Endogenous hydrogen sulfide promotes human preimplantation embryonic development by regulating metabolism-related gene expression.

Hydrogen sulfide (H2S) as an endogenous gaseous signaling molecule had been proved to play a vital role in gametes physiology, covering meiosis, maturation and aging. However, little is known about H2S involvement in embryonic development. The present study explored the positive effect of H2S on human early embryonic development. Results validated that the two H2S producing enzymes, CBS and CSE mRNA and proteins were identified in donated human cleavage and blastocyst-stage embryos. The l-cysteine incubation produced endogenous H2S in human blastocysts. NaHS positively affected in vitro blastulation. Single-cell RNA-seq analysis identified 228 differentially expressed genes (DEGs) after NaHS treatment versus the control. The Gene Ontology (GO) enrichment analysis of DEGs showed that genes for protein modification and metabolism were significantly enriched in the NaHS treatment group. For the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, 2-oxocarboxylic acid metabolism, glycosaminoglycan biosynthesis-keratan sulfate, steroid biosynthesis, carbon metabolism, and biosynthesis of amino acids were significantly enriched. Six DEGs, including Neural EGFL like 1 (NELL1), aconitase 1 (ACO1), phosphoglycerate mutase 1 (PGAM1), TP53 induced glycolysis regulatory phosphatase (TIGAR), UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 2 (B3GNT2), and carbohydrate Sulfotransferase 4 (CHST4) were validate by real-time RT-PCR. These findings suggest that H2S is a positive regulator of early embryonic development and may alter the transcription of embryonic genes for protein modification and metabolism in human embryos.

A nomogram to assist blastocyst selection in vitrified-warmed embryo transfer cycles.

AIM: The purpose of the study was to establish a mathematical model to help rank the order of blastocysts and assist selection of which blastocysts to warm in vitrified-thawed embryo transfer cycles. DESIGN: A total of 2862 women who underwent first vitrified-thawed single blastocyst transfer (SBT) between July 2015 and July 2019 were retrospectively recruited and randomized into a training set (n = 2289) and testing set (n = 573). Least absolute shrinkage and selection operator (LASSO) regression was used to screen the factors critical to live birth (LB). Subsequently, a nomogram model was established to convert the effect of each factor on LB into a measurable score. The efficacy of the model was then evaluated by the receiver operating characteristic and calibration curve. The performance of the model was also internally tested in the testing set. RESULTS: Maternal age, endometrial thickness, oocyte number, day-3 embryo quality, blastocyst morphology, and blastulation day were selected as the critical predictors of LB in the vitrified-thawed SBT cycle and fitted into a nomogram model. The area under the curve (AUC) of the model was 0.67, and the AUC in the testing set was 0.64, which indicates moderate discrimination. The calibration curve showed good concordance between prediction and observation. Importantly, the score of each variable in the nomogram helped to rank the order of the blastocysts resulting in LB. CONCLUSIONS: The nomogram model can provide guidance for embryo selection in vitrified-thawed blastocyst transfer cycles, which may help to optimize the LB rate.

Association between the number of top-quality blastocysts and live births after single blastocyst transfer in the first fresh or vitrified-warmed IVF/ICSI cycle.

RESEARCH QUESTION: Is there an association between the total number of top-quality blastocysts (TQB) developed in the first IVF/intracytoplasmic sperm injection cycle (ICSI) and live births after a single blastocyst transfer (SBT)? DESIGN: Pregnancy outcomes from 1336 infertile women who had undergone their first IVF/ICSI treatment and accepted a first-time embryo transfer with a single fresh or vitrified-warmed blastocyst between January 2016 and August 2018 were assessed retrospectively. The restricted cubic splines method was used to evaluate the association between the number of TQB, and ongoing pregnancies and live births. RESULTS: A significant non-linear functional form was found between the number of TQB and the ongoing pregnancies and live births (P < 0.05). The odds of an ongoing pregnancy or live birth were similar, at about 11% or higher for each additional TQB up to five TQB (odds ratio [OR] 1.11; 95% confidence interval [CI] 1.01-1.21). After this, pregnancy outcomes nearly plateaued, indicating that the number of TQB was not related to pregnancy when it was greater than five. CONCLUSIONS: The quantity of TQB available for transfer or cryopreservation can provide important predictors for pregnancy and live birth after the first embryo transfer cycle with a single blastocyst. This valuable information may assist with the future application of SBT.

A visualized clinical model predicting good quality blastocyst development in the first IVF/ICSI cycle.

RESEARCH QUESTION: Is it possible to establish a visualized clinical model predicting good quality blastocyst (GQB) formation for patients in their first IVF/intracytoplasmic sperm injection (ICSI) cycle? DESIGN: A total of 4783 patients in their first IVF/ICSI cycle between January 2015 and December 2019 were retrospectively included and randomly divided into the training set (n = 3826) and the testing set (n = 957) in an 8:2 ratio. The least absolute shrinkage and selection operator (LASSO) regression was adopted to select the most critical predictors for GQB formation to construct a visualized nomogram model based on the data of patients in the training set. Receiver operating characteristic and calibration curves were used to evaluate the predictive accuracy and discriminative ability. The performance of the model was also validated on independent data from patients treated in the testing set. RESULTS: Maternal age, maternal serum anti-Müllerian hormone (MsAMH) concentration and the number of oocytes retrieved were highlighted as critical predictors of GQB development and were incorporated into the nomogram model. Based on the area under the curve (AUC) values, the predictive ability for ≥1, ≥3 and ≥5 GQB were 0.831, 0.734 and 0.748, respectively. The calibration curve also showed high concordance between the observed and predicted results. The AUC for predicting ≥1, ≥3 and ≥5 GQB in the testing set were 0.805, 0.695 and 0.707, respectively, which were similar to those for the training set. CONCLUSIONS: The visualized nomogram model provides great predictive value for GQB development in patients in their first IVF/ICSI cycle and can be used to improve clinical counselling.

Perinatal and neonatal outcomes of pregnancies after early rescue intracytoplasmic sperm injection in women with primary infertility compared with conventional intracytoplasmic sperm injection: a retrospective 6-year study.

BACKGROUND: Early rescue intracytoplasmic sperm injection (ICSI) has been used in clinic as appropriate currently. While the outcomes of children born after this method were not well assessed. The purpose of this study was to evaluate the effect of early rescue ICSI on women with primary infertility. METHODS: Fresh embryo transfer cycles after rescue (n = 214) and conventional (n = 546) ICSI were retrospectively evaluated from women with primary infertility who underwent their first assisted reproductive technology cycles at our center in 2012-2017. The conventional ICSI group was subdivided into ICSI-1 (semen suitable for in vitro fertilization, IVF) and ICSI-2 (poor semen quality) to minimize bias from differences in semen quality. Pregnancy, delivery and neonatal outcomes were compared between groups. RESULTS: There was a higher rate of polyspermy and a lower rate of top-quality embryos (TQE) on day 3 for oocytes subject to rescue ICSI compared with conventional ICSI. This reduced the total number of TQE and the number of TQE transferred in the rescue ICSI group. There was no significant difference between groups in clinical pregnancy, ongoing pregnancy, early miscarriage and live birth. For pregnant women, gestational age, route of delivery, risk of preterm birth and gestational diabetes mellitus were also comparable. Neonatal outcomes including sex ratio, birth weight, neonatal intensive care unit admission and birth defects were also similar after rescue and conventional ICSI. Moreover, no differences were observed with the different ICSI subgroups. CONCLUSIONS: For women with primary infertility who have a high risk of IVF fertilization failure (FF), rescue ICSI provides a safe and efficient alternative to minimize FF after initial IVF, but results in fewer TQE on day 3.

Comparative secretome profile analysis of cultured immortalized human endometrial stromal cells supplemented with implanted versus nonimplanted blastocyst-conditioned medium: A preliminary analysis.

AIM: Human endometrial stromal cells (HESCs) were previously shown to be capable of discriminating embryos with different qualities. Here we aimed to compare the specific response of the HESC secretome to implanted blastocyst-conditioned medium (BCM) versus nonimplanted medium and identify cytokine candidates useful for the assessment of blastocyst implantation. METHODS: Cleavage embryos were individually cultured in one microdrop of medium for blastocyst formation. The BCM was collected after fresh blastocyst transfer on day 5 and used to supplement HESC culture medium. A high-throughput antibody array covering 440 cytokines was used to detect the secretory proteins of HESCs supplemented with implanted or nonimplanted BCM. RESULTS: A total of 22 differentially expressed proteins were found out of 440 cytokines in the supernatant of HESCs supplemented with BCM from the implanted group compared to the nonimplanted group, including seven upregulated and 15 downregulated proteins. Gene Ontology enrichment analysis showed that the differentially expressed proteins were mainly involved in cell chemotaxis and motility, and ERK1/2 cascade regulation. Kyoto Encyclopedia of Genes and Genomes analysis suggested that the mitogen-activated protein kinase and phosphatidylinositol 3 kinase/Akt pathways were mainly involved. CONCLUSION: HESCs specifically responded to BCM from different quality blastocysts, a finding that can be used to develop a novel approach for blastocyst quality assessment.

Detection of IL-6, IL-10, and TNF-α level in human single-blastocyst conditioned medium using ultrasensitive Single Molecule Array platform and its relationship with embryo quality and implantation: a pilot study.

PURPOSE: The purpose was to investigate the association between embryonic development or implantation and the content of interleukin-6 and 10 (IL-6, IL-10) and tumor necrosis factor-α (TNF-α) in single-blastocyst conditioned medium (SBCM). METHODS: Thirty-eight SBCM samples (SBCMs) were collected from blastocysts with different morphological scores. IL-6, IL-10, and TNF-α concentration in 38 SBCMs was detected by Single Molecule Array and compared according to the blastocyst quality: top-quality (TQ) and non-top quality (NTQ), or blastulation time: day 5 (D5) and day 6 (D6). In another experiment, 61 SBCMs were collected from TQ blastocyst transplanted on D5, and IL-6 concentration in SBCM was compared based on whether embryos are implanted or not (implanted and non-implanted). RESULTS: In the first experiment, IL-6, IL-10, and TNF-α concentration was not significantly different between the TQ-SBCM and NTQ-SBCM. The D6-SBCM had a higher IL-6 concentration compared with the D5-SBCM, while IL-10 and TNF-α concentration was not significantly different between the D5-SBCM and D6-SBCM. The IL-6 concentration in D5-NTQ or D6-TQ SBCM was higher than that in D5-TQ or D6-NTQ SBCM (P < 0.05), respectively. Furthermore, the spearman analysis demonstrated that IL-6 concentration in SBCM was negatively correlated with the blastocyst quality on D5 and positively correlated with the blastocyst quality on D6. In the second experiment, no significant difference in IL-6 concentration was found between SBCM from implanted and non-implanted blastocyst. CONCLUSION: IL-6 concentration in SBCM was associated with embryo quality depending on the blastulation time, although it might not be associated with the blastocyst implantation.

Obstetric and perinatal outcomes of pregnancies according to initial maternal serum HCG concentrations after vitrified-warmed single blastocyst transfer.

RESEARCH QUESTION: Do pregnancy, obstetric and perinatal outcomes differ according to initial maternal serum human chorionic gonadotrophin (HCG) level measured on day 11 after single blastocyst transfer? DESIGN: Vitrified-warmed single blastocyst transfer cycles (n = 640) were collected between 1 January 2013 and 30 April 2017 with positive HCG values and retrospectively analysed by receiver operating characteristic curves to predict clinical pregnancy, ongoing pregnancy and delivery. Cycles were divided into a low HCG group (n = 155) and high HCG group (n = 485) based on cut-off value of live birth prediction. Cycles in the HCG group were subdivided into a low-high subgroup (n = 162), medium-high subgroup (n = 162) and high-high subgroup (n = 161) based on tertile points. Pregnancy rates and obstetric and perinatal outcomes were compared. RESULTS: The area under curves for clinical pregnancy, ongoing pregnancy and live birth prediction were 0.95, 0.81 and 0.79, respectively; corresponding cut-off values were 152.2 IU/l, 211.9 IU/l and 211.9 IU/l; HCG less than 211.9 IU/l indicated an extremely low clinical pregnancy rate (34.84%), a high early miscarriage rate (61.11%) and a low live birth rate (12.26%). Rates of gestational diabetes mellitus (GDM) (P = 0.007) and female neonates (P = 0.001) were significantly higher in the LHG group compared with the HHG group; no significant differences were observed in the low versus high HCG group overall. CONCLUSIONS: Lower initial maternal serum HCG levels indicated poorer clinical outcomes. Within the high HCG group, a lower initial maternal HCG level was found to be associated with GDM occurrence and proportion of female neonates.

Clinical outcomes after transfer of blastocysts derived from frozen-thawed cleavage embryos: a retrospective propensity-matched cohort study.

PURPOSE: To evaluate the clinical outcomes after fresh transfer of blastocysts cultured from vitrified-thawed cleavage embryos (VTCE) compared with conventional frozen-thawed blastocysts transfer (FBT), or with the usual fresh blastocysts transfer (FRBT). METHODS: A total of 155 cycles undergoing fresh transfer of VTCE blastocysts, 4904 cycles undergoing FBT, and 1014 cycles undergoing FRBT were retrospectively analyzed from August 2014 to July 2017. Pregnancy, delivery, and neonatal outcomes were compared after propensity score matching. RESULTS: VTCE blastocysts' transfer resulted in a lower risk of early miscarriage (8.82% versus 19.70%, P < 0.05) and a decreased fetal birth weight (2611.90 ± 618.65 g versus 2931.86 ± 546.52 g, P < 0.01) compared to FBT. No significant difference was found regarding live birth rate, gestational age, and cesarean section. Correspondingly, VTCE blastocysts' transfer led to significantly compromised pregnancy outcomes regarding clinical pregnancy rate and implantation, and even a slightly compromised live birth rate when compared with FRBT. Moreover, a higher occurrence of cesarean Section (88.89% versus 71.29%, P < 0.05) and a shorter gestational age (262.04 ± 14.99 days versus 268.06 ± 14.07, P < 0.05) were also found. Nevertheless, the risk of small for gestational age and large for gestational age, and the neonatal birth weight were comparable. CONCLUSIONS: VTCE blastocysts' transfer results in a comprehensively moderate outcome, which is an acceptable option for patients. Our results can provide efficient value for patients' counseling. Furthermore, these findings indicate directions for exploring the mechanisms of low birth weight and short gestational age.

[IVOS â ¡ versus Sperm Class Analyzer in the results of semen analysis].

OBJECTIVE: To compare the results obtained from the computer-aided sperm analysis (CASA) systems of the two fully-automated commercial sperm quality analyzers, Hamilton-Thorn IVOS Ⅱ (IVOS Ⅱ) and Spanish Sperm Class Analyzer (SCA). METHODS: A total of 99 semen samples were collected in the Center of Reproduction of Shenzhen Zhongshan Urology Hospital from September 2018 to October 2018 and, according to the sperm concentration, divided into groups A (<15 ×106/ml), B (15－50 ×106/ml) and C (>50 ×106/ml). IVOS Ⅱ, SCA and manual microscopy were used for the examination of each sample, followed by comparison of the sperm concentration, sperm motility and percentage of progressively motile sperm (PMS) obtained from IVOS Ⅱ and SCA. RESULTS: The sperm concentrations derived from IVOS Ⅱ and SCA were significantly higher than that from manual microscopy in group A (ï¼»10.24 ± 4.60ï¼½ and ï¼»10.20 ± 5.11ï¼½ vs ï¼»8.45 ± 4.15ï¼½ ×106/ml, P < 0.05), but showed no statistically significant difference in group B (ï¼»30.95 ± 11.84ï¼½ and ï¼»31.81 ± 12.90ï¼½ vs ï¼»29.14 ± 10.65ï¼½ ×106/ml, P > 0.05) or C (ï¼»102.14 ± 45.97ï¼½ and ï¼»109.48 ± 46.32ï¼½ vs ï¼»104.74 ± 41.87ï¼½ ×106/ml, P > 0.05). Significant differences were not observed between IVOS Ⅱ and SCA in the percentage of PMS (ï¼»24.21 ± 14.62ï¼½% vs ï¼»23.92 ± 15.42ï¼½%, P > 0.05) or sperm motility (ï¼»37.48 ± 19.34ï¼½% vs ï¼»37.69 ± 16.61ï¼½%, P > 0.05) in group B, nor in group C (PMS: ï¼»30.80 ± 12.06ï¼½% vs ï¼»32.98 ± 16.10ï¼½%, P > 0.05; sperm motility: ï¼»44.50 ± 15.62ï¼½% vs ï¼»47.26 ± 17.46ï¼½%, P > 0.05). Both the percentage of PMS and sperm motility obtained from IVOS Ⅱ were remarkably lower than those derived from SCA in group A (PMS: ï¼»18.54 ± 12.96ï¼½% vs ï¼»22.90 ± 12.88ï¼½%, P < 0.05; sperm motility: ï¼»26.97 ± 14.05ï¼½% vs ï¼»34.90 ± 15.18ï¼½%, P < 0.05). IVOS Ⅱ and SCA both showed a high repeatability (CV <15%), and the former exhibited an even higher one than the latter, in detection of sperm concentration, sperm motility and the percentage of PMS. CONCLUSIONS: IVOS Ⅱ and SCA both had a good consistency in the results of sperm concentration, motility and progressive motility, but showed a poor comparability with low-concentration semen samples.