Nuclear status of day 2 preembryos affects embryo quality and implantation potential / 中华男科学杂志

<p><b>OBJECTIVE</b>To assess the effects of the nuclear status of day 2 preembryos on day 3 embryo quality and implantation potential and to weigh its clinical value in the human in-vitro fertilization-embryo transfer (IVF-ET) program.</p><p><b>METHODS</b>Embryos obtained from 409 fresh conventional IVF-ET/ICSI cycles from July to October 2006 were assessed retrospectively. Day 2 preembryos were classified according to the number of nuclei in each blastomere in 3 groups: grade A with only mononucleated blastomeres, grade B with one or more blastomeres containing no visible nucleus, and grade C with one or more multinucleated blastomeres. Comparisons were made of the rates of arrested embryos and excellent embryos on day 3 as well as of the pregnancy outcome and implantation potential of those in whom cohorts of similar nuclear scoring embryos were transferred.</p><p><b>RESULTS</b>There were fewer arrested embryos and more excellent embryos on day 3 in grade A than in grade B and C (P < 0.01), and so were there in grade B than in grade C (P < 0.01). Among the 234 cycles in which all the transfer embryos were derived from a similar day 2 nuclear scoring, 51 cycles originated from grade A embryos (group A) and 183 from grade B (group B), with similar clinical pregnancy rates between the two groups, while the implantation rate was higher in group A than in B (P < 0.05).</p><p><b>CONCLUSION</b>Day 2 nuclear scoring can be used to predict the devel- opment and implantation potential of embryos. A combined evaluation of day 2 nuclear scoring and day 3 embryo morphology helps identify the most viable embryos and reduce the number of embryos for transfer.</p>

Cryopreservation by cryoloop damages human immature oocytes / 中华男科学杂志

<p><b>OBJECTIVE</b>To examine the influence of cryoloop on the spindle and chromosome configurations of human oocytes cryopreserved in the germinal vesicle (GV) and metaphase II (M II) stages, as well as on the survival rate and potential for in vitro maturation (IVM).</p><p><b>METHODS</b>GV oocytes were randomly assigned into a control group (matured in vitro into the M II stage), a GV cryopreserved group (cryopreserved in the GV stage and then matured in vitro), and an M II cryopreserved group (matured in vitro and cryopreserved in the M II stage). After cryopreservation and IVM, immunostaining of the tubulin and chromatin was performed followed by visualization using laser scanning confocal microscopy (LSCM).</p><p><b>RESULTS</b>A significantly higher survival rate was observed in the GV cryopreserved group than in the M II , but the maturation rate showed no significant difference between the GV cryopreserved group and the control (P > 0.05). Compared with the control group, there was a statistically significant decrease in the rates of normal meiotic spindles and chromosomes in the GV cryopreserved group (P < 0.05). A significantly lower rate of normal spindles was noted in the M II cryopreserved group than in the control, but no statistical difference was shown in the rate of normal meiotic chromosomes between the two groups (P > 0.05).</p><p><b>CONCLUSION</b>Cryopreservation by cryoloop has a damaging effect on the spindle and chromosome of human oocytes in the GV and M II stages.</p>

Influence of maternal age on meiotic spindle and chromosome configuration of oocytes / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the effect of maternal age on meiotic spindle and chromosome configuration of oocytes.</p><p><b>METHODS</b>Spindle and chromosome configuration was examined in day 1 unfertilized human oocytes after in vitro fertilization (IVF) and intracytoplasmic sperm injection(ICSI) by immunocytochemistry and visualized by laser confocal microscopy.</p><p><b>RESULTS</b>Statistically significant differences were observed on normal spindle and chromosome configurations of oocytes between 25-29 maternal age group (33% and 31%, respectively), and 30-34 age group (P< 0.05) as well as 35-40 age group(0%, P<0.01). The incidence of abnormal spindle and chromosome configurations of oocytes from 30-34 and 35-40 maternal age groups was much higher than that of oocytes from 25-29 age group (P<0.01, P<0.05).</p><p><b>CONCLUSION</b>Incidence of abnormal spindle and chromosome configuration of oocytes is related to maternal age. It could be an important reason of age related oocyte aneuploidy.</p>

Comparison between the results of ICSI with fresh and with frozen-thawed sperm obtained by PESA to treat azoospermia / 中华男科学杂志

<p><b>OBJECTIVE</b>Retrospective study of the results of ICSI (intracytoplasmic sperm insemination) with frozen sperm obtained by PESA (percutaneous epididymal sperm aspiration) was performed in 27 patients.</p><p><b>METHODS</b>With conventional freezing method, sperm from diagnosing PESA and the remaining motile sperm after treating cycle were frozen. After frozen-thawed and ICSI process, fertilization rate, implantation rate, clinical pregnancy rate were compared and other outcomes including pregnant combinations and parameters of newborns of experimental group (which used frozen-thawed sperm) and control group (which used fresh PESA sperm) were analyzed respectively.</p><p><b>RESULTS</b>One hundred and sixty three and 1 157 oocytes of stage M II were injected respectively in the experimental group (15 cycles) and control group (100 cycles), and fertilization rate of experimental group was prominently higher than that of control group (84.05% vs 73.29%, P < 0.05), while implantation rate and clinical pregnancy rate were of no difference from the control, respectively (23.07% vs 15.73%; 53.33% vs 37.00%, P > 0.05). The differences in newborn's weights between two groups were of no statistical significance (P > 0.05). In the experimental group, eight clinical pregnancies were achieved including 5 live deliveries and 3 ongoing pregnancies, 37 clinical pregnancies including 30 deliveries with only 1 fetal death, 3 ongoing pregnancies and 4 abortions in the control group. Neither vital pregnant combinations nor neonate malformations were found in both groups.</p><p><b>CONCLUSION</b>ICSI using frozen-thawed sperm obtained by PESA is an economic effective and safe method to treat azoospermia. Recovering rates of frozen sperm form PESA should be further increased.</p>

Influence of postovulatory ageing on balanced predivision of sister chromatid in mouse oocytes / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the impact of postovulatory ageing to balanced predivision of oocyte sister chromatid.</p><p><b>METHODS</b>The mouse oocytes were cultured 0-72 h. Then chromosome 16 was detected by fluorescence in situ hybridization (FISH). The oocyte spindle and chromosome configuration were examined by immunocytochemistry.</p><p><b>RESULTS</b>For freshly ovulated mouse oocyte, the balanced predivision of sister chromatid occurred only at 7%. However, for oocytes cultured for 24 h, 48 h and 72 h in vitro, the balanced predivision of sister chromatid occurred up to at 32%, 51% or 62% respectively (P< 0.01). The abnormal cell spindle and chromosome configuration occurred at 9% of freshly ovulated oocytes, but it increased to 63%, 83% and 98% when the oocytes were cultured in vitro for 24 h, 48 h or 72 h respectively (P< 0.01).</p><p><b>CONCLUSION</b>The occurrence of balanced predivision of oocyte sister chromatid may result during postovulatory ageing, and may be related to change of oocyte spindle and chromosome configuration.</p>

Expression of p57 and homeobox A10 during decidualization of endometrial stromal cell in vitro / 生理学报

In order to elucidate the function of homeobox A10 gene (HOXA10) and p57 during decidualization our present study was designed to observe the change of HOXA10 and p57 expression and subcellular localization of HOXA10 in the process of endometrial stromal cell (ESC) differentiation in vitro. Decidualization was induced by 0.5 mmol/L 8-Bromo-cAMP (8-Br-cAMP) together with 1x10(-6) mol/L medroxyprogesterone acetate (MPA). Expression of p57 and HOXA10 was detected by RT-PCR and Western blot after 1-day, 2-day, and 4-day treatment (D1, D2, D4). ESCs cultured in 2%FBS for 1 and 4 d were used as control (C1, C4). The location of HOXA10 was detected by indirect immunofluorescence and HOXA10-GFP transfection. The results are as follows: (1) The expression of HOXA10 decreased progressively during the course of decidualization, and showed significant difference compared to control group C4 after 2-day treatment (D2). (2) On the contrary, the expression of p57 increased progressively and also showed significant difference compared to the control group C4 after 2-day treatment (D2). (3) There was no significant change of HOXA10 and p57 expression after culturing ESCs in 2%FBS for 4 d (C1, C4). (4) HOXA10 located in the nucleus throughout the course. Cytoplasm and nucleus shuttle was not detected in the experiment. Our results suggest that the down-regulation of HOXA10 may contribute to the increase of p57 and that the up-regulation of p57 likely plays an important role in ESC differentiation. Progesterone receptor (PR) pathway may participate in promoting ESCs to exit cell cycle and enter differentiation.

Study on the mechanism of oocyte aneuploidy by spectral karyotyping / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish the method of detecting oocyte aneuploidy by spectral karyotyping (SKY).</p><p><b>METHODS</b>The unfertilized oocytes were fixed 1-2 days after oocyte retrieval. Spectral karyotyping was performed according to the protocol.</p><p><b>RESULTS</b>64% of oocytes were normal, 36% of oocytes were aneuploidy, of which 22% were due to nondisjunction and 14% unbalanced predivision.</p><p><b>CONCLUSION</b>SKY is an effective method for detecting oocyte aneuploidy. Both nondisjunction and unbalanced predivision are involved in oocyte aneuploidy formation.</p>

Establishment of two human embryonic stem cell lines from cleavage arrested embryos / 中华妇产科杂志

Objective To determine whether cleavage developmentally retarded embryos have not cleaved during a 24 hour period could develop into blastocysts and produce hESC cell lines.Methods A total of 120 such embryos were cultured to blastocyst stage by sequential culture.Blastocysts formation rate and quality of blastocyst were detected under microscope.The relation between blastocyst formation rate and blastomere number,the fragment of blastomere and blastomere symmetry were analyzed by stepwise Logistical regression analysis.Inner cell masses(ICMs)were isolated by immunosurgery.Colonies derived from the ICMs were passed every 4-7 days and the derivatives were passaged and identified.Results A total of 22 blastocysts were obtained from 120 embryos.The blastulation rate was 18.7%.Early blatocyst, blastocyst,full blastocyst,expanded blastocyst,hatching blastoeyst and hatched blastocyst accounted for 5.9%,23.5%,35.3%,23.5%,5.9%,and 5.9% respectively.The grade of ICM and trophoblast was mostly scored C or B.Blastocyst formation rate was related to cell number and blastomere symmetry but not fragment.Immunosurgery resulted in the formation of 7 ICMs and 3 primary colonies,which produced 2 cell lines.The cell lines satisfied the criteria that characterize pluripotent hESC cells.Undifferentiated cells were positive for AKP,SSEA-4,TRA-1-60,and TRA-1-81.It could continue to proliferate in vitro and form embryoid bodies when cultured in suspension.It had capability to form teratoma in SCID mice.Both cell lines had normal karyotypes after 45 and 34 passages respectively.Conclusions Our results suggest that a subset of developmentally retarded embryos can form blastocysts and give rise to hESC cell lines.