miR-188-3p-targeted regulation of ATG7 affects cell autophagy in patients with nonobstructive azoospermia.

BACKGROUND: Nonobstructive azoospermia (NOA) is one of the most difficult forms of male infertility to treat, and its pathogenesis is still unclear. miRNAs can regulate autophagy by affecting their target gene expression. Our previous study found that miR-188-3p expression in NOA patients was low. There are potential binding sites between the autophagy gene ATG7 and miR-188-3p. This study aimed to verify the binding site between miR-188-3p and ATG7 and whether miR-188-3p affects autophagy and participates in NOA by regulating ATG7 to influence the autophagy marker genes LC3 and Beclin-1. METHODS: Testicular tissue from 16 NOA patients and 16 patients with normal spermatogenesis and 5 cases in each group of pathological sections were collected. High-throughput sequencing was performed to detect mRNA expression differences. Quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting, immunohistochemical staining and immunofluorescence were used to detect protein localization and expression. Autophagosome changes were detected by electron microscopy. The targeting relationship between miR-188-3p and ATG7 was confirmed by a luciferase assay. RESULTS: ATG7 protein was localized in the cytoplasm of spermatogenic cells at all levels, and the ATG7 gene (p = 0.019) and protein (p = 0.000) were more highly expressed in the NOA group. ATG7 expression after overexpression/inhibition of miR-188-3p was significantly lower (p = 0.029)/higher (p = 0.021) than in the control group. After overexpression of miR-188-3p, the ATG7 3'UTR-WT luciferase activity was impeded (p = 0.004), while the ATG7 3'UTR-MUT luciferase activity showed no significant difference (p = 0.46). LC3 (p = 0.023) and Beclin-1 (p = 0.041) expression in the NOA group was significantly higher. LC3 and Beclin-1 gene expression after miR-188-3p overexpression/inhibition was significantly lower (p = 0.010 and 0.024, respectively) and higher (p = 0.024 and 0.049, respectively). LC3 punctate aggregation in the cytoplasm decreased after overexpression of miR-188-3p, while the LC3 punctate aggregation in the miR-188-3p inhibitor group was higher. The number of autophagosomes in the miR-188-3p mimic group was lower than the number of autophagosomes in the mimic NC group. CONCLUSIONS: LC3 and Beclin-1 were more highly expressed in NOA testes and negatively correlated with the expression of miR-188-3p, suggesting that miR-188-3p may be involved in the process of autophagy in NOA. miR-188-3p may regulate its target gene ATG7 to participate in autophagy anDual luciferase experiment d affect the development of NOA.

Effects of the distance between small intramural uterine fibroids and the endometrium on the pregnancy outcomes of in vitro fertilization-embryo transfer.

AIM: To explore the effects of the distance between small intramural uterine fibroids (≤4 cm) and the endometrium on the outcomes of in vitro fertilization-embryo transfer (IVF-ET). METHODS: We prospectively analyzed pregnancy outcomes in 117 infertile women with small intramural uterine fibroids and 117 infertile women without uterine fibroids who all underwent IVF-ET. The size and number of small intramural uterine fibroids and the shortest distance between the small intramural uterine fibroids and the endometrium were measured by transvaginal three-dimensional ultrasound. The endometrial and subendometrial blood flow parameters, implantation rate, clinical pregnancy rate, abortion rate and live birth rate were compared between the women with and without small uterine fibroids and among the different shortest distances (≤1, 1-3 and >3 mm). The effects of the size and number of small intramural uterine fibroids on IVF-ET outcomes were observed. RESULTS: The endometrial volume on ET day, the implantation rate and the live birth rate were significantly lower, but the abortion rate was significantly higher, in the women with small intramural uterine fibroids than in those without uterine fibroids (p < 0.05). The endometrial flow index was higher in the shortest distance ≤1-mm group than in the groups with 1-3 and >3 mm, and the implantation rate was higher in ≤1-mm group than in the >3-mm group (p < 0.05). There were no significant differences in clinical outcomes between different sizes and numbers of small intramural uterine fibroids. CONCLUSION: Small intramural uterine fibroids can affect IVF-ET outcomes. Compared with other shortest distances (1-3 and >3 mm), the shortest distance of ≤1 mm has a higher implantation rate.

Effect of modified single cell fixation method on cell-nuclear areas and fluorescence in-situ hybridization signals.

BACKGROUND: Fluorescence in situ hybridization (FISH) is an irreplaceable method in pre-implantation genetic diagnosis. We explored the effects of a modified single cell fixation method on the cell-nuclear area and FISH signal. METHODS: From January 2006 to March 2008, the blastomeres with marked nuclei from D3 embryos were selected. Cells were fixed with three different methods. The effects of the three methods on the cell-nuclear areas and FISH signals were then analyzed. RESULTS: The cell fixation rate was higher in conventional (Group B, 94.85%) and modified (Group C, 95.79%) Tween-20/HCl + methanol/glacial acetic acid methods than in the methanol/glacial acetic acid method (Group A, 86.73%) with p < 0.05. The complete signal rates in group A, B, and C were 95.3%, 93.5%, and 93.4%, respectively, with p > 0.05. The mean cell-nuclear areas in groups A, B, and C were 55.3, 46.2, and 49.5 microm3, respectively, with p < 0.05 in group A compared with group B or C, but with p > 0.05 between Group B and C. There was no significant difference in signal overlap and splitting rates between the three groups. CONCLUSIONS: Modified Tween-20/HCl + methanol/glacial acetic acid method fails to increase FISH signal overlap and splitting rates. It is simple and its fixation time is short. It can be widely used in clinical practice.

[Study on the incidence and influences on ectopic pregnancy from embryo transfer of fresh cycles and frozen-thawed cycles].

OBJECTIVE: To investigate ectopic pregnancy from embryo transfer (ET)of in-vitro fertilization (IVF) cycle and intracytoplasmic sperm injection (ICSI) cycle and frozen-thawed (FET) cycle. METHODS: From Jan.2005 to Dec. 2010, a total of 9037 IVF-ET or ICSI-ET cycles and 4034 FET cycles were performed in our reproductive medicine center, Affiliated First Hospital of Zhengzhou University. The incidence of ectopic pregnancy rate was studied in fresh cycles IVF-ET (5998) and ICSI-ET (3039) cycles, and natural FET (2198) and hormone replacement (E-P) FET (1836) cycles. RESULTS: Of 4034 FET cycles, 1090 clinical pregnancies and 26 ectopic pregnancies were observed, the incidence of ectopic pregnancy was 2.38% (26/1090). Of 9037 fresh cycles, 3602 cycles were clinical pregnancy, and 133 cycles were ectopic pregnancy, and the incidence of ectopic pregnancy was 3.69% (133/3602). The ectopic pregnancy rate in FET cycles was lower than in fresh cycles significantly (P < 0.05). Of 3039 fresh ICSI-ET cycles, the incidence of ectopic pregnancy was 2.62% (34/1298) in 1298 clinical pregnancies. Of 5998 IVF-ET cycles, 2304 clinical pregnancies were observed, the incidence of ectopic pregnancy was 4.30% (99/2304). Ectopic pregnancy rate in the fresh ICSI-ET cycles was lower than that of IVF-ET group significantly (P < 0.01). The ectopic pregnancy rate in the natural FET cycles was 1.46% (8/547), which was significantly lower than 3.31% (18/543) in E-P group (P < 0.05). CONCLUSIONS: The incidence of ectopic pregnancy of FET cycles was significantly lower than that of fresh embryo transfer cycles. The application of exogenous sex hormones in assisted reproductive cycles might increase occurrence of ectopic pregnancy.

Abnormal BMI in Male and/or Female Partners Are Deleterious for Embryonic Development and Pregnancy Outcome During ART Process: A Retrospective Study.

Objective: To investigate the effect of BMI in male and/or female partners on embryo development and clinical pregnancy outcome during ART. Methods: Data of 11,130 cycles between January 2018 and December 2020 were retrospectively analyzed. They were divided into Group A, B, C, and D based on couples' BMI values, also were divided into Group I, II, III and IV in IVF cycles and Group i, ii, iii, and iv in ICSI cycles. After grouping, inter-group indicators comparisons and logistic regression analysis were performed. Results: In IVF cycles, CPR in Group I and Group III were higher than Group IV. In Group III, it was higher than Group II. The AR in Group IV was higher, but the LBR was lower than Group I, Group II, and Group III. Logistic regression analysis results suggested that AR in Group IV was higher than that in Group I in IVF cycles, whereas LBR was lower.In ICSI cycles, high-quality embryo rate in Group i and Group ii were both higher than that in Group iii and Group iv. The CPR in Group i was higher than Group ii and Group iv, and in Group iii was higher than Group ii and Group iv. The AR in Group i was lower than Group iii and Group iv, and AR in Group ii was lower than Group iv. LBR, in Group I it was higher than Group ii, Group iii, and Group iv. Logistic regression analysis results suggested CPR in Group ii was significantly lower than that in Group i. AR in Group iii was considerably higher than that in Group i. LBR in Group ii and Group iv were significantly lower than that in Group i. Conclusion: Female higher BMI was not conducive to the formation of high-quality embryos in ICSI cycle. Female and/or male BMI affected AR and LBR more than CPR not only in IVF cycles, but also in ICSI cycles.

Reproductive Outcomes of Single Embryo Transfer in Women with Previous Cesarean Section.

The aim of this study is to evaluate the reproductive outcomes of patients with previous cesarean section (CS) undergoing single-embryo transfer (SET). A total of 5479 patients previously underwent CS or vaginal delivery (VD) were included. The patients with previous CS included single/double cleavage (SCT/DCT), single/double blastocyst stage embryo transfer (SBT/DBT). The comparison of reproductive outcomes between CS and VD, SET, and double-embryo transfer of CS was conducted. The main outcome measures included clinical pregnancy rate (CPR), multiple pregnancy rate (MPR), live birth rate (LBR), and cumulative live birth rate (CLBR). There were no differences in CPR and LBR between patients previously underwent CS and VD, and the preterm birth rate of multiple births in patients underwent CS were significantly higher than underwent VD. As for patients underwent CS, the CPR, MPR, and LBR were lower in patients treated with SCT than DCT in IVF/ICSI cycles. The CPR and LBR of patients treated with SCT were not statistically different compared with DCT and SBT when patients' age were younger than 35 years in FET cycles, and the MPR of patients treated with SCT was lower. The CPR and LBR of patients treated with SCT were lower than DCT/SBT when patients' age were 35 years or older in FET cycles. The CPR, LBR, and CLBR of patients treated with SBT were not lower than DCT in IVF/ICSI-ET, FET, and complete cycles, but the MPR was lower. Reducing the number of embryos transferred should be considered seriously for CS. SCT under the 35 years of FET cycles is feasible. SBT is an effective strategy to reduce MPR of IVF/ICSI, FET, and complete cycles regardless of overall or age stratification.

[Effect of endometrial stimulation on pregnancy outcome of in vitro fertilization patients].

OBJECTIVE: To analyze the effect of endometrial stimulation on the pregnancy outcome of patients undergoing in vitro fertilization-embryo transfer (IVF-ET). METHODS: A total of 759 subjects were treated by fresh IVF at our hospital in 2007. Sixty-eight subjects received endometrial stimulation at Days 4-5 of menstruation before IVF while other 691 were controls without treatment. No significant difference was found between two groups in terms of duration and cause for infertility, day and dosage of Gn, number of retrieved oocytes and transferred embryos and fertilization rate. The clinical pregnancy rate of two groups were analyzed. Firstly the clinical outcomes for the patients of primary or secondary infertility were compared in treatment and control groups respectively. Then the clinical outcomes of the patients in first cycle and later cycles were compared in treatment and control groups respectively. RESULTS: There were no significant differences in clinical pregnancy rate and incidence of abortion and ectopic pregnancy. For first cyclers, the clinical pregnancy rate showed no significant difference between two groups (38.89% vs 37.16%). But for later cyclers, the clinical pregnancy rate was higher in the treatment group than that in the control group (53.13% vs 34.33%). CONCLUSION: Endometrial stimulation may improve endometrial receptivity to some extent. And it can boost the clinical pregnancy rate for those patients with a repeated implantation failure.

[Effects of cryopreservation time and thawing method of human oocyte vitrification on the outcome of assisted reproduction].

OBJECTIVE: To evaluate the effects on pregnancy outcome of freezing time from oocyte retrieval and thawing method for metaphaseII human oocytes vitrification. METHODS: From Mar 2007 to Mar 2009, the clinical outcome of 30 infertile women undergoing vitrified-thawing oocytes of in vitro fertilization-embryo transfer (IVF-ET) in the Reproductive Medical Center of the First Affiliated Hospital of Zhengzhou University was studied retrospectively, including 21 women with double fallopian tube obstruction and 9 women's husband azoospermia. All infertile women were divided into three groups, including 5 cases in group A (freezing between 4 and 5 hours from oocyte retrieval and conventional thawing method), 9 cases in group B (freezing within 2 hours from retrieval and conventional thawing method) and 16 cases in group C (freezing within 2 hours from retrieval and improved thawing method). The vitrified oocytes were preserved for 2 months to 1 year and thawed for Intracytoplasmic sperm injection (ICSI) and embryo transfer. The outcome of IVF and pregnancy were recorded. RESULTS: (1) The rates of oocyte survival was (65 ± 33)% in group B and (72 ± 23)% in group C and the rate of transfer cycle was 9/9 in group B and 16/16 in group C, which were all significantly higher than (16 ± 17)% of oocyte survival and 1/5 of transfer cycle in group A (P = 0.001, 0.021). However, the rate of oocyte survival and transfer cycle between group B and group C did not reach statistical difference (P > 0.05). The rate of implantation and clinical pregnancy of (33 ± 38)% and 9/16 in group C were significantly higher (4 ± 11)% and 1/9 in group B (P = 0.033, 0.040). (2) The mean age of women in group C were (28.6 ± 2.1) in oneself oocyte, (28.0 ± 4.6) in donor oocyte and (28.1 ± 3.4) in donor sperm. The rate of oocyte survival was (73 ± 25)%, (88 ± 10)% and (66 ± 25)%. The rate of fertilization rate was (84.6 ± 0.9)%, (79.3 ± 2.0)% and (82.8 ± 15.0)%. The rate of implantation was (20.0 ± 44.7)%, (33.0 ± 0.1)%, (41.6 ± 41.7)%. The rate of clinical pregnancy was 1/5 in oneself cycles, 3/3 in donor oocyte cycles, 5/8 banked donor sperm cycles in group C. All above clinical parameters were not statistically different (P > 0.05). (3) In group A, one women underwent IVF-ET and no clinical pregnancy was observed. One women pregnancy was terminated at two months in group B. The clinical pregnancies rate of group C was 9/16, late abortion occurred in 1 woman, the other 8 women underwent term pregnancy, including 5 male infants and 4 female infants. All of infants showed normal Karyotype. Live-birth rates per warmed oocyte was 5.9%(8/135). The mean gestational weeks and birth weight of the infants were (39.4 ± 0.9) weeks and (3574 ± 569) g, respectively. CONCLUSIONS: Embryo quality and clinical outcome of thawing cycles could be significantly improved when oocyte vitrification was performed within 2 hours from oocyte retrieval and improved thawing method.

[Outcomes of ICSI with microamount frozen-thawed sperm obtained by PESA or TESA in the treatment of azoospermia].

OBJECTIVE: To investigate the effects and clinical pregnancy outcomes of intracytoplasmic sperm insemination (ICSI) with microamount frozen-thawed sperm obtained by percutaneous epididymal sperm aspiration (PESA) or testicular sperm aspiration (TESA) in azoospermia patients. METHODS: We divided 365 azoospermia patients treated by ICSI into an experimental group (n = 123) and a control group (n = 242) , the former with microamount frozen-thawed sperm, and the latter fresh sperm obtained by PESA or TESA. The rates of fertilization, good embryos, clinical pregnancy, miscarriage, ectopic pregnancy and multiple pregnancy were analyzed and compared between the two groups. RESULTS: With PESA, the experimental group showed no statistically significant differences from the control group in the rates of fertilization (75.67% vs 76.49%), good embryos (64.96% vs 66.09%), clinical pregnancy (55.21% vs 57.22%), clinical miscarriage (13.21% vs 12.61%), ectopic pregnancy (3. 77% vs 5.41%) and multiple pregnancy (37.74% vs 37.84%) (P > 0.05); nor with TESA (74.41% vs 76.43%, 64.63% vs 66.35%, 46.81% vs 53.39%, 18.18% vs 14.55%, 4.55% vs 1.82%, 37.74% vs 37.84%, P > 0.05). The revival rate of the frozen-thawed sperm from PESA was 70.07%, not significantly different from that of TESA (62.67%) (P > 0.05). CONCLUSION: ICSI with frozen-thawed micro-amount sperm obtained by PESA or TESA is a safe, economic and effective method for the treatment of azoospermia. The techniques for reviving frozen sperm from PESA or TESA remain to be optimized, and whether these techniques may result in long-term genetic risks in the offspring deserves further investigation.

[Clinical application of oocyte vitrification in failed testicular sperm extraction cycles: report of 8 cases].

OBJECTIVE: To evaluate the clinical application value of oocyte vitrification in failed testicular sperm extraction cycles in non-obstructive azoospermia (NOA) patients. METHODS: We retrospectively analyzed the clinical data of 8 women undergoing oocyte frozen-thawing cycles by vitrification because of failed testicular sperm extraction from their NOA husbands and no banked donor sperm on the day of oocyte retrieval. The oocytes were cryopreserved by vitrification with cryotop and thawed 2 months later. The surviving metaphase II (MII) oocytes were injected with the banked donor sperm of the same blood type as the husbands by intracytoplasmic sperm injection (ICSI) for fertilization. The rates of oocyte survival, fertilization, cleavage, good embryos and pregnancy were evaluated. RESULTS: Sixty oocytes were vitrified and 47 (78.3%) survived after thawing, of which 41 MII oocytes underwent ICSI and 33 (80.5%) of them were fertilized. The rates of cleavage and good embryos were 81.8% (27/33) and 59.3% (16/27) respectively. Fifteen of the embryos were transferred to the 8 patients, with 1.9 +/- 0.8 per cycle, of which 5 (33.3%) were confirmed by ultrasound to have been implanted and 5 resulted in clinical pregnancy (62.5%), all singleton without miscarriage. Three normal boys and 1 normal girl were already born, with the pregnancy time of (39 + 4 +/- 0.4) wk and newborn body weight of (3787.5 +/- 513.7) g, respectively. CONCLUSION: Vitrification of oocytes in failed testicular sperm extraction cycles is a promising technique for preserving female fertility, which, with ICSI of banked donor sperm, may result in satisfactory clinical outcomes.

Follicle-stimulating hormone may predict sperm retrieval rate and guide surgical approach in patients with non-obstructive azoospermia.

Testicular sperm aspiration- (TESA) or micro-dissection testicular sperm extraction- (MD-TESE) combined intracytoplasmic sperm injection (ICSI) was the only option for non-obstructive azoospermia (NOA) patients to have a biological offspring and they had different success rates in sperm retrieval. Our study aimed to find predictor(s) for predicting the sperm retrieval rate (SRR) in NOAs and guide clinicians in choosing different surgical approaches, TESA or MD-TESE for NOAs. 294 NOAs who had undergone TESA or MD-TESE were divided into TESA group and MD-TESE group. Depending on sperm retrieval, each group was divided into two subgroups: successful subgroups and failure subgroups. They respectively were 24 cases and 131 cases, 53 cases and 86 cases. Clinical data, including body mass index (BMI), testicular volume, and serum hormone levels, were analyzed in a retrospective manner. The results showed that follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels and SRR were lower in TESA group as compared to these in MD-TESE group, while testicular volume was higher (P < 0.05). The surgical approach of sperm retrieval significantly affected the SRR (P < 0.05). In TESA subgroups, testicular volume, FSH and LH differed significantly (P < 0.05). In MD-TESE subgroups, the level of FSH and LH differed significantly between both groups (P < 0.05). Using logistics regression, we found a negative correlation (ß=-0.083) between FSH and the SRR in TESA group but a positive correlation (ß = 0.064) in MD-TESE group (P < 0.05). In conclusion, serum FSH level can predict the SRR of NOAs and guide the clinicians while selecting the suitable surgery approach for NOAs.

[Analysis of 1540 neonates with birth defects related to different assisted reproductive technique].

OBJECTIVE: To investigate the incidence of and clinical factors influencing neonatal birth defects from different assisted reproductive technology. METHODS: Between October 1998 and December 2006, 1271 newborns from mothers treated by in vitro fertilization techniques [including in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI) and thaw embryo transfer (Thaw-ET)] matched with 269 newborns from mothers treated by artificial insemination were enrolled in Reproductive Medicine Center in First Hospital Affiliated to Zhengzhou University. Their medical information was analyzed retrospectively to compared neonatal characteristics, the incidence of birth defect and anomalous organs involved between in vitro fertilization group and artificial insemination group. RESULTS: In group of in vitro fertilization, those newborns with low birth weight from IVF, ICSI and Thaw-ET were 20.0% (134/671), 22.4% (92/410), 18.9% (36/190) respectively, which were more than 11.5% (31/269) cases in group of artifical semination with statistical significance (P < 0.05). The rates of multiple pregnancy of 23.8% (160/671), 25.4% (104/410), 21.1% (40/190) in subgroup of IVF, ICSI and Thaw-ET were significantly higher than 10.0% (27/269) in group of artifical insemination (P < 0.05). The rate of macrosomia in group of in vitro fertilization was significantly lower than that of artificial insemination group (3.9% vs 8.2%, P < 0.05). However, the incidence of birth defect involved in various organs did not show significant difference between two groups (P > 0.05). CONCLUSIONS: The incidence of multiple pregnancy demonstrated obviously increasing trends born with various In Vitro Fertilization techniques, which pave the way to high risk pregnancy. However, the incidence of newborn birth defect was not increased significantly. Thus, to lower occurrence of multiple pregnancy was the key approach to obtain neonates health.

Factors predicting clinical pregnancy rate of in vitro fertilization-embryo transfer (a STROBE-compliant article).

The aim of this study was to investigate the factors predicting clinical pregnancy rate of in vitro fertilization-embryo transfer (IVF-ET).The data of 9960 patients receiving IVF-ET fresh cycle at our Reproductive Center from January 2009 to December 2017 were first divided into pregnant group and non-pregnant group to find the clinical pregnancy rate-related factors. According to the serum HCG levels at 36 hours and 12 hours after HCG trigger, all patients were divided into 4 groups including <50 mIU/ml, ≥50 and <100 mIU/ml, ≥100 and <200 mIU/ml, and ≥200 mIU/ml groups to know whether the HCG levels at 36 hours and 12 hours affect the pregnancy rate. According to the serum HCG ratio at 36 hours to 12 hours (36 h/12 h) after HCG trigger, all patients were divided into three groups including <0.88, 0.88-1.06 and >1.06 groups to observe whether the serum HCG ratio (36 h/12 h) affects the clinical pregnancy rate. According to different assisted pregnancy modes, all patients were divided into 3 groups including IVF, ICSI, and IVF/ICSI groups to observe whether the assisted pregnancy mode affects the clinical pregnancy rate. The correlation of the clinical pregnancy rate with pregnancy rate-related factors obtained above was analyzed using logistic regression analysis model.The clinical pregnancy rate significantly increased (P < .01) in the HCG ratio (36 h/12 h) >1.06 group as compared with the HCG ratio (36 h/12 h) < 0.88 and 0.88-1.06 groups. The serum estrogen (E2) level at 36 hours was significantly lower and the number of retrieved oocytes was significantly higher in the HCG ratio (36 h/12 h) >1.06 group than in the HCG ratio (36 h/12 h) <0.88 and 0.88-1.06 groups (P = .000).The serum HCG ratio (36 h/12 h) may be used as a predictor of IVF-ET clinical pregnancy rate. High clinical pregnancy rate is probably associated with E2 down-regulation in the HCG ratio (36 h/12 h) >1.06 group.

Clinical analysis of spontaneous pregnancy reduction in the patients with multiple pregnancies undergoing in vitro fertilization/intracytoplasmic sperm injection-embryo transfer.

OBJECTIVE: To investigate the spontaneous pregnancy reduction (SPR) rate, SPR-related factors and the effects of SPR on pregnancy outcomes in the patients with multiple pregnancies undergoing in vitro fertilization/intracytoplasmic sperm injection-embryo transfer (IVF/ICSI-ET). METHODS: Between January 1998 and December 2010, 3957 patients undergoing fresh/frozen-thawed cycles (IVF/ICSI-ET) and their 5106 neonates were enrolled in this study. According to spontaneous pregnancy reduction (SPR), this study included singleton originating from twins [(2→1) group] or from triplets [(3→1) group], and twins originating from triplets [(3→2) group]. According to SPR time, this study included ≤8 week, 8-18 week and ≥18 week's groups. Outcome measures were SPR rate, preterm rate, mean birth weight and the rates of low birth weight and very low birth weight. RESULTS: SPR rate was higher in triplets group than in twins group, in frozen-thawed cycles than in fresh cycles, in the patients ≥35 years than in the patients <35 years (all P<0.05). Compared with ≤8 week group, preterm rate was significantly increased in 8-18 week group (P<0.05). Pregnancy outcomes were better in (2→1) group than in twins group, in (3→1) group than in triplets group (all P<0.05). After multi-fetal pregnancy reduction (MFPR), the mean birth weight was higher and low birth weight was lower in SPR group than in only MFPR group (all P<0.05). CONCLUSION: SPR rate is related to age and the initial number of gestational sacs. Both SPR and MFPR can improve pregnancy outcomes. The later the SPR occurs, the worse the neonatal outcomes are. Due to the possibility of SPR, it is necessary to appropriately delay MFPR until 8 gestational weeks.

[Effects of intrauterine contraceptive device on expression of vascular endothelial growth factor, kinase insert domain-containing receptor and microvessel density in endometrium].

OBJECTIVE: To investigate the expression of vascular endothelial growth factor (VEGF) and its receptor, kinase insert domain-containing receptor(KDR) and microvessel density (MVD) in endometrium from women wearing fixed copper-intrauterine contraceptive device (IUD, FCu-IUD) or fixed indomethacin-releasing copper-IUD (FICu-IUD). METHODS: Twenty healthy women were divided into two study groups: 10 cases wearing the FCu-IUD (FCu-IUD group), 10 cases wearing the FICu-IUD (FICu-IUD group). Immunohistochemical technique was used to determine the expression of VEGF and KDR in endometrium, and the microvessel density (MVD) was counted. The expression of VEGF mRNA was determined by in situ-hybridization. RESULTS: Before insertion of FCu-IUD, the expression of VEGF and KDR proteins was 0.357 +/- 0.032 and 0.215 +/- 0.029 respectively. After insertion of FCu-IUD, the expression of VEGF and KDR proteins was 0.568 +/- 0.027 and 0.244 +/- 0.022 respectively, significantly higher than before insertion (P < 0.05). The expression of VEGF mRNA was 0.359 +/- 0.022 before insertion of FCu-IUD, after insertion of FCu-IUD, the expression of VEGF mRNA was 0.425 +/- 0.019 (P < 0.05). There were no significant changes in the level of VEGF protein and mRNA, as well as KDR in endometrium before and after insertion of FICu-IUD. Compared with before insertion of FCu-IUD 15.4 +/- 2.8, a significant increase in MVD was observed after insertion of FCu-IUD 19.8 +/- 4.8, and the expression of VEGF protein was positively correlated with MVD (r = 0.847, P < 0.01). MVD counts were not different significantly before and after insertion of FICu-IUD. CONCLUSIONS: FCu-IUD can enhance the expression of VEGF and KDR in the endometrium. FICu-IUD can inhibit the activity of VEGF and KDR by releasing indomethacin. VEGF and KDR may be related to the structural and functional changes of microvessels in endometrium after insertion of FCu-IUD or FICu-IUD.

An research on the isolation methods of frozen-thawed human ovarian preantral follicles.

OBJECTIVE: To explore the effective isolation method for preantral follicles from human frozen-thawed ovarian tissue. METHODS: The ovarian cortical tissue was frozen by direct cover vitrification (DCV). The frozen-thawed ovarian tissue was used for isolation of preantral follicles with collagenase combined with mechanical method and mechanical method alone, respectively. RESULTS: 1. There was no statistical difference in the survival rates of follicles in various stages between before and after freezing (P > 0.05). 2. The survival rate of secondary follicles was higher, but the survival rate of primordial follicles was lower in mechanical method alone than in collagenase combined with mechanical method (all P < 0.05). 3. The diameters of follicles were larger and E2 levels were higher in mecha-nical method alone than that in collagenase combined with mechanical method (all P < 0.05). CONCLUSION: After the frozen-thawed ovarian tissue was cultured for 6 days, compared with collagenase combined with mechanical method, mechanical method alone can obtain higher survival rate of secondary follicles, greater follicular diameter and higher E2 level, which are conducive to follicular subsequent development.

Ectopic pregnancy in frozen-thawed embryo transfer: a retrospective analysis of 4,034 cycles and related factors.

We analyzed the incidence of ectopic pregnancy in frozen-thawed embryo transfer (FET) as a function of natural and hormone replacement cycles and ectopic pregnancy-related factors. In this study, there were 4,034 FET cycles performed in our center between January 2005 and December 2010, and the rates of ectopic pregnancy were compared between natural and hormone replacement cycles. The analysis of ectopic pregnancy-related factors in FET was performed with 1:4 age-matched chi-square tests. The rate of ectopic pregnancy was lower in natural FET cycles (1.46%) than in hormone replacement FET cycles (3.31%) with a statistical significance (P < 0.05). Many factors were associated with ectopic pregnancy in FET, but only treatment protocols were considered as a controllable factor. We conclude that the incidence of ectopic pregnancy is significantly lower in natural FET cycles than in hormone replacement FET cycles. The application of exogenous sex hormones in assisted reproductive cycles may be an important factor to cause ectopic pregnancy in FET. This suggests that care should be taken when selecting the treatment protocol in order to avoid ectopic pregnancy.

Follow-up of children born after intracytoplasmic sperm injection with epididymal and testicular spermatozoa.

BACKGROUND: To evaluate the safety of intracytoplasmic sperm injection (ICSI) with epididymal or testicular sperm, this study compared children born after ICSI treatment with epididymal or testicular sperm with children conceived after ICSI with ejaculated sperm. METHODS: This retrospective study included 317 children born after ICSI with percutaneous epididymal sperm aspiration (PESA), 103 children born after ICSI with testicular sperm aspiration (TESA), and a control group of 1008 children born after ICSI with ejaculated sperm. All of the patients received their assisted reproductive treatment in the Reproductive Medicine Center of the First Affiliated Hospital of Zhengzhou University from January 2004 to December 2011. Data, such as the rate of stillbirths, perinatal mortality, gestational age, birth weight, and the rate of congenital malformations of the three groups, were compared. RESULTS: PESA and TESA children were not different from ICSI children in the rate of stillbirths, perinatal mortality, infant mortality rate, gestational age, the rate of prematurity, and the rate of malformations (P > 0.05). A slight increase in birth defects was reported in the TESA group compared with those in the control group, but there was no significant difference between the groups (P > 0.05). CONCLUSION: ICSI with epididymal or testicular sperm does not lead to more stillbirths or congenital malformations compared with ICSI using ejaculated sperm.

Comparative study of pregnancy outcomes between day 3 embryo transfer and day 5 blastocyst transfer in patients with progesterone elevation.

OBJECTIVE: To explore the effects of progesterone (P) elevation on pregnancy outcomes of day 3 embryo and day 5 blastocyst transfer. METHODS: Clinical outcomes (pregnancy and ectopic pregnancy rates) following day 3 embryo and day 5 blastocyst transfer cycles were retrospectively analysed. Day 3 embryo and day 5 blastocyst transfer cycles were divided into normal P level (P ≤ 1.5 ng/ml) and P elevation group (P > 1.5 ng/ml), based on the serum P level on the day of human chorionic gonadotropin (hCG) administration. RESULTS: A total of 2868 cycles were analysed. In day 3 embryo transfer cycles (n = 2345), the clinical pregnancy rate was significantly higher in the normal P level group compared with the P elevation group (55.4% versus 46.7%, respectively) and the ectopic pregnancy rate was significantly lower in the normal P level group compared with the P elevation group (2.8% versus 7.9%, respectively). In day 5 blastocyst transfer cycles (n = 523), there were no significant differences in the clinical pregnancy and ectopic pregnancy rates between the two groups, based on the P level. CONCLUSION: These preliminary findings suggest that day 5 blastocyst transfer should be adopted for patients with P elevation on the day of hCG administration.