Evaluation of microRNA expression profiles in human sperm frozen using permeable cryoprotectant-free droplet vitrification and conventional methods.

ABSTRACT: For sperm cryopreservation, the conventional method, which requires glycerol, has been used for a long time. In addition, the permeable cryoprotectant-free vitrification method has been continuously studied. Although the differences of cryopreservation effects between the two methods have being studied, differences in microRNA (miRNA) profiles between them remain unclear. In this study, we investigated the differences in miRNA expression profiles among conventional freezing sperm, droplet vitrification freezing sperm and fresh human sperm. We also analyzed the differences between these methods in terms of differentially expressed miRNAs (DEmiRs) related to early embryonic development and paternal epigenetics. Our results showed no significant differences between the cryopreservation methods in terms of sperm motility ratio, plasma membrane integrity, DNA integrity, mitochondrial membrane potential, acrosome integrity, and ultrastructural damage. However, sperm miRNA-sequencing showed differences between the two methods in terms of the numbers of DEmiRs (28 and 19 with vitrification using a nonpermeable cryoprotectant and the conventional method, respectively) in postthaw and fresh sperm specimens. DEmiRs related to early embryonic development and paternal epigenetics mainly included common DEmiRs between the groups. Our results showed that the differences between conventional freezing and droplet vitrification were minimal in terms of miRNA expression related to embryonic development and epigenetics. Changes in sperm miRNA expression due to freezing are not always detrimental to embryonic development. This study compared differences in miRNA expression profiles before and after cryopreservation between cryopreservation by conventional and vitrification methods. It offers a new perspective to evaluate various methods of sperm cryopreservation.

[Meta-analysis of the efficacy of bone anchorage and maxillary facemask protraction devices in treating skeletal class â ¢ malocclusion in adolescents].

OBJECTIVE: To assess the efficacy of bone anchorage and maxillary facemask protraction devices in treating skeletal class Ⅲ malocclusion in adolescents. METHODS: Articles relating to the use of bone anchorage and maxillary facemask protraction devices for treating skeletal class Ⅲ malocclusion in adolescents were searched from the databases of Cochrane Library, PubMed, EmBase, CNKI, and Wanfang database. Several inclusion and exclusion criteria were developed for the article screening. The clinical data were extracted, and the quality of the selected articles was evaluated. A Meta-analysis of SNA, SNB, ANB, ANS-Me, Wits, and U1-PP change was performed by using RevMan 5.3. RESULTS: Seven studies (264 patients) were included in the Meta-analysis. Among these studies, three were randomized controlled trials, and four were non-randomized controlled trials. Compared with the maxillary facemask protraction device group, the bone ancho-rage device group had higher SNA changes and lower ANS-Me, Wits, and U1-PP changes (P<0.05). No significant differences were observed in the SNB and ANB changes between these two groups. CONCLUSIONS: Compared with the maxillary facemask protraction device, the bone anchorage device can increase the extent of protraction of the maxilla and has better controls for the labial inclination of the maxillary anterior teeth in treating skeletal class Ⅲ malocclusion among adolescents. However, additional high-quality randomized controlled trials must be performed to verify the results.

A brief exposure to cadmium impairs Leydig cell regeneration in the adult rat testis.

Cadmium is an endocrine disruptor, impairing male reproduction. The objective of this study is to investigate whether cadmium affects rat Leydig cell regeneration and to dissect the underlying mechanism. Adult male Sprague-Dawley rats received a single intraperitoneal injection (i.p.) of 0, 0.5 or 1.0 mg/kg of cadmium chloride, followed by ethane dimethane sulfonate (EDS) treatment to eliminate adult Leydig cells 20 days later. Compared to control (0 dose), cadmium treatment reduced serum testosterone levels by days 21, 35, and 56 after EDS treatment. Serum luteinizing hormone (LH) levels were also affected by day 56, the only time point examined. There were fewer regenerated Leydig cells in the cadmium-treated testis on days 35 and 56 after EDS treatment. Further studies demonstrated that the mRNA or protein levels of Leydig (Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, and Hsd11b1), non-Leydig (Fshr and Dhh), and gonadotroph (Lhb) cells were also significantly lower in cadmium-treated animals. Since LH and desert hedgehog (DHH) are critical factors for Leydig cell differentiation, our result demonstrated that the lower doses of cadmium exposure, even briefly, may permanently damage Leydig cell regeneration.

Leukemia inhibitory factor stimulates steroidogenesis of rat immature Leydig cells via increasing the expression of steroidogenic acute regulatory protein.

Leukemia inhibitory factor (LIF) has many physiological roles. However, its effects on Leydig cell development are still unclear. Rat immature and adult Leydig cells were cultured with different concentrations of LIF alone or in combination with luteinizing hormone (LH) for 24 h. LIF (1 and 10 ng/ml) significantly increased androgen production in immature Leydig cells, but had no effects on testosterone production in adult Leydig cells. Further studies revealed that LIF dose-dependently increased Star and Hsd17b3 expression levels in immature Leydig cells. Gene microarray revealed that the upregulation of anti-oxidative genes and Star might contribute to LIF-induced androgen production. In conclusion, LIF has stimulatory effects on androgen production in rat immature Leydig cells.

Blastocyst-stage versus cleavage-stage embryo transfer in the first frozen cycles of OHSS-risk patients who deferred from fresh embryo transfer.

Elective cryopreservation of all embryos has been the most effective means to avoid developing ovarian hyperstimulation syndrome (OHSS). However, it is still unknown which stage is optimal for freezing and transferring into uterus in OHSS-risk patients. This study was undertaken to evaluate whether OHSS-risk patients could benefit from transferring blastocysts. A total of 162 women were allocated to cleavage-stage embryo transfer (ET) (group A = 70) and blastocysts transfer (group B = 92) on the basis of patients' voluntary in their first frozen cycles. Although the mean number of transferred embryos in group A was significantly more than those in group B (2.37 ± 0.52 versus 2.11 ± 0.52, p < 0.05), the clinical pregnancy rates, implantation rates and live birth rates in group B were significantly higher than those in group A (47.83% versus 31.43%, p < 0.05; 31.44% versus 18.67%, p < 0.05; 40.21% versus 27.14%, p < 0.05), and the multiple pregnancy rates in both groups were comparable (34.09% versus 36.36%, p > 0.05). The observed results in OHSS-risk population allow us to take a position in favor of blastocyst transfer, thus pregnancy and live birth could be achieved with fewer ETs and in a shorter time frame.

[Evaluation of facial aesthetics in young population with beautiful faces in Nanchang city].

PURPOSE: To analyze the full-face and lateral aesthetical standards among fine looking young people in Jiangxi province, in order to provide reference for orthodontic treatment. METHODS: Fourteen young males and fifteen young females from universities in Nanchang city were selected through audition of young beauty within Jiangxi province. Their full-face and lateral pictures were then taken. Ten orthodontists and 85 non-orthodontists in different ages and sexuality who were randomly selected were asked to rank all of the selected pictures in descending order of attractiveness independently and also pick out the most beautiful male and female photos according to their own judgments. Comparisons were carried out to select the universally recognized beautiful males and females and analyze their soft tissue facial profile. RESULTS: Orthodontists held significantly different opinions on the facial esthetic profile for males and females, especially for males. On the other hand, although the non-professionals also had different views on the profiles, the evaluations for males were quite consistent. To be specific, young people selected No.13 male and No.1 female while the old people preferred to select No.7 male and the No.9 female. At the same time, men selected No.7 and No.13 male, and No.1 and No.9 female while women selected No.13 male and No.1 female. CONCLUSIONS: Both orthodontists and non-orthodontists have different opinions on the facial esthetic profile for young males and females. During orthodontic treatments, orthodontists should respect the opinions of the patients and their family members on the matter of facial improvements.

Live birth following vitrification of in vitro matured oocytes derived from sibling smaller follicles at follicle selection phase in the context of in vitro fertilization.

In ovarian stimulation, a 31-year-old woman with polycystic ovary syndrome was at the risk of developing ovarian hyperstimulation syndrome, follicle aspiration was performed, and eight immature oocytes were collected from follicle fluids. After 28 h in vitro culture, six of them reached MII and were vitrified. The patient failed to conceive in her fresh in vitro fertilization cycle and next two replacement cycles. In the third replacement cycle, a successful pregnancy was obtained by vitrified-thawed oocytes. This case demonstrates that follicular aspiration during follicle selection phase has protective effects against developing ovarian hyperstimulation syndrome, and rescued immature oocytes are viable and could produce promising embryos for live birth.

Chronic hepatitis B virus infection in women is not associated with IVF/ICSI outcomes.

OBJECTIVE: This study was to evaluate whether chronic HBV (Hepatitis B virus) infection in women is associated with poor performance following IVF/ICSI (in vitro fertilization/intracytoplasmic sperm injection) treatments. MATERIALS AND METHODS: 123 cycles with female chronic HBV infection were compared with 246 cycles with no-infected couples, matched for female age, D3 serum FSH (follicles stimulation hormone) levels, body mass index and assisted reproductive technology approach used (IVF or ICSI), in a ratio of 1:2. RESULTS: The details in IVF/ICSI cycles, including the dosage of gonadotrophin used, the serum estradiol levels and the endometrial thickness on the day of hCG (human chorionic gonadotrophin) injection, the mean number of oocytes retrieved, and the embryology data, were similar among seropositive and seronegative women. And there was no significant differences in implantation rates and live birth rates between seropositive women group and matched control (30.52 versus 28.34% per transfer; 42.28 versus 40.65%). CONCLUSIONS: The results indicated that women with chronic HBV infection is not associated with outcomes of IVF/ICSI treatments.

[Suppression of mitochondrial oxidative phosphorylation on in vitro maturation, fertilization and developmental competence of oocytes].

OBJECTIVE: To investigate the roles of mitochondrial oxidative phosphorylation (OXPHOS) capacity in oocyte maturation, fertilization and embryo development. METHODS: Carbonyl cyanide p- (tri-fluromethoxy) phenyl-hydrazone (FCCP), a metabolic inhibitor of mitochondria, was introduced into culture medium. The integrity of spindle and chromosome alignment, reactive oxygen species (ROS) levels, and rates of maturation, germinal vesicle breakdown, fertilization and blastulation were assessed in vitro. RESULTS: Significant decreases were detected in the percentages of oocytes with nuclear maturation, normal spindle formation and chromosome alignment, ROS levels and capable for blastocyst formation between oocytes treated with FCCP and non-treated (control group), 55.8%, 37.9%, 0.67 and 57.9% (FCCP 10 nmol/L group), 47.3%,34.7%, 0.59 and 41.8% (FCCP 100 nmol/L group) versus 62.9%, 61.9%,0.94 and 68.3% (control group) respectively, P<0.05. However, No significant differences were found in the rates of GVBD and fertilization in oocytes from the FCCP treated and the control. CONCLUSION: Inhibition of mitochondrial metabolic capacity resulted in decreased the percentages of oocytes with nuclear maturation, normal spindle formation and chromosome alignment, ROS levels and capable for blastocyst formation. But the treatment of FCCP did not affect the rate of fertilization.

[Effects of controlled ovarian hyperstimulation on mitochondria copy number and functions in murine oocytes].

OBJECTIVE: To investigate the effect of whether controlled ovarian hyperstimulation (COH) on mitochondrial DNA (mtDNA) copy number, mitochondrial function, distribution, the level of reactive oxygen species (ROS) in oocytes and the mechanism of oocyte loss in COH. METHODS: Matured murine oocytes were classified into COH group and natural cycles (NC) group. The copies of mtDNA, the magnitude of mitochondrial membrane potential (Δφm) and oocyte adenosine triphosphate (ATP) content, pattern of mitochondrial distribution, and ROS levels were evaluated by realtime PCR, immunofluorescence and fluorescence-luciferase mensuration. RESULTS: The copies of mtDNA, the levels of Δφm, and ATP content in oocytes between COH and NC groups showed statistical difference [(1.15 ± 0.01)×10(5), 0.34 ± 0.03 and (241 ± 20) fmol/oocyte (COH)] versus [(2.15 ± 0.19)×10(5), 0.82 ± 0.07 and (325 ± 11) fmol/oocyte (NC)], respectively(P < 0.05). However, there were no significant differences in the rate of evenly distributed mitochondria and the level of ROS in oocytes from COH and NC [(76.5% (78/102) in COH versus 82.1% (69/84)]; 1.07 ± 0.07 in COH versus 0.93 ± 0.08 in NC (P > 0.05). CONCLUSION: It was indicated that non-physiological COH treatments inhibit mtDNA replication, alter mitochondrial function, which might partly be involved in the low development potential of COH oocyte.

In vitro maturation and fertilization of oocytes from unstimulated ovaries in infertile women with polycystic ovary syndrome.

OBJECTIVE: To evaluate the effects of in vitro maturation and fertilization of oocytes from unstimulated ovaries in infertile women with polycystic ovary syndrome (PCOS). DESIGN: Retrospective study. SETTING: Reproductive Medicine Center, First Affiliated Hospital of Wenzhou Medical College, People's Republic of China. PATIENT(S): One hundred eighteen women with PCOS undergoing 152 cycles of in vitro maturation treatment. INTERVENTION(S): Oocyte retrieval was carried out by ultrasound-guided puncture on days 9-14 of the cycle. The oocytes were cultured in vitro using maturation culture medium, which consisted of M-199 + 20% fetal bovine serum (FBS) + 75 mIU/mL recombinant FSH +/- 0.5 IU/mL hCG. After the oocytes had matured in vitro, fertilization and embryo transfer were performed. MAIN OUTCOME MEASURE(S): Rates of clinical pregnancy, multiple pregnancies, and live birth. RESULT(S): Relatively optimal laboratory results were obtained in this study. Embryo transfer was performed in 140 cycles, with a clinical pregnancy rate (PR) of 40.0% per transfer. Fifty-six babies have been born and there are 10 ongoing pregnancies. The overall multiple PR was 33.93%. CONCLUSION(S): Our results show that using in vitro matured oocytes from unstimulated ovaries could be offered as an alternative to conventional IVF in women with PCOS, and future work should address ways to decrease the incidence of multiple pregnancies.

Impact of assisted hatching on fresh and frozen-thawed embryo transfer cycles: a prospective, randomized study.

The aim of this study was to determine if assisted hatching (AH) could improve the rates of pregnancy and implantation for both fresh and frozen-thawed embryo transfer cycles. A total of 760 fresh embryo transfer cycles and 200 frozen-thawed embryo transfer cycles were randomly assigned to either the treatment group (AH) or the control group (no AH). Zona thinning by laser was performed just before embryo transfer. In fresh embryo transfer cycles, the AH group and control group results were comparable. There were no significant differences in the rates of positive human chorionic gonadotrophin (HCG; 47.5 versus 48.8%), clinical pregnancy (42.4 versus 42.6%), or implantation (26.3 versus 25.2%) between the two groups. However, in frozen-thawed embryo transfer cycles, the rates of positive HCG (32.0 versus 17.0%), clinical pregnancy (25.0 versus 14.0%) and implantation (16.7 versus 7.3%) were significantly greater in the AH group than in the control group (P <: 0.05). The results of this investigation show that in the fresh embryo transfer cycles, laser-assisted hatching by zona thinning has no impact on the rates of positive HCG, clinical pregnancy and implantation, whereas in frozen-thawed cycles, assisted hatching by zona thinning significantly increases all three of these rates.

Exposure to human chorionic gonadotropin during in vitro maturation does not improve the maturation rate and developmental potential of immature oocytes from patients with polycystic ovary syndrome.

OBJECTIVE: To investigate the effects of human chorionic gonadotropin (hCG) in culture medium on the in vitro maturation (IVM) and subsequent developmental potential of human immature oocytes. DESIGN: Prospective, randomized study. SETTING: Reproductive Medicine Center, First Affiliated Hospital of Wenzhou Medical College, People's Republic of China. PATIENT(S): 62 women with polycystic ovary syndrome (PCOS) undergoing IVM treatment. INTERVENTION(S): Immature oocytes were retrieved from unstimulated ovaries of women with PCOS. We tried three different culture systems in this experiment. In group A, oocytes were cultured in the medium containing M199 + 20% fetal bovine serum (FBS) + 75 mIU/mL recombinant follicle-stimulating hormone (FSH) + 0.5 IU/mL recombinant hCG. In group B, oocytes were cultured in hCG-free IVM medium (M199 + 20% FBS + 75 mIU/mL recombinant FSH) for 10 hours, then transferred to the same medium as used for group A. In group C, oocytes were cultured only in hCG-free IVM medium. After the oocytes had matured in vitro, fertilization and embryo transfer were performed. MAIN OUTCOME MEASURE(S): Rates of maturation, fertilization, cleavage, implantation, clinical pregnancy, miscarriage, and live birth. RESULT(S): For groups A, B, and C, the maturation rates at 32 and 48 hours were 46.02% and 69.25%; 43.72% and 64.51%; and 51.87% and 67.51%, respectively. Relatively satisfactory clinical results and implantation rates were obtained in all three groups. No statistically significant differences among groups A, B, and C were found in the rates of maturation, fertilization, cleavage, implantation, clinical pregnancy, miscarriage, or live birth. CONCLUSION(S): The results of our study indicated that the addition of hCG to in vitro culture medium did not improve the maturation rate or development potential of immature oocytes. For the IVM and development of immature oocytes from women with PCOS, hCG appears to be unnecessary.

[In vitro maturation and fertilization of unstimulated immature oocyte for the treatment of infertile women].

OBJECTIVE: To evaluate the efficacy of in vitro maturation (IVM) and fertilization of unstimulated immature oocytes for the treatment of infertile women. METHODS: Fifty-four cycles of IVM were carried out in 40 patients including 26 women with infertility due to polycystic ovary syndrome (PCOS), and 14 patients with history of assisted reproductive technology (ART) failure. Transvaginal ultrasound-guided oocyte collection was performed during menstrual cycle days 9 - 12 without pretreatment of gonadotropins. After 24 - 48 hours of culture, the metaphase II stage oocytes were inseminated by intracytoplasmic sperm injection (ICSI). Embryo transfer was performed 2 or 3 days after ICSI. Laser assisted hatching was done before embryo transfer. RESULTS: Seven cycles were cancelled and the cancel rate was 13% (7/54). A total of 857 immature oocytes were obtained with the mean numbers of 18.2 per cycle, and 632 oocytes developed at MII stage (73.7%, 632/857). A total of 476 oocytes were fertilized by ICSI (75.3%, 476/632), with a cleavage rate of 91.2% (434/476). Embryo transfer was performed in 47 cycles and the mean number of embryos transferred were 4.3 per cycle (range: 2 - 6). The mean endometrial thickness on the day of embryo transfer was 8.9 mm. Nineteen clinical pregnancies were obtained, giving a pregnancy rate of 35% (19/54) per start cycle and 40% (19/47) per transfer cycle. CONCLUSIONS: IVM of unstimulated immature oocytes for the treatment of women with various causes of infertility especially due to PCOS is an effective alternative method. The clinical pregnancy rate of 40% (19/47) is similar to that by conventional in vitro fertilization treatment in our unit.