A compact, high-throughput semi-automated embryo vitrification system based on hydrogel.

RESEARCH QUESTION: What is the efficiency and efficacy of the novel Biorocks semi-automated vitrification system, which is based on a hydrogel? DESIGN: This comparative experimental laboratory study used mouse model and human day 6 blastocysts. Mouse oocytes and embryos were quality assessed post-vitrification. RESULTS: The Biorocks system successfully automated the solution exchanges during the vitrification process, achieving a significantly improved throughput of up to 36 embryos/oocytes per hour. Using hydrogel for cryoprotective agent delivery, 12 vessels could be processed simultaneously, fitting comfortably within an assisted reproductive technology (ART) workstation. In tests involving the cryopreservation of oocytes and embryos, the system yielded outcomes equivalent to the manual Cryotop method. For example, the survival rate for mouse oocytes was 98% with the Biorocks vitrification system (n = 46) and 95% for the manual Cryotop method (n = 39), of which 46% and 41%, respectively, progressed to blastocysts on day 5 after IVF. CC-grade day 6 human blastocysts processed with the Biorocks system (n = 39) were associated with a 92% 2 h re-expansion rate, equivalent to the 90% with Cryotop (n = 30). The cooling/warming rates achieved by the Biorocks system were 31,900°C/minute and 24,700°C/minute, respectively. Oocyte quality was comparable or better post-vitrification for Biorocks than Cryotop. CONCLUSIONS: The Biorocks semi-automated vitrification system offers enhanced throughput without compromising the survival and developmental potential of oocytes and embryos. This innovative system may help to increase the efficiency and standardization of vitrification in ART clinics. Further investigations are needed to confirm its efficacy in a broader clinical context.

Embryological characteristics and clinical outcomes of oocytes with different degrees of abnormal zona pellucida during assisted reproductive treatment.

Abnormalities in the zona pellucida (ZP) adversely affect oocyte maturation, embryo development and pregnancy outcomes. However, the assessment of severity is challenging. To evaluate the effects of different degrees of ZP abnormalities on embryo development and clinical outcomes, in total, 590 retrieval cycles were scored and divided into four categories (control, mild, moderate and severe) based on three parameters: perivitelline space, percentage of immature oocytes and percentage of oocytes with abnormal morphology. As the severity of abnormal ZP increased, both the number of retrieved oocytes and mature oocytes decreased. The fertilization rate did not differ significantly among groups. The rates of embryo cleavage and day-3 high-quality embryos in the mild group and the moderate group did not vary significantly between the two groups but were significantly higher than those in the severe group. The blastulation rates of the abnormal ZP groups were similar; however, they were lower than those of the control group. Moreover, the cycle cancellation rate of the severe abnormal ZP group was as high as 66.20%, which was significantly higher than that of the other three groups. Although the rates of cumulative clinical pregnancy and live births were lower than those in the control group, they were comparable among the abnormal ZP groups. There were no differences in the neonatal outcomes of the different groups. Together, ZP abnormalities show various degrees of severity, and in all patients regardless of the degree of ZP abnormalities who achieve available embryos, there will be an opportunity to eventually give birth.

Decreased HAT1 expression in granulosa cells disturbs oocyte meiosis during mouse ovarian aging.

BACKGROUND: With advanced maternal age, abnormalities during oocyte meiosis increase significantly. Aneuploidy is an important reason for the reduction in the quality of aged oocytes. However, the molecular mechanism of aneuploidy in aged oocytes is far from understood. Histone acetyltransferase 1 (HAT1) has been reported to be essential for mammalian development and genome stability, and involved in multiple organ aging. Whether HAT1 is involved in ovarian aging and the detailed mechanisms remain to be elucidated. METHODS: The level of HAT1 in aged mice ovaries was detected by immunohistochemical and immunoblotting. To explore the function of HAT1 in the process of mouse oocyte maturation, we used Anacardic Acid (AA) and small interfering RNAs (siRNA) to culture cumulus-oocyte complexes (COCs) from ICR female mice in vitro and gathered statistics of germinal vesicle breakdown (GVBD), the first polar body extrusion (PBE), meiotic defects, aneuploidy, 2-cell embryos formation, and blastocyst formation rate. Moreover, the human granulosa cell (GC)-like line KGN cells were used to investigate the mechanisms of HAT1 in this progress. RESULTS: HAT1 was highly expressed in ovarian granulosa cells (GCs) from young mice and the expression of HAT1 was significantly decreased in aged GCs. AA and siRNAs mediated inhibition of HAT1 in GCs decreased the PBE rate, and increased meiotic defects and aneuploidy in oocytes. Further studies showed that HAT1 could acetylate Forkhead box transcription factor O1 (FoxO1), leading to the translocation of FoxO1 into the nucleus. Resultantly, the translocation of acetylated FoxO1 increased the expression of amphiregulin (AREG) in GCs, which plays a significant role in oocyte meiosis. CONCLUSION: The present study suggests that decreased expression of HAT1 in GCs is a potential reason corresponding to oocyte age-related meiotic defects and provides a potential therapeutic target for clinical intervention to reduce aneuploid oocytes.

A novel homozygous variant in PADI6 is associate with human cleavage-stage embryonic arrest.

Repeated absence of useable embryos is a difficult problem for infertility patients. Among them, embryonic developmental arrest is more common, but the genetic cause is not known. The embryos of a patient who came to our hospital three times could not develop beyond the four-cell stage. In addition to recording the developmental details of the embryos by daily photo-taking, the PADI6 R132C homozygous variants was further confirmed by whole-exome sequencing. Subsequently, PADI6 R132C was analyzed by bioinformatics methods for conservativeness across species. In addition, the possible impact of the pathogenic mutation on the structure of the protein PADI6 were also assessed. Generally, we identified a homozygous variants [NM_207421.4, c.394C>T(p.R132C] in the middle protein-arginine deiminase domain in PADI6 gene. The homozygous variant is highly conserved across species. Homozygous variant in PADI6 R132C could cause a human cleavage-stage embryonic arrest in female patients. These findings provide further evidence for the important roles of the homozygous PADI6R132C variant in embryonic development. Our findings contribute to a deeper understanding of the molecular genetic basis of female infertility.

Granulosa cell mevalonate pathway abnormalities contribute to oocyte meiotic defects and aneuploidy.

With aging, abnormalities during oocyte meiosis become more prevalent. However, the mechanisms of aging-related oocyte aneuploidy are not fully understood. Here we performed Hi-C and SMART-seq of oocytes from young and old mice and reveal decreases in chromosome condensation and disrupted meiosis-associated gene expression in metaphase I oocytes from aged mice. Further transcriptomic analysis showed that meiotic maturation in young oocytes was correlated with robust increases in mevalonate (MVA) pathway gene expression in oocyte-surrounding granulosa cells (GCs), which was largely downregulated in aged GCs. Inhibition of MVA metabolism in GCs by statins resulted in marked meiotic defects and aneuploidy in young cumulus-oocyte complexes. Correspondingly, supplementation with the MVA isoprenoid geranylgeraniol ameliorated oocyte meiotic defects and aneuploidy in aged mice. Mechanically, we showed that geranylgeraniol activated LHR/EGF signaling in aged GCs and enhanced the meiosis-associated gene expression in oocytes. Collectively, we demonstrate that the MVA pathway in GCs is a critical regulator of meiotic maturation and euploidy in oocytes, and age-associated MVA pathway abnormalities contribute to oocyte meiotic defects and aneuploidy.

The effect of the day 3 embryo cell number on the neonatal outcomes of day 5 single blastocyst transfer in frozen embryo transfer cycles.

OBJECTIVE: To evaluate the influence of the day 3 embryo cell number on the neonatal outcomes of day 5 single blastocyst transfer in frozen embryo transfer (FET) cycles. METHODS: This retrospective study analysed a total of 2315 delivery cycles of day 5 single blastocyst transfer in FET cycles, including 489, 761 and 1103 live-born infants segregated according to a day 3 embryo cell number of <8, 8 and >8 cells, respectively. The neonatal outcomes of the three groups were compared. RESULTS: The day 3 embryo cell number did not significantly affect the incidence of monozygotic twins. The sex ratio increased as the day 3 embryo cell number increased, but the difference was not statistically significant. There were no significant differences in the rates of preterm birth or low birth weight among the three groups. The rates of stillbirths and neonatal deaths were also not significantly different among the three groups. Moreover, the day 3 embryo cell number did not increase the risk of birth defects in newborns. CONCLUSIONS: The day 3 embryo cell number did not significantly affect neonatal outcomes.

Maternal circadian disruption before pregnancy impairs the ovarian function of female offspring in mice.

Circadian disturbance brought on by shift employment, nighttime light pollution, and other factors is quite prevalent in contemporary culture. However, the effect of maternal circadian disruption before pregnancy on the reproduction of offspring in mice requires further research. Herein, we exposed female ICR mice to constant light to establish a model of preconceptional circadian disruption and then checked the ovarian function of female offspring (named the CLE group below). Our results revealed obesity, abnormal lipid metabolism and earlier puberty onset in the CLE group. Additionally, impaired ovarian follicle development, oocyte quality and preimplantation embryo development were shown in the CLE group. Moreover, the expression levels of Gnrh1 in the hypothalamus and Cyp17a1, Bmper, Bdnf and Lyve1 in ovaries, as well as circadian clock genes, including Clock, Cry1, Nr1d2 and Per2, were significantly downregulated in the CLE group. Mechanistically, immune responses, including the interleukin-17 (IL-17) signalling pathway, cytokine-cytokine receptor interaction and the chemokine signalling pathway, were altered in the CLE group, which may be responsible for the damaged ovarian function.

The influence of day 3 embryo cell number on the clinical pregnancy and live birth rates of day 5 single blastocyst transfer from frozen embryo transfer cycles.

BACKGROUND: To evaluate the influence of day 3 embryo cell number on the clinical pregnancy and live birth rates of day 5 single blastocyst transfer in frozen embryo transfer (FET) cycles. METHODS: Our retrospective study included 3761 day 5 single blastocyst FET cycles between January 2015 and December 2019. These FET cycles were divided into three groups according to the day 3 embryo cell number: 939 cycles in the < 8-cell group, 1224 cycles in the 8-cell group and 1598 cycles in the > 8-cell group. The clinical pregnancy and live birth rates were compared among the three groups. RESULTS: The clinical pregnancy rate of day 5 single blastocyst transfer in FET cycles increased significantly as the day 3 embryo cell number increased (52.2%, 61.4% and 66.8%, P < 0.001). Similarly, the live birth rate increased significantly as the day 3 embryo cell number increased (42.7%, 49.8% and 54.9%, P < 0.001). The results of the subgroup analysis showed that the clinical pregnancy and live birth rates were not significantly different among the three groups when good-quality blastocysts were transferred. The clinical pregnancy and live birth rates increased significantly as the day 3 embryo cell number increased when fair- and poor-quality blastocysts were transferred. CONCLUSION: The day 3 embryo cell number needs to be considered when day 5 single blastocyst transfer is performed in FET cycles, especially when fair- and poor-quality blastocysts are used for transfer. The transfer of a day 5 single blastocyst derived from an embryo with faster development on day 3 may shorten the time to achieving a live birth.

Circular RNA circFoxo3 Promotes Granulosa Cell Apoptosis Under Oxidative Stress Through Regulation of FOXO3 Protein.

Oxidative stress leads to ovarian functional decline by inducing granulosa cell (GC) apoptosis. Circular RNA circFoxo3 acts as a critical factor in regulating cell cycle and apoptosis, and cellular senescence in tumor cells. However, function of circFoxo3 is little understood in oxidative stress-induced injury of follicular GCs. In this study, we aimed to illustrate the regulation pattern of circFoxo3 in GCs under oxidative stress. CircFoxo3 was confirmed to be expressed in both human and mouse GCs by amplification with divergent primers and sequencing. In vitro and in vivo ovarian oxidative stress model, the expression of circFoxo3, FOXO3 protein, and its downstream targets were examined by quantitative real-time PCR and Western blotting, respectively. Knockdown of circFoxo3 was performed to evaluate the effects of circFoxo3-mediated GC apoptosis in vitro. RNA pull-down was used to discover the protein that interacted with circFoxo3 so as to illustrate the mechanism of circFoxo3 in GCs. Our results demonstrated that circFoxo3 was significantly upregulated in hydrogen peroxide (H2O2)-treated GCs and a 3-nitropropionic acid (3-NP)-induced mouse model of ovarian oxidative stress. Protein level of transcriptional factor FOXO3 was also remarkably increased in both in vitro and in vivo oxidative stress model, but FOXO3 mRNA expression revealed no significant difference. Knockdown of endogenous circFoxo3 downregulated FOXO3 protein level and blocked H2O2-induced cell apoptosis. CircFoxo3 could pull down high levels of MDM2 protein that induced FOXO3 ubiquitination and degradation. Furthermore, knockdown of MDM2 and circFoxo3 showed remarkably higher level of apoptosis when compared with the knockdown of circFoxo3 alone. Our study suggested that circFoxo3 regulated FOXO3 protein level in GCs by reducing interactions between FOXO3 and MDM2. In conclusion, circFoxo3 was positively associated with FOXO3 protein and jointly played crucial roles in mediating GC apoptosis induced by oxidative stress.

Blastocyst development rate influences singleton gestational age of similarly graded blastocysts after vitrified-warmed single embryo transfer cycles.

RESEARCH QUESTION: Does the development rate of blastocysts influence neonatal outcomes after blastocyst transfer cycles when the morphological score of the transferred blastocysts is similar? DESIGN: A retrospective study involving singleton live births born to 1280 women undergoing single frozen blastocyst transfers (FBTs) between January 2016 and December 2018 at a tertiary care centre. Patients were grouped into day-5 or day-6 groups depending on the development rate of blastocysts. These were further grouped into four groups based on the blastocyst inner cell mass and trophectoderm scoring: excellent (AA); good (AB or BA); average (AC, CA or BB); and poor (BC or CB). The primary outcomes were gestational age and singleton birth weight. RESULTS: Singletons resulting from day-5 single FBT were at a lower risk of preterm birth than those resulting from day-6 single FBT (adjusted OR 0.63, 95% CI 0.41 to 0.97, P = 0.035). In the day-5 good-quality blastocyst group and day-5 average-quality blastocyst group, singletons were at a lower risk of preterm birth than those resulting from day-6 groups, respectively (adjusted OR 0.22, 95% CI 0.08 to 0.63, P = 0.005 and adjusted OR 0.52, 95% CI 0.29 to 0.94, P = 0.03). CONCLUSIONS: Day-6 single FBT was associated with a higher risk of preterm birth compared with day-5 single FBT in good and average morphological scoring blastocysts. Our analysis was restricted to women with singleton births from single FBTs. Future prospective studies are required to confirm the findings.

The effects of the day of trophectoderm biopsy and blastocyst grade on the clinical and neonatal outcomes of preimplantation genetic testing-frozen embryo transfer cycles.

This study analyzed the effects of the day of trophectoderm (TE) biopsy and blastocyst grade on clinical and neonatal outcomes. The results showed that the implantation and live birth rates of day 5 (D5) TE biopsy were significantly higher compared with those of D6 TE biopsy. The miscarriage rate of the former was lower than that of the latter, but there was no statistically significant difference. Higher quality blastocysts can achieve better implantation and live birth rates. Among good quality blastocysts, the implantation and live birth rates of D5 and D6 TE biopsy were not significantly different. Among fair quality and poor quality blastocysts, the implantation and live birth rates of D5 TE biopsy were significantly higher compared with those of D6 TE biopsy. Neither blastocyst grade nor the day of TE biopsy significantly affected the miscarriage rate. Neonatal outcomes, including newborn sex, gestational age, preterm birth, birth weight and low birth weight in the D5 and D6 TE biopsies were not significantly different. Both blastocyst grade and the day of TE biopsy must be considered at the same time when performing preimplantation genetic testing-frozen embryo transfer.

Trophectoderm Mitochondrial DNA Content Associated with Embryo Quality and Day-5 Euploid Blastocyst Transfer Outcomes.

Mitochondria play a critical role in cell function and embryo development. Recently, increasing studies have investigated whether mitochondrial DNA (mtDNA) can be used as a predictive biomarker of embryo implantation. However, the results of its effect on implantation are still controversial. To further understand the clinical application value of mtDNA content for reproductive potential, we analyzed the influence of relative mtDNA quantity on embryo quality and transfer outcomes based on the results of second-generation sequencing of preimplantation genetic testing patients in our center. Biopsied trophectoderm (TE) from aneuploid blastocysts contained much larger amounts of mtDNA than those from euploid blastocysts (p < 0.000). In an analysis of only euploid blastocysts (n = 769), female age had no effect on mtDNA content (p = 0.216). TE cells biopsied on day 5 (n = 355) contained significantly higher amounts of mtDNA compared to those biopsied on day 6 (n = 388) or day 7 (n = 26) (p < 0.000). Higher quality trophoblast was associated with lower mtDNA content (p = 0.026), but quality of inner cell mass was not correlated with quantity of mtDNA (p = 0.112). For transferred embryos, the biopsied date and mtDNA content were significantly associated with embryo implantation and live birth outcomes. Day-5 euploid blastocysts with lower quantities of mtDNA exhibited higher implantation rate and live birth rate. However, our data indicated that mtDNA content may not be considered an independent predictive marker, it may be a useful reference for the selection of day-5 transferred euploid blastocysts.

Effects of the sperm DNA fragmentation index on the clinical and neonatal outcomes of intracytoplasmic sperm injection cycles.

BACKGROUND: Most studies have mainly focused on the effects of the sperm DNA fragmentation index (DFI) on fertilization, embryonic developmental potential and aneuploidy, pregnancy and abortion rates after in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) and have remained controversial. However, few studies have reported the effects of sperm DFI on neonatal outcomes, including stillbirths, neonatal deaths, sex, gestational age, prematurity, birthweight, low birth weight (LBW) and birth defects in newborns. Our objective was to evaluate the effects of sperm DFI on the clinical and neonatal outcomes of ICSI cycles. METHODS: This retrospective study analysed a total of 2067 oocyte retrieval, 1139 transfer and 713 delivery cycles from conventional ICSI cycles, including 301, 469, and 214 live-born infants in groups segregated according to sperm DFI as the < 15%, 15-30% and >  30% groups, respectively. The clinical and neonatal outcomes were compared among the three groups. RESULTS: Sperm DFI did not significantly affect the rates of fertilization, clinical pregnancy, miscarriage or ongoing pregnancy. Sperm DFI did not increase the risk of stillbirths or neonatal deaths. The rates of stillbirths and neonatal deaths were not significantly different among the three groups. The sex, gestational age, prematurity, birthweight and LBW of newborns in the three groups were not significantly affected by sperm DFI. Moreover, sperm DFI did not increase the number of birth defects in children. CONCLUSIONS: Sperm DFI did not affect the clinical or neonatal outcomes of ICSI cycles.

Effects of the normal sperm morphology rate on the clinical and neonatal outcomes of conventional IVF cycles.

The present study assessed the influences of the normal sperm morphology rate on the clinical and neonatal outcomes of conventional in vitro fertilisation cycles. This retrospective study analysed 427 and 2,728 cycles from the normal sperm morphology rate <4% and ≥4% group respectively. The clinical (total fertilisation failure, clinical pregnancy, implantation and abortion) and neonatal (sex, gestational age, preterm birth, birthweight, low birth weight, live births and birth defects of newborns) outcomes were compared. The rate of total fertilisation failure in the normal sperm morphology rate <4% group was significantly higher compared with that in the normal sperm morphology rate ≥4% group (2.8% versus 1.2%, p = .012). Total fertilisation failure was completely resolved by early rescue intracytoplasmic sperm injection. The clinical pregnancy, implantation and abortion rates were not significantly different between the two groups. Additionally, the sex, preterm birth, low birth weight, live births and birth defect rates, gestational age and birthweight of newborns were not significantly different between the two groups. Thus, normal sperm morphology rate <4% significantly increased the total fertilisation failure rate but did not affect the clinical or neonatal outcomes.

Effect of frozen and fresh embryo transfers on the birthweight of live-born twins.

OBJECTIVE: To explore the influence of frozen embryo transfer (FET) and fresh embryo transfer (Fresh) on the birthweight of live-born twins. STUDY DESIGN: A total of 8482 live-born twins were studied. The proportions of small for gestational age (SGA) and large for gestational age (LGA), the mean birthweight and the z score of live-born twins in the two groups were compared. Multiple linear regression analysis was used to evaluate the relationship between confounding factors and the birthweight of live-born twins. RESULTS: The proportion of SGA infants significantly decreased as BMI increased (BMI < 20, 6.1 %; 20 ≤ BMI≤25, 4.1 %; BMI > 25, 3.6 %; P<0.05). The proportion of LGA infants significantly increased as BMI increased (BMI < 20, 20.5 %; 20 ≤ BMI≤25, 25.2 %; BMI > 25, 30.7 %; P<0.0001). The proportion of SGA infants was significantly lower in the FET group than in the Fresh group, whereas the proportion of LGA infants was significantly higher in the former than in the latter. The absolute mean birthweight of live-born twins was significantly higher in the FET group compared with the Fresh group (2579 ±â€¯458 vs. 2534 ±â€¯465, P < 0.0001). The mean z score of the FET group was also significantly higher than that of the Fresh group (0.420 vs. 0.240, P < 0.0001). Multiple linear regression analysis indicated that FET was a more significant factor than fresh embryo transfer in influencing the birthweight of live-born twins. CONCLUSION: FET significantly increased the birthweight of live-born twins compared with fresh embryo transfer.

The impact of follicle-flushing during oocyte collection on embryo development of in-vitro fertilization.

BACKGROUND: To evaluate the impact of follicle-flushing during oocyte collection on embryo development potential retrospectively. METHODS: A total of 1714 cases, including 133 who experienced retrieval difficulty (repeated follicle-flushing) on the day of oocyte retrieval (difficulty group) and the control 1581 cases (control group), were assessed in this retrospective study. The number of oocytes recovered, two pro-nuclei fertilization (2PN-fertilization), day 3 good-quality embryo and day 5/6 blastocyst utilization rates were compared between the difficulty group and control group correspondingly. Embryo implantation, clinical pregnancy and neonatal outcomes were further analyzed between the two groups in the fresh day- 3 embryo transfer cycles. RESULTS: The number of oocytes recovered in the difficulty group (9.08 ± 4.65) were significantly reduced compared with the control group (12.13 ± 5.27),P < 0.001; The 2PN-fertilization, day 3 good-quality embryo and blastocyst utilization rates were significantly lower in the difficulty group compared with controls (71.7% vs. 75.7%; 52.7% vs. 56.5%; 31.9% vs. 37.0%, all P < 0.05). Embryo implantation in the difficulty group was 53.2%, which was lower than the control value of 58.7%, although not reaching statistical significance. The rate of fresh embryo transfer cycles in the difficulty group was lower than normal ones (51.88% vs. 61.99%, P = 0.026). The pregnancy and live birth rates were similar between the two groups. But the rate of spontaneous miscarriages of the difficulty group was higher than the control group, although not reaching statistical significance. The neonatal outcomes had no statistical difference between the two groups. CONCLUSIONS: Oocyte retrieval difficulty, which include repeated flushing and the corresponded extending time required for oocyte recovery, significantly reduced day 3 good-quality embryo and blastocyst utilization rates of these patients. But the live birth rate had no difference between the difficulty group and the normal ones.

HDAC3 maintains oocyte meiosis arrest by repressing amphiregulin expression before the LH surge.

It is known that granulosa cells (GCs) mediate gonadotropin-induced oocyte meiosis resumption by releasing EGF-like factors in mammals, however, the detailed molecular mechanisms remain unclear. Here, we demonstrate that luteinizing hormone (LH) surge-induced histone deacetylase 3 (HDAC3) downregulation in GCs is essential for oocyte maturation. Before the LH surge, HDAC3 is highly expressed in GCs. Transcription factors, such as FOXO1, mediate recruitment of HDAC3 to the amphiregulin (Areg) promoter, which suppresses AREG expression. With the LH surge, decreased HDAC3 in GCs enables histone H3K14 acetylation and binding of the SP1 transcription factor to the Areg promoter to initiate AREG transcription and oocyte maturation. Conditional knockout of Hdac3 in granulosa cells in vivo or inhibition of HDAC3 activity in vitro promotes the maturation of oocytes independent of LH. Taking together, HDAC3 in GCs within ovarian follicles acts as a negative regulator of EGF-like growth factor expression before the LH surge.

Comparison of the pregnancy and obstetric outcomes between single cleavage-stage embryo transfer and single blastocyst transfer by time-lapse selection of embryos.

To compare the pregnancy and obstetric outcomes following single cleavage-stage embryo transfer (SCT) and single blastocyst transfer (SBT) using time-lapse imaging (TLI), a total of 2066 normally fertilized and cleaved embryos from 233 patients were divided into Day 3 SCT group (n = 171) and Day 5 SBT group (n = 62) according to patient's willingness. Embryo selection criteria were based on embryo cleavage patterns, timing parameters, and blastocyst quality. The pregnancy and obstetric outcomes of each group were evaluated. There were no statistically significant differences with regard to pregnancy outcomes including the implantation rate, early abortion rate, ongoing pregnancy rate and live birth rate, and obstetric outcomes including preterm birth rate, gestational week, birth height, birth weight and fetal malformation rate between SCT group and SBT group. SBT group had significantly higher monozygotic twinning (MZT) rates than SCT group (6.98% vs. 0, p < .05). Although not statistically significant, there was a trend of higher proportion of male-to-female sex ratio at birth in SBT group than SCT group (1.38 vs. 1.05). Based on the combination of cleavage patterns and timing parameters, SCT may be an alternative to SBT because it can provide similar pregnancy and obstetric outcomes and meanwhile lower monozygotic twinning rates.

Preliminary functional inquiry of lncRNA ENST00000433673 in embryo implantation using bioinformatics analysis.

Long non-coding RNAs (lncRNAs), a class of non-coding RNA, have been shown to be essential in many diseases, such as infertility. Here, we found three candidate lncRNAs, ENST00000414116, ENST00000433673, and ENST00000448179, that are highly expressed in the uterus endometrial tissues of normal patients compared to the tissues of patients with adenomyosis, endometriosis, and recurrent implantation failure. lncRNAs ENST00000414116 and ENST00000433673 showed high expression in endometrial stromal cells (ESCs) and endometrial epithelial cells (EECs), respectively, and lncRNA ENST00000448179 was specifically expressed in ESCs. The bioinformatics analysis results indicated that the target mRNAs of lncRNA ENST00000433673 were related to biological adhesion. Interestingly, intercellular adhesion molecule 1 (ICAM1), an interacting mRNA of the target mRNA integrin subunit alpha L (ITGAL), has been reported be an important regulator of embryo implantation. Further studies found that the target mRNA ITGAL and the interacting mRNA ICAM1 were highly expressed in the uterus endometrial tissues and EECs of normal patients. Based on our results, our study indicates that lncRNA ENST00000433673 might mediate the high expression of the target mRNA ITGAL, thereby promoting the expression of the interacting mRNA ICAM1 and the adhesion of EECs, which facilitates adhesion and implantation between the embryo and the mater. Abbreviations: AMs: adenomyosis; EMs: endometriosis; RIF: recurrent implantation failure; miRNAs: microRNAs; lncRNAs: Long non-coding RNAs; RT-qPCR: real-time quantitative PCR; ESCs: endometrial stromal cells; EECs: endometrial epithelial cells; BFE: free binding energy; PCDHB9: protocadherin beta 9; PARVG: parvin gamma; MAPK6: mitogen-activated protein kinase 6; LAF1: lymphocyte function-associated antigen 1.