Shengli Lin

The DNA methylation landscape of human early embryos

DNA methylation is a crucial element in the epigenetic regulation of mammalian embryonic development. However, its dynamic patterns have not been analysed at the genome scale in human pre-implantation embryos due to technical difficulties and the scarcity of required materials. Here we systematically profile the methylome of human early embryos from the zygotic stage through to post-implantation by reduced representation bisulphite sequencing and whole-genome bisulphite sequencing. We show that the major wave of genome-wide demethylation is complete at the 2-cell stage, contrary to previous observations in mice. Moreover, the demethylation of the paternal genome is much faster than that of the maternal genome, and by the end of the zygotic stage the genome-wide methylation level in male pronuclei is already lower than that in female pronuclei. The inverse correlation between promoter methylation and gene expression gradually strengthens during early embryonic development, reaching its peak at the post-implantation stage. Furthermore, we show that active genes, with the trimethylation of histone H3 at lysine 4 (H3K4me3) mark at the promoter regions in pluripotent human embryonic stem cells, are essentially devoid of DNA methylation in both mature gametes and throughout pre-implantation development. Finally, we also show that long interspersed nuclear elements or short interspersed nuclear elements that are evolutionarily young are demethylated to a milder extent compared to older elements in the same family and have higher abundance of transcripts, indicating that early embryos tend to retain higher residual methylation at the evolutionarily younger and more active transposable elements. Our work provides insights into the critical features of the methylome of human early embryos, as well as its functional relation to the regulation of gene expression and the repression of transposable elements.

Effect of in vitro culture period on birthweight of singleton newborns

STUDY QUESTION Does prolonged in vitro culture influence newborn birthweight? SUMMARY ANSWER The absolute mean birthweight and gestational age- and gender-adjusted birthweight (Z scores) of singletons born from blastocyst transfer are higher than singletons born from Day 3 transfer. WHAT IS KNOWN ALREADY An increased proportion of large-for-gestational age (LGA) newborns occurs after blastocyst transfer compared with Day 2 transfer, and Z scores for newborns after blastocyst transfer are higher than newborns after transfer on Day 2 or Day 3. STUDY DESIGN, SIZE AND DURATION This study was a retrospective analysis of newborn birthweight, including 2929 singletons at the Reproductive Medical Center of Peking University Third Hospital between January 2009 and June 2012. The number of singletons after Day 3 transfer was 2833 and the number of singletons after blastocyst transfer (Day 5-6) was 96. PARTICIPANTS/MATERIALS, SETTING, AND METHODS Only cycles with fresh embryo transfer were included. Patients ≤40 years of age with a BMI < 30 kg/m(2) were analyzed. Only data from singleton newborns born alive after the 20th week of gestation were included in the data analysis. Patients with more than one fetal sac diagnosed by ultrasound but who delivered singletons were excluded. Patients who received PGD and cycles with donor oocytes were excluded. Multiple linear regression analysis was performed to determine the significance of individual factors on absolute birthweight of singleton newborns. The absolute birthweight and Z scores of singletons were compared. MAIN RESULTS AND THE ROLE OF CHANCE Multiple linear regression analysis indicated that maternal age, maternal BMI, paternal BMI, type of infertility, gestational age, infant gender and culture period were significantly associated with birthweight. The absolute birthweight for singletons resulting from blastocyst transfer was significantly greater than singletons resulting from Day 3 transfer (3465.31 ± 51.36 versus 3319.82 ± 10.04 g respectively, P = 0.009). The Z scores for singletons after blastocyst transfer were significantly higher than singletons after Day 3 transfer (0.347 versus 0.029 respectively, P = 0.016). LIMITATIONS AND REASONS FOR CAUTION In our clinic, blastocyst culture is mainly offered to patients with unsuccessful IVF cycles but also to patients with uterine malformations, and therefore this protocol introduced a potential selection bias in our study. Moreover, as certain culture media are associated with fetal overgrowth, the media used may be also a confounding factor, even though the absolute birthweights of singletons were comparable. WIDER IMPLICATIONS OF THESE FINDINGS Our study suggests that a prolonged (5-6 days) in vitro culture period has a significant effect on the mean absolute birthweight and Z scores of singleton newborns. The effect of prolonged in vitro culture on epigenetic changes in the embryo needs further study. STUDY FUNDING/COMPETING INTEREST(S) National Natural Science Foundation of China for Young Scholars (81300483). The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER Not applicable.

No effect of embryo culture media on birthweight and length of newborns

STUDY QUESTION Does the type of media used to culture embryos for IVF influence the birthweight and length of neonates? SUMMARY ANSWER No significant differences were observed in birthweight and length among the three embryo culture media used for in vitro embryo culture. WHAT IS KNOWN ALREADY Since the establishment of IVF as an assisted reproductive technology (ART), many different culture systems have been used for the development of human embryos. Some studies have shown that the types of culture media influence the newborn birthweight; however, other studies have shown no effect. To further explore this contradictory issue, we compared the birthweight and length of neonates born after the transfer of embryos cultured in one of three commercially available media. STUDY DESIGN, SIZE AND DURATION This retrospective analysis of birthweight and length of newborns included 1201 women who delivered singletons and 445 women who delivered twins. The following three commercially available culture media were used: G5™, Global and Quinns advantage media. Women who underwent IVF-ET cycles between 2008 and 2010 were analyzed. PARTICIPANTS/MATERIALS, SETTING AND METHODS Patients younger than 40 years of age with a body mass index (BMI) <30 kg/m(2) were analyzed. Only data from singletons and twins born alive after the 20th week of gestation were included in the data analysis. Patients who received preimplantation genetic diagnosis (PGD) and donor oocytes were excluded. MAIN RESULTS AND THE ROLE OF CHANCE The analysis of 1201 singletons and 445 sets of twins showed no significant association between mean birthweight or mean birth length and the type of embryo culture medium. Inter-twin mean birthweight and length disparities were analyzed, but were not shown to be significantly different. Multiple linear regression analysis showed that maternal weight, maternal height, gestational age and infant gender were significantly related to birthweight, and paternal height, gestational age and newborn complications were significantly associated with birth length. LIMITATIONS AND REASONS FOR CAUTION The current study showed that birthweight and length of newborns were not associated with the embryo culture medium. More research needs to be performed to analyze the effects of other culture medium formulations and to evaluate the long-term effects of embryo culture medium on the health of children conceived through ART. WIDER IMPLICATIONS OF THESE FINDINGS: Our retrospective study suggests that embryo culture medium does not influence neonatal birthweight and length; however, the effects of culture medium on epigenetic variation of embryos need to be studied further.

Effect of ABO blood type on ovarian reserve in Chinese women

OBJECTIVE To explore the effect of ABO blood type on ovarian reserve in Chinese women. DESIGN Retrospective analysis. SETTING University-affiliated IVF center. PATIENT(S) The retrospective analysis involved 35,479 women who underwent in vitro fertilization and embryo transfer (IVF-ET) cycles between 2006 and 2012. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) The association between ABO blood types and diminished ovarian reserve (DOR). RESULT(S) Among 35,479 Chinese women, 11,395 (32.12%) had blood type B, 10,583 (29.83%) had blood type O, 9,861 (27.79%) had blood type A, and 3,640 (10.26%) had blood type AB. There was a statistically significantly higher percentage of blood type O among those with follicle-stimulating hormone (FSH) levels ≤10 IU/L compared with those with FSH levels >10 IU/L. Conversely, among the women with DOR, there was statistically significantly higher percentage of those with blood types B and AB. Blood type A was not associated with DOR occurrence. Multivariate logistic regression analysis showed that blood type O was statistically significantly less often associated with DOR occurrence, whereas the B antigen (blood type B or AB) was statistically significantly associated with an increased risk of DOR. CONCLUSION(S) Our results have shown that there is an association between ABO blood type and DOR occurrence in Chinese women. Women with blood type O were statistically significantly less likely to have DOR, whereas those with B antigen (blood type B or AB) were statistically significantly more likely to have DOR. Blood type A was not associated with ovarian reserve.

Altered amphiregulin expression induced by diverse luteinizing hormone receptor reactivity in granulosa cells affects IVF outcomes

The expression of specific genes (LHR, AREG, EREG, EGFR, NPPC and NPR2) involved in peri-ovulatory signalling pathways induced by LH surge in granulosa cells was investigated, and their relationships with IVF outcomes analysed. mRNA levels of the genes of 147 infertile women undergoing IVF and intracytoplasmic sperm injection (ICSI) with embryo transfer were evaluated. Compared with non-pregnant women, amphiregulin (AREG) mRNA levels in mural and cumulus graunulosa cells were significantly higher (P < 0.05) in pregnant women, and were positively correlated with number of oocytes retrieved and good-quality embryos. No significant differences were found between the two groups in the remaining detected genes. To investigate the reason for the differences in AREG expression, mural granulosa cells were cultured and stimulated with human chorionic gonadotrophin (HCG) for 2-24 h. At 4 h after HCG stimulation, AREG and epiregulin mRNA expression peaked, with much greater increases in the pregnant group. The fold-change of AREG expression was positively correlated with number of good-quality embryos. No obvious correlation, however, was found between NPPC/Npr2 expression levels in granulosa cells and IVF outcomes. Altered AREG expression induced by diverse luteinizing hormone receptor reactivity in granulosa cells may provide a useful marker for oocyte developmental competency.

Value of transferring embryos that show no evidence of fertilization at the time of fertilization assessment

OBJECTIVE To determine the value of transferring embryos formed from nonpronuclear (0PN) zygotes. DESIGN A case-control study. SETTING Not applicable. PATIENT(S) The current study was a retrospective analysis of embryo transfers of just 0PN embryos using fresh cleavage-stage embryos (0PN cleavage fresh), frozen-thawed cleavage-stage 0PN embryos (0PN cleavage frozen), and frozen 0PN blastocyst-stage embryos (0PN blast frozen). INTERVENTION(S) To study the effect of 0PN transfer, comparison groups were used: fresh cycles of 2PN (2PN cleavage fresh-C) and frozen-thawed cycles cleavage-stage (2PN cleavage frozen-C) and blastocyst-stage (2PN blast frozen-C). Comparison groups were matched for cycle and patient characteristics to the 0PN group. MAIN OUTCOME MEASURE(S) Implantation rate (IR), pregnancy rate, and transferable embryo rate. RESULT(S) For fresh cycles, the IR in the 0PN cleavage fresh was lower than that in the 2PN cleavage fresh-C (8.04% vs. 19.50%, respectively). For frozen-thawed cycles, the IR in the 0PN cleavage frozen was lower than that in the 2PN cleavage frozen-C (15.38% vs. 28.24%, respectively), but the IR in 0PN blast frozen was comparable to that of 2PN blast frozen-C (39.56% vs. 48.18%, respectively). CONCLUSION(S) Transfer of 0PN embryos from fresh or frozen-thawed cycles results in pregnancies and live births. Nonpronuclear embryos have a lower IR than 2PN embryos, but if the embryos are cultured to the blastocyst stage and then are frozen, their IRs approach that of 2PN embryos in subsequent frozen-thawed cycles. The culture of 0PN embryos to the blastocyst stage may select for embryos with a near-normal IR.

Increased incidence of ectopic pregnancy after in vitro fertilization in women with decreased ovarian reserve

The incidence of ectopic pregnancy after assisted reproductive technology is increased approximately 2.5–5-fold compared with natural conceptions. Strategies were used to decrease the incidence of ectopic pregnancy, but ectopic pregnancy still occurs. In the present study, women were selected with decreased ovarian reserve (defined as FSH > 10 IU/L) aged 20 to 38 years who underwent IVF-ET between 2009 and 2014. These 2,061 women were age-matched with an equal number of women with normal ovarian reserve (defined as FSH ≤ 10 IU/L). During cycles following fresh embryo transfer, 93 patients were diagnosed with ectopic pregnancy. The incidence of ectopic pregnancy in clinical pregnancies was significantly higher in the decreased ovarian reserve than in the normal ovarian reserve group (5.51% vs. 2.99%). After adjusting for confounding factors, the incidence of ectopic pregnancy was significantly associated with decreased ovarian reserve. Our results showed that decreased ovarian reserve is an independent risk factor for ectopic pregnancy after in vitro fertilization-embryo transfer.

Fractalkine restores the decreased expression of StAR and progesterone in granulosa cells from patients with polycystic ovary syndrome.

Low progesterone levels are associated with luteal phase deficiency in women with polycystic ovary syndrome (PCOS). The mechanisms regulating progesterone biosynthesis in the granulosa cells from women with PCOS is largely unknown. Fractalkine is expressed in human ovaries, and is reported to regulate progesterone production in granulosa cells of healthy women. In the current study, we aimed to examine the role of fractalkine in women with PCOS. Reduced fractalkine levels were found in follicular fluid and granulosa cells, accompanied by decreased progesterone production and reduced steroidogenic acute regulatory protein (StAR) expression in the granulosa cells of patients with PCOS. Administration of fractalkine reversed the inhibition of progesterone and StAR expression. The mechanism mediating these effects may be associated with the inhibition of ERK activity in the granulosa cells from women with PCOS. Our findings revealed that fractalkine regulated steroidogenesis in follicular granulosa cells of women with PCOS.

Influence of embryo culture medium on incidence of ectopic pregnancy in in vitro fertilization

OBJECTIVE To explore the effect of type of media used to culture embryos for IVF on the incidence of ectopic pregnancy (EP). DESIGN Retrospective analysis. SETTING University-affiliated IVF center. PATIENT(S) The retrospective analysis involved 23,481 women who underwent IVF-ET cycles between 2011 and 2013. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) There was an association between EP and the culture medium. RESULT(S) During 23,481 fresh transfer cycles, 364 patients were diagnosed with EP. The EP to clinical pregnancy rate was 3.01% in the G5 group, 3.89% in the G5 Plus group, and 4.04% in the Global group. The EP to clinical pregnancy rates were significantly higher in the G5 Plus and Global groups than in the G5 group. After adjusting for confounding factors, the incidence of EP was significantly associated with the G5 Plus and Global media. CONCLUSION(S) Our results showed that there is an association between incidence of EP and the culture medium. The rates of EP to clinical pregnancy were significantly higher in the G5 Plus and Global media than in the G5 medium.

Evaluation of syphilis serostatus on the safety of IVF treatment.

An increasing number of infertile syphilis-infected individuals have turned to assisted reproductive technology; however, the safety of syphilis carrier serostatus on IVF and embryo transfer outcomes has not been evaluated. Data from 482 patients who delivered singletons were analysed. In the retrospective study, the rate of IVF and intracytoplasmic sperm injection fertilization was 79.50% ± 17.57%/78.72% ± 16.66% in the Treponema pallidum particle agglutination assay negative (TPPA-negative) and rapid plasma reagin negative (RPR-negative) group, 76.12% ± 22.99%/74.05% ± 20.31% in the TPPA-positive and RPR-negative group, and 75.66% ± 21.72%/70.90% ± 16.11% in the TPPA-positive and RPR-positive group. The clinical pregnancy rate was 39.79% in the TPPA-negative and RPR-negative group, 46.30% in the TPPA-positive and RPR-negative group, and 36.59% in the TPPA-positive and RPR-positive group. No significant differences were found between the groups. The neonatal gestational age and mean birth weight were not significantly different between the TPPA-negative and TPPA-positive groups. Multiple linear regression analysis also showed no association between TPPA serostatus and newborn birth weight and gestational age. The present retrospective study showed that TPPA and RPR serostatus did not affect the outcomes of IVF and embryo transfer. Syphilis-infected individuals can undergo IVF and embryo transfer cycles after penicillin treatment.