Factors associated with spontaneous miscarriage risk in IUI treatment: A retrospectively cohort of 31,933 cycles.

To determine the factors associated with intrauterine insemination (IUI) miscarriages and reduce the IUI miscarriage rate, a retrospective study was performed by reviewing 31,933 IUI cycles from 2006 to 2018. The overall there were 14.50% clinical pregnancies, and 16.74% miscarriages. Logistic regression revealed the following three predictive variables: females aged ≥ 35 years (odds ratio [OR] = 2.131; p < 0.001), spontaneous miscarriage history (OR = 1.513; p = 0.005), and ovarian stimulation schemes such as clomiphene citrate (CC) (OR = 1.459; p = 0.003). The natural cycle led to a lower miscarriage rate for patients without spontaneous miscarriage history both for those over 35 years old (OR = 0.402; p = 0.034) and for those under 35 years old (OR = 0.806; p = 0.017). Gonadotropin (Gn) showed the lowest miscarriage rate for patients without abortion history, though no significant differences were found. Patients under 35 with a history of miscarriage were protected from miscarriage by using CC and Gn together (OR = 0.516; p = 0.032). No significant differences were found between various ovarian protocols when patients with abortion history were aged ≥ 35 years (p = 0.606). CC + Gn showed the lowest miscarriage rate. In conclusion, the natural cycle could be suggested for infertility couples to minimize abortion risk. When ovarian induction is required, CC + Gn had the lowest miscarriage rate for women with a history of spontaneous miscarriage while Gn is more successful for individuals without such a history.

How to Attain a Popularity Goal? Examining the Mediation Effects of Popularity Determinants and Behaviors.

Popularity has been examined extensively in recent years, particularly regarding its behavioral correlates. However, much less is known about the social cognitive processes related to popularity and the strategies to attain popularity. This study examined the longitudinal association between popularity goal and popularity status by focusing on the mediation effects of perceived contributing behaviors for popularity (i.e., popularity determinants) and actual behaviors in a sample of 5th and 6th graders (N = 382; 47% girls) in China. The results revealed that participants' popularity goal indirectly related to aggression, academic performance, and prosocial behaviors through the mediation of the corresponding popularity determinant perceptions. Furthermore, participants' popularity goal longitudinally predicted their later popularity status changes through the mediation of perceptions of prosocial behaviors as a popularity determinant and prosocial behaviors. The findings of this study were discussed in relationship to their cultural context.

[Autoradiography observation on cochlea and organs in guinea pigs after intra-abdominal injection of bFGF].

OBJECTIVE: To observe whether bFGF could cross the blood-labyrinth barrier (BLB) after intra-abdominal injection and to establish an experimental basis for its clinical applications. METHOD: Thirty guinea pigs were divided into three groups. Animals in group 1 were administered o I-bFGF, while animals in group 2 and 3 were administered 125 and saline, respectively, via intra-abdominal injection. The both cochlea, blood, liver, brain, thyroid gland and kidney were collected and weighted. A radioimmunoassay analyzer was employed to measure counts per minute (CPM) of each sample, and autoradiography was performed on both cochlea. RESULT: The CPM value of organ samples in the 125I group was higher than that in other groups, and radioactive grain was observed in cochlear samples of this group. In the 125I-bFGF group, blood demonstrated the highest CPM value, while cochlea and brain demonstrated the lowest CPM value, with no radioactive grain observed in cochlear samples. CONCLUSION: bFGF has some difficulties in getting across BLB, so the way of bFGF application in clinics need further study.

Chondrocyte-specific Smad4 gene conditional knockout results in hearing loss and inner ear malformation in mice.

Smad4 is the central intracellular mediator of transforming growth factor-beta (TGF-beta) signaling, which plays crucial roles in tissue regeneration, cell differentiation, embryonic development, and regulation of the immune system. Conventional Smad4 gene knockout results in embryonic lethality, precluding its use in studies of the role of Smad4 in inner ear development. We used chondrocyte-specific Smad4 knockout mice (Smad4Co/Co) to investigate the function of Smad4 in inner ear development. Smad4Co/Co mice were characterized by a smaller cochlear volume, bone malformation, and abnormalities of the osseous spiral lamina and basilar membrane. The development of the hair cells was also abnormal, as evidenced by the disorganized stereocilia and reduced density of the neuronal processes beneath the hair cells. Auditory function tests revealed the homozygous Smad4Co/Co mice suffered from severe sensorineural hearing loss. Our results suggest that Smad4 is required for inner ear development and normal auditory function in mammals.

Smad5 haploinsufficiency leads to hair cell and hearing loss.

The Smads are a group of related intracellular proteins critical for transmitting the signals to the nucleus from the transforming growth factor-beta superfamily at the cell surface. Knockout of the Smad5 is embryonic lethal. However, the Smad5 knockout of single allele (+/-) could survive. We used Smad5 heterozygous knockout (+/-) to determine the role of Smad5 in the development of inner ear morphology and function. In situ hybridization showed that Smad5 was expressed predominantly in hair cells, spiral ganglion, and supporting cells. Measurements of hearing thresholds using auditory brainstem response showed that Smad5 defect resulted in progressive hearing loss between 4 and 24 weeks after birth. Morphological examination revealed apoptosis in the inner ear, with significant loss of outer hair cells in adult Smad5 mutant mice. Our results indicated that deficiency in the Smad5-mediated signaling resulted in apoptosis of hair cells, suggesting Smad5 is a gene that may be related with presbycusis.

[Overexpression of Hath1 induces production of hair cell-like cells in greater epithelial ridge cell cultures from postnatal rat cochlea].

OBJECTIVE: To investigate the effect of Hath1 (human atonal homolog 1) overexpression on greater epithelial ridge (GER) cells from postnatal rat cochlea in vitro. METHODS: GER cells were isolated by using a combinatorial approach of enzymatic digestion and mechanical separation from P1 rat cochlear. The GER cell cultures were infected by adenovirus containing Hath1 and enhanced green fluorescent protein (ad-Hath1-EGFP), while transfecting EGFP(ad-EGFP) was as controls. Immunostaining were performed at different time points after adenovirus infection. RESULTS: Some of the infected GER cells became myosin VIIa-positive following ad-Hath1-EGFP infection. The earliest time point to see induction of hair cell differentiation (hair cell marker expression) by ad-Hathl was 5 days post-infection. In contrast, infection of the GER sheet cultures with ad-EGFP control virus did not show any myosin VIIa-positive cells at 3-12 days post-infection in all cultures examined. CONCLUSIONS: GER cells may potentially serve as hair cell progenitors and they are capable of differentiating hair cell-like cells when forced to express Hath1.

[Relevance between clinical behavior and bionomical findings of acoustic neuromas].

OBJECTIVE: To evaluate the relationship between labeling index (LI) Ki-67, proliferating cell nuclear antigen (PCNA) and transforming growth factor-beta1 (TGF-beta1) with the clinical behavior of acoustic neuroma. METHODS: Expression of Ki-67, PCNA and TGF-beta1 was detected by immunohistochemistry in 53 specimens of acoustic neuromas. The relationship among tumor proliferation, histological representation, size of tumor, clinical proliferation index of tumor and tumor proliferation activity were analyzed. RESULTS: In all 53 cases, the positive rate of Ki-67 was 77.4% (41/53) but the positive rate of PCNA was 84.9% (45/53). There was significant difference between the proliferate index, clinic growth rate and course of disease (t = 2.14, t = 2.70; P < 0.05). The positive rate of TGF-beta1 was 83.0% (44/53). The correlation of TGF-beta1 with LI (Ki-67) was significant difference (r = 0.36, P < 0.05). Cystic degeneration often occurred in large-size tumor (Z = 4.44, P < 0.05). There was no significant relationship between the expression of LI (Ki-67), LI (PCNA) and TGF-beta1 and the course of disease as well as between the cystic degeneration and the non-cystic degeneration. Although clinic growth rate of cystic degeneration was bigger than that of non-cystic degeneration, there was not statistically significant. CONCLUSIONS: Ki-67 and PCNA are reflected proliferation activities of tumor cells in acoustic neuromas. Cell proliferation-labeling index LI (PCNA) was related with clinical growth rates. TGF-beta1 might participate in the biological behavior of acoustic neuroma. Cystic degeneration was one of special pattern of acoustic neuroma, however, tumor enlargement might due to the volume of the cystic but unrelated to fast proliferation of parenchyma cell.

Isolation, growth and differentiation of hair cell progenitors from the newborn rat cochlear greater epithelial ridge.

Mammalian cochlear hair cell loss is irreversible and leads to permanent hearing loss. To restore hearing physiologically, it is necessary to generate new functional hair cells either from endogenous cells or from exogenously transplanted hair cells/progenitors. Previous studies suggest that cochlear greater epithelial ridge (GER) and lesser epithelial ridge (LER) cells are capable of differentiating into hair cells. While it was recently possible to obtain and culture pure LER progenitors, isolation of pure GER progenitors has not been reported. Here we describe a method that allows isolation of pure GER cells from neonatal rat cochleae. The cochlear epithelial sheet (CES) containing GER progenitor cells was mechanically separated from the underlying mesenchymal tissue after digestion with thermolysin. The GER area could then be dissected following mechanical removal of organ of Corti as well as all the lateral area. The isolated GER cells showed significant proliferation and expressed markers for GER cells but not markers for hair cells or LER. When the GER cells were cultured in serum-free medium containing epidermal growth factor, spheres were formed where they continued to proliferate. Furthermore, when GER cells were induced to express Hath1 or co-cultured with mesenchymal cells prepared from neonate rat cochleae, they showed the potential to differentiate into hair cell-like cells. Successful isolation, culture and differentiation of GER hair cell progenitors will shed additional light on the mechanism of hair cell differentiation and potential hair cell replacement.

[In vitro culture of greater epithelial ridge cells from rat cochleae].

OBJECTIVE: To establish in vitro culture systems of greater epithelial ridge (GER) cells from rat cochlear and to investigate the characterization, growth pattern and ultrastructure of GER cells. METHODS: Using a combinatorial approach of enzymatic digestion and mechanical separation to allow isolation and culture of GER cells from P1 rat cochleae. The dissociated GER cells were cultured in serum-free or 10% fetal bovine serum DMEM respectively. BrdU, phalloidin, ZO1, calretinin and myosin VIIa immunostaining and scanning electron microscope observation were performed in GER cell cultures. RESULTS: The dissociated GER cell cultures showed positive to ZO1, phalloidin and BrdU staining, but negative to myosin VIIa and calretinin. They assumed a polygonal morphology which was similar to epithelial cells and grew in islands-like patches in medium containing 10% fetal bovine serum while forming spheres in serum-free medium. The GER cells presented significant ability to proliferate in both conditions. Scanning electron microscope showed that there was microvillus and centre bodies but not hair cell specific stereociliary bundles on the surface of GER cultures. CONCLUSIONS: The GER cell cultures showed significant ability to proliferate and grew in islands-like patches in medium containing 10% fetal bovine serum while forming spheres in serum-free medium. The dissociated GER cells expressed epithelial cell specific marker but not marker of hair cells.

[Expression of three kinds of transcription factors in greater epithelial ridge cells of rat cochlear].

OBJECTIVE: To detect the expression of Math1, Hes1 and Hes5 in greater epithelial ridge (GER) cells of rat cochlear and explore their influence on hair cell differentiation. METHODS: Postnatal day 0 (P0), day 1 (P1) , day 3 (P3) day 4 (P4) and day 5 (P5) rat cochlear were dissected respectively and then pure GER cells were separated by a combinatorial approach of attachment and mechanical separation. The total RNA of GER cells was extracted by Trizol one step method and the expression of Math1, Hes1 and Hes5 in GER cells was detected with reverse transcription polymerase chain reaction. RESULTS: Math1 was expressed in P0 - P5 rat GER cells and Hes1 was expressed only in PO - P3 rat GER cells, while there was no expression of Hes5 in P0 - P5 rat GER cells. CONCLUSIONS: Probably only when the expression of Math1 reaches a certain level can it induce GER cells to differentiate into hair cells. Meanwhile this process might controlled by Hes1 to some extent.

[Isolation and identification of greater epithelial ridge in rat cochlea].

OBJECTIVE: To investigate the method for isolating greater epithelial ridge by use of thermolysin digestion combined with dissection under microscope. METHOD: The basement membrane prepared from postnatal day 0 to postnatal day 3 rat was placed into D-Hanks solution with thermolysin and DNase, then incubated at 37 degrees C for 25 minute and dissected under microscope. After GER had been isolated, morphological comparison, immunohistochemistry, RT-PCR and GER culture were performed for the purpose of identification of GER. RESULT: Immunohistochemistry indicated that the isolated GER was purely epithelial tissue. RT-PCR analysis certified that the isolated GER expressed Hes1 which was selectively expressed in GER at early postnatal in cochlea. The isolated GER could be successfully cultured in the presence of 5% serum and could survive at least 12 day in the serum-free medium. CONCLUSION: GER isolation can be successfully performed by use of thermolysin combined with microdissection. This method may provide a good technique to establish the GER cell lines or perform series of gene transfection experiments.

Isolation and culture of hair cell progenitors from postnatal rat cochleae.

Cochlear hair cells are a terminally differentiated cell population that is crucial for hearing. Although recent work suggests that there are hair cell progenitors in postnatal mammalian cochleae, isolation and culture of pure hair cell progenitors from a well-defined cochlear area have not been reported. Here we present an experimental method that allows isolation and culture of hair cell progenitors from postnatal rat cochleae. These progenitor cells are isolated from the lesser epithelial ridge (LER, or outer spiral sulcus cell) area of pre-plated neonatal rat cochlear segments. They express the same markers as LER cells in vivo, including ZO1, Islet1, Hes1, and Hes5. When these cells are induced to express Hath1, they show the potential to differentiate into hair cell-like cells. Interestingly, these cells can be lifted from monolayer cultures and maintained in aggregate cultures in which spheres can be formed. Hair cell progenitors in the spheres display their proliferating capability and express only epithelial markers. Furthermore, when these spheres are mixed with dissociated mesenchymal cells prepared from postnatal rat utricular whole mounts, and replated onto a collagen substratum, the epithelial progenitor cells are able to differentiate into cells expressing markers of hair cells and supporting cells in epithelial islands, which mirrors the inner ear sensory epithelium in vivo. Successful isolation and culture of hair cell progenitors from the mammalian cochlea will facilitate studies on gene expression profiling and mechanism of differentiation/regeneration of hair cells, which are crucial for repairing hearing loss.

[Expression of aquaporin-1,3 in the cochlea and endolymphatic sac of guinea pig].

OBJECTIVE: To detect the expression of Aquaporin-1,3 in the cochlea and endolymphatic sac of guinea pig. METHOD: Two-step immunohistochemical method and immunofluorescence was used to examine the expression of Aquaporin-1,3. RESULT: Aquaporin-1 was expressed on the basilar part of spiral ligament, basal membrane of Corti's organ and epithelialis of scala tympani, and basilar part under the cellular epithelialis in endolymphatic sac. Aquaporin-3 was expressed on stria vascularis, spiral ligament, Corti's organ, spiral ganglion and, the basilar part and the cellular epithelialis in endolymphatic sac. CONCLUSION: Aquaporin-1,3 were widely expressed in the cochlea and endolymphatic sac of guinea pig.

[The expression pattern of L-type voltage-gated Ca2+ channel in rat cochlea].

OBJECTIVE: To investigate the molecular basis of the voltage-gated Ca2+ channel (VGCC) which controls the afferent synaptic neurotransmitters release in inner hair cell and their cellular expression. METHOD: In this study we used different and complementary approaches to determine which types of 4 subunits of VGCC were expressed in the organ of Corti. RESULT: RT-PCR analysis showed that alpha1C and alpha1D were present in the organ of Corti total RNA extract, and the corresponding antisense riboprobes could detect the expression of alpha1C and alpha1D in IHCs and OHCs. CONCLUSION: The voltage-gated Ca2+ channel expressed in sensory cell in organ of Corti are alpha1C and alpha1D subtypes.

[Construction of bicistronic eukaryotic vector containing basic fibroblast growth factor and study of their functions in gene therapy for hearing impairment].

OBJECTIVE: To construct basic fibroblast growth factor(bFGR) and enhance green fluorescence protein(EGFP) fusion gene eukaryotic expression vector internal ribosome entry site (pIRES)-bFGF-GFP and to evaluate the effect of transduction bFGF gene on noise induced hearing-loss in inner ear hair cells of guinea pigs. METHODS: Human bFGF cDNA was inserted into mammalian expressed plasmid pIRES-EGFP. The recombinant expression plasmid pIRES-bFGF-EGFP was transfected into inner ear of guinea pigs, using lipofectin method. The transduced bFGF gene was mediated by SA lipidsome. IRES- bFGF-GFP was administered into the round window as rescue agent at the same time of noise exposure or as a protective agent 7 days before. RESULTS: SA liposome-mediated bFGF expressed at a high level in the cochlea of guinea pigs, and in the rescue group, a significant lower hearing thresholds was displayed. pIRES- bFGF-EGFP could protect hair cells. It demonstrated that pIRES- bFGF-GFP could protect the inner ear both structurally and functionally. bFGF/EGFP gene could be transcripted and translated into inner ear hair cells of guinea pig. bFGF/EGFP gene could express a specific protein. The recombinant bFGF/EGFP had significant protective effect as well as manifestation of autonomous fluorescence. CONCLUSION: bFGF/EGFP fusion protein not only expressed in hair cells of guinea pig but also showed significant bFGF activity and autonomous fluorescence. IRES induced exogenous gene could enter the hair cells.

Effects of alpha-tocopherol on noise-induced hearing loss in guinea pigs.

Preventing noise-induced hearing loss (NIHL) by antioxidants is based on the hypothesis that generation of reactive oxygen species is one of the causes of NIHL. alpha-Tocopherol is a naturally occurring antioxidant with no noticeable side effects. In this study, we attempted to protect guinea pigs from developing NIHL by administering alpha-tocopherol. Pigmented male guinea pigs were exposed to a noise (4 kHz octave band, 100 dB SPL), 8 h/day for 3 days consecutively. alpha-Tocopherol (10 mg/kg or 50 mg/kg daily) was given by intraperitoneal injection from 3 days before through 3 days after the noise exposure. Auditory evoked brainstem response (ABR) thresholds at 2, 4 and 8 kHz were recorded prior to the experiment, immediately post-noise, 2 and 8 days post-noise. On day 8 post-noise, after the ABR recording, guinea pigs were decapitated and the cochleae were removed for cochlear surface preparations and scanning electron microscope (SEM) study. ABR threshold shifts of groups receiving alpha-tocopherol were significantly smaller than those of groups not receiving alpha-tocopherol at all frequencies and all time points tested except that of group 3 at 8 kHz 8 days post-noise. No hair cell loss was seen on the surface preparations, but stereocilia loss was found by SEM study. The noise-induced stereocilia loss was significantly decreased by alpha-tocopherol. These results indicate that alpha-tocopherol can attenuate the noise-induced cochlear damage. Further investigations on the preventive effect of alpha-tocopherol on NIHL in noise-exposed workers are necessary.

[Effects of noise on antioxidant enzymes of cochlea in guinea pigs].

OBJECTIVE: To investigate the effect of noise on the antioxidant enzymes of cochleae. METHODS: 16 male pigmented guinea pigs (250 - 300 g) were randomly divided into 2 groups, control group and noise group. Each group had 8 animals. The animals in noise group were performed auditory evoked brainstem responses (ABR) recording before and after exposure to a continuous noise (4 kHz, octave band, 100 dB, SPL) 8 h/d for 3 consecutive days. Immediately at the end of the third day's noise exposure after ABR recording, guinea pigs were decapitated. Both the right and the left cochlea with the bony capsule removed were homogenized, and the supernatants were prepared for assays. Reactive oxygen species (ROS), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) were measured. RESULTS: ROS level of the noise group [(281.2 +/- 3.5) U/mg pro] was significantly higher than that of the control group [(273.0 +/- 3.2) U/mg pro, P < 0.05] and SOD, CAT and GSH-Px activities of the noise group [(206.5 +/- 5.1) NU/mg pro, (47.0 +/- 9.0) U/g pro, (14.1 +/- 2.5) U/mg pro respectively] were significantly lower than that of the control group [(221.8 +/- 4.8) NU/mg pro, (60.8 +/- 9.9) U/g pro, (21.1 +/- 3.1) U/mg pro respectively, P < 0.05]. CONCLUSION: Noise may damage the defensive system of antioxidant enzymes in cochlea.

[Protective and rescue effects of transgenic bFGF/GFP expression mediated by cationic liposome on gentamicin-induced guinea pig cochlear toxicity].

OBJECTIVE: To observe the expression of cationic stearylamine (SA) liposome mediated basic fibroblast growth factor/green fluorescence protein (bFGF/GFP) gene in the cochlea of guinea pig, and evaluate the protection and rescue action of bFGF against the damage caused by gentamicin. METHODS: Thirty-six guinea pigs were divided into 3 experimental groups. Prevention group with inoculation of SA-bFGF/GFP complexes through the round window of right ear and injection of gentamicin 150 mg.kg(-1).d(-1) one day after for 8 days and rescue group with injection of gentamicin for 8 days and infusion of SA-bFGF/GFP complexes in the same way on the ninth day, and control group with only injection of gentamicin for 8 days. Auditory brainstem responses (ABR) was measured prior to and after the administration and before the animals were killed respectively. The animals were killed after the experiment and ABR test, and specimens and slices of chochleae were made to examine the absence of outer and inner hair cells. The expression of GFP in cochlea shown by green fluorescence was observed with fluorescent microscopy. RESULTS: Fluorescent microscopy showed green fluorescence in the cochleae of guinea pigs in prevention and rescue groups. There was no significant difference in ABR threshold between the left and right ears of animals in each group before and after experiment, among both ears of animals in the 3 groups before experiment, and between prevention and rescue group groups before killing (all P > 0.05). However, the ABR thresholds in prevention group and rescue group were significantly lower than that in control group before the animals were killed (P < 0.01 and P < 0.05). The average number of lost outer hair cells was 5 106 +/- 299 cells and 5 605 +/- 109 cells in prevention group and rescue group respectively, without a significant difference between them (P > 0.05). The average amount of missing inner hair cells was 301 +/- 64 cells and 487 +/- 92 cells in prevention group and rescue group respectively, without a significant difference between them (P > 0.05), and significantly lower than that in control group (1 062 +/- 67, P < 0.01 and P < 0.05). The average amount of missing outer hair cells in control group was 6 248 +/- 119 cells, significantly higher than that in prevention group (5 106 +/- 299, P < 0.01) and that in rescue group (5 605 +/- 109, P < 0.05). CONCLUSION: SA-liposome mediated bFGF/GFP gene, which was perfused in one ear, can be expressed highly in both cochleae of the guinea pig, and may protect and rescue cochlea against gentamicin ototoxicity.

[Protective effect of adenoviral-mediated brain derived neurotrophic factor gene on spiral ganglion].

OBJECTIVE: To observe the expression of brain derived neurotrophic factor (BDNF) in the cochlea of guinea pig, and assess the activity of BDNF in spiral ganglion cell following the damage of noise. METHODS: Twenty-seven guinea pigs were exposed to a 4 kHz narrow band noise at 135 dB SPL for 4 hours. At seven days after the noise exposure, twelve guinea pigs were inoculated with ad-BDNF, and twelve guinea pigs were inoculated with ad-LacZ, while three guinea pigs were inoculated with artificial perilymphatic fluid. The animals were sacrificed After 1 week, 4 weeks and 8 weeks, respectively. The cochlea was stained with immunohistochemisty (ABC method) to examine the expression of BDNF. The number of spiral ganglion cell was counted to assess the activity of BDNF. RESULTS: BDNF gene was expressed in whole cochlea, and the expression was obviously elevated 1 week after the adenovirus administration and reduced thereafter. The number of degenerated cells in ad-BDNF group in the eight weeks was lower than that in another two groups and the different between them was statistical significant (P < 0.01). CONCLUSION: Adenoviral-mediated brain derived neurotrophic factor can be expressed in a high level in the cochlea of guinea pig, and may prevent the cell of spiral ganglion from the damage of noise. This study lays the groundwork for alleviation of hearing loss using gene therapy.