A moderately extended time interval between hCG administration and oocyte retrieval is good for most patients with oocyte retrieval scheduled on the same day: a retrospective cohort study.

Evidence is limited regarding the time intervals between human chorionic gonadotropin (hCG) administration and oocyte retrieval in controlled ovarian hyperstimulation cycles, and it is difficult to determine proper schedules to optimise outcomes for patients undergoing oocyte retrieval on the same day. We aimed to identify correlations between factors pertaining to treatment outcomes and time intervals to facilitate working schedules of ART centres. Our study included 2509 patients who underwent ICSI cycles. Based on different time intervals between hCG administration and oocyte retrieval, all cycles were divided into four groups: group 1 (34.00-35.99 hours), group 2 (36.00-36.99 hours), group 3 (37.00-37.99 hours) and group 4 (38.00-39.32 hours). Female age, basal FSH level, Gn starting stimulation dosage and total Gn dosage of group 1 were significantly higher than those of other groups. E2 level on hCG day and number of follicles aspirated were significantly higher in group 4 than in the other groups. Number of oocytes retrieved, oocyte retrieval rate, cleavage rate and number of usable embryos were positively correlated with the time interval, even after adjusting for female age, basal FSH level, E2 on hCG day and number of follicles aspirated. A fixed hCG administration time matching arranged oocyte retrieval is good enough for most patients to achieve maximal treatment outcomes. For patients with lower treatment expectations (expected no. of oocyte retrieval ≤3), moderately delayed oocyte retrieval would be more appropriate.Impact StatementWhat is already known on this subject? The time interval between hCG administration and OPU during COH is essential for ART treatment outcomes, but different intervals were reported in previous studies.What the results of this study add? Fixed hCG administration time matching arranged OPU is good enough for most patients to achieve maximal outcomes. For patients with lower treatment expectations (expected no. of oocyte retrieval ≤3), moderately delayed oocyte retrieval would be required.What the implications are of these findings for clinical practice and/or further research? We studied whether the oocytes and pregnancy outcomes changed along with extended time intervals, and there is no need to adhere to an exact interval for every patient. Therefore, it would help clinicians develop more reasonable time schedules for fertility centres and patients undergoing oocyte recovery on the same day.

No impact of treated hyperprolactinemia on cumulative live birth rate and perinatal outcomes in in vitro fertilization-embryo transfer.

AIM: To investigate whether treated hyperprolactinemia has an impact on pregnancy outcomes in in vitro fertilization/intracytoplasmic sperm injection-embryo transfer (IVF/ICSI-ET). METHODS: A retrospective cohort study was conducted on 535 women who underwent IVF/ICSI-ET between January 2012 and December 2016, of which 123 had treated hyperprolactinemia (case group), 369 were matched controls. Besides, 43 remained hyperprolactinemic after treatment consisted of abnormal group. Cumulative live birth rate (CLBR) after one oocyte retrieval cycle was taken as the primary outcome. A time-to-event analysis using Fine and Gray's test was used to compare CLBR between case and control groups. RESULTS: The median prolactin level was 80.00 ng/mL before dopamine agonist treatment in case group, and it reduced to 14.80 ng/mL after the treatment, similar to the level of control group (15.17 ng/mL, P = 0.316). No significant differences in baseline characteristics were found between case and control groups. The CLBR after one oocyte retrieval cycle were 69.1% (85/123) and 66.4% (245/369) in the case group and control group, respectively (P = 0.580). No significant differences were found between case and control groups in perinatal outcomes. Pregnancy and perinatal outcomes of abnormal group were similar to those of case and control groups. CONCLUSION: Impact of treated hyperprolactinemia on CLBR and perinatal outcomes in IVF-ET was not evident.

What was the fate of human embryos following long-term cryopreservation (≥12 years) and frozen embryo transfer?

STUDY QUESTION: Do human embryos survive long-term cryopreservation (CP) (≥12 years) and implant after frozen embryo transfer (ET)? SUMMARY ANSWER: Human embryos remain usable after long-term CP. WHAT IS KNOWN ALREADY: Several cohort studies have reported the live birth rate or neonatal outcomes of human embryos after CP for up to 5 years. Only a few case reports have described successful live births from human embryos after long-term CP up to 12 years. STUDY DESIGN, SIZE, DURATION: This retrospective observational study in China included 20 patients (128 embryos) from March 2016 to April 2017. PARTICIPANTS/MATERIALS, SETTING, METHODS: Twenty patients who had at least one live birth during their previous IVF/ICSI treatments and had surplus embryos cryopreserved were observed. Data concerning frozen embryo recovery, pregnancy and obstetric outcomes following frozen ET were recorded. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 128 embryos of 20 patients were observed. The embryo storage duration was 12.0-17.1 years, with a mean of 13.9 ± 1.73 years. In all, 115 embryos were thawed to transfer, with a survival rate of 74%. Sixty embryos were further cultured, which resulted in 20 blastocysts with a blastocyst formation rate of 33%. There were 21 cleavage-stage embryos and 13 blastocysts transferred in a total of 12 and 11 cycles, respectively, which resulted in one biochemical pregnancy, one first trimester miscarriage, two ectopic pregnancies, three singletons and one case of twins, with a clinical pregnancy rate of 25% (D3 ET) and 36% (blastocyst transfer) and a live birth rate of 17% (D3 ET) and 27% (blastocyst transfer). Two of the four patients who had live birth developed gestational diabetes mellitus. One of the five live births was a preterm delivery. LIMITATIONS, REASONS FOR CAUTION: The sample size was small due to the unique study population, and all the embryos underwent slow freezing. The fate of long-term cryopreserved embryos after vitrification is still unclear. WIDER IMPLICATIONS OF THE FINDINGS: The results provide evidence to support the use of embryos after extended CP to preserve patients' fertility. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the National Key Research and Development Programme of China (2016YC1000205) and the Guangzhou Scientific Programme (201508020006). None of the authors has any conflicts of interest to declare.

Number of blastocysts biopsied as a predictive indicator to obtain at least one normal/balanced embryo following preimplantation genetic diagnosis with single nucleotide polymorphism microarray in translocation cases.

PURPOSE: The aim of this study is to investigate the minimum number of blastocysts for biopsy to increase the likelihood of obtaining at least one normal/balanced embryo in preimplantation genetic diagnosis (PGD) for translocation carriers. METHODS: This blinded retrospective study included 55 PGD cycles for Robertsonian translocation (RT) and 181 cycles for reciprocal translocation (rcp) to indicate when only one of the couples carried a translocation. Single-nucleotide polymorphism microarray after trophectoderm biopsy was performed. RESULTS: Reliable results were obtained for 355/379 (93.7 %) biopsied blastocysts in RT group and 986/1053 (93.6 %) in rcp group. Mean numbers of biopsied embryos per patient, normal/balanced embryos per patient, and mean normal/balanced embryo rate per patient were 7.4, 3.1, and 40.7 % in RT group and 8.0, 2.1, and 27.3 %, respectively, in rcp group. In a regression model, three factors significantly affected the number of genetically transferrable embryos: number of biopsied embryos (P = 0.001), basal FSH level (P = 0.040), and maternal age (P = 0.027). ROC analysis with a cutoff of 1.5 was calculated for the number of biopsied embryos required to obtain at least one normal/balanced embryo for RT carriers. For rcp carriers, the cutoff was 3.5. The clinical pregnancy rate per embryo transfer was 44.2 and 42.6 % in RT and rcp groups (P = 0.836). CONCLUSIONS: The minimum numbers of blastocysts to obtain at least one normal/balanced embryo for RT and rcp were 2 and 4 under the conditions of female age < 37 years with a basal FSH level < 11.4 IU/L.

The effects of embryo culture media on the birthweight of singletons via fresh or frozen-thawed embryo transfer: a large-scale retrospective study.

BACKGROUND: Embryo culture media used for IVF treatment might affect fetal growth and thus birthweight of the newborns. METHODS: A retrospective study was conducted in South China using data from 2370 singleton neonates born after IVF/ICSI between 2009 and 2012. Two culture media, i.e., either Vitrolife or SAGE were used as embryo culture media during the study period. Neonates' birthweights were compared between the two embryo culture media groups. RESULTS: Among the 2370 singletons, 1755 cases came from fresh cleavage embryo transfer while 615 were from frozen-thawed cleavage embryo transfer. Within the fresh embryo transfer newborns, no statistical difference was observed in either birthweight (mean ± SD: 3196.0 ± 468.9 versus 3168.4 ± 462.0g, p > 0.05) or adjusted birthweight controlled for gestational age and gender (z-score mean ± SD: 0.11 ± 1.02 versus 0.11 ± 0.99 g, P > 0.05) between the Vitrolife (n = 419) and the SAGE group (n = 1336). Likewise within frozen embryo transfer neotates, no statistical difference of the birthweight (3300.6 ± 441.3 vs.3256.0 ± 466.7 g, P > 0.05) and adjusted birthweight (0.30 ± 0.99 g versus 0.29 ± 0.97 g, P > 0.05) was found between the Vitrolife (n = 202) and the SAGE group (n = 413). The sex ratio [OR1.17, 95 % CI (0.94-1.46)/OR1.1, 95 % CI (0.78-1.54)], rate of small for gestational age [OR1.14, 95 % CI (0.82-1.59)/OR1.06, 95 % CI (0.56-2.02)] and large for gestational age [OR1.07, 95 % CI (0.64-1.76)/OR0.98, 95 % CI (0.47-2.02)] in fresh and frozen-thawed subgourps are all comparable respectively between the two culture media. No group differences were found in the rate of low birthweight and macosomia. Multiple linear regression analysis demonstrated that maternal weight, gestational age, frozen-thawed embryo transfer and infant gender were significantly related to neonatal birthweight (P < 0.001). CONCLUSIONS: It appears that embryos cultured in SAGE or Vitrolife media after fresh or frozen-thawed cleavage embryo transfer did not affect neonate's birthweight.

Rotating shift work and menstrual characteristics in a cohort of Chinese nurses.

BACKGROUND: Shift work disrupts the circadian rhythm and may cause menstruation disorders. This study assessed the impact of shift work on menstrual cycle in a population of Chinese nurses. METHODS: Questionnaires on menstrual characteristics and shift schedules were sent to female nurses of the First Affiliated Hospital of Sun Yat-sen University (FAHSYSU) and Guanghua Hospital of Stomatology (GHHS), affiliated to Sun Yat-sen University. Part I was a cross-sectional study and included 139 nurses in GHHS who had regular 8:00-17:30 working (non-shift group), and 334 nurses from FAHSYSU who worked shifts, a response rate of 67.5 % and 59.6 %, respectively (age ≤ 50 years). Menstrual patterns were compared and age-adjusted relative risks of shift work were analyzed. Part II was a nested case-control study. Cases were nurses in Part I who had regular cycle with mean cycle length (MCL) of 25-31 days and but at least 3 days variation in MCL after starting shift work (n = 45). Controls consisted of 67 nurses with matching shift patterns and age, but no MCL changes. A control non-shift age-matched group consisted of 30 GHHS nurses with no MCL changes. A follow-up second questionnaire was sent 2 years later. RESULTS: In Part I, the shift group had a significantly higher proportion of nurses with menstrual cycle irregularity. The proportion of nurses with a cycle of 25-31 days decreased from 81.7 to 67.8 % after changing to shift work. Logistic regression analysis showed that night shift frequency was the only risk factor associated with cycle shortening. After adjusting for age, MCL was shorter when night work was performed > 7 times per month. In Part II, the mean change in MCL in the case group, including prolongation or shortening, was 4.115 ± 2.084 days after shift working. In the 2 years' follow-up, the MCL of the study group did not recover to the original length. CONCLUSIONS: Rotating shift work can increase the prevalence of menstrual cycle irregularity. Night shift frequency was the only risk factor associated with cycle reduced. Changes in MCL did not show recovery over a follow-up period of 2 years.

Influence of menstrual cycle on the expression of clock genes in peripheral blood mononuclear cells in Macaca fascicularis.

OBJECTIVE: To investigate whether the expression patterns of periphery clock genes were influenced by menstrual cycle in a monkey model. STUDY DESIGN: In this preliminary study, the expression patterns of four clock genes (Bmal1, Clock, Cry1 and Per2) in peripheral blood mononuclear cells (PBMCs) from 6 female Macaca fascicularis in menstrual, late follicular and mid luteal phases of menstrual cycle were determined by qrt-PCR. RESULTS: Bmal1 and Per2 mRNA levels were found to exhibit significant diurnal rhythms in all phases of the menstrual cycle. The expression of Cry1 mRNA was statistically rhythmic in late follicular and mid luteal phases. A main effect of menstrual cycle existed on the rhythms of Bmal1, Cry1 and Per2 expression, but not Clock expression. No significant differences were detected between menstrual phase and late follicular phase in all clock genes. Significant differences were found on the expression of Bmal1, Cry1 or Per2 mRNA between late follicular phase and mid luteal phase, when no difference existed in estrogen level, indicating the role of progesterone on biological clock gene expression. Furthermore, the peak of Bmal1 mRNA level slightly advanced in mid luteal phase compared with that in menstrual and late follicular phases. CONCLUSION: The expression patterns of clock genes in PBMCs were influenced by menstrual cycle, potentially by the change of progesterone levels, and this effect maybe correlated with early pregnancy.

[A follow-up study on children conceived through intracytoplasmic sperm injection].

OBJECTIVE: To investigate the perinatal complications, birth defects and growth of children conceived through intracytoplasmic sperm injection (ICSI). METHODS: A total of 575 children conceived by ICSI in our reproductive medical center, were studied. The follow-up study would include items as pregnant complications, neonatal complications, birth defects in perinatal period, subsequently detected birth defects, body weight and body length/height growth. RESULTS: Prematurity and low birth weight of ICSI children were higher in the multiple births than in the singleton births. The rates of materal gestational hypertension, neonatal asphyxia, respiratory distress syndrome, infection diseases were higher in the multiple pregnancies than in the singleton pregnancies (P < 0.05). Eleven ICSI children had died. Ten of them died in the neonatal period and they were preterm infants. One fullterm singleton ICSI child died of hepatoblastoma at the age of 2. The rate of birth defects in perinatal period was higher in ICSI children of multiple pregnancies than in the general population (P < 0.05). The body weight and body length/height of most ICSI children had obtained the standard range between 1 to 3 year-olds. CONCLUSION: The higher rates of perinatal complications in ICSI children were closely related to multiple pregnancies.

[Clinical analysis of intracytoplasmic sperm injection in patients with previous fertilization failure after conventional IVF].

OBJECTIVE: To analyze the clinical outcome of intracytoplasmic sperm injection (ICSI) in patients with previous fertilization failure after conventional IVF. METHODS: Data from 20 ICSI cases (22 ICSI cycles) with previous complete failure of fertilization or with fertilization rate < or = 20% between January 2002 and December 2004 were retrospectively analyzed. The control group consisted of 100 consecutive ICSI cycles for male factor infertility in the same period. RESULTS: The fertilization rate dramatically increased from 5.4% after conventional IVF to 76.9% after ICSI treatment (chi-squared = 264.66, P < 0.001). However, the fertilization rate in the subgroup with previous low fertilization was significantly lower than those in the control and in the subgroup without previous fertilization (67.9% vs 77.5%, 67.9% vs 84.2%). Compared with the control group, the subgroup without previous fertilization had a higher pregnancy rate and implantation rate, but only the difference in the implantation rate was statistically significant (40.5% vs 18.9%). CONCLUSION: ICSI can overcome previous fertilization failure with conventional in vitro fertilization and thus improve the clinical outcome.

[Successful preimplantation genetic diagnosis for beta-thalassemia using multiplex nested polymerase chain reaction].

OBJECTIVE: To develop single-cell multiplex nested polymerase chain reaction (PCR) assays for preimplantation genetic diagnosis (PGD) in couples at risk of having child with beta-thalassemia. METHODS: Primers were designed and synthesized according to the documented mutation sites common among Chinese. Venous blood was collected from 4 pairs of husband and wife, all heterozygotes for beta-thalassemia, and underwent multiple nested PCR. Intraooplasmic sperm injection and mechanical bio psy was used to obtain single blastomere. Multiplex nested PCR was used to detect the CD41-42 mutation and the closely linked polymorphic marker, HumTHO1 gene or CD41-42, CD41-28, IVSII654 mutation and HumTHO1 gene in the single blastomeres from four clinical PGD cycles. The normal embryos with high scores capable of continuing to divide were transplanted into the uteri. The process of gestation was observed. RESULTS: 200 lymphocytes were amplified by nested PCR. The average amplification rate of the most common 16 beta-thalassemia mutations in Chinese population was 91.3% and the average rate of allele drop out for different sites was 17.0% without differences between any 2 sites. During the 4 PGD cycles 33 embryos underwent bioassay with a success rate of 100%. 33 blastomeres were obtained to undergo PCR, of which 30 were successfully amplified with an amplification rate of 90.9%. Explicit diagnosis was obtained in 26 of the 30 embryos: 7 normal homozygotes, 11 heterozygotes, and 8 abnormal or complex heterozygotes. One or more embryos were transferred back into the uteri of the 4 women and clinical pregnancy occurred in one woman. Five weeks after the implantation B-mode ultrasonography showed monocyesis, and in the 17th week of gestational period paracentesis of cord blood showed normal homozygote. At last a normal female infant confirming the PGD result had been born, which was the first reported unaffected pregnancy resulting from PGD using multiplex nested PCR for couples as beta-thalassemia gene carriers. The results of diagnosis for embryo all corresponded to those for blastomere. The average ADO rate of blastomere was 13.3% (4/30). CONCLUSION: PGD using multiplex nested PCR, as an alternative to prenatal diagnosis, is a reliable and effective way to help couples-carriers of pathogenetic genes to get a healthy baby.

[Expression of soluble human leukocyte antigen G and its relationship to embryo development].

OBJECTIVE: To find out the expression of soluble human leukocyte antigen G (sHLA-G) and its relationship to the cleavage embryo development. METHODS: One hundred and seventy-seven day 3 cleavage embryos were detected for sHLA-G by immunohistochemistry. RESULTS: sHLA-G was detected in 57.1% cleavage embryos. The positive rate of sHLA-G in cleavage embryos developed from dipronucleate fertilized eggs was 66.2% (90/136), that developed from tripronucleate fertilized eggs was 26.8% (11/41). There was significant difference between these two groups (P < 0.01). The positive rate of sHLA-G in the grade 1 cleavage embryos developed from dipronucleate fertilized eggs was 64.3% (18/28), that in the grade 2 cleavage embryos developed from dipronucleate fertilized eggs was 91.7% (66/72), that in the grade 3 cleavage embryos developed from dipronucleate fertilized eggs was 16.7% (6/36), there were significant differences between these different embryo grades (P < 0.01), and the intensity of sHLA-G had negative relationship with the embryo grades (r = -0.503). The positive rate of sHLA-G in the first class cleavage embryos developed from tripronucleate fertilized eggs was 88.9% (32/36), that from dipronucleate fertilized eggs was 64.3% (18/28). There was significant difference in these two groups (P < 0.01). The intensity of sHLA-G in cleavage embryos developed from tripronucleate fertilized eggs was higher than that from dipronucleate fertilized eggs. The positive rate of sHLA-G in the cleavage embryos developed from dipronucleate fertilized eggs whose cell number was less than 4 was 56.7% (34/60), that from dipronucleate fertilized eggs whose cell number ranged from 5 to 6 was 67.9% (36/53), and that from dipronucleate fertilized eggs whose cell number ranged from 7 to 8 was 87.0% (20/23). There were no significant differences in these three groups. The intensity of sHLA-G had no significant difference between embryos with different cell number (P > 0.05), but it had relationship with the cell number (r = 0.267). CONCLUSION: Cleavage embryos express sHLA-G which is related with the embryo development.

[Preimplantation genetic diagnosis for beta-thalassemia using whole genome amplification].

OBJECTIVE: To achieve pregnancy with unaffected embryo using in vitro fertilization and embryo transfer (IVF-ET) and preimplantation genetic diagnosis(PGD) for the couples at risk of having children with beta-thalassemia. METHODS: A couple carrying different thalassemia mutations of codon 41/42 and codon IVS2 position 654 received standard IVF treatment and intracytoplasmic sperm injection, embryo biopsy, single cell polymerase chain reaction and DNA analyses, and only the unaffected or carrier embryos were transferred to uterus. Pregnancy confirmation, and prenatal diagnosis were done at 20 week's gestation. RESULTS: A total of 13 embryos were analyzed in the IVF cycle. PGD indicated that 2 were normal 18.1 , 3 were affected 27.3 , and 6 were carriers 54.5 ; diagnosis was not possible in 2. Three embryos were transferred to uterus on the third day after oocyte retrieval. Ultrasonography showed twin pregnancy with one blighted ovum. The prenatal diagnoses revealed that both fetuses were unaffected, one normal baby and one carrier were born. CONCLUSION: These studies represent the successful application of PGD for beta-thalassemia in China.

[Successful preimplantation genetic diagnosis for beta-thalassemia using primer extension preamplification].

OBJECTIVE: To achieve preimplantation genetic diagnosis (PGD) of the couples at risk of having children with beta-thalassemia, as an alternative to prenatal diagnosis. METHODS: Two couples carrying different thalassemia mutations of codon 41/42 and codon intervening sequence 2 position 654 received standard in vitro fertilization treatment and intracytoplasmic sperm injection, embryo biopsy and the whole genome was amplified by primer extension preamplification (PEP). Nested polymerase chain reaction was then used to amplify two mutation sites separately. Both were detected by reverse dot-blot. RESULTS: A total of 35 oocytes were retrieved from the two patients. Among them, 87% showed two pronuclei, and embryo biopsy was performed on 16 of these embryos and 25 blastomeres were obtained. The amplification efficacy was 84%. The genotype study of non-transferred and surplus embryos showed 15% of allele drop-out rate. Five embryos were transferred to the uterus of both patients. One pregnancy achieved, resulted in live healthy twin births, which confirmed the results of PGD. CONCLUSIONS: This unaffected pregnancy resulting from PGD by PEP for beta-thalassemia demonstrates that this technique can be a effective diagnostic tool for carrier couples who desire a healthy child.

[Preimplantation genetic diagnosis for beta-thalassemia].

OBJECTIVE: To investigate the effect of in vitro fertilization and embryo transfer (IVF-ET) and preimplantation genetic diagnosis (PGD) for the couples at risk of having children with beta-thalassemia. METHODS: Four couples carrying different thalassemia mutations received standard IVF treatment. Embryo biopsy was conducted. Single blastomeres were genotyped by a protocol involving primer extension preamplification, nested polymerase chain reaction and reverse dot-blot analysis. Only the unaffected embryos were transferred to the uterus. RESULTS: A total of 97 oocytes were retrieved from the four female carriers. Among them, 83% showed two pronuclei. Embryo biopsy was performed on 47 of these embryos. The amplification efficiency was 84.8%. The average ADO rate was 14.9%. Ten unaffected embryos were transferred. A twin pregnancy with one blighted ovum was confirmed at 7 weeks' gestation by ultrasonography and one normal baby and one carrier of thalassemia mutation were born finally. CONCLUSION: This unaffected pregnancy resulting from PGD for beta-thalassemia demonstrates that PGD technique can be a powerful diagnostic tool for couples carrying beta-thalassemia mutations who desire a healthy child and wish to avoid abortion of an affected fetus.

[Influence of advanced age on the outcome of in vitro fertilization and embryo transfer].

OBJECTIVE: To determine the influence of age on the outcome of In vitro fertilization and embryo transfer (IVF-ET). METHODS: A retrospective study of 139 cycles of conventional IVF and 69 intracytoplasmic sperm injection (ICSI) cycles was performed between January 1999 and December 1999. RESULTS: A total of 208 patients (age 36 to 45 years) after IVF or ICSI were divided into five age groups (36 years group, n = 78; 37 years group, n = 49; 38 years group, n = 50; 39 years group, n = 18; 40 approximately 45 years group, n = 13). Pregnancy rate is 23.1%. There appear to be no significant difference between infertility duration, IVF cycles, fertilization rate and cleavage rate, the number and the quality of embryos transferred, and the prenatal outcome in each age group. But with the age growing, the number of follicles in each group decreased significantly (14.7 +/- 1.2, 13.0 +/- 2.0, 11.3 +/- 0.9, 9.7 +/- 0.9 and 6.5 +/- 1.9 respectively), the pregnancy rate were significantly lower (24.1, 20.5, 13.2, 11.1 and 9.8% respectively), implantation rate were significantly lower too (15.6, 11.2, 10.5, 6.5 and 2.2% respectively). But the abortion rate increase significantly (23.0, 27.2, 33.4, 41.2 and 43.3% respectively), and multiple pregnancy rate decreased (31.2, 27.3, 15.4, 6.7 and 0.0% respectively). The pregnancy rates of the ICSI and IVF were similar after stratification by age. CONCLUSIONS: The fecundity reduct significantly at the age older than 36 years old. It reduct more obviously when the age older than 40 years old. Transferring four or more embryos may increase pregnancy rate, but without increasing multiple pregnancy in women older than 40 years.