Hai-Yan Zheng

Combined analysis of endometrial thickness and pattern in predicting outcome of in vitro fertilization and embryo transfer: a retrospective cohort study

ObjectiveTo evaluate the combined effect of endometrial thickness and pattern on clinical outcome in patients undergoing in vitro fertilization/intracytoplasmic sperm injection and embryo transfer (IVF/ICSI-ET).MethodsCycles of IVF/ICSI-ET conducted between January 2003 and December 2008 at a university-based reproductive center were reviewed retrospectively. Endometrial ultrasonographic characteristics were recorded on the day of hCG administration. In the combined analysis, endometrial thickness groups (group 1: equal or <7 mm; group 2: 7-14 mm; group 3: >14 mm) were subdivided into two endometrial patterns (pattern A: triple-line; pattern B: no-triple line). Clinical pregnancy rate (CPR) and early miscarriage rate in different groups were analyzed.ResultsA total of 2896 cycles were reviewed. Clinical pregnancy rate (CPR) was 24.4% in group1-A. There were no second trimester pregnancies in group 1-B. Miscarriage rate in group 2-A was significantly lower compared to group 2-B (P < 0.01), although CPR did not show any significant differences between the groups. A no-triple line endometrial pattern with moderate endometrial thickness (7-14 mm) had a detrimental effect on pregnancy outcome, but not the occurrence of pregnancy. In group 3, there was no difference in CPR and miscarriage rates between the two patterns; adequate endometrial thickness (>14 mm) seemed to mitigate the detrimental impact (high miscarriage rate) of pattern B.ConclusionCombined analysis of endometrial thickness and pattern on the day of hCG administration was a better predictor of the outcome of IVF/ICSI-ET and may be more helpful for patient counseling than the separate analyses.

Aberrant DNA methylation of imprinted H19 gene in human preimplantation embryos

DNA methylation patterns of the H19 differentially methylated region were successfully determined in 32 of 50 human day 3 embryos produced and discarded after assisted reproductive technology procedures. We found methylation patterns similar to those of somatic cells in 81.3% of embryos and demethylation or hypomethylation patterns in 18.7% of embryos.

Aberrant DNA methylation of imprinted loci in human spontaneous abortions after assisted reproduction techniques and natural conception

STUDY QUESTION Do assisted reproduction techniques (ARTs) affect DNA methylation of imprinted genes and does aberrant methylation of imprinted genes account for the incidence of human spontaneous abortion (SA)? SUMMARY ANSWER Our results show that imprinting errors of imprinted genes may contribute to human SA, and the occurrence of aberrant methylation of imprinted genes in ART pregnancies was comparable with that in natural pregnancies. WHAT IS KNOWN ALREADY Animal data and human studies demonstrated that in vitro culture of embryos can cause methylation defects in individual genes, which might affect subsequent embryonic development and contribute to SA. However, our previous studies showed an abnormal methylation pattern of PEG1 in human aborted chrionic villus samples (CVS) but an increased occurrence of aberrant methylation in CVS from ART-derived pregnancies was not observed. STUDY DESIGN, SIZE AND DURATION CVS were collected from women who underwent abortion procedures in the Department of Gynecology and Obstetrics in Nanfang Hospital from May 2008 to July 2011. Muscle samples (MS) were obtained from aborted fetuses and stillbirths. The samples were divided into four experimental groups: (A) SA/stillbirth after ART (n = 75), (B) multi-fetal reduction after ART (n = 73), (C) SA/stillbirth of natural pregnancies (n = 90) and (D) induced abortion (IA) of natural pregnancies (n = 82). PARTICIPANTS/MATERIALS, SETTING AND METHODS The mean ± SD age of patients was 31.0 ± 4.1 (range: 18-45 years). The DNA methylation patterns of one paternally methylated (H19) and two maternally methylated (LIT1 and SNRPN) genes were analyzed in CVS and MS using pyrosequencing and bisulfite sequencing PCR. MAIN RESULTS AND THE ROLE OF CHANCE Clear hypo-methylation (<10%) or hyper-methylation (>90%) were not detected in LIT1 and SNRPN but two regions of hyper-methylation (91.7 and 91.4%) were observed in H19. The mean percentage of methylation in the SA samples (groups A and C) was higher than that in the IA samples (groups B and D; P<0.05). Box plot analyses showed that in the 165 SA samples, methylation values for 40/495 (8.1%) differentially methylated regions of the three genes represented outliers. The incidence of outlier was highest for LIT1 (13.3%, 22/165). In contrast, no outliers were found in the 155 IA samples. The receiver operating characteristic curve analyses showed a positive correlation between percentage methylation of all three genes and incidence of SA (P<0.05). In addition, the conception modes (natural versus ART) and the fertilization methods used in ART (IVF and ICSI) did not affect the methylation patterns of the imprinted genes. No increase in the rate of abnormal methylation was found in the ART samples. LIMITATIONS AND REASONS FOR CAUTION The studied loci represent only a small fraction of developmentally important genes. Further studies are needed to evaluate changes in the expression and the methylation status of other genes that may lead to SA. WIDER IMPLICATIONS OF THE FINDINGS The findings provide new insights into the etiology of human SA. The possibility that the abnormal methylation seen is a consequence of the defect that led to the SA cannot be excluded. STUDY FUNDING/COMPETING INTEREST(S) None of the authors has any competing interest. This study was supported by National Natural Science Foundation of China (81170574), The National Key Basic Research Development Plan of China (973 Program) (2007CB948104), Comprehensive strategic sciences cooperation projects of Guangdong Province and Chinese Academy (04020416) and Guangzhou Science and Technology Program key projects (11C22120737).

Ovarian damage after laparoscopic endometrioma excision might be related to the size of cyst

OBJECTIVE To investigate the relationship between the size of an excised endometrioma and the magnitude of damage to the ovary after the surgery. DESIGN A retrospective, controlled study. SETTING A university hospital. PATIENT(S) Eighty-five women with a history of laparoscopic excision of unilateral endometrioma who underwent in vitro fertilization (IVF). INTERVENTION(S) IVF-embryo transfer procedures. MAIN OUTCOME MEASURE(S) Antral follicle counts (AFC), number of dominant follicles (follicles ≥ 15 mm), and number of oocytes retrieved. RESULT(S) In the group with cyst diameters of ≥ 4 cm and group with cyst diameters of <4 cm, the AFC, number of dominant follicles, and number of oocytes retrieved were decreased in the operated ovaries when compared with those in intact ovaries; in the former group, a statistically significant reduction was observed. The differences of AFC, number of dominant follicles, and number of oocytes retrieved from both ovaries were further compared among the two groups: the decrease in the group with cyst diameters of ≥ 4 cm was higher than in the group with cyst diameters of <4 cm. After adjusting for age and AFC in intact ovaries, similar results were obtained, although AFC only showed a tendency. In addition, the receiver operating characteristic curve analysis revealed a statistically significant, positive correlation between the size of excised cysts and the incidence of fewer than four oocytes retrieved from an operated ovary. CONCLUSION(S) The magnitude of the ovarian damage after laparoscopic endometrioma excision might be related to the size of cyst; the damage to ovaries is more severe when an endometrioma ≥ 4 cm is excised.

Circulating luteinizing hormone level after triggering oocyte maturation with GnRH agonist may predict oocyte yield in flexible GnRH antagonist protocol

BACKGROUND The use of gonadotrophin-releasing hormone (GnRH) agonist for triggering final oocyte maturation and ovulation can reduce ovarian hyperstimulation syndrome (OHSS) in high-risk patients. LH levels post-trigger with GnRH agonist might be correlated with oocyte yield and maturity. Our aim was to evaluate the relationship between serum LH level at 12-h post-trigger and oocyte yield, maturity and fertilization rate in patients at high risk of OHSS and therefore who were treated with a flexible GnRH antagonist protocol in which final oocyte maturation was triggered with GnRH agonist. METHODS In a prospective cohort study, 91 patients at high risk of OHSS were treated with a flexible GnRH antagonist protocol and divided into six groups according to their serum LH levels at 12-h after GnRH agonist administration: ≤15.0, 15.1-30.0, 30.1-45.0, 45.1-60.0, 60.1-75.0 and >75.0 IU/l. The oocyte yield, maturity, fertilization rate and clinical outcomes for each LH interval were analyzed. RESULTS There was a statistically significant reduction in oocyte yield with a concentration of serum LH ≤15.0 IU/l (P < 0.05), whereas no statistically significant differences in the oocyte maturity and fertilization rate among the six groups (P > 0.05) were seen. Only 5 out of 91 patients (5.5%) had a serum LH ≤15.0 IU/l at 12-h post-trigger with GnRH agonist. In addition, no statistically significant difference was seen regarding high-quality embryos, implantation rate, clinical pregnancy rate and early miscarriage between patients with LH ≤15.0 IU/l and >15.0 IU/l (P > 0.05). CONCLUSIONS Serum LH level at 12-h post-trigger with GnRHa <15.0 IU/l is associated with a dramatically lower oocyte yield but not with the oocyte maturity and fertilization rate. Serum LH levels post-trigger with GnRH agonist do not affect clinical outcomes.

Abnormal methylation patterns at the IGF2/H19 imprinting control region in phenotypically normal babies conceived by assisted reproductive technologies

OBJECTIVE To evaluate epigenetic risks linked to assisted reproductive technologies (ART) by determining the methylation status of the IGF2/H19 imprinting control region (ICR) in offspring born after ART. STUDY DESIGN A combined bisulfite restriction analysis (COBRA) and sequencing technique were used to determine the methylation status of the IGF2/H19 ICR in 61 phenotypically normal newborns conceived by ART. Thirty naturally conceived newborns were studied as controls. RESULTS There was no significant difference in methylation between ART newborns and naturally conceived newborns (46.7 ± 8.2% vs. 48.5 ± 8.7%, p>0.05). Abnormal demethylation patterns of the IGF2/H19 ICR were found in three dizygotic twins conceived by intracytoplasmic sperm injection (ICSI), but all three were phenotypically normal, and their sibling twins exhibited normal methylation patterns. CONCLUSION We hypothesize that the aberrant methylation patterns probably resulted from imprinting errors of paternal gametes or from in vitro embryo culture. Further investigation to determine whether gene expression can be regulated by other mechanisms in addition to DNA methylation would be beneficial.

Assisted reproductive technologies do not increase risk of abnormal methylation of PEG1/MEST in human early pregnancy loss

OBJECTIVE To evaluate the epigenetic risk linked to assisted reproductive technology (ART) by analyzing the methylation patterns of imprinted PEG1 gene in aborted human chorionic villus. DESIGN Experimental research study. SETTING Research laboratory. PATIENT(S) Four patients groups were tested: spontaneous abortion after ART (n = 44), multifetal reduction after ART (n = 22), spontaneous abortion of natural pregnancies (n = 45), and induced abortion of natural pregnancies (n = 47). INTERVENTION(S) Methylation patterns of PEG1 in the aborted chorionic villus were determined. MAIN OUTCOME MEASURE(S) The DNA methylation patterns were analyzed using pyrosequencing and bisulfite sequencing polymerase chain reaction. The percentage of methylation was compared in chorionic villus from the four experimental groups. RESULT(S) Regardless of conception method, the PEG1 methylation percentage in chorionic villus from spontaneous abortions was significantly higher than in villus from induced abortions and multifetal reduction. In the spontaneous abortions groups, the percent methylation of PEG1 was similar in the villus derived from ART and from natural pregnancies. The two fertilization methods (IVF and intracytoplasmic sperm injection) did not show significant differences either. However, receiver operating characteristic curve analysis revealed a significant positive correlation between PEG1 methylation percentage and rate of early spontaneous abortions. CONCLUSION(S) As some studies have suggested, imprinting errors of PEG1 may contribute to spontaneous abortion, but ART procedures might not increase the occurrence of aberrant PEG1 methylation patterns.

Abnormal DNA Methylation of Imprinted Loci in Human Preimplantation Embryos

Purpose: To evaluate the epigenetic risk linked to assisted reproductive technology (ART) at single-embryo level by analyzing the methylation status of imprinted H19, PEG1, and KvDMR1 in human preimplantation embryos. Methods: A total of 254 human day 3 embryos produced by ART procedures were collected. All embryos were normally fertilized but unsuitable for transfer or freezing due to poor quality. Pyrosequencing with confirmatory routine bisulfite sequencing were used to determine the DNA methylation patterns of H19 differentially methylated region (DMR) in 63 embryos, PEG1 DMR in 65 embryos, and KvDMR1 in 67 embryos. Results: Aberrant methylation patterns were found in 8.0% embryos at H19 DMR, 16.9% embryos at PEG1 DMR, and 10.4% embryos at KvDMR1. No methylation errors were found in corresponding sperm samples. Conclusions: We hypothesized that the use of poor-quality embryos may increase the risk of imprinting defects because they might have methylation errors.

Aberrant DNA methylation of imprinted loci in human in vitro matured oocytes after long agonist stimulation

OBJECTIVE To evaluate the epigenetic risk linked to assisted reproductive technology at oocyte level by analyzing methylation status of imprinted H19, PEG1 and KvDMR1 in human MII oocytes rescue-matured from MI/GV oocytes after long agonist stimulation. STUDY DESIGN 580 MI/GV oocytes from 275 patients receiving an ICSI procedure were additionally cultured for 24h, and 221 rescue-matured MII oocytes were obtained. Pyrosequencing with confirmatory routine bisulfite sequencing were used to determine the methylation status of H19 DMR in 35 oocytes, PEG1 DMR in 47 oocytes, and KvDMR1 in 34 oocytes. RESULTS Abnormal methylation status were found in 22.9% oocytes at H19 DMR, 17.0% oocytes at PEG1 DMR and 8.8% oocytes at KvDMR1. CONCLUSION We hypothesize that the use of MII-rescue oocytes may increase the risk of imprinting defects because they might not have completed full imprinting programme.