De-Sheng Ye

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.

Misdiagnosis and Delayed Diagnosis for Ectopic and Heterotopic Pregnancies after In Vitro Fertilization and Embryo Transfer

This study examined the misdiagnosis and delayed diagnosis factors for ectopic pregnancy (EP) and heterotopic pregnancy (HP) after in vitro fertilization and embryo transfer (IVF-ET) in an attempt to reduce the diagnostic error. Clinical data of patients who underwent IVF-ET treatment and had clinical pregnancy from 12463 cycles were retrospectively analyzed. Their findings of serum β-hCG test and transvaginal ultrasonography were also obtained during follow-up. These patients were divided into two groups according to the diagnosis accuracy of EP/HP: early diagnosis and misdiagnosis/delayed diagnosis. The results showed that the incidence of EP and HP was 3.8% (125/3286) and 0.8% (27/3286) respectively for IVF/ICSI-ET cycle, and 3.8% (55/1431) and 0.7% (10/1431) respectively for frozen- thawed embryo transfer (FET) cycle. Ruptured EP occurred in 28 patients due to initial misdiagnosis or delayed diagnosis. Related factors fell in 3 categories: (1) clinician factors: misunderstanding of patients’ medical history, insufficient training in ultrasonography and unawareness of EP and HP; (2) patient factors: noncompliance with medical orders and lack of communication with clinicians; (3) complicated conditions of EP: atypical symptoms, delayed elevation of serum β-hCG level, early rupture of cornual EP, asymptomatic in early gestation and pregnancy of unknown location. All the factors were interwoven, contributing to the occurrence of EP and HP. It was concluded that complicated conditions are more likely to affect the diagnosis accuracy of EP/HP after IVF-ET. Transvaginal ultrasonography should be performed at 5 weeks of gestation. Intensive follow-up including repeated ultrasonography and serial serum β-hCG tests should be performed in patients with a suspicious diagnosis at admission.SummaryThis study examined the misdiagnosis and delayed diagnosis factors for ectopic pregnancy (EP) and heterotopic pregnancy (HP) after in vitro fertilization and embryo transfer (IVF-ET) in an attempt to reduce the diagnostic error. Clinical data of patients who underwent IVF-ET treatment and had clinical pregnancy from 12463 cycles were retrospectively analyzed. Their findings of serum β-hCG test and transvaginal ultrasonography were also obtained during follow-up. These patients were divided into two groups according to the diagnosis accuracy of EP/HP: early diagnosis and misdiagnosis/delayed diagnosis. The results showed that the incidence of EP and HP was 3.8% (125/3286) and 0.8% (27/3286) respectively for IVF/ICSI-ET cycle, and 3.8% (55/1431) and 0.7% (10/1431) respectively for frozen- thawed embryo transfer (FET) cycle. Ruptured EP occurred in 28 patients due to initial misdiagnosis or delayed diagnosis. Related factors fell in 3 categories: (1) clinician factors: misunderstanding of patients’ medical history, insufficient training in ultrasonography and unawareness of EP and HP; (2) patient factors: noncompliance with medical orders and lack of communication with clinicians; (3) complicated conditions of EP: atypical symptoms, delayed elevation of serum β-hCG level, early rupture of cornual EP, asymptomatic in early gestation and pregnancy of unknown location. All the factors were interwoven, contributing to the occurrence of EP and HP. It was concluded that complicated conditions are more likely to affect the diagnosis accuracy of EP/HP after IVF-ET. Transvaginal ultrasonography should be performed at 5 weeks of gestation. Intensive follow-up including repeated ultrasonography and serial serum β-hCG tests should be performed in patients with a suspicious diagnosis at admission.

Cumulative live birth rate after three ovarian stimulation IVF cycles for poor ovarian responders according to the bologna criteria

SummaryThis study explored the cumulative live birth rate after three ovarian stimulation in vitro fertilization (IVF) cycles for poor ovarian responders according to the Bologna criteria. In this retrospective cohort study, 479 poor ovarian responders according to the Bologna criteria in the first ovarian stimulation IVF cycle between July 2006 and January 2012 in our IVF centre were included. The cumulative live birth rate was calculated by optimistic and pessimistic methods. The cumulative live birth rate after three ovarian stimulation IVF cycles for poor ovarian responders according to the Bologna criteria was 12.7%–20.5%. The three-cycle cumulative live birth rate was 18.5%–24.5%, 13.2%–27.4% and 8.6%–14.9% for poor responders aged ≤35 years, 36–39 years and ≥40 years, respectively. In conclusion, poor responders according to the Bologna criteria can receive an acceptable cumulative live birth rate after three ovarian stimulation IVF cycles, especially poor responders aged <40 years.This study explored the cumulative live birth rate after three ovarian stimulation in vitro fertilization (IVF) cycles for poor ovarian responders according to the Bologna criteria. In this retrospective cohort study, 479 poor ovarian responders according to the Bologna criteria in the first ovarian stimulation IVF cycle between July 2006 and January 2012 in our IVF centre were included. The cumulative live birth rate was calculated by optimistic and pessimistic methods. The cumulative live birth rate after three ovarian stimulation IVF cycles for poor ovarian responders according to the Bologna criteria was 12.7%–20.5%. The three-cycle cumulative live birth rate was 18.5%–24.5%, 13.2%–27.4% and 8.6%–14.9% for poor responders aged ≤35 years, 36–39 years and ≥40 years, respectively. In conclusion, poor responders according to the Bologna criteria can receive an acceptable cumulative live birth rate after three ovarian stimulation IVF cycles, especially poor responders aged <40 years.

Minimum dose of hCG to trigger final oocyte maturation and prevent OHSS in a long GnRHa protocol.

This paper was aimed to study the minimum dose of human chorionic gonadotropin (hCG) to effectively trigger maturation of oocytes and prevent ovarian hyperstimulation syndrome (OHSS) in a series of hyper-responders treated with a long gonadotropin releasing hormone agonist (GnRHa) protocol. Six women at high risk of developing severe OHSS in a long GnRHa protocol were enrolled into this study. Serum hormone levels on the day of and after hCG administration, antral follicle count, oocyte retrieval number and quality were determined. In total, 6 women aged between 29 and 36 years and at risk of developing severe OHSS, received 2000 U hCG. Five of them were treated with coasting for 1 day and the rest one for 4 days. The mean number of oocytes collected was 19 (range 14–27) and the fertilization rate per collected oocyte was 72.81%. Of the 6 women in the study, only one cancelled embryos transfer and all embryos were frozen, and then she delivered two health boys on term in the subsequent frozen-thawed embryo transfer (FET) cycle. Pregnancies and births were achieved in 3 patients out of 5 in vitro fertilization-embryo transfer (IVF-ET) cycles. No woman developed moderate or severe OHSS. Triggering with 2000 U hCG is feasible to prevent OHSS in unpredicted hyper-responders undergoing IVF in a long GnRHa protocol.SummaryThis paper was aimed to study the minimum dose of human chorionic gonadotropin (hCG) to effectively trigger maturation of oocytes and prevent ovarian hyperstimulation syndrome (OHSS) in a series of hyper-responders treated with a long gonadotropin releasing hormone agonist (GnRHa) protocol. Six women at high risk of developing severe OHSS in a long GnRHa protocol were enrolled into this study. Serum hormone levels on the day of and after hCG administration, antral follicle count, oocyte retrieval number and quality were determined. In total, 6 women aged between 29 and 36 years and at risk of developing severe OHSS, received 2000 U hCG. Five of them were treated with coasting for 1 day and the rest one for 4 days. The mean number of oocytes collected was 19 (range 14–27) and the fertilization rate per collected oocyte was 72.81%. Of the 6 women in the study, only one cancelled embryos transfer and all embryos were frozen, and then she delivered two health boys on term in the subsequent frozen-thawed embryo transfer (FET) cycle. Pregnancies and births were achieved in 3 patients out of 5 in vitro fertilization-embryo transfer (IVF-ET) cycles. No woman developed moderate or severe OHSS. Triggering with 2000 U hCG is feasible to prevent OHSS in unpredicted hyper-responders undergoing IVF in a long GnRHa protocol.

Impact of Abnormal DNA Methylation of Imprinted Loci on Human Spontaneous Abortion

Currently, there is a growing concern regarding the safety of assisted reproductive technology (ART) due to increased risk of spontaneous abortion (SA) and imprinting disorders in ART-conceived offspring. Early investigations suggested that aberrant genetic imprinting may be related to pregnancy loss; however, few studies have used human tissue specimens. Here the DNA methylation patterns of 3 imprinted genes, including maternally inherited GRB10 and the paternally inherited IGF2 and PEG3 genes, were evaluated in human chorionic villus samples by pyrosequencing and bisulfite sequencing polymerase chain reaction. The samples were divided into 4 groups: (1) SA of natural conception (NC; n = 84), (2) induced abortion of NC (n = 94), (3) SA after ART (n = 73), and (4) fetal reduction after ART (n = 86). The methylation levels and the percentages of abnormal methylation of the IGF2, GRB10, and PEG3 genes between the ART group and the NC group showed no significant difference. Both IGF2 and GRB10 genes showed higher methylation levels in the SA group compared to the non-SA group. Additionally, determining the single-nucleotide polymorphisms of 4 loci, including IGF2 rs3741205, rs3741206, rs3741211, and GRB10 rs2237457, showed that the TC+CC genotype of IGF2 rs3741211 had a 1.91-fold increased risk of SA after ART. However, there was no association between the mutant genotype of IGF2 rs3741211 and the methylation levels of IGF2 and H19, and ART might not affect the distribution of the abovementioned genotypes. It provides support for the opinion that genetic imprinting defects may be associated with SA, which might not be due to ART treatments.

Retrospective analysis of reproductive outcomes in women with primary ovarian insufficiency showing intermittent follicular development

The aim of this retrospective study was to explore the reproductive outcomes of IVF treatment in women with primary ovarian insufficiency (POI) showing intermittent follicular development. A total of 44 POI women with normal karyotype and absent autoimmunity, attending the centre for fertility treatment at Nanfang Hospital, Guangzhou from March 2009 to March 2011, were identified as suitable for inclusion in this study. Out of 44 women, 20 (20/44; 45.5%) had growing follicles and 13 underwent 27 oocyte retrievals. The empty follicle rate per oocyte retrieval was 70.4% (19/27); eight oocytes were recovered: one (12.5%) germinal vesicle (GV), two (25.0%) metaphase I (MI), one (12.5%) metaphase II (MII), and four (50.0%) atretic. One MI oocyte matured in vitro and two women had embryo transfer. Only the woman with the MI oocyte matured in vitro conceived, giving birth to a healthy baby at term. These results suggest that intermittent follicular development is common in women with POI but most of the developed follicles are empty or contain atretic oocytes. The pregnancy rate remains very low for IVF treatment.

The association between serum β-hCG on day 24 of pregnancy and ongoing pregnancy in frozen embryo transfer cycles.

To determine the relationship between serum levels of the β‐subunit of human chorionic gonadotropin (β‐hCG) on day 24 of pregnancy during a frozen embryo transfer (FET) cycle and ongoing pregnancy.

Down-regulation of long non-coding RNA MALAT1 inhibits granulosa cell proliferation in endometriosis by up-regulating P21 via activation of the ERK/MAPK pathway

STUDY QUESTION Is there a specific mechanism underlying the association between lung adenocarcinoma transcript 1 (MALAT1) and endometriosis-related infertility? SUMMARY ANSWER The down-regulation of MALAT1 in endometriosis granulosa cells (GCs) may have an adverse effect on the growth and development of oocytes by inhibiting GC proliferation, due to cell cycle-dependent mechanisms that enhance P21 expression through activation of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway. WHAT IS KNOWN ALREADY The association between endometriosis and infertility is well supported throughout the literature, and endometriosis per se and its surgical treatment have an adverse effect on the ovarian reserve and on oocyte development. MALAT1, one of the most extensively expressed and evolutionarily conserved transcripts, has been implicated to play a role in human development and many diseases. However, little is known about the role of MALAT1 long non-coding RNA (lncRNA) in endometriosis and its associated infertility. STUDY DESIGN, SIZE, DURATION We measured MALAT1 lncRNA expression levels in GCs from 52 endometriosis patients and 52 controls. Also, MALAT1 was knocked down in a human GC tumor-derived cell line, KGN, to investigate the role of MALAT1 and its molecular mechanism in cell proliferation. PARTICIPANTS/MATERIALS, SETTING, METHODS GCs were collected from women with or without endometriosis undergoing IVF or ICSI treatment. All endometriosis patients were diagnosed by laparoscopy or laparotomy, and control patients were limited to male factor or tubal disease and had a normal ovarian reserve. Quantitative real-time PCR (qRT-PCR) was used to measure the differential expression levels of MALAT1 lncRNA between endometriosis patients and controls. The receiver operating characteristic (ROC) curve was drawn to evaluate the diagnostic values of MALAT1 in endometriosis. In the KGN cell line, MALAT1 was knocked down with locked nucleic acid GapmeRs. Cell counting kit-8 assays, ethynyl-2-deoxyuridine assays and flow cytometry were used to study the role of MALAT1 in cell proliferation and cell-cycle progression, and western blotting was performed to detect the potential underlying mechanism. MAIN RESULTS AND THE ROLE OF CHANCE We first found that MALAT1 lncRNA was significantly down-regulated in endometriosis GCs and was associated with the antral follicle count (R = 0.376, P < 0.001 versus control). In addition, MALAT1 lncRNA levels were significantly lower in the GCs of infertile women with advanced stages of endometriosis (P = 0.01 versus control). The ROC curves illustrated strong separation between all the endometriosis patients and the control group (AUC: 0.705; 95% CI: 0.606-0.804; P < 0.001), Stage I-II and control group (AUC: 0.651; 95% CI: 0.536-0.767; P = 0.016), and Stage III-IV and control group (AUC: 0.827; 95% CI: 0.718-0.936; P < 0.001). MALAT1 lncRNA was primarily localized in the nuclei of GCs. We found a negative correlation between MALAT1 lncRNA and P21 mRNA in the GCs from patients (R = -0.628; P < 0.001). MALAT1 knockdown in KGN cells inhibited cell proliferation and cell-cycle progression. In addition, MALAT1 knockdown induced an increase in both the mRNA and protein levels of P21, and of P53, phosphorylated ERK1/2 (p-ERK1/2) and phosphorylated c-Jun N-terminal protein kinase (p-JNK) protein levels, as well as causing a decrease in cyclin dependent kinase 2 (CDK2), cyclin D1 and p-P38 MAPK protein levels. Furthermore, inhibition of the ERK/MAPK pathway with U0126, the up-regulation of p-ERK1/2, P21 and P53, and the down-regulation of CDK2 and cyclin D1 by the knockdown of MALAT1 were all attenuated by MALAT1 knockdown. Therefore, MALAT1 may regulate GC proliferation through P21/P53-dependent control of the cell cycle, and the ERK/MAPK pathway participates in this process. LARGE SCALE DATA None. LIMITATIONS, REASONS FOR CAUTION The hormonal treatment used in IVF and surgical removal of endometriotic lesions may have altered MALAT1 expression in GCs. The ovarian granulosa-like tumor cell line, KGN, was used for further functional and mechanistic studies due to the difficulties in obtaining human GCs in sizable amounts and maintaining primary cultures. WIDER IMPLICATIONS OF THE FINDINGS Our finding represents the first example of an lncRNA-based mechanism in endometriosis GCs. Women with endometriosis show altered MALAT1 expression levels in GCs that may impair fertility by regulating the function of GCs. Therefore, analysis of MALAT1 and its molecular mechanisms of action provide new insights into the pathogenesis of endometriosis and its associated infertility. STUDY FUNDING/COMPETING INTEREST(s) This work was supported by the National Natural Science Foundation of China (grant number: 81671524) and the National key research and development program of China (grant number: 2017YFC1001100). The authors declare there is no conflict of interest.