancy rates are expected.

Effects of fertility preservation in patients with breast cancer: A retrospective two-centers study.

Purpose: To assess the efficacy of fertility preservation (FP) and the impact of chemotherapy on the reproductive potential of Japanese patients with breast cancer. Methods: Sixty-two patients with breast cancer visited the authors' centers from October, 2003 to June, 2015. They were divided into two groups according to the treatment: oocyte or embryo vitrification for FP before cancer treatment (group A) or infertility treatment after cancer treatment (group B). Group B was divided into two subgroups, B1 (no chemotherapy) and B2 (postchemotherapy), in order to analyze the effect of anticancer drugs on ovarian reserves and assisted reproductive technology outcomes. The number of retrieved oocytes, vitrified oocytes or embryos, and pregnancy rates were analyzed and compared: group A compared to group B1 compared to group B2. Results: The patients in groups A and B1 underwent egg collection without any chemotherapy. The numbers of collected oocytes and vitrified embryos were significantly higher in groups A and B1 than in group B2. Nearly 50% of the in vitro fertilization patients who underwent an embryo transfer (ET) became pregnant, including two patients in group A who underwent a vitrified-warmed ET. Among the pregnant women, 70% did not have chemotherapy. Conclusion: For patients with breast cancer, FP with unfertilized oocytes or embryos before chemotherapy seems to be promising for achieving higher pregnancy rates, with no risk of minimal residual disease.

A live birth from vitrified-warmed oocytes in a Philadelphia chromosome-positive acute lymphoid leukemia patient 5 years following allogenic bone marrow transplantation and after a magnitude 9.0 earthquake in Japan.

PURPOSE: To report a live birth from vitrified-warmed oocytes for a Philadelphia chromosome-positive acute lymphoid leukemia (Ph-ALL) patient. METHODS: A 20-year-old single woman with Ph-ALL requested oocyte cryopreservation at a private fertility clinic using assisted reproduction technology (ART). In cases of leukemia, there is a very short time before chemotherapy, follwed shortly by total body irradiation (TBI), and although she had already received the chemotherapy, ten oocytes were vitrified and stored for 59 months before warming. Soon after the oocyte cryopreservation, she received TBI and bone marrow transplant (BMT). During the storage, a magnitude 9.0 earthquake occurred making oocyte transport necessary. The embryo transfer was planned in a hormone replacement cycle, and intracytoplasmic sperm injection (ICSI) was performed on the vitrified-warmed oocytes. On day 3, two embryos were transferred. RESULTS: The patient became pregnant and delivered a healthy girl after ICSI using vitrified-warmed oocytes. CONCLUSIONS: Oocyte cryopreservation is the best option for fertility preservation of young single women with leukemia. Oncologists and gynecologists who conduct ART should cooperate to improve the quality of life of cancer patients.

Potential indications for ovarian autotransplantation based on the analysis of 5,571 autopsy findings of females under the age of 40 in Japan.

Ovarian cryopreservation and autotransplantation could be of potential value for preservation of fertility in the patients with various malignancies. Ovarian tissue should be cryopreserved actively for fertility preservation, but stored tissue should be autotransplanted with much caution until reliable methods are established to detect minimal residual disease in grafts in precise and reproducible manners.

A birth of twins-one boy and one girl-from a single embryo transfer and a possible natural pregnancy.

PURPOSE: To describe a rare case of a birth of dizygotic twins with different-sex infants from a single embryo transfer. METHODS AND RESULTS: A patient, who had her right ovary and tube removed, and her husband were treated with ICSI and a single embryo transfer. When a single fresh embryo was transferred on day 4, following oocyte retrieval using GnRH agonist-long protocol, two gestational sacs were recognized at 8 weeks of gestation. Healthy twins with a boy and a girl were delivered at 37 weeks 0 days of gestation by a cesarean section. The boy's weight was 2096g, and his height was 45.0 cm, while the girl's weight was 1988g, and her height was 41.5 cm. Peripheral lymphocyte chromosome analysis of the two infants showed normal karyotype, 46, XY (boy) and 46, XX (girl). CONCLUSIONS: A single embryo transfer could produce different-sex twins.

Birth of a healthy male infant after transfer of vitrified-warmed blastocysts derived from intracytoplasmic sperm injection with vitrified-warmed oocytes and frozen-thawed spermatozoa.

PURPOSE: To report a successful delivery of a healthy baby after transfer of vitrified-warmed blastocysts derived from introcytoplasmic sperm injection (ICSI) with vitrified-warmed oocytes and frozen-thawed sperm. METHODS: A female patient and her husband with non-obstructive azoospermia received a transfer of vitrified-warmed blastocysts from vitrified-warmed oocytes and frozen-thawed sperm. The main outcome measures were fertilization, pregnancy and birth. RESULTS: Nine oocytes were matured and vitrified. When the vitrified oocytes were warmed, six survived with good quality morphology. Using ICSI, frozen-thawed sperm was injected into the six warmed oocytes that survived, and the fertilization rate was 100%. The zygotes were cultured, and five of six early embryos became blastocysts. One of them was transferred, but pregnancy was not achieved. The second time around, two vitrified-warmed blastocysts were transferred resulting in pregnancy, and a healthy boy was delivered. CONCLUSIONS: This is a rare case of a successful birth using a vitrified-warmed blastocyst grown after ICSI with a vitrified-warmed oocyte and frozen-thawed sperm.

Raloxifene increases proliferation and up-regulates telomerase activity in human umbilical vein endothelial cells.

Vascular endothelial senescence is involved in human atherosclerosis. Telomerase activity is known to be critical in cellular senescence and its level is modulated by regulation of telomerase catalytic subunit (telomerase reverse transcriptase (TERT)) at both the transcriptional and post-transcriptional levels. Since the cardioprotective effect of estrogen itself has not been ruled out, we examined that of raloxifene, which has been classified as a selective estrogen receptor modulator, on the proliferation and telomerase activity of human umbilical vein endothelial cells (HUVECs). Raloxifene, like estrogen, clearly induced the telomerase activity and human TERT (hTERT) expression via estrogen receptor (ER) alpha and ERbeta. Treatment with raloxifene for 5 days significantly induced cell growth, and either cotreatment with a telomerase inhibitor, 3'-azido-3'-deoxythymidine, or transfection with hTERT-specific small interfering RNA significantly attenuated the raloxifene-induced cell growth. Raloxifene also induced the phosphorylation of Akt, and pretreatment with a phosphatidylinositol 3-kinase inhibitor, LY294002, significantly attenuated the raloxifene-induced telomerase activity. In addition, raloxifene induced both the phosphorylation of hTERT and IkappaB. Moreover, cotreatment with an IkappaBalpha phosphorylation inhibitor, BAY-11-7082, or a specific NFkappaB nuclear translocation inhibitor, SN50, significantly attenuated the raloxifene-induced telomerase activity and the association of NFkappaB with hTERT. These results show that raloxifene induced the up-regulation of telomerase activity not only by the transcriptional regulation of hTERT but also by post-translational regulation of the phosphorylation of Akt and hTERT and the association of hTERT with NFkappaB in HUVECs. Thus, the up-regulation of telomerase activity in vascular endothelial cells might be one mechanism contributing to the potential atheroprotective effect of raloxifene.

Inhibition of phosphatidylinositol 3-kinase increases efficacy of cisplatin in in vivo ovarian cancer models.

The phosphatidylinositol 3-kinase (PI3K)/Akt cascade has an important role in the resistance of ovarian cancer cells to cisplatin in vitro; however, there have been no reports about whether blocking the PI3K/Akt cascade enhances the sensitivity to cisplatin in vivo. We investigated whether inhibition of PI3K increased the efficacy of cisplatin in an in vivo ovarian cancer model. Blocking the PI3K/Akt cascade with a PI3K inhibitor (wortmannin) increased the efficacy of cisplatin-induced inhibition of intraabdominal dissemination and production of ascites in athymic nude mice inoculated ip with the Caov-3 human ovarian cancer cell line. In addition, wortmannin increased the efficacy of cisplatin-induced apoptosis in tumors cells. There were no detectable side effects in mice treated with wortmannin. Moreover, the antitumor effect of cisplatin detected in mice inoculated with Caov-3 cells stably transfected with empty vector was significantly attenuated, compared with mice inoculated with Caov-3 cells stably transfected with a dominant-negative Akt, K179M-Akt. We confirmed that wortmannin blocked Akt phosphorylation and the downstream targets of the PI3K/Akt cascade, such as BAD (Bcl-2-associated death protein) and nuclear factor-kappaB in vivo by immunohistochemical staining and Western blotting. In accordance with the previously reported in vitro results, these in vivo results support the idea that combination therapy with cisplatin and a PI3K inhibitor would increase the therapeutic efficacy of cisplatin.

Medroxyprogesterone acetate induces cell proliferation through up-regulation of cyclin D1 expression via phosphatidylinositol 3-kinase/Akt/nuclear factor-kappaB cascade in human breast cancer cells.

The mechanism of medroxyprogesterone acetate (MPA)-induced cell proliferation in human breast cancer cells remains elusive. We examined the mechanism by which MPA affects the cyclin D1 expression in progesterone receptor (PR)-positive T47D human breast cancer cells. MPA (10 nM) treatment for 48 h induced proliferation of the cells (1.6-fold induction). MPA induced cyclin D1 expression (3.3-fold induction), and RU486, a selective PR antagonist, blocked the MPA-induced cell proliferation and cyclin D1 expression (23% inhibition). MPA increased both the protein level (2.2-fold induction) and promoter activity (2.7-fold induction) of cyclin D1 in MCF-7 cells transfected with PRB but not with PRA. Although MPA transcriptionally activated cyclin D1 expression, cyclin D1 promoter does not have progesterone-responsive element-related sequence. We further examined the mechanism for the regulation of the cyclin D1 expression. Because the cyclin D1 promoter contains three putative nuclear factor-kappaB (NFkappaB)-binding motifs and NFkappaB is a substrate of Akt, we investigated the effect of the phosphatidylinositol 3-kinase (PI3K)/Akt/NFkappaB cascade on the responses of cyclin D1 to MPA. MPA induced the transient phosphorylation of Akt (2.7-fold induction at 5 min), and treatment with PI3K inhibitor (wortmannin) attenuated the MPA-induced up-regulation of cyclin D1 expression (40% inhibition) and cell proliferation (40% inhibition). MPA also induced phosphorylation of inhibitor of NFkappaBalpha (IkappaBalpha) (2.3-fold induction), and treatment with wortmannin attenuated the MPA-induced IkappaBalpha phosphorylation (60% inhibition). Treatment with an IkappaBalpha phosphorylation inhibitor (BAY 11-7085) or a specific NFkappaB nuclear translocation inhibitor (SN-50) attenuated the MPA-induced up-regulation of both cyclin D1 expression (80 and 50% inhibition, respectively) and cell proliferation (55 and 34% inhibition, respectively). Because MPA induced a transient phosphorylation of Akt and the cyclin D1 promoter contains no progesterone-responsive element-related sequence, the MPA-induced cell proliferation through PRB by up-regulation of cyclin D1 expression via the PI3K/Akt/NFkappaB cascade may be a nongenomic mechanism.

Growth factors change nuclear distribution of estrogen receptor-alpha via mitogen-activated protein kinase or phosphatidylinositol 3-kinase cascade in a human breast cancer cell line.

In the present study, to examine the dynamic changes in the localization of nuclear estrogen receptor (ER)alpha induced by growth factors, we used time-lapse confocal microscopy to directly visualized ERalpha fused with green fluorescent protein (GFP-ERalpha) in single living cells treated with epidermal growth factor (EGF) or IGF-I. We observed that 17beta-estradiol (E2) changed the normally diffuse distribution of GFP-ERalpha throughout the nucleoplasm to a hyperspeckled distribution within 10 min. Both EGF and IGF-I also changed the nuclear distribution of GFP-ERalpha, similarly to E2 treatment. However, the time courses of the nuclear redistribution of GFP-ERalpha induced by EGF or IGF-I were different from that induced by E2 treatment. In the EGF-treated cells, the GFP-ERalpha nuclear redistribution was observed at 30 min and reached a maximum at 60 min, whereas in the IGF-I-treated cells, the GFP-ERalpha nuclear redistribution was observed at 60 min and reached a maximum at 90 min. The EGF-induced redistribution of GFP-ERalpha was blocked by pretreatment with a MAPK cascade inhibitor, PD98059, whereas the IGF-I-induced redistribution of GFP-ERalpha was blocked by pretreatment with a phosphatidylinositol 3-kinase inhibitor, LY294002. Analysis using an activation function-2 domain deletion mutant of GFP-ERalpha showed that the change in the distribution of GFP-ERalpha was not induced by E2, EGF, or IGF-I treatment. These data suggest that MAPK and phosphatidylinositol 3-kinase cascades are involved in the nuclear redistribution of ERalpha by EGF and IGF-I, respectively, and that the activation function-2 domain of ERalpha may be needed for the nuclear redistribution of ERalpha.

Both estrogen and raloxifene protect against beta-amyloid-induced neurotoxicity in estrogen receptor alpha-transfected PC12 cells by activation of telomerase activity via Akt cascade.

Although estrogen is known to protect against beta-amyloid (Abeta)-induced neurotoxicity, the mechanisms responsible for this effect are only beginning to be elucidated. In addition, the effect of raloxifene on Abeta-induced neuro-toxicity remains unknown. Here we investigated whether raloxifene exhibits similar neuro-protective effects to estrogen against Abeta-induced neurotoxicity and the mechanism of the effects of these agents in PC12 cells transfected with the full-length human estrogen receptor (ER) alpha gene (PCER). Raloxifene, like 17beta-estradiol (E2), significantly inhibited Abeta-induced apoptosis in PCER cells, but not in a control line of cells transfected with vector DNA alone (PCCON). Since telomerase activity, the level of which is modulated by regulation of telomerase catalytic subunit (TERT) at both the transcriptional and post-transcriptional levels, is known to be involved in suppressing apoptosis in neurons, we examined the effect of E2 and raloxifene on telomerase activity. Although both E2 and raloxifene induced telomerase activity in PCER cells, but not in PCCON cells, treated with Abeta, they had no effect on the level of TERT expression. These results suggest that neither E2 nor raloxifene affects the telomerase activity at the transcriptional level. We therefore studied the mechanism by which E2 and raloxifene induce the telomerase activity at the post-transcriptional level. Both E2 and raloxifene induced the phosphorylation of Akt, and pre-treatment with a phosphatidylinositol 3-kinase inhibitor, LY294002, attenuated both E2- and raloxifene-induced activation of the telomerase activity. Moreover, both E2 and raloxifene induced both the phosphorylation of TERT at a putative Akt phosphorylation site and the association of nuclear factor kappaB with TERT. Our findings suggest that and raloxifene exert neuroprotective effects by E2 telomerase activation via a post-transcriptional cascade in an experimental model relevant to Alzheimer's disease.

Lowering intracellular and extracellular calcium contents prevents cytotoxic effects of ethylene glycol-based vitrification solution in unfertilized mouse oocytes.

We investigated the characteristics of the changes in intracellular calcium (Ca2+) concentration ([Ca2+](i)) and the viability of the unfertilized mouse oocytes exposed to various concentrations of ethylene glycol (EG)-containing solutions or vitrification solutions. Oocytes exposed to EG (1, 5, 10, 20, and 40% (v/v)) exhibited a rapid and dose-dependent increase in [Ca2+](i). The survival rate was 100% when oocytes were exposed to the EG concentration up to 5% through 5 min, while all oocytes were dead within 3 min when exposed to 10, 20, or 40% EG. When extracellular Ca2+ was removed, increase in [Ca2+](i) at 10 and 20% EG was less than that at the same concentrations of EG with extracellular Ca2+. The survival rates of the oocytes exposed to 10, 20, and 40% EG at 3 min were 100, 97, and 0%, respectively. In the presence of 20 microM 1,2-bis(o-aminopheoxy)ethane-N,N,N',N'-tetraacetic acid tetra acetoxymethyl ester (BAPTA-AM), a Ca2+ chelator, a small increase in [Ca2+](i) exposed to 10, 20, and 40% EG was observed until 4 min. Subsequently prolonged elevation of the [Ca2+](i) was observed in the oocytes exposed to 40% EG but not with 10 and 20% EG. The survival rate of the oocytes, in the presence of 20 microM BAPTA-AM, exposed to 10 and 20% EG was 100% throughout 5 min, while the oocytes exposed to 40% EG were alive only for 3 min. Treatment by the vitrification solution with various concentrations of EG (10, 20, and 40%) caused a smaller increase in [Ca2+](i), while the survival rates were higher compared to those without vitrification solution at the same concentrations of EG. These data suggested that the sustained [Ca2+](i) rises by EG in unfertilized mouse oocytes resulted in cell death. Therefore, the lowering of [Ca2+](i) in the oocytes exposed to the cryoprotectant may improve the viability of cryopreserved unfertilized oocytes.