Direct Effects of Mifepristone on Mice Embryogenesis: An In Vitro Evaluation by Single-Embryo RNA Sequencing Analysis.

The clinical use of mifepristone for medical abortions has been established in 1987 in France and since 2000 in the United States. Mifepristone has a limited medical period that lasts <9 weeks of gestation, and the incidence of mifepristone treatment failure increases with gestation time. Mifepristone functions as an antagonist for progesterone and glucocorticoid receptors. Studies have confirmed that mifepristone treatments can directly contribute to endometrium disability by interfering with the endometrial receptivity of the embryo, thus causing decidual endometrial degeneration. However, whether mifepristone efficacy directly affects embryo survival and growth is still an open question. Some women choose to continue their pregnancy after mifepristone treatment fails, and some women express regret and seek medically unapproved mifepristone antagonization with high doses of progesterone. These unapproved treatments raise the potential risk of embryonic fatality and developmental anomalies. Accordingly, in the present study, we collected mouse blastocysts ex vivo and treated implanted blastocysts with mifepristone for 24 h. The embryos were further cultured to day 8 in vitro to finish their growth in the early somite stage, and the embryos were then collected for RNA sequencing (control n = 3, mifepristone n = 3). When we performed a gene set enrichment analysis, our data indicated that mifepristone treatment considerably altered the cellular pathways of embryos in terms of viability, proliferation, and development. The data indicated that mifepristone was involved in hallmark gene sets of protein secretion, mTORC1, fatty acid metabolism, IL-2-STAT5 signaling, adipogenesis, peroxisome, glycolysis, E2F targets, and heme metabolism. The data further revealed that mifepristone interfered with normal embryonic development. In sum, our data suggest that continuing a pregnancy after mifepristone treatment fails is inappropriate and infeasible. The results of our study reveal a high risk of fetus fatality and developmental problems when pregnancies are continued after mifepristone treatment fails.

High levels estradiol affect blastocyst implantation and post-implantation development directly in mice.

BACKGROUND: Previous studies have demonstrated that high levels of estradiol (E2) impair blastocyst implantation through effects on the endometrium; however, whether high E2 directly affects blastocysts is not well established. The present study sought to clarify the direct impacts of high E2 levels on blastocysts in vitro. METHODS: ICR virgin albino mice were used. Using an in-vitro 8-day blastocyst culture model, immunofluorescence staining for the estrogen receptor (ER), blastocyst outgrowth assays, differential staining and TUNEL assays of blastocysts, and embryo transfer, we investigated the main outcomes of exposure to different E2 concentrations (10-7 to 10-4 M) in vitro and in vivo. RESULTS: ERα and ERß expression were detected in pre-implantation stage embryos. In vitro exposure of blastocysts to 10-4 M E2 for 24 h followed by 7 days culture in the absence of E2 caused severe inhibition of implantation and post-implantation development. The late adverse effects of E2 on post-implantation development still occurred at concentrations of 10-7 to 10-5 M. In addition, blastocyst proliferation was reduced and apoptotic cells were increased following exposure to 10-4 M E2. Using an in vivo embryo-transfer model, we also showed that treatment with high E2 resulted in fewer implantation sites (38% vs. 72% in control) and greater resorption of implanted blastocysts (81% vs. 38% in control). CONCLUSION: Exposure to high E2 concentrations in vitro is deleterious to blastocyst implantation and early post-implantation development, mainly owing to direct impacts of E2 on implanting blastocysts. In clinical assisted reproductive technique (ART), high serum E2 concentrations not only affects the endometrium, but also affects blastocysts directly at the period of implantation.

Disposition of embryos from women who only produced morphologically poor embryos on day three.

BACKGROUND: The presence of only morphologically poor embryos (MPEs) on day3 is common in autologous in vitro fertilization (IVF), particularly among p Tel: 886-7-7317123 Ext. 8916. Fax: 886-7-7322915.atients who have advanced maternal age or are poor responders. However, there are limited data regarding the disposition of embryos from patients who only produced MPEs on day3. The present study was designed to investigate the possible benefits of extended culturing MPEs. Try to detect whether the extended culture (day4 or day5 culture) can improve the live birth rate per cycle? METHODS: This retrospective, observational, single-center, cohort study examined 224 IVF/intracytoplasmic sperm injection (ICSI) cycles between January 2010 and June 2015, in which women only produced MPEs on day3. A total of 544 MPEs were analyzed. The defines a day3 embryo as an MPE if it fails to develop to eight cells, blastomeres of equal size, and less than 20% cytoplasmic fragments. Of the 224 cycles, 89 (39.7%) underwent fresh embryo transfer on day3, and 135 (60.3%) underwent extended culture. Of the 135 extended cultures, 54 cycles (40.0%) experienced day4, or day5 embryo transfer, 16 cycles (11.9%) had all embryos frozen, and 65 cycles (48.1%) had total embryo arrest. RESULTS: Analysis of patient baseline demographic data, cycle characteristics, and cycle outcomes for day3 transfer group and extended culture group indicated that a higher body mass index in the day3 transfer group was the only significant difference (p = 0.006). Both fresh transfer groups had low live birth rates (LBRs) (4.5% vs. 7.4% p = 0.46). After extended culture, 65 cycles (48.1%) were cancelled because the embryos exhibited developmental arrest and 70 cycles (51.9%) grew to day4 or day5. Thirteen frozen embryo transfer (FET) cycles and 22 frozen blastocysts derived from MPEs were thawed. There were more high-quality embryos (p < 0.001), higher implantation rates (IRs) (p = 0.038), and higher LBRs (p = 0.042) for embryos that underwent FET cycles. MPES in extended culture transfer have favorable survival than MPES in day3 transfer. CONCLUSION: The extended culture of MPEs in fresh transfer cycles did not increase the LBR. However, younger females with the extended culture of MPEs followed by FET resulted in significantly higher LBRs and may be a feasible strategy to improve outcomes for patients with poor embryo quality. However, day3 embryo transfer may be a better choice if a fresh transfer is unrestricted and avoid the cycle cancellation. Extended culture may decrease to the transfer of developmental potential arrest embryos to patients.

Non-embryotoxic dosage of alternariol aggravates ochratoxin A-triggered deleterious effects on embryonic development through ROS-dependent apoptotic processes.

Alternariol (AOH) and ochratoxin A (OTA), two mycotoxins found in many foods worldwide, exhibit cytotoxicity and embryotoxicity, triggering apoptosis and cell cycle arrest in several mammalian cells and mouse embryos. The absorption rate of AOH from dietary foodstuff is low, meaning that the amount of AOH obtained from the diet rarely approaches the cytotoxic threshold. Thus, the potential harm of dietary consumption of AOH is generally neglected. However, previous findings from our group and others led us to question whether a low dosage of AOH could aggravate the cytotoxicity of other mycotoxins. In the present study, we examined how low dosages of AOH affected OTA-triggered apoptosis and embryotoxicity and investigated the underlying regulatory mechanism in mouse blastocysts. Our results revealed that non-cytotoxic concentrations of AOH (1 and 2 µM) could enhance OTA (8 µM)-triggered apoptotic processes and embryotoxicity in mouse blastocysts. We also found that AOH can enhance OTA-evoked intracellular reactive oxygen species (ROS) generation and that this could be prevented by pretreatment with the potent ROS scavenger, N-acetylcysteine. Finally, we observed that this ROS generation acts as a key inducer of caspase-dependent apoptotic processes and subsequent impairments of embryo implantation and pre- and post-implantation embryonic development. In sum, our results show that non-cytotoxic dosages of AOH can aggravate OTA-triggered apoptosis and embryotoxicity through ROS- and caspase-dependent signaling pathways.

Mifepristone Directly Disrupts Mouse Embryonic Development in Terms of Cellular Proliferation and Maturation In Vitro.

Mifepristone (RU-486), a synthetic steroid with potent antiprogestogen and anti-glucocorticoid properties, has been widely used in clinical practice. Its effect on the endometrium, ovary, and fallopian tube has been well reported in many human and animal studies. However, its direct impact on post-implantation embryos remains underexplored. Additionally, some women choose to keep their pregnancy after mifepristone treatment fails. Thus, the potential risk remains controversial. Hence, this study investigated the direct effects of mifepristone on the development of mice blastocysts in vitro in terms of implantation and post-implantation. We detected the level of progesterone (P4) associated with ovulation in vivo. The presence of progesterone receptors (PRs) in blastocysts and post-implantation embryos was also evaluated. Cultured embryos were treated directly with mifepristone. We further examined embryonic implantation and post-implantation of blastocysts in vitro to evaluate the direct effects of mifepristone on embryos by the assessment of embryonic outgrowth and differential cell staining. In the oviduct lumen, the P4 level dramatically increased at 48 h and slightly decreased at 72 and 96 h following ovulation. PR was expressed in blastocysts not only in the preimplantation stage but also in the early post-implantation period. In the evaluation of developmental stages, mifepristone significantly reduced the successful ratio of developing into the late egg cylinder and the early somite stage. In addition, it further decreased the cell number of the embryos' inner cell mass and trophectoderm. We herein provide evidence that mifepristone affects blastocyst viability directly and inhibits post-implantation embryo development in vitro. Furthermore, our data reveal a potential risk of fetus fatality and developmental problems when pregnancies are continued after mifepristone treatment fails.

Monopronucleated (1PN) and tripronuclear (3PN) zygotes formation during assisted reproduction in POSEIDON group 4 patients: Emphasizing on polar bodies.

AIM: Abnormal fertilization (1PN/3PN) and its accompanying polar body (PB) conditions have been less discussed in poor ovarian responders. By observing the PBs, we analyzed the mechanisms of abnormal fertilization and aimed to explore the role of intracytoplasmic sperm injection/in vitro fertilization (ICSI/IVF) in POSEIDON group 4 patients. METHODS: An observational study. All fresh IVF/ICSI cycles from January 2018 to December 2019 were evaluated. The inclusion criteria were POSEIDON group 4. Fertilization and PB conditions were assessed 16-18 h post-insemination. Primary observation endpoints including normal fertilization, abnormal fertilization, and total fertilization failure rate. RESULTS: A total of 351 cycles involving 180 patients met the inclusion criteria. Of these, 15 cycles reported no retrieved oocytes. Finally, 336 cycles (IVF, n = 267; ICSI, n = 69) were included. A total of 1005 oocytes and 939 embryos were assessed. The mean female age was 40.8 years, and the mean AMH level was 0.6 ng/mL. The normal fertilization rate was 69.7%. The zygote distribution was 18.7% 0PN, 3.9% 1PN, 66.9% 2PN, 9.5% 3PN, and 1.0% ≥4PN. For 1PN zygotes, 59% were denoted as 1PN2PB. The mean 3PN rate was 8.9%. CONCLUSIONS: In POSEIDON group 4, most of the monopronucleated zygotes were 1PN2PB. Digyny (3PN1PB), due to failure to extrude the second PB, was the major cause of triploidy in which ICSI could not circumvent. The distribution of abnormally fertilized zygotes was similar in IVF and ICSI. To investigate the mechanisms of abnormal fertilization and assess whether ICSI is necessary, analysis of PB will provide important clues.

Risk of major adverse cardiovascular and cerebrovascular events in Taiwanese women with endometriosis.

BACKGROUND/PURPOSE: Endometriosis (EM) is linked to cardiovascular disease (CVD). However, whether this finding can be applied to the Taiwanese population remained unanswered. To investigate the association between EM and major adverse cardiovascular and cerebrovascular events (MACCE) and the therapeutic effect on the risk of MACCE in Asian women with EM. A retrospective population-based cohort study was performed. METHODS: A total of 17 543 patients with EM aged between 18 and 50 years were identified from a general population of 1 million Taiwanese after excluding diagnoses of major CVD and cerebrovascular accident (CVA) prior to EM. The comparison group (n = 70 172) without EM was selected by matching the study cohort with age, sex, and income and urbanization levels in a 4:1 ratio. RESULTS: During a median follow-up period of 9.2 years, Taiwanese women with EM had a significantly higher frequency of comorbidities, medical and surgical treatment, and MACCE than did their non-EM counterparts (2.76% vs 2.18%, P < .0001). After adjustment for comorbidities, patients with EM had an approximately 1.2-fold increased risk of MACCE (95% CI 1.05-1.29; P = .0053) and a higher cumulative incidence of MACCE compared with the normal population. Neither medical nor surgical treatment increased the risk of MACCE. Furthermore, medical treatment for EM appeared to be protective against MACCE. CONCLUSION: Taiwanese women with EM not only had a substantially higher frequency of comorbidities but also an increased risk of MACCE compared with the general population.

Planned conservative management of placenta increta and percreta with prophylactic transcatheter arterial embolization and leaving placenta in situ for women who desire fertility preservation.

INTRODUCTION: The objective of our study was to demonstrate planned conservative management of placenta increta and percreta in a single tertiary center. METHODS: From April 2005 to July 2019, patients with placenta increta and percreta were managed conservatively at the Kaohsiung Chang Gung Memorial Hospital in Taiwan. The severity of placenta invasion was diagnosed by magnetic resonance imaging (MRI). After delivery of the neonate, prophylactic transcatheter arterial embolization (TAE) was performed immediately. The placenta was left in situ and prophylactic antibiotics were administered during hospitalization. The patient profiles, outcomes, and complications were retrospectively reviewed. RESULTS: Based on the MRI findings, twenty-one patients with placenta increta or percreta were included. With prophylactic TAE, the mean surgical blood loss was 854.7 ± 478.2 mL. The mean natural resorption time of residual placenta was 4.69 ± 1.65 months. Regarding maternal complications, 4 patients (19%) had delayed postpartum hemorrhage (PPH), 12 patients (57.1%) developed postpartum infections, 3 patients (14.3%) progressed to sepsis, 4 patients (19%) underwent surgical evacuation, and 4 patients (19%) underwent hysterectomy. No maternal mortality was reported. Main neonatal complications were prematurity and respiratory distress. Regarding fertility, 16 (76.1%) patients had return of menstruation, and one (4.7%) had a subsequent pregnancy resulting in a live birth. DISCUSSION: Planned conservative management with prophylactic TAE and leaving placenta in situ is feasible and safe for women with placenta increta or percreta who desire fertility preservation. Delayed PPH and postpartum infection are common complications after conservative treatment.

Developmental potential of surplus morulas with delayed and/or incomplete compaction after freezing-thawing procedures.

BACKGROUND: Morulas with delayed growth sometimes coexist with blastocysts. There is still limited evidence regarding the optimal disposal of surplus morulas. With the advancement of vitrification, the freezing-thawing technique has been widely applied to zygotes with 2 pronuclei, as well as embryos at the cleavage and blastocyst stages. The freezing of morulas, however, has rarely been discussed. The purpose of this study was to investigate whether these poor-quality and slow-growing morulas are worthy of cryopreservation. METHODS: This is a retrospective, observational, proof-of-concept study. A total of 1033 day 5/6 surplus morulas were cryopreserved from January 2015 to December 2018. The study included 167 women undergoing 180 frozen embryo transfer cycles. After the morulas underwent freezing-thawing procedures, their development was monitored for an additional day. The primary outcome was the blastocyst formation rate. Secondary outcomes were clinical pregnancy rate, live birth rate and abortion rate. RESULTS: A total of 347 surplus morulas were thawed. All studied morulas showed delayed compaction (day 5, n = 329; day 6, n = 18) and were graded as having low (M1, n = 54), medium (M2, n = 138) or high (M3, n = 155) fragmentation. The post-thaw survival rate was 79.3%. After 1 day in extended culture, the blastocyst formation rate was 66.6%, and the top-quality blastocyst formation rate was 23.6%. The day 5 morulas graded as M1, M2, and M3 had blastocyst formation rates of 88.9, 74.0, and 52.8% (p < 0.001), respectively, and the top-quality blastocyst formation rates were 64.8, 25.2, and 9.0% (p < 0.001), respectively. The clinical pregnancy rate was 33.6%. CONCLUSIONS: The post-thaw blastocyst formation rate was satisfactory, with approximately one-half of heavily fragmented morulas (M3) developing into blastocysts. Most of the poor-quality morulas were worth to freeze, with the reasonable goal of obtaining pregnancy and live birth. This alternative strategy may be a feasible approach for coping with poor-quality surplus morulas in non-PGS (preimplantation genetic screening) cycles.

Age is a major prognosticator in extremely low oocyte retrieval cycles.

OBJECTIVE: Clinical prognosis appears to be varied in females with poor ovarian response (POR), and poor responders defined by the Bologna criteria might not be sufficiently homogeneous. The aim of this study was to determine the major predictor of reproductive outcomes in extremely low oocyte retrieval cycles. MATERIALS AND METHODS: A cohort of fresh in vitro fertilization/intracytoplasmic sperm injection cycles (n = 858) was analyzed from January 2001 to September 2014. Females from whom zero, one, two, or three oocytes were retrieved following ovarian stimulation were examined. Univariate analyses were performed to determine the association of pregnancy rate with potential confounding variables. Multiple logistic regression analysis was subsequently performed to identify factors that affected the occurrence of pregnancy. RESULTS: The clinical pregnancy rate was higher in women aged < 40 years, long protocol, and high embryo score in univariate analysis. After adjusting for confounding factors in multivariate analysis, the maternal age [odds ratio (OR) = 0.91], primary or secondary infertility (OR = 1.99), number of matured oocytes retrieved (OR = 0.64), and score of embryos transferred (OR = 1.39) were significantly associated with the clinical pregnancy rate per cycle and per transfer. In the age subgroup analysis, POR females aged < 35 years significantly demonstrated the highest number of matured oocytes, embryo scores, and clinical pregnancy rates compared with POR females aged 35-40 years and ≥ 40 years. CONCLUSION: This study highlights the predictive value of maternal age and embryo quality on the probability of pregnancy in females with extremely low oocyte retrieval cycles. Young females with few eggs collected can still achieve acceptable pregnancy probability as long as they have good-quality embryos. Future randomized control trials for POR using the Bologna criteria should first stratify patients into different age groups.

Reassessing the feasibility of the zygote score for predicting embryo viability in IVF/ICSI using the GnRH antagonist protocol compared to the long protocol.

BACKGROUND: Many factors from the oocyte/sperm or the process of fertilization may affect the zygote formation. The zygote score (Z-score) describes the quality of a human zygote based on its pronuclear morphology, nucleolar precursor bodies, and alignment of polar bodies, and it can be used in the selection process at the zygote stage for embryo transfer or cryopreservation. OBJECTIVE: The aim of this retrospective cohort study was to investigate the relationship between different controlled ovarian stimulation (COS) protocols and the zygote score (Z-score) and to assess the feasibility of the Z-score for predicting embryo survival in the GnRH-antagonist (GnRH-ant) protocol. METHODS: It is a retrospective, single-center cohort study. A total of 3,826 zygotes with normal fertilization were analyzed from 744 in vitro fertilization /intra-cytoplasmic sperm injection (IVF/ICSI) cycles (long protocol n = 392; GnRH-ant n = 352) between Jan 2010 and April 2014 in the IVF unit of Chang-Gung Memorial Hospital Kaohsiung Medical Center. RESULTS: The Z-score distribution differed significantly between these two protocols. The overall Z-score was poorer for zygotes from GnRH-ant cycles (p<0.05). Univariate and multivariate analyses indicated the type of COS protocol is one of the main determinants of Z-score grading. Our study found good-quality day 3 embryo/blastocyst formation and the cumulative embryo survival rate were correlated with the Z-score but not the COS protocol. With the GnRH-ant protocol, the number of Z1 in the transferred cohort embryos was significantly correlated with the clinical pregnancy rate (r = 0.976; p = 0.024) and live birth rate (r = 0.971; p = 0.029). This correlation was not seen with the long protocol. CONCLUSIONS: The Z-score distribution for the GnRH antagonist cycles was poorer than that of the long protocol, but the Z-score system is a valuable parameter for predicting embryo viability in the GnRH-ant protocol, providing a strong correlation with the clinical pregnancy rate and live birth rate.

Apoptotic effects on maturation of mouse oocytes, fertilization and fetal development by puerarin.

Previously we identified puerarin, an isoflavone compound, as a risk factor for normal embryonic development that triggers apoptotic processes in the inner cell mass of mouse blastocysts, leading to retardation of embryonic development and cell viability. In the current study, we investigated whether puerarin exerts deleterious effects on mouse oocyte maturation, in vitro fertilization (IVF) and subsequent pre- and post-implantation development, both in vitro and in vivo. Notably, puerarin caused significant impairment of these processes in vitro. Pre-incubation of oocytes with puerarin during in vitro maturation led to increased post-implantation embryo resorption and decreased mouse fetal weight. In an in vivo animal model, intravenous injection with or without puerarin (1, 3 and 5 mg/kg body weight/day) for 4 days caused a decrease in oocyte maturation and IVF, and led to deleterious effects on early embryonic development. Importantly, pre-incubation of oocytes with a caspase-3-specific inhibitor effectively blocked puerarin-triggered deleterious effects, clearly implying that embryonic injury induced by puerarin is mediated by a caspase-dependent apoptotic mechanism. These results clearly demonstrate that puerarin has deleterious effects on mouse oocyte maturation, fertilization and subsequent embryonic development in vitro and in vivo.

Effects of ochratoxin a on mouse oocyte maturation and fertilization, and apoptosis during fetal development.

We previously reported that ochratoxin A (OTA), a mycotoxin found in many foods worldwide, causes nephrotoxicity, hepatotoxicity, and immunotoxicity, and is a risk factor for abnormal embryonic development. More specifically, OTA triggers apoptotic processes in the inner cell mass of mouse blastocysts, decreasing cell viability and embryonic development. In the current study, we investigated the deleterious effects of OTA on mouse oocyte maturation, in vitro fertilization (IVF), and subsequent pre- and postimplantation development both in vitro and in vivo. Notably, OTA significantly impaired mouse oocyte maturation, decreased IVF rates, and inhibited subsequent embryonic development in vitro. Preincubation of oocytes with OTA during in vitro maturation increased postimplantation embryonic resorption and decreased mouse fetal weight. In an in vivo animal model, provision of 1-10 µM OTA in the drinking water or intravenous injection of 1 or 2 mg/kg body weight of OTA decreased oocyte maturation and IVF, and had deleterious effects on early embryonic development. Importantly, preincubation of oocytes with a caspase-3-specific inhibitor effectively blocked these OTA-triggered deleterious effects, suggesting that the embryonic injury induced by OTA is mediated via a caspase-dependent apoptotic mechanism. Furthermore, OTA upregulated the levels of p53 and p21 in blastocyst cells derived from OTA-pretreated oocytes, indicating that such cells undergo apoptosis via p53-, p21-, and caspase-3-dependent regulatory mechanisms. This could have deleterious effects on embryonic implantation and fetal survival rates, as seen in our animal models. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 724-735, 2016.

Ectopic pregnancy following in vitro fertilization with embryo transfer: A single-center experience during 15 years.

OBJECTIVE: Ectopic pregnancy is an obstetrical disease that is potentially associated with maternal death in the first trimester. It is one of the well-known complications following in vitro fertilization (IVF) with embryo transfer (ET). The incidence of ectopic pregnancy is estimated to be 2.1-8.6% of clinical pregnancy after IVF-ET, which is higher than natural conceptions (incidence rate 2%). This study aimed to re-evaluate the ectopic pregnancy rate in patients undergoing IVF-ET and to investigate the effects of embryo stage and frozen-thawed blastocyst transfer and ET during full bladder distention on ectopic pregnancy rate. MATERIALS AND METHODS: This retrospective study reviewed women who achieved a clinical pregnancy after IVF-ET at the Department of Obstetrics and Gynecology, Kaohsiung Chang Gung Memorial Hospital between 1999 and 2013. We compared ectopic pregnancy rate following Day 3 ET with Day 5 ET, and after fresh ET with thawed ET. Besides, multivariate analysis was used to clarify the factors affecting ectopic pregnancy after IVF-ET. RESULTS: Of the total 1213 clinical pregnancies after fresh ET, 18 (1.5%) were verified as ectopic, which is similar to the rate following natural conception. The ectopic pregnancy rates were similar for Day 3 (1.2%) and Day 5 (1.7%) ETs. The incidence of ectopic pregnancy in thawed ET cycles (0.6%) was not significantly reduced than fresh ET cycles (1.5%). Tubal ET (TET) and ET under full bladder distention had a significant effect on ectopic pregnancy. CONCLUSION: Thawed ET was not associated with a lower incidence of ectopic pregnancy than fresh ET, and embryo stage did not affect the rate of ectopic pregnancy. In addition, TET and ET under conditions of full bladder distention may increase the ectopic pregnancy rate.

The impact of previous ovarian surgery on ovarian reserve in patients with endometriosis.

BACKGROUND: To investigate the impact of previous ovarian surgery on ovarian reserve in patients with endometriosis. METHODS: A total of 829 female patients were recruited. Their medical records were reviewed retrospectively. Patients who had diagnoses of endometriosis or endometrioma were defined as the endometriosis group, and those without endometriosis were as the control group. We further divided these patients into four groups according to whether they had received ovarian surgeries before. Group 1: control group without previous surgery; Group 2: control group with previous surgery; Group 3: endometriosis group without previous surgery; Group 4: endometriosis group with previous surgery. The subgroups with endometrioma or not and different operative procedures were also analyzed. The parameters for comparison included age, body mass index, serum estradiol, follicle-stimulating hormone, luteinizing hormone, cancer antigen 125, and anti-Müllerian hormone (AMH) level. RESULTS: The level of serum AMH was highest in group 1 and lowest in group 4. The decline was significant between group 1 and group 4 (p < 0.05). The serum AMH level was lower in group 4 than in group 3 but no significant difference. Serum estradiol level was significantly higher in group 3 than in group 2 (p < 0.05). Cancer antigen 125 levels were both significantly higher in group 3 and group 4 as compared with group 1 and group 2 (p < 0.05). CONCLUSIONS: Performing repeated ovarian surgery in patients with recurrent endometriosis needs careful consideration and adequate patient counselling because of the predictable deteriorating ovarian reserve.

Clinical outcomes and development of children born to couples with obstructive and nonobstructive azoospermia undergoing testicular sperm extraction-intracytoplasmic sperm injection: A comparative study.

OBJECTIVE: To evaluate and compare the clinical outcomes and development of children born between obstructive azoospermia (OA) couples and nonobstructive azoospermia couples (NOA) after testicular sperm extraction (TESE) and intracytoplasmic sperm injection (ICSI). MATERIALS AND METHODS: Data were collected from infertile couples suffering from azoospermia who underwent TESE and ICSI from January 2001 to December 2009 at Chang Gung Memorial Hospital, Taiwan. A total of 154 ICSI cycles were performed using extracted testicular sperm from men with obstructive azoospermia (67 ICSI cycles) and men with nonobstructive azoospermia (87 ICSI cycles). Retrospective analysis of clinical outcomes and development of children born after TESE-ICSE between obstructive azoospermia couples and nonobstructive azoospermia couples. RESULTS: The assisted reproductive technology (ART) result between OA and NOA groups, including age, E2 level on hCG day, number of oocytes retrieved, normal fertilization rate, zygote Grade 1 score distribution, number of top-quality embryos transferred, clinical pregnancy rate per transfer, chemical pregnancy rate per transfer, implantation rate, live birth rate per transfer, and abortion rate per transfer, were all similar. Thirty-one live births resulted from 67 ICSE cycles in the OA group and 33 live births from 87 ICSE cycles in the NOA group. The obstetric and perinatal outcomes were similar between the groups, and children conceived by using ICSI were generally healthy without raised tendency of major birth defect and development impairment. CONCLUSION: In our study, there were no differences in the fertility rate and clinic pregnancy rate between the OA and NOA groups using TESE-ICSI. Also, the clinical outcomes and development of children were similar between the OA and the NOA groups using TESE-ICSI.

Reproducibility and clinical significance of pre-ovulatory serum progesterone level and progesterone/estradiol ratio on the day of human chorionic gonadotropin administration in infertile women undergoing repeated in vitro fertilization cycles.

BACKGROUND: The objective of this study was to explore the cycle-to-cycle reproducibility of serum progesterone level and progesterone/estradiol (P/E2) ratio in the final step of triggering oocyte maturation in patients undergoing repeated consecutive controlled ovarian hyperstimulation for in vitro fertilization (COH-IVF) treatment and to investigate the clinical parameters associated with serum progesterone concentration and P/E2 ratio. METHODS: We retrospectively studied 524 cycles in 203 infertile women who underwent two or more fresh COH-IVF cycles from July 1998 to May 2012 in a university hospital IVF unit. The patients were divided into groups according to the number (2, 3 or >=4) of total successive IVF cycles with successful oocyte retrieval. The within-subject reproducibility of serum P and P/E2 was tested by calculating intra-class correlation coefficients (ICCs). Multiple linear regression analysis was used to assess the association between patient variables and pre-ovulatory serum P level and P/E2 ratio. RESULTS: The ICCs in women who underwent 2, 3 and >=4 IVF cycles were -0.052, 0.163 and 0.212, respectively, for serum P concentration and 0.180, 0.168 and 0.148, respectively, for P/E2 ratio. All ICCs for both serum P and P/E2 ratio were indicative of poor reproducibility. The number of oocytes was significantly positively related to P concentration, and endometrial thickness was significantly negatively related to P concentration and P/E2 ratio. CONCLUSION: The cycle-to-cycle reproducibility of pre-ovulatory serum P concentration and P/E2 ratio was poor in individual patients, and these fluctuations were more cycle- than patient-dependent. The number of oocytes was the most significant factor relating to P concentration. By using milder stimulation approach to produce fewer oocytes in the next cycle is a strategy to overcome the high serum P concentration, while clinicians should consider each patient's general condition including the age, ovarian reserve, embryo grading and the capacity of frozen-thawed embryo transfer.

Progesterone elevation on the day of human chorionic gonadotropin administration is not the only factor determining outcomes of in vitro fertilization.

OBJECTIVE: To assess whether progesterone elevation is the only factor in determining outcomes of in vitro fertilization (IVF). DESIGN: Retrospective cohort study. SETTING: Infertility clinic at Kaohsiung Chang Gung Memorial Hospital, Taiwan. PATIENT(S): One thousand five hundred eight women undergoing a total of 1,508 IVF cycles. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Clinical pregnancy and live-birth rates. RESULT(S): Patients were classified into four subgroups according to their progesterone concentration on the day of human chorionic gonadotropin (hCG) triggering. The clinical pregnancy and live-birth rates were statistically significantly associated with the age of the woman, the day of embryo transfer, the progesterone concentration on the day of hCG administration, the number of transferred embryos, and the number of top-quality embryos transferred. However, after omitting the women with the highest progesterone concentration (≥1.94 ng/mL), only four factors-patient age, day of embryo transfer, number of transferred embryos, and number of top-quality embryos transferred-were statistically significantly associated with the clinical pregnancy and live-birth rates. CONCLUSION(S): Progesterone concentration on the day of hCG administration is not the only factor determining the clinical pregnancy and live-birth rates. Fresh embryos from women should be frozen with extremely high progesterone concentrations. Each patient's general condition and the capacity for frozen-thawed embryo transfer should be considered before implantation.

Evaluation of serum anti-Mullerian hormone as a biomarker of early ovarian aging in young women undergoing IVF/ICSI cycle.

OBJECTIVE: To determine whether or not the level of serum anti-Müllerian hormone (AMH) is related to early ovarian aging in young women (< 35 years of age) undergoing in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) cycles. DESIGN: Retrospective cohort study. SETTING: An IVF laboratory in a university hospital in Taiwan. Patient (s): 70 young women (< 35 years of age) with low level of serum AMH (< 2 ng/ml) and 104 young women with level of serum AMH (≥ 2 ng/ml) who underwent IVF/ICSI cycles between January 2011 and November 2012 were enrolled. Intervention (s): None. Main outcome measure (s): Number of oocytes, fertilization rate, embryo quality, cycle cancellation rate, clinical pregnancy/abortion rate, and perinatal/infant outcomes. RESULTS: The clinical pregnancy rate per transfer was favorable (low AMH group vs. normal AMH group [47.2% and 47.9%]) for women < 35 years of age, including women with a low serum AMH. Similarly, the live birth rate per transfer (low AMH group vs. normal AMH group [37.7% and 35.4%]) and perinatal outcomes were also comparable between the two groups. A significantly higher cycle cancellation was noted in the low AMH group than the normal AMH group (24.2% vs. 7.6%). CONCLUSION: Although early ovarian aging should be taken into consideration for young and infertile women with low AMH level than expected, our results suggest that low serum AMH level may suggest early ovarian aging in accelerated oocyte loss only, but may not fully represent "early ovarian aging" based on the favorable outcomes of pregnancy.

Characterization of apoptosis induced by emodin and related regulatory mechanisms in human neuroblastoma cells.

Emodin (1,3,8-trihydroxy-6-methylanthraquinone), a major constituent of rhubarb, has a wide range of therapeutic applications. Recent studies have shown that emodin can induce or prevent cell apoptosis, although the precise molecular mechanisms underlying these effects are unknown. Experiments from the current study revealed that emodin (10-20 µM) induces apoptotic processes in the human neuroblastoma cell line, IMR-32, but exerts no injury effects at treatment doses below 10 µM. Treatment with emodin at concentrations of 10-20 µM led to a direct increase in the reactive oxygen species (ROS) content in IMR-32 cells, along with significant elevation of cytoplasmic free calcium and nitric oxide (NO) levels, loss of mitochondrial membrane potential (MMP), activation of caspases-9 and -3, and cell death. Pretreatment with nitric oxide (NO) scavengers suppressed the apoptotic biochemical changes induced by 20 µM emodin, and attenuated emodin-induced p53 and p21 expression involved in apoptotic signaling. Our results collectively indicate that emodin at concentrations of 10-20 µM triggers apoptosis of IMR-32 cells via a mechanism involving both ROS and NO. Based on the collective results, we propose a model for an emodin-triggered apoptotic signaling cascade that sequentially involves ROS, Ca²âº, NO, p53, caspase-9 and caspase-3.