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The zona pellucida (ZP) is an extracellular matrix surrounding mammalian oocytes. It is composed of three to four glycoproteins, ZP1-ZP4. ZP3 is essential for sperm binding and zona matrix formation. Here, we identified a novel heterozygous mutation (NM_001110354.2:c.502_504delGAG) of ZP3, occurring in a pair of sisters with empty follicle syndrome (EFS). A mouse model with the same mutation was established using the CRISPR/Cas9 gene-editing system. As in the above family, F0 -, F1 -, and F2 -generation female mice with the mutation were all infertile. Further analysis using the Chinese hamster ovary cells (CHO-K1) also showed that this mutation weakens the strength of binding between ZP3 and ZP2, which hinders the assembly of ZP and results in unstable ZP formation. Immunohistochemical analysis using ovarian serial sections in both humans and mice demonstrated that the ZP of preantral follicles was thinner than normal control, or even absent. Our study presents a new gene mutation that leads to EFS, providing new evidence and support for the genetic diagnosis of infertile individuals with similar phenotypes. Our results also show that the loop of ZP3 is not only a linker between two amphiphilic helices but may play a critical role in specifying the correct heterodimerization partner. This article is protected by copyright. All rights reserved.

Summary Objective Compare outcomes of patients in whom only one or two oocytes were retrieved who underwent conventional in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). Materials and methods Patients who received IVF and only one to two oocytes were obtained on the day of oocyte retrieval. Fertilization rate, good-quality embryo rate, pregnancy Demographic data and clinical characteristics were recorded and analyzed. Results Of 194 patients, 118 received conventional IVF and 76 ICSI. There were no significant differences in age (mean age, 36 years in both groups), infertility parameters, and number of oocytes retrieved between the groups. ICSI patients had a higher fertilization rate (82.7% vs. 67.0%) and 2PN fertilization rate (78.8% vs. 59.7%); however, no difference in good-quality embryo rate or PR was noted. For women less than 35 years of age, there was no significant difference in these outcome parameters between the groups. For patients greater or equal to 35 years of age ICSI was associated with a higher fertilization rate (83.1% vs. 62.4%) and 2PN fertilization rate (80.3% vs. 55.0%); however, there was no difference in PR. Conclusions ICSI did not improve the good-quality embryo rate or clinical PR rate compared to conventional IVF using semen with normal parameters in women with poor ovarian reserve.

To explore the related factors of remission and relapse in lupus nephritis (LN) patients.A retrospective study was conducted for proliferation and membrane LN patients diagnosed from 2003 to 2010. Their clinical, laboratory and pathological parameters were collected. According to the response to treatment, they were divided into 3 groups of complete remission (CR), partial remission (PR) and no response (NR). Those in remission were divided into 2 groups of relapsing and non-relapsing during maintenance period. Associated factors for remission and relapse were analyzed.(1) Among a total of 105 patients, there were 99 females and 6 males with an average follow-up period of (51 ± 30) months. Eighty-six patients achieved remission after 6-month treatment. (2) The outcomes were CR (n = 36), PR (n = 50) and NR (n = 19). Proteinuria in PR group was higher than that in CR group (4.7 (3.1-7.6) vs 1.7 (1.4-3.8), P0.01) while proteinuria of CR group was lower than that of NR group (1.7 (1.4-3.8) vs 3.0 (2.3-5.9), P0.01). Serum albumin level of CR group was significantly higher than those of PR (30.6 (27.8-34.6) vs 22.4 (19.3-29.4), P0.01) and NR groups (30.6 (27.8-34.6) vs 23.1 (18.9-28.6), P0.01). Serum creatinine was significantly higher in NR group than those of CR (128.9 (69.9-184.3) vs 58.1 (53.0-70.9), P0.01) and PR group (128.9 (69.9-184.3) vs 67.5 (53.5-129.1), P0.05). Acute index (AI) and chronic index (CI) were lower in CR group than those of PR and NR groups. (3) A total of 86 cases achieved remission (CR/PR) while 20 cases (23.3%) had relapse. During the maintenance period, the relapse rate was higher in the group on prednisone alone than those on combined therapy of prednisone plus immunosuppressant (P0.05). Sixty patients (90.9%) in non-relapse group and 12 cases (60.0%) in recurrence group had good compliance.Initial proteinuria, serum creatinine, serum albumin, estimated glomerular filtration rate and AI were related with remission of induction period. Prednisone-alone therapy is an independent risk factor for relapse during maintenance period. Poor compliance of patients may be one of the risk factors for relapse during maintenance period. It may be useful to maintain sustained remission and reduce relapse in LN patients by improving their compliance and using steroids plus immunosuppressant during maintenance period.

Endometritis is an inflammation of the uterine lining that is commonly initiated at parturition. The uterine epithelial cells play an important role in defending against invading pathogens. Magnolol, a hydroxylated biphenyl compound isolated from Magnolia officinalis, has been shown to have anti-inflammatory effects. The aim of this study was to investigate the anti-inflammatory effect of magnolol in modifying lipopolysaccharide (LPS)-induced signal pathways in mouse uterine epithelial cells. We found that magnolol inhibited TNF-α and IL-6 production in LPS-stimulated mouse uterine epithelial cells. We also found that magnolol inhibited LPS-induced NF-κB activation, IκBα degradation, phosphorylation of ERK, JNK, and P38. Furthermore, magnolol could significantly inhibit the expression of TLR4 stimulating by LPS. These results suggest that magnolol exerts an anti-inflammatory property by downregulating the expression of TLR4 upregulated by LPS, thereby attenuating TLR4-mediated NF-κB and MAPK signaling and the release of pro-inflammatory cytokines. These findings suggest that magnolol may be a therapeutic agent against endometritis.

To investigate the expression pattern and significance of two important oocyte-secreted factors: growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) during oocyte maturation in women with polycystic ovary syndrome (PCOS) and infertile women due to husband factors.Total of 25 oocytes [9 at germinal vesicle GV stage, 9 at MI stage and 7 at MII stage] were obtained from 12 patients with PCOS and 82 oocytes (29 at GV stage, 26 at MI stage and 27 at MIIstage) were from 56 controls. The nested quantitative real time (RT) PCR was used to detect the abundance of GDF9 and BMP15 mRNA in each oocyte.(1) The expression level of GDF9 mRNA at the GV stage, MI stage and MII stage in PCOS group were 44.8 (4.2 - 529.0), 27.6 (9.8 - 172.7) and 49.0 (0.2 - 65.9) respectively, the expression in were 149.9 (55.4 - 387.9), 29.9 (2.5 - 205.8) and 657.8 (149.4 - 1376.2) in control group, respectively. The expression of GDF9 mRNA at MII stage was significantly lower in PCOS group than in controls (P0.01), however, the differences didn't reach statistical significant at GV or MI stage between the two groups (P0.05). The expression of GDF9 mRNA displayed some changes at different maturation stage in controls (P0.05, P0.01), however, the expression didn't demonstrate any dynamic changes in PCOS group (P0.05). (2) The expression level BMP15 mRNA at the GV stage, MI stage and MII stage in PCOS group were 0.1 (0.1 - 22.0), 3.2 (0.6 - 55.0) and 6.4 (3.2 - 8.5), respectively, the expression were 41.6 (6.5 - 96.1), 4.0 (2.0 - 10.4) and 49.7 (2.3 - 139.5) in control group, respectively. The expression of BMP15 mRNA at GV stage was significantly lower in PCOS group than in controls (P0.01), however, the differences were not significant at MI or MII stage between the two groups (P0.05). The expression of BMP15 mRNA also displayed some changes at different maturation stage in controls (P0.05), however, the level didn't demonstrate any dynamic changes in PCOS group (P0.05).It was suggested that the low expression of oocyte secreted factors in mature oocytes from PCOS patients might be associated with impaired oocyte quality and developmental competence in PCOS.

To compare the amino acid differences of changes of frozen-thawed early-stage human embryos and fresh cultured early-stage human embryos.Discarded embryos and their in vitro culture medium of patients who underwent in vitro fertilization-embryo transfer (IVF-ET) at the Research Center for Reproductive Medicine, the Sixth Affiliated Hospital of Sun Yat-sen University, from September 2010 to April 2011 were collected. Amino acid levels were determined by high performance liquid chromatography.The amino acid differences of changes in the culture medium of fresh embryos (661.50 μmol/L) were significantly higher than in the medium of post-thawed embryos (232.00 μmol/L) at 0.5 h (P 0.001). At 1 and 2 h, no significant difference of change was found in all amino acids. Differences in the concentration of amino acids between post-thawed embryos and blank control medium were already present beginning at 1 h.The level of amino acid metabolism of frozen-thawed early-stage human embryos has already recovered from the state of metabolic stagnation during cryopreservation at 1 h of incubation after thawing, and the amino acid metabolism level at that time approximates that in fresh embryos before freezing. This may be established as the optimal embryo transfer time in IVF-ET.

To investigate the epidemiology and the risk factors of acute kidney injury (AKI) in hospitalized patients in order to help clinicians better understand and prevent AKI.All patients hospitalized in Renji Hospital of Shanghai Jiao Tong University, which is a three-level General Hospital in Shanghai, during January to December of 2008 were screened by Lab Administration Network. Study group was comprised of the patients with full clinical data of AKI, as defined by Acute Kidney Injury Network (AKIN). The incidence, etiology and distribution characteristics of hospitalized patients with AKI were retrospectively analyzed. Logistic regression analysis was used to investigate the risk factors in severity of AKI.Nine hundred and thirty-four patients suffering from AKI for 1 001 episodes were enrolled. The incidence of AKI in hospitalized patients was 2.4% (934/38 734). The ratio of male to female was 1.88:1. The mean age was (60.82 ± 16.94) years old. Higher incidence was seen with an increase in age. Three hundred and thirty-one(35.4%) patients with AKI were found in medical department, 592(63.4%) patients in surgical department and 11(1.2%) patients in department of gynecologic and obstetrics. Analysis of the causes of AKI showed that pre-AKI accounted for 52.0%, followed by renal parenchyma AKI (44.7%) and postrenal AKI (3.3%). The most common reason for AKI was acute tubular necrosis (ATN, 37.5%), followed by absolute (33.6%) and relative inadequacy of blood volume (13.4%). Multivariate logistic regression analysis showed that chronic kidney disease (CKD) [odds ratio (OR)=2.085, 95% confidence interval (95%CI): 1.536-2.830,P0.01], renal injurious drugs (OR=1.438, 95%CI: 1.087-1.901 ,P0.05), and failure of organs other than kidney (OR=1.327, 95%CI: 1.014-1.737,P0.05) were independent risk factors for stage II-III AKI.AKI is one of the most common clinical syndromes in hospitalized patients. With the increase of age, the incidence increases gradually. The most common reasons for hospitalized AKI are pre-AKI and ATN. CKD, renal injurious drugs and failure of other organs are independent risk factors of medium to serious AKI.

Objective To investigate the expression pattern of two oocyte-secreted factors—growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BM15)—during oocyte maturation in women with polycystic ovary syndrome (PCOS) and in controls and to evaluate the expression differences in oocytes between the two groups. Design Case-control study. Setting University-affiliated hospital. Patient(s) Twenty-five oocytes were obtained from 12 patients with PCOS and 82 oocytes from 56 controls. Intervention(s) None. Main Outcome Measure(s) The abundance of GDF9 and BMP15 mRNA in oocytes of the germinal vesicle (GV), metaphase I (MI), and MII stage. Result(s) The expression of GDF9 and BMP15 mRNA displayed dynamic changes during oocyte maturation in controls after ovarian stimulation, with a decline at the MI stage and an increase to the peak at the MII stage. However, their expression in oocytes from patients with PCOS demonstrated a reduced state without any dynamic change. Conclusion(s) The results suggest that the expression of GDF9 and BMP15 in oocytes from patients with PCOS cannot reach the normal level even after ovarian stimulation and that the expression pattern is abnormal during oocyte maturation, which may be associated with impaired oocyte quality and developmental competence in PCOS.

To evaluate the application value and feasibility of various cervical screening methods and to explore a rapid and efficient cervical cancer screening program for the women in the rural areas of China.We sequentially conducted human papillomavirus (HPV) DNA test by hybrid capture-2 (hc2) with cervical cells, liquid-based thinprep cytology test (TCT), visual inspection with acetic acid (VIA), visual inspection with iodine (VILI), colposcopy respectively for the 2499 married women between 30 and 49 years from Xiushui county of Jiangxi province. All the detection methods were performed independently under double-blind design. Women who were diagnosed positive for having any VIA,VILI and colposcopy inspection or for those women who were diagnosed negative for VIA, VILI and colposcopy but with positive result of HPV or TCT test underwent cervical biopsy directly and endocervical curettage (ECC)when necessary. We performed cervical biopsy endocervical curettage within two weeks to observe the sensitive (SE), specificity (SP), negative predict value (NPV) and positive predict value (PPV) of these detection methods when used alone or combined each other, including HPV test, TCT inspection, VIA, VILI, and colposcopy, the pathological diagnosises of cervical tissue were confirmed by IARC (International Agency for Research on Cancer) while the cytological findings were underegone through the updated program of TBS (The Bethesda System) in 2001.A total of 2499 women underwent the screening and found 443 women who were diagnosed as HPV positive, 337 women with abnormal cervical cytology and 27 women with ASC-H, 157 cases with ASCUS; 103 cases with HSIL, 49 cases with LSIL and 1 cervical cancer. According to the pathological findings. There were 181 women diagnosed as cervical intraepithelial neoplasia (CIN) or cervical cancer, including 81 cases with CIN1 37 cases with CIN2,60 case cervical cancer. The sensitivity rates of HPV, TCT, HPV+ TCT, VIA, VILI, VIA+VILI and colposcopy were 96.67%, 89.47%, 97.98%, 56.57%, 36.36%, 63.64% and 39.39%, and the specificity rates were 85.00%, 96.91%, 86.97%, 94.60%, 96.23%, 92.97% and 98.14% respectively.HPV + TCT seemed to be more sensitive than other screening methods in the cervical cancer screening program.

To analyze the relationship between meiotic spindle location and embryo developmental potential of in vivo and in vitro matured human oocytes.One hundred and thirty-four in vivo matured oocytes and 45 in vitro matured oocytes were observed with polscope at the time of intracytoplasm sperm injection (ICSI).Meiotic spindle was detected in 83.6% (112/134) and 82.2% (37/45) in in vivo and in vitro matured oocytes respectively. In vivo matured oocytes which showed a minimal angle (0-5 degrees ) between the meiotic spindle and the first polar body had a higher fertilization rate (93.3%) than the others. The frequency of the oocytes which had a 0-5 degrees spindle angle in in vivo and in vitro matured oocytes was 22.4% and 17.8%, respectively, and that of oocytes which had a 6 degrees - 45 degrees, 46 degrees-90 degrees and90 degrees spindle angle was 55.2% vs 51.1%, 3.0% vs 8.9%, and 3.0% vs 4.4%. No significant difference was found between them. No relationship was found between the position of meiotic spindle and embryo quality.There is some relationship between the angle of the meiotic spindle with the first polar body and fertilization rate. No significant difference is found in the position of the meiotic spindle between in vivo and in vitro matured human oocytes.

To detect whether the depth of embryo transfer has influence on pregnancy outcome in in vitro fertilization-embryo transfer (IVF-ET).The distance between the high echogenic transfer dot and the fundal endometrium was measured under guidance of transabdominal ultrasound. The average distance 0.75 cm was used to divide patients into two groups. Group 1 included 44 patients with a distance0.75 cm, while group 2 had 48 patients with a distance/= 0.75 cm.There were no significant differences in the average age (32 +/- 3 versus 31 +/- 4), the mean number of oocytes retrieved (14 +/- 7 versus 14 +/- 6), and the mean number of embryos transferred (2.3 +/- 0.6 versus 2.2 +/- 0.5) between groups 1 and 2. The distance to the fundal endometrium in group 2 was (0.50 +/- 0.21) cm, significantly smaller than that of group 1 (1.02 +/- 0.21) cm (P0.01). However, pregnancy rate was significantly higher (P0.05) in group 2 (58.3%, 28/48) than in group 1 (36.4%, 16/44). Although implantation rate in group 2 (33.0%, 35/106) was higher than group 1 (22.2%, 22/99), the difference was not significant.Pregnancy outcome may be affected by the depth of embryo transfer, and the fundal endometrium is a favorable region for embryo deposition.

To investigate the constitution of abnormal spermatozoa from patients with sex chromosome anomalies.Triple color fluorescence in situ hybridization (FISH) was used to determine the sex chromosome constitution of spermatozoa from three patients with sex chromosome anomalies (case 1:46,XY/47,XXY, case 2:45,XO/46,X,Yqh-, case 3:47,XXY). The preimplantation genetic diagnosis (PGD) was performed to case 2.An increased ratio (2.05 vs 1) of X-bearing to Y-bearing spermatozoa was only observed in case 2, who also had an increased incidence of total abnormal spermatozoa (29.71%). An increased incidence of total abnormal spermatozoa (4.91%) was also observed in case 3. Among the constitution of abnormal spermatozoa, case 2 had the increased proportions of XY18 disomy, O18 monosomy and XO monosomy, while case 3 had an increase proportion of XY18 disomy (1.87%). PGD was performed to case 2 and one embryo with XX1818 was selected for implanting.Using FISH to detect the sperm sex chromosomes in patients with sex chromosome anomalies can provide the useful information to evaluate the risk of sex chromosome anomalies in preimplantation embryos.

To analyze the clinical outcome of intracytoplasmic sperm injection (ICSI) in patients with previous fertilization failure after conventional IVF.Data from 20 ICSI cases (22 ICSI cycles) with previous complete failure of fertilization or with fertilization rateor = 20% between January 2002 and December 2004 were retrospectively analyzed. The control group consisted of 100 consecutive ICSI cycles for male factor infertility in the same period.The fertilization rate dramatically increased from 5.4% after conventional IVF to 76.9% after ICSI treatment (chi-squared = 264.66, P0.001). However, the fertilization rate in the subgroup with previous low fertilization was significantly lower than those in the control and in the subgroup without previous fertilization (67.9% vs 77.5%, 67.9% vs 84.2%). Compared with the control group, the subgroup without previous fertilization had a higher pregnancy rate and implantation rate, but only the difference in the implantation rate was statistically significant (40.5% vs 18.9%).ICSI can overcome previous fertilization failure with conventional in vitro fertilization and thus improve the clinical outcome.

To achieve pregnancy with unaffected embryo using in vitro fertilization and embryo transfer (IVF-ET) and preimplantation genetic diagnosis(PGD) for the couples at risk of having children with beta-thalassemia.A couple carrying different thalassemia mutations of codon 41/42 and codon IVS2 position 654 received standard IVF treatment and intracytoplasmic sperm injection, embryo biopsy, single cell polymerase chain reaction and DNA analyses, and only the unaffected or carrier embryos were transferred to uterus. Pregnancy confirmation, and prenatal diagnosis were done at 20 week's gestation.A total of 13 embryos were analyzed in the IVF cycle. PGD indicated that 2 were normal 18.1 , 3 were affected 27.3 , and 6 were carriers 54.5 ; diagnosis was not possible in 2. Three embryos were transferred to uterus on the third day after oocyte retrieval. Ultrasonography showed twin pregnancy with one blighted ovum. The prenatal diagnoses revealed that both fetuses were unaffected, one normal baby and one carrier were born.These studies represent the successful application of PGD for beta-thalassemia in China.

To achieve preimplantation genetic diagnosis (PGD) of the couples at risk of having children with beta-thalassemia, as an alternative to prenatal diagnosis.Two couples carrying different thalassemia mutations of codon 41/42 and codon intervening sequence 2 position 654 received standard in vitro fertilization treatment and intracytoplasmic sperm injection, embryo biopsy and the whole genome was amplified by primer extension preamplification (PEP). Nested polymerase chain reaction was then used to amplify two mutation sites separately. Both were detected by reverse dot-blot.A total of 35 oocytes were retrieved from the two patients. Among them, 87% showed two pronuclei, and embryo biopsy was performed on 16 of these embryos and 25 blastomeres were obtained. The amplification efficacy was 84%. The genotype study of non-transferred and surplus embryos showed 15% of allele drop-out rate. Five embryos were transferred to the uterus of both patients. One pregnancy achieved, resulted in live healthy twin births, which confirmed the results of PGD.This unaffected pregnancy resulting from PGD by PEP for beta-thalassemia demonstrates that this technique can be a effective diagnostic tool for carrier couples who desire a healthy child.

To investigate the effect of in vitro fertilization and embryo transfer (IVF-ET) and preimplantation genetic diagnosis (PGD) for the couples at risk of having children with beta-thalassemia.Four couples carrying different thalassemia mutations received standard IVF treatment. Embryo biopsy was conducted. Single blastomeres were genotyped by a protocol involving primer extension preamplification, nested polymerase chain reaction and reverse dot-blot analysis. Only the unaffected embryos were transferred to the uterus.A total of 97 oocytes were retrieved from the four female carriers. Among them, 83% showed two pronuclei. Embryo biopsy was performed on 47 of these embryos. The amplification efficiency was 84.8%. The average ADO rate was 14.9%. Ten unaffected embryos were transferred. A twin pregnancy with one blighted ovum was confirmed at 7 weeks' gestation by ultrasonography and one normal baby and one carrier of thalassemia mutation were born finally.This unaffected pregnancy resulting from PGD for beta-thalassemia demonstrates that PGD technique can be a powerful diagnostic tool for couples carrying beta-thalassemia mutations who desire a healthy child and wish to avoid abortion of an affected fetus.

To review the retrospective treatment results of the azoospermia patients during January 2001 to January 2002 in the fertility center.One hundred males attempted intracytoplasmic sperm injection (ICSI) cycle for treatment of azoospermia. All patients were undergone sperm retrieval by percutaneous epididymal sperm aspiration (PESA) or testicular sperm extraction (TESE) while their wives received conventional ovarian hyperstimulation. The hormone levels, testicular histology, the rates of sperm retrieval, fertilization, implantation and pregnancy were analysed and evaluated.Sperm were retrieved by PESA in 76 of 100 (76%) and by TESE in 23 of 100 (23%) men of azoospermia. The fertilization rate, implantation rate and clinical pregnancy rate were 71.3%, 20.35% and 42.11% respectively in PESA group, and 75.18%, 22.05% and 41.60% respectively in TESA group. Thirty-two clinical pregnancies were achieved with 15 ongoing pregnancies and subsequent live delivery for 15 cases in PESA group, and 2 cases of miscarriage, while 10 clinical pregnancies were achieved with 6 ongoing pregnancies, 2 cases of live delivery and 2 cases of miscarriage in TESA group. One case failed to retrieve sperm by TESE and canceled.Hormonal levels and testicular histology are unable to predict which men with azoospermia will have sperm retrieved by PESA and TESE. PESA and TESE with ICSI are effective methods to treat azoospermia. There were no significant differences in fertilization, implantation and clinical pregnancy rate between two groups.