Reduced PATL2 Impairs the Proliferation of Ovarian Granulosa Cells by Decreasing ADM2 Expression in Patients with PCOS.

It is recognized that PCOS patients are often accompanied with aberrant follicular development, which is an important factor leading to infertility in patients. However, the relevant regulatory mechanisms of abnormal follicular development are not well understood. In the present study, by collecting human ovarian granulosa cells (GCs) from PCOS patients who underwent in vitro fertilization (IVF), we found that the proliferation ability of GCs in PCOS patients was significantly reduced. Surprisingly, PATL2 and adrenomedullin 2 (ADM2) were obviously decreased in the GCs of PCOS patients. To further explore the potential roles of PATL2 and ADM2 on GC, we transfected PATL2 siRNA into KGN cells to knock down the expression of PATL2. The results showed that the growth of GCs remarkably repressed after knocking down the PATL2, and ADM2 expression was also weakened. Subsequently, to study the relationship between PATL2 and ADM2, we constructed PATL2 mutant plasmid lacking the PAT construct and transfected it into KGN cells. The cells showed the normal PATL2 expression, but attenuated ADM2 expression and impaired proliferative ability of GCs. Finally, the rat PCOS model experiments further confirmed our findings in KGN cells. In conclusion, our study suggests that PATL2 promoted the proliferation of ovarian GCs by stabilizing the expression of ADM2 through "PAT" structure, which is beneficial to follicular development, whereas, in the ovary with polycystic lesions, reduction of PATL2 could result in the decreased expression of ADM2, subsequently weakened the proliferation ability of GCs and finally led to the occurrence of aberrant follicles.

Efficacy and Safety of the Chinese Herbal Compound TJAOA101 in Treating Diminished Ovarian Reserve: A Protocol for Multicenter, Prospective, and Pre-Post Study.

OBJECTIVE: Diminished ovarian reserve (DOR) can lead to early menopause, poor fecundity, and an increased risk of disorders such as osteoporosis, cardiovascular disease, and cognitive impairment, seriously affecting the physical and mental health of women. There is still no safe and effective strategy or method to combat DOR. We have developed a novel Chinese herbal formula, Tongji anti-ovarian aging 101 (TJAOA101), to treat DOR. However, its safety and efficacy need to be further validated. METHODS: In this prospective and pre-post clinical trial, 100 eligible patients aged 18-45 diagnosed with DOR will be recruited. All participants receive TJAOA101 twice a day for 3 months. Then, comparisons before and after treatment will be analyzed, and the outcomes, including anti-mullerian hormone (AMH) and follicle-stimulating hormone (FSH) levels and the antral follicle count (AFC), the recovery rate of menopause, and the Kupperman index (KMI), will be assessed at baseline, every month during medication (the intervention period), and 1, 3 months after medication (the follow-up period). Assessments for adverse events will be performed during the intervention and follow-up periods. CONCLUSION: A multicenter, prospective study will be conducted to further confirm the safety and efficacy of TJAOA101 in treating DOR and to provide new therapeutic strategies for improving the quality of life in DOR patients.

Aberrant BMP15/HIF-1α/SCF signaling pathway in human granulosa cells is involved in the PCOS related abnormal follicular development.

AIMS: To investigate the regulatory mechanism of SCF expression in human GCs of PCOS related follicles. MATERIALS AND METHODS: SCF, BMP15 and HIF-1α were evaluated in human serums, follicular fluids (FFs) and GCs, which were collected from 69 PCOS patients and 74 normal ovulatory patients. KGN cell line was used in this study. RESULTS: Our results showed that the rate of MII oocyte and 2PN fertilization was lower in PCOS group, though PCOS patients retrieved much more oocytes. The level of BMP15 in FF and the level of SCF in serum and FF were also lower in PCOS patients. We found a weakened expression of HIF-1α and SCF in GCs from PCOS patients when compared with the non-PCOS patients. The expression of HIF-1α and SCF was significantly increased in KGN cells after treating cells with rhBMP15, however, this promotion effects of BMP15 on HIF-1α and SCF expression were obviously abolished by co-treatment with BMP-I receptor inhibitor (DM). Moreover, knock down of HIF-1α expression in KGN cells significantly reduced the expression of SCF in human GCs, in spite of activating BMP15 signaling pathway. CONCLUSIONS: The present study suggest that BMP15 could induce SCF expression by up-regulating HIF-1α expression in human GCs, the aberrance of this signaling pathway might be involved in the PCOS related abnormal follicular development.

A novel heterozygous variant in PANX1 is associated with oocyte death and female infertility.

PURPOSE: Oocyte death is a severe clinical phenotype that causes female infertility and recurrent in vitro fertilization and intracytoplasmic sperm injection failure. We aimed to identify pathogenic variants in a female infertility patient with oocyte death phenotype. METHODS: Sanger sequencing was performed to screen PANX1 variants in the affected patient. Western blot analysis was used to check the effect of the variant on PANX1 glycosylation pattern in vitro. RESULTS: We identified a novel PANX1 variant (NM_015368.4 c.86G > A, (p. Arg29Gln)) associated with the phenotype of oocyte death in a non-consanguineous family. This variant displayed an autosomal dominant inheritance pattern with reduced penetrance. Western blot analysis confirmed that the missense mutation of PANX1 (c.86G > A) altered the glycosylation pattern in HeLa cells. Moreover, the mutation effects on the function of PANX1 were weaker than recently reported variants. CONCLUSION: Our findings expand the inheritance pattern of PANX1 variants to an autosomal dominant mode with reduced penetrance and enrich the variational spectrum of PANX1. These results help us to better understand the genetic basis of female infertility with oocyte death.

Acute Kidney Injury and 3-Year Mortality in Elderly Patients After Non-cardiac Surgery.

Objective: The present study aimed to investigate whether acute kidney injury (AKI) was associated with 3-year mortality in elderly patients after non-cardiac surgery. Methods: The present study was a 3-year follow-up study of two randomized controlled trials. A total of 1,319 elderly patients who received non-cardiac surgery under general anesthesia were screened. AKI was diagnosed by the elevation of serum creatinine within a 7-day postoperative period according to Kidney Disease: Improving Global Outcomes (KDIGO) guidelines. A long-term telephonic follow-up was undertaken by investigators who were not involved in the previous two trials and had no access to the study group assignment. The date of death was taken from the official medical death certificate. The primary outcome was to investigate the association between AKI and postoperative 3-year mortality using the multivariable Cox regression risk model. Results: Of the 1,297 elderly patients (mean age 71.8 ± 7.2 years old) who were included in the study, the incidence of AKI was 15.5% (201/1297). Of the patients with AKI, 85% (170/201) were at stage 1, 10% (20/201) at stage 2, and 5% (11/201) at stage 3. The 3-year all-cause mortality was 28.9% (58/201) in patients with AKI and 24.0% (263/1,096) in patients without AKI (hazard ratio 1.247, 95% confidence interval 0.939-1.657, P = 0.128). The multivariable Cox regression showed that AKI was not associated with 3-year mortality after adjustment of confounding factors (adjusted hazard ratio 1.045, 95% confidence interval 0.780-1.401, P = 0.766). Conclusions: AKI was a common postoperative complication, but it was not associated with 3-year mortality in elderly patients who underwent non-cardiac surgery. The low incidence of severe AKI might underestimate its underlying association with long-term mortality.

GnRH antagonist weakens endometrial stromal cells growth ability by decreasing c-kit receptor expression.

BACKGROUND: Several surveys have reported that patients treated with gonadotropin-releasing hormone antagonist (GnRH-ant) protocol showed a significantly lower rate of implantation and clinical pregnancy compared to GnRH agonist (GnRH-a) protocol during in vitro fertilization-fresh embryo transfer. Subsequent studies imputed this poor outcome to the negative effects of GnRH-ant on endometrial receptive. However, the mechanisms were not fully understood. METHODS: The clinical data of 2815 patients undergoing fresh embryo transfer in our center were analyzed. Human endometrial stromal cells (ESCs) from healthy women undergoing elective pregnancy termination of a normal pregnancy at 8-10 weeks gestation were treated with GnRH-analogs or imatinib (c-kit receptor inhibitor). CCK8 and Flow cytometry were used to investigated the growth ability of ESCs. Immunofluorescence staining and western blot was used to detected the target proteins. RESULTS: The clinical data showed that the endometrial thickness on HCG Day were significantly lower in GnRH-ant group. Although no difference of embryo quality in these two groups, GnRH-ant group showed remarkably decreased rate of HCG positive, embryo implantation and pregnancy. Moreover, GnRH-ant significantly reduced the proliferation and induced the apoptosis of ESCs. Furthermore, the expression and activation of c-kit receptor, which played pivotal roles during embryo implantation, were observably decreased by GnRH-ant. Inhibiting the activation of c-kit by imatinib remarkably suppressed the proliferation and promoted the apoptosis of ESCs. Additionally, the phosphorylation of AKT and expression of Cyclin D1, which were closely related with cellular growth, were distinctly lessened after treating with imatinib. CONCLUSIONS: In summary, our study showed that GnRH-ant weakened the activization of c-kit receptor by decreasing its expression, causing the impaired growth ability of ESCs. Our findings provided a new insight into the effects of GnRH-ant on endometrium.

PATL2 regulated the apoptosis of ovarian granulosa cells in patients with PCOS.

AIM: PCOS often showed abnormal follicular development. Previous studies have found that the increased apoptosis of granulosa cells (GCs) is one of the key factors leading to follicular dysplasia. It has been found that the decrease or deletion of PATL2 function can significantly inhibit the development and maturation of human oocytes. We found that PATL2 was also expressed in human ovarian GCs, suggesting that PATL2 may be involved in the regulation of related biological events in GCs. This study aims to explore the function of PATL2 on regulation of GCs apoptosis, and the potential role of PATL2 in the development of PCOS-related abnormal follicles. MATERIALS AND METHODS: The follicular GCs of PCOS patients and normal ovulating female patients were collected. Moreover, human granular cell line (KGN) was used for in vitro experiments. RESULTS: (1) The maturation rate and fertilization rate of oocytes in the PCOS group were significantly lower than those in the normal control group (p＜0.05). (2) Flow cytometry and TUNEL staining showed that the apoptosis level of GCs in the PCOS group was significantly increased. (3) Immunofluorescence and Western Blot showed that the PATL2 expression level of GCs in the PCOS group was significantly reduced. (4) Knocking down the expression of PATL2 by siRNA significantly prevented the apoptosis of GCs. CONCLUSIONS: Reduced PATL2 could resulted in the increased apoptosis level of ovarian GCs, which might be closely related to the occurrence and development of abnormal follicles in PCOS.

Impact of goal-directed hemodynamic management on the incidence of acute kidney injury in patients undergoing partial nephrectomy: a pilot randomized controlled trial.

BACKGROUND: The incidence of acute kidney injury (AKI) remains high after partial nephrectomy. Ischemia-reperfusion injury produced by renal hilum clamping during surgery might have contributed to the development of AKI. In this study we tested the hypothesis that goal-directed fluid and blood pressure management may reduce AKI in patients following partial nephrectomy. METHODS: This was a pilot randomized controlled trial. Adult patients who were scheduled to undergo partial nephrectomy were randomized into two groups. In the intervention group, goal-directed hemodynamic management was performed from renal hilum clamping until end of surgery; the target was to maintain stroke volume variation < 6%, cardiac index 3.0-4.0 L/min/m2 and mean arterial pressure > 95 mmHg with crystalloid fluids and infusion of dobutamine and/or norepinephrine. In the control group, hemodynamic management was performed according to routine practice. The primary outcome was the incidence of AKI within the first 3 postoperative days. RESULTS: From June 2016 to January 2017, 144 patients were enrolled and randomized (intervention group, n = 72; control group, n = 72). AKI developed in 12.5% of patients in the intervention group and in 20.8% of patients in the control group; the relative reduction of AKI was 39.9% in the intervention group but the difference was not statistically significant (relative risk 0.60, 95% confidence interval [CI] 0.28-1.28; P = 0.180). No significant differences were found regarding AKI classification, change of estimated glomerular filtration rate over time, incidence of postoperative 30-day complications, postoperative length of hospital stay, as well as 30-day and 6-month mortality between the two groups. CONCLUSION: For patients undergoing partial nephrectomy, goal-directed circulatory management during surgery reduced postoperative AKI by about 40%, although not significantly so. The trial was underpowered. Large sample size randomized trials are needed to confirm our results. TRIAL REGISTRATION: Clinicaltrials.gov identifier: NCT02803372 . Date of registration: June 6, 2016.

Mild starting dosage ovarian stimulation combined with a modified prolonged GnRH-a protocol improved IVF/ICSI outcomes in normal ovarian responders.

INTRODUCTION: Controlled ovarian hyperstimulation (COH) is essential for artificial reproduction technology (ART). This study aimed to evaluate the effects of a mild starting dosage of r-FSH ovarian stimulation after the modified prolonged GnRH-a down-regulation protocol for COH on the clinical outcomes in normal ovarian responders undergoing in vitro fertilization/intracytoplasmic sperm injection-embryo transfer (IVF/ICSI-ET). MATERIAL AND METHODS: In the retrospective study, the patients were separated into two groups according to the starting dosage of r-FSH: a mild dosage group (75 IU ≤ r-FSH < 150 IU, n = 858) and a conventional dosage group (150 IU ≤ r-FSH ≤ 225 IU, n = 535). Data were collected from clinical records. The baseline characteristics and clinical outcomes were compared between the two groups. RESULTS: Although the duration of r-FSH treatment was a little longer in the mild dosage group, the total r-FSH dosage and the cost of ovarian stimulation were significantly lower than those in the conventional dosage group. Furthermore, compared to the conventional dosage group, the number of retrieved oocytes was also lower in the mild dosage group, whereas the rates of two pronuclei (2PN) fertilized oocytes and good-quality embryos were remarkable higher. The implantation rate, clinical pregnancy rate and live birth rate were significantly higher in the mild dosage group. There was no difference in early miscarriages rate, incidence of moderate and severe ovarian hyper-stimulation syndrome (OHSS) or incidence of ectopic pregnancy between the two groups. CONCLUSIONS: The modified prolonged GnRH-a pituitary down-regulation regimen combined with mild r-FSH starting dosage improved IVF/ICSI outcomes and reduced the financial cost in normal ovarian responders.

C-kit signaling promotes human pre-implantation 3PN embryonic development and blastocyst formation.

BACKGROUND: Although in vitro culture system has been optimized in the past few decades, the problem of few or no high quality embryos has been still not completely solved. Accordingly, fully understanding the regulatory mechanism of pre-implantation embryonic development would be beneficial to further optimize the in vitro embryo culture system. Recent studies have found the expression of c-kit in mouse embryo and its promotion effects on mouse embryonic development. However, it is unclear the expression, the role and the related molecular regulatory mechanism of c-kit in human pre-implantation embryo development. Therefore, the present study is to determine whether c-kit is expressed in human pre-implantation embryos, and to investigate the possible regulatory mechanism of c-kit signaling in the process of embryonic development. METHODS: The present study includes human immature oocytes and three pronucleus (3PN) embryos collected from 768 women (28-32 ages) undergoing IVF, and normal 2PN embryos collected from ICR mice. Samples were distributed randomly into three different experimental groups: SCF group: G-1™ (medium for culture of embryos from the pro-nucleate stage to day 3) or G-2™ (medium for culture of embryos from day3 to blastocyst stage) + HSA (Human serum album) solution + rhSCF; SCF + imanitib (c-kit inhibitor) group: G-1™ or G-2™ + HSA solution + rhSCF + imanitib; SCF + U0126 (MEK/ERK inhibitor) group: G-1™ or G-2™ + HSA solution + rhSCF + U0126; Control group: G-1™ or G-2™ + HSA solution + PBS; The rate of good quality embryos at day 3, blastulation at day 6 and good quality blastulation at day 6 were analysis. RT-PCR, western blot and immunofluorescence staining were applied to detect the target genes and proteins in samples collected from human or mice, respectively. RESULTS: c-kit was expressed ubiquitously in all human immature oocytes, 3PN embryos and 3PN blastocysts. In the experiment of human 3PN embryos, compared with other groups, SCF group showed obviously higher rate of good quality at day 3, better rate of blastocyst formation at day 6 and higher rate of good quality blastocyst formation at day 6. Furthermore, we observed a higher ETV5 expression in SCF group than that in other groups. Similar results were also found in animal experiment. Interestingly, we also found a higher phosphorylation level of MEK/ERK signal molecule in mice embryos from SCF group than those from other groups. Moreover, inhibition of MEK/ERK signaling would remarkably impeded the mice embryonic development, which might be due to the reduced ETV5 expression. CONCLUSIONS: The present study firstly revealed that c-kit signaling might promote the human pre-implantation embryonic development and blastocyst formation by up-regulating the expression of ETV5 via MEK/ERK pathway. Our findings provide a new idea for optimizing the in vitro embryo culture condition during ART program, which is beneficial to obtain high quality embryos for infertile patients.

Outcomes of neonates born following transfers of frozen-thawed cleavage-stage embryos with blastomere loss: a prospective, multicenter, cohort study.

BACKGROUND: Despite limited information on neonatal safety, the transfer of frozen-thawed cleavage-stage embryos with blastomere loss is common in women undergoing in vitro fertilization. We aimed to evaluate the pregnancy outcomes and safety of frozen-thawed cleavage-stage embryos with blastomere loss. METHODS: This prospective, multicenter, cohort study included all frozen-thawed cleavage-stage embryo transfer (FET) cycles between 2002 and 2012. Pregnancy outcomes and subsequent neonatal outcomes were compared between FET cycles with intact embryos and those with blastomere loss. RESULTS: A total of 12,105 FET cycles were included in the analysis (2259 cycles in the blastomere loss group and 9846 cycles in the intact embryo group). The blastomere loss group showed significantly poorer outcomes with respect to implantation, pregnancy, and live birth rates than the intact embryo group. However, following embryo implantation, the two groups were similar with respect to live birth rates per clinical pregnancy. Among multiple pregnancies (4229 neonates), neonates from the blastomere loss group were at an increased risk of being small for gestational age (aOR = 1.50, 95% CI 1.00-2.25) compared to those from the intact group. A similar trend was observed among singletons (aOR = 1.84, 95% CI 0.99-3.37). No associations were found between blastomere loss and the subsequent occurrence of congenital anomalies or neonatal mortality. However, neonates from the blastomere loss group were at an increased risk of transient tachypnea of the newborn (aOR = 5.21, 95% CI 2.42-11.22). CONCLUSIONS: The transfer of embryos with blastomere loss is associated with reduced conception rates. Once the damaged embryos have implanted, pregnancies appear to have the same probability of progressing to live birth but with an increased risk of small for gestational age neonates and transient tachypnea of the newborn. STUDY REGISTRATION: This study was retrospectively registered at Chinese Clinical Trial Registry. Registration number: ChiCTR-OOC-16007753 . Registration date: 13 January 2016.

Increased SCF in Follicular Fluid and Granulosa Cells Positively Correlates With Oocyte Maturation, Fertilization, and Embryo Quality in Humans.

Stem cell factor (SCF), which is derived from granulosa cells (GCs), plays a key role in the process of follicular development and oocyte maturation. The present study aimed to explore whether the levels of SCF in follicular fluid (FF) and GCs can be used as a potential marker for predicting oocyte developmental potential. Follicular fluid and GC samples from 150 female patients undergoing intracytoplasmic sperm injection were collected in this study. The SCF concentrations in FFs and SCF messenger RNA (mRNA) in GCs were evaluated by using enzyme-linked immunosorbent assay and real-time polymerase chain reaction, respectively. The results showed that the levels of SCF protein and mRNA were significantly associated with oocyte maturation, normal fertilization, cleavage, and embryo quality. Moreover, the levels of SCF protein and mRNA in pregnancy group were also higher than those in the nonpregnancy group. The cutoff value of SCF in FF for predicting high-quality embryo was 1.346, with a sensitivity of 57.8% and a specificity of 72.4%, and the cutoff value of SCF in GCs for predicting high-quality embryo was 6.650, with a sensitivity of 64.4% and a specificity of 78.1%. In conclusion, our results showed a positive and statistically significant relationship between SCF level and oocyte maturation, normal fertilization, cleavage, embryo quality, and clinical pregnancy. Therefore, the levels of SCF in FF and GCs might be considered as a new marker for predicting oocyte developmental potential.

ICSI improves fertilization in isolated teratozoospermic men: a study with strictly controlled external factors and WHO-5 standard.

A clear clinical management pathway (conventional in vitro fertilization, IVF or intracytoplasmic sperm injection, ICSI) for treating patients with teratozoospermia is lacking. Here we conducted a retrospective study of fertility indices in 2,178 IVF/ICSI cycles in order to reevaluate clinical management of couples with isolated teratozoospermia (< 4% morphologically normal sperms and normal sperm concentration and motility with the standard of WHO-5).We strictly controlled external factors that could affect oocyte quality or endometrial receptivity to minimize the impact of confounders. Fertilization, total fertilization failure, embryo quality, blastocyst formation rate, and pregnancy rate were studied. Retrospectively, in conventional IVF cycles a significantly lower fertilization rate and higher total fertilization failure rate were observed in couples with isolated teratozoospermia as compared to couples with a normal semen profile. Furthermore, when ICSI was used to treat these teratozoospermic couples, improvement in fertilization was noted. However, the embryo quality, blastocyst formation rate, and pregnancy of couples with isolated teratozoospermia were not enhanced by ICSI. Multiple variable analysis showed that many factors including percentage of morphologically normal sperm are statistically correlated with fertilization rate and total fertilization failure in conventional IVF cycles. In addition the insemination method was correlated with fertilization rate in cases with isolated teratozoospermia. Further studies are warranted to compare outcomes of conventional IVF and ICSI in cases of isolated teratozoospermia, where less than 5 oocytes are retrieved.

(4-Carb-oxy-2-sulfonato-benzoato-κO,O)bis-(1,10-phenanthroline-κN,N')manganese(II).

In the title complex, [Mn(C(8)H(4)O(7)S)(C(12)H(8)N(2))(2)], the Mn(II) atom is chelated by one 4-carb-oxy-2-sulfonato-benzoate anion and two phenathroline (phen) ligands in a distorted octa-hedral MnN(4)O(2) geometry. The benzene ring of the 4-carb-oxy-2-sulfonato-benzoate anion is twisted with respect to the two phen ring systems at dihedral angles of 66.38 (9) and 53.56 (9)°. In the crystal, inter-molecular O-H⋯O and C-H⋯O hydrogen bonding links the mol-ecules into chains running parallel to [100]. Inter-molecular π-π stacking is also observed between parallel phen ring systems, the face-to-face distance being 3.432 (6) Å.