Association between TSH Level and Pregnancy Outcomes in Euthyroid Women Undergoing IVF/ICSI: A Retrospective Study and Meta-analysis.

The aim of this study was to determine the association between thyroid-stimulating hormone (TSH) level and pregnancy outcomes in euthyroid women undergoing in vitro fertilization (IVF)/intra-cytoplasmic sperm injection (ICSI). A total of 1185 women were enrolled in the retrospective study, and 12 studies with a total of 6624 women were included in the meta-analysis (including the data of the present retrospective study). Participants in the retrospective study were divided into two groups in terms of their serum TSH levels: TSH ≤2.5 mIU/L group (n=830) and TSH >2.5 mIU/L group (n=355). They were monitored for the status of clinical pregnancy or miscarriage. In the TSH ≤2.5 mIU/L group, 441 (53.1%) women achieved clinical pregnancy, while 48 (5.8%) had early pregnancy loss and 12 (1.4%) had ectopic pregnancy. In the TSH >2.5 mIU/L group, 175 (49.3%) women achieved clinical pregnancy, while 21 (5.9%) had early pregnancy loss and 3 (0.8%) had ectopic pregnancy. No significant differences were observed between the two groups in pregnancy outcomes (P=0.126, P=0.512, P=0.297). The meta-analysis also revealed no significant difference in the clinical pregnancy rate and the miscarriage rate between women with serum TSH ≤2.5 mIU/L and those with serum TSH >2.5 mIU/L. In conclusion, high TSH levels (TSH level >2.5 mIU/L) did not affect clinical pregnancy rate or increase miscarriage rate in euthyroid women undergoing IVF/ICSI.

Developmental Potential and Clinical Value of Embryos with Abnormal Cleavage Rate.

To investigate the developmental potential and clinical value of embryos with abnormal cleavage rate, a retrospective analysis was performed on 66 635 2-prokaryotic (2PN) and 1-pronuclear (1PN) embryos. The embryos were given conventionally in vitro fertilization (IVF) treatment and continuously cultured on the day 3 (D3) at the Reproductive Medicine Center, Tongji Medical College, Huazhong University of Science and Technology from January 2016 to December 2017. The embryos were separated into the day-2 (D2) undivided group with 106 cases, the arrested development group with 3482 cases, the blastomere reduction group with 541 cases, and the control group with 62 506 cases, respectively. The blastocyst utilization rates of these three abnormal groups were 2.83%, 10.86% and 6.84%, respectively, which were significantly different from that in control group (39.46%). Furthermore, 2 cases of anabiosis and 1 case of live birth were found in D2 undivided group. In arrested development group, there were 55 cases of anabiosis, 11 cases of clinical pregnancy in single-embryo transplantation (including 6 cases of live birth), and 25 cases of clinical pregnancy in combination with one normal embryo transplantation (including 23 cases of live births and 15 cases of dizygotic twins under B-ultrasound). There were 13 case of anabiosis in blastomere reduction group: there was 1 case of single embryo transplantation and clinical pregnancy was obtained; there were also 6 cases of clinical pregnancy in combination with one single normal embryo transplantation (including 5 cases of live births and 2 cases of dizygotic twins under B-ultrasound). In conclusion, embryos with abnormal cleavage rate still have the potential to continue to develop, and have certain blastocyst utilization rate and live birth.

Analysis of birth defects among children 3 years after conception through assisted reproductive technology in China.

BACKGROUND: Previous studies inconsistently suggest that assisted reproduction technology (ART) may increase the risk of birth defects in children. METHOD(S): Live birth infants, conceived by in vitro fertilization fresh embryo transfer (IVF), intracytoplasmic sperm injection fresh embryo transfer (ICSI), or frozen-thawed embryo transfer (FET) in Reproductive Center of Tongji Hospital (Wuhan, China) between 1997 and 2008, were followed up at birth and after 3 years. Preterm pregnancy, multiple pregnancy, sex ratio (male/female), congenital malformation were compared. RESULT(S): A total of 4,236 children were born after ART (IVF 2,543, ICSI 908, FET 785). Compared with IVF, the rate of preterm pregnancy and sex ratio in ICSI were lower (p < 0.05); the rate of multiple pregnancy in ICSI and FET were all lower than IVF (p < 0.05). Congenital defects were comparable in all groups at birth. In total, 2,908 children participated in the second follow-up from 34 months to 60 months with an average of 40 months, and the cases of birth defects had doubled (3 years: 5.16%, birth: 2.22%). The birth defect rate in boys conceived through ICSI was significantly higher than the IVF group after 3-year follow-up (ICSI boys: 8.62%, IVF boys: 5.21% [p < 0.05]), even though there was no significant difference at birth. CONCLUSION(S): Compared with IVF, FET may not increase risk of birth defects. Children conceived through ICSI, especially males, had higher rates of congenital malformations that were inapparent at birth. So longitudinal monitoring may provide insights into the risks of ART.

[Effect of domestic highly purified urinary follicle stimulating hormone on outcomes of in vitro fertilization-embryo transfer in controlled ovarian stimulation].

OBJECTIVE: To investigate the effect of domestic urine-derived high-purity follicle- stimulating hormone (HP-FSH, Lishenbao) on the outcome of in vitro fertilization(IVF) embryo transfer (ET) in controlled ovarian stimulation (COS). METHODS: From 1 September 2010 to 31 March 2011, total of 3178 infertility patients from 14 Reproductive Center with IVF or intracytoplasmic sperm injection (ICSI) indications who accepted first IVF or ICSI cycle were studied retrospectively. Their causes of infertility include all infertility factors except ovulatory dysfunction infertility and uterine factor infertility. The only long luteal phase gonadotropin-releasing hormone agonist (GnRH-a) protocol was included. Patients were divided into 2 groups according to the type of follicle-stimulating hormone (FSH) agents used: 1932 cases in HP-FSH group and 1246 cases in recombinant FSH (rFSH)group. Patients in both groups were combined with human menopausal gonadotropin (hMG) at doses of 150 U when follicle with diameter reached to 14-16 mm. When 3 dominate follicle with diameter reached 18 mm, hCG at dose of 5000 to 10 000 U or recombinant hCG at dose of 250 µg was administered by intramuscular injection. After 34 to 36 hours, oocytes were obtained guided by ultrasound, then IVF-ET were underwent in their Reproductive Center. The primary endpoint was comparison of live birth rate between the two groups. The secondary endpoints were comparisons of clinical pregnancy rate, miscarriage rate, and implantation rate, as well as COS and IVF outcome between the two groups. RESULTS: (1) There were significantly differences in baseline characteristics of the patients between two groups. The mean age was elder(32 ± 4 versus 30 ± 4, P < 0.01) , the infertility duration was longer (5 ± 4 versus 5 ± 3, P < 0.01) , and antral follicle count (AFC) was less (11 ± 5 versus 13 ± 7, P < 0.01) in patients of HP-FSH group compared with those in patients of rFSH group. (2) As compared with rFSH, the total doses of gonadotropin needed was (2348 ± 1011) U in HP-FSH group versus (2022 ± 659) U in rFSH group, the number of oocytes 13 ± 6 in HP-FSH group and 14 ± 7 in rFSH group, the rate of embryo frozen cycle of 66.30% (1281/1932) in HP-FSH group and 74.88% (933/1246) in rFSH group, which all reached statistical difference (P < 0.01). However, there were no significant different implantation rate [30.49% (1111/3644) versus 32.45% (737/2271)] between two groups. The other clinical parameters did not show significant difference, including clinical pregnancy rate per started cycle [41.61% (804/1932) versus 41.97% (523/1246) ] , clinical pregnancy rate per ET cycle[46.58% (804/1726) versus 48.47% (523/1079)], live birth rate per started cycle[34.21% (661/1932) versus 34.19% (426/1246)], live birth rate per ET cycle [38.30% (661/1726) versus 39.48% (426/1079)], miscarriage rate[13.6% (109/804) versus 16.4% (86/523)], and moderate/severe ovarian hyperstimulation syndrome (OHSS) rate [5.80% (112/1932) versus 7.78% (97/1246)](P > 0.05).(3) Treatment cost: the cost of gonadotropins needed for the patients in HP-FSH group was lower than that in rFSH group (4005 ± 1650 versus 6482 ± 2095, P < 0.01). CONCLUSION: In IVF/ICSI treatment cycles, domestic HP-FSH has similar live birth rate and lower financial burden when compared with rFSH.

[MicroRNA expression and its role in the cell cycle regulation in decidualized endometrial stromal cells in vitro].

OBJECTIVE: To study microRNA (miRNA) expression and role of cell cycle regulation in decidualized endometrial stormal cells (ESC) in vitro. METHODS: ESC was induced decasualization in vitro and matched with non-decidualized cells as controls. The expression repertoire of miRNA was measured by microarray chip and was validated by real-time PCR. Flow cytometry was used to identify ESC cycle during decidual reaction in vitro and after miRNA222 inhibitor was transfected into it. RESULTS: (1) Between decidualized and undecidualized stromal cells, there were 49 miRNAs significantly different expression by microarray chip, including 16 miRNA up-regulation and 33 miRNA down-regulation.hsa-miR-27b, 30c, 143, 101, 181b, 29b, 30d, 507, 23a, 222, 221 exhibited significantly differential expression between decicualized and undecidualized stromal cells by real-time PCR (P < 0.05). (2) After miRNA222 inhibitor (NC-FAM) transfection to decidual ESC, ESC were cultured by FBS medium for 24 hours, the rate of transfection was 70%. ESC were transfected with miRNA 222 inhibitor and cultured for 48 hours, the percentage of ESC at S-phase of (6.2 ± 0.7)% were significantly lower than (10.9 ± 0.8)% in control group (P < 0.05);the percentage of ESC at G(0)/G(1) phase increased at transfection group [(77.5 ± 1.3)% vs. (73.0 ± 1.6)% at control group], but there was no significant difference (P > 0.05). Decasualization ESC were transfected with miRNA 222 inhibitor and cultured for 48 h, the percentage of ESC at S-phase was (3.3 ± 0.6)% in transfection group, which were significantly lower than (7.8 ± 0.9)% in control group (P < 0.05). The percentage of ESC at G(0)/G(1) phase was (80.7 ± 1.6)% in transfection group and (74.9 ± 1.1)%. In control group, which did not reached statistical difference (P > 0.05). CONCLUSION: miRNA was involved in ESC decidual process in vitro by regulating cell cycle.

[Cryopreservation by cryoloop damages human immature oocytes].

OBJECTIVE: 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). METHODS: 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). RESULTS: 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). CONCLUSION: Cryopreservation by cryoloop has a damaging effect on the spindle and chromosome of human oocytes in the GV and M II stages.

[Nuclear status of day 2 preembryos affects embryo quality and implantation potential].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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). CONCLUSION: 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.

[Establishment of two human embryonic stem cell lines from cleavage arrested embryos].

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 blastocyst 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.

[Influence of maternal age on meiotic spindle and chromosome configuration of oocytes].

OBJECTIVE: To study the effect of maternal age on meiotic spindle and chromosome configuration of oocytes. METHODS: 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. RESULTS: 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). CONCLUSION: 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.

[Comparison between the results of ICSI with fresh and with frozen-thawed sperm obtained by PESA to treat azoospermia].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[Influence of postovulatory ageing on balanced predivision of sister chromatid in mouse oocytes].

OBJECTIVE: To study the impact of postovulatory ageing to balanced predivision of oocyte sister chromatid. METHODS: 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. RESULTS: 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). CONCLUSION: 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.

[Vitrification of human cleaved embryos in vitro fertilization-embryo transfer].

OBJECTIVE: To assess the outcome of thawing human cleaved embryos from cryopreservation by vitrification. METHODS: A total of 957 day 2 or day 3 embryos from 219 patients were thawed after vitrification with ethylene glycol 5.5 solution and 0.25 ml straw between Jan 2003 and Jun 2005, 514 embryos were recovered and transferred in 178 patients. RESULTS: The survival rate of thawing embryos and the clinical pregnancy rate after transfer was 72.2% and 19.7% respectively. Twenty-two healthy babies were born from 16 deliveries, including 12 girls and 10 boys, 6 pregnancies ended in miscarriage and another 13 are ongoing. CONCLUSION: Vitrification method is an alternative for cryopreservation of human cleaved embryos because of high effectiveness, convenience and good cost efficiency.

[Study on the mechanism of oocyte aneuploidy by spectral karyotyping].

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

[Expression of p57 and homeobox A10 during decidualization of endometrial stromal cell in vitro.].

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.

[Microorganism contamination in in vitro fertilization-embryo transfer system and their sources].

OBJECTIVE: To investigate the incidence of microorganism contamination in in vitro fertilization-embryo transfer (IVF-ET) and to determine the sources of microorganism. METHODS: Two thousand one hundred and seventy-four cycles of in vitro fertilization from January 1999 to June 2003 were evaluated retrospectively and bacterial cultures were performed in 61 semen samples from asymptomatic men with normal semen parameters and in 34 follicle fluid samples from infertility women through oocyte picking up procedures. RESULTS: Microorganisms were found in 11 cases. The incidence of their contamination in IVF culture system was 0.51% and the most common microorganisms were Escherichia coli and fungi. Microorganisms were detected in 97% of unprocessed semen, 10% in processed semen, 6% in semen mixed with media and 9% in follicle fluid. CONCLUSIONS: The incidence of microorganism contamination was 0.51% and the most common microorganisms were Escherichia coli and fungi. Semen may have the potential to contaminate IVF culture system.

[Expression of telomerase in human ovarian luteinized granulosa cells and its relationship to ovarian function].

OBJECTIVE: To detect the expression of telomerase catalytic subunit (TERT), telomerase in human ovarian luteinized granulosa cells and to investigate the relationship between telomerase expression and the ovarian fecundity. METHODS: Ovarian luteinized granulosa cells were recovered from 22 women with regular menses who underwent in vitro fertilization/intracytoplasmic sperm injection programme. We carried out in situ hybridization histochemistry on luteinized granulosa cells to detect TERT mRNA expression, and telomeric repeat amplification protocol to detect telomerase activity. RESULTS: TERT mRNA were present in luteinized granulosa cells. The expression of TERT mRNA and telomerase activity in ovarian luteinized granulosa cells decreased with increase of age, basal serum follicle stimulating hormone levels. CONCLUSIONS: These results suggest that telomerase may play an active role in ovarian function.A reduced telomerase activity of granulosa cells may be one of the important mechanisms involved in decreased ovarian function in women.