Objective:To explore the differences of metabolomic profiles in day 3 (D3) culture medium of embryos with varying developmental potentials, in order to provide a theoretical foundation for the establishment of embryo selection technology platform using metabolomics.Methods:Eight patients who received in vitro fertilization and embryo transfer (IVF-ET) treatment at Reproductive Medicine Center of Guangxi Zhuang Autonomous Region Maternal and Child Health Hospital between November 13 and December 5, 2023 were selected as the study subjects. The D3 culture medium from patient embryos was collected and divided into high-quality blastocysts ( n=42), non-high-quality blastocysts ( n=33), and embryos that failed to form blastocysts (non-formation group, n=43) according to the formation of day 5 blastocysts. High-performance liquid chromatography-mass spectrometry was employed to perform non-targeted metabolomic analysis in the D3 culture medium from three distinct groups. Results:1) The metabolites in D3 culture medium of embryos with varying developmental potentials exhibit significant differences. Specifically, 79 differential metabolites were identified between the blastocyst formation group and the non-blastocyst formation group (all P<0.05); additionally, 73 differential metabolites were found between the high-quality blastocyst group and the non-high-quality blastocyst group (all P<0.05). 2) The area under the receiver operating characteristic curve of significantly differential metabolites for predicting potential of D3 embryo blastocyst formation and high-quality blastocyst formation were both greater than 0.9, demonstrating excellent predictive performance. 3) KEGG pathway enrichment analysis revealed that differential metabolites associated with blastocyst formation potential were primarily enriched in pathways including D-amino acid metabolism, glycine-serine-threonine metabolism, arginine biosynthesis, and histidine metabolism ( P<0.05). For high-quality blastocyst formation, the differential metabolites were predominantly enriched in pathways related to tryptophan metabolism, D-amino acid metabolism, serotonergic synapses, and protein digestion and absorption ( P<0.05). Conclusion:Embryos with different developmental potentials have significantly different metabolic profiles, and it is feasible to predict the developmental potential of D3 embryos by metabolomics analysis.

Objective:To explore the differences of metabolomic profiles in day 3 (D3) culture medium of embryos with varying developmental potentials, in order to provide a theoretical foundation for the establishment of embryo selection technology platform using metabolomics.Methods:Eight patients who received in vitro fertilization and embryo transfer (IVF-ET) treatment at Reproductive Medicine Center of Guangxi Zhuang Autonomous Region Maternal and Child Health Hospital between November 13 and December 5, 2023 were selected as the study subjects. The D3 culture medium from patient embryos was collected and divided into high-quality blastocysts ( n=42), non-high-quality blastocysts ( n=33), and embryos that failed to form blastocysts (non-formation group, n=43) according to the formation of day 5 blastocysts. High-performance liquid chromatography-mass spectrometry was employed to perform non-targeted metabolomic analysis in the D3 culture medium from three distinct groups. Results:1) The metabolites in D3 culture medium of embryos with varying developmental potentials exhibit significant differences. Specifically, 79 differential metabolites were identified between the blastocyst formation group and the non-blastocyst formation group (all P<0.05); additionally, 73 differential metabolites were found between the high-quality blastocyst group and the non-high-quality blastocyst group (all P<0.05). 2) The area under the receiver operating characteristic curve of significantly differential metabolites for predicting potential of D3 embryo blastocyst formation and high-quality blastocyst formation were both greater than 0.9, demonstrating excellent predictive performance. 3) KEGG pathway enrichment analysis revealed that differential metabolites associated with blastocyst formation potential were primarily enriched in pathways including D-amino acid metabolism, glycine-serine-threonine metabolism, arginine biosynthesis, and histidine metabolism ( P<0.05). For high-quality blastocyst formation, the differential metabolites were predominantly enriched in pathways related to tryptophan metabolism, D-amino acid metabolism, serotonergic synapses, and protein digestion and absorption ( P<0.05). Conclusion:Embryos with different developmental potentials have significantly different metabolic profiles, and it is feasible to predict the developmental potential of D3 embryos by metabolomics analysis.

OBJECTIVE: To carry out preimplantation genetic testing for monogenic/single gene disorders (PGT-M) for a Chinese family affected with Molybdenum co-factor deficiency due to pathogenic variant of MOCS2 gene. METHODS: A family with molybdenum co-factor deficiency who attended to the Maternal and Child Health Care Hospital of Guangxi Zhuang Autonomous Region in April 2020 was selected as the research subject. Trophoblast cells were biopsied from blastocysts fertilized by intracytoplasmic sperm injection. Embryos carrying the MOCS2 gene variant and chromosome copy number variation (CNV) of more than 4 Mb were detected by single-cell whole genome amplification, high-throughput sequencing and single nucleotide polymorphism typing. Embryos without or carrying the heterozygous variant and without abnormal chromosome CNV were transplanted. During mid-pregnancy, amniotic fluid sample was collected for prenatal diagnosis to verify the results of PGT-M. RESULTS: Eleven oocytes were obtained, among which three blastocysts were formed through culturing. Results of genetic testing suggested that one embryo was heterozygous for the maternally derived MOCS2 gene variant and without chromosomal CNV. Following embryo transfer, intrauterine singleton pregnancy was attained. Prenatal diagnosis by amniocentesis at 18 weeks of gestation revealed that the MOCS2 gene variant and chromosomal analysis results were both consistent with that of PGT-M, and a healthy male infant was born at 37⁺⁵ weeks of gestation. CONCLUSION PGT-M has helped the couple carrying the MOCS2 gene variant to have a healthy offspring, and may become an important method for couples carrying other pathogenic genetic variants.
Female ; Humans ; Pregnancy ; Aneuploidy ; China ; DNA Copy Number Variations ; Genetic Testing/methods* ; Preimplantation Diagnosis/methods* ; Metal Metabolism, Inborn Errors/genetics*

Objective:To evaluate the detection ability and efficiency of single nucleotide polymorphisms array (SNP-array) and next generation sequencing (NGS) in preimplantation genetic testing (PGT).Methods:Totally 188 couples who carried pathogenic gene mutation and requested preimplantation genetic testing for monogenic (PGT-M) treatment were retrospectively analyzed in the Reproductive Center of Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region during January 2020 and August 2022. After ovulation induction, insemination was conducted by intracytoplasmic sperm injection (ICSI) and cultured in vitro, 995 blastocysts were harvested and biopsied. After whole genome amplification (WGA) of the genetic material from embryonic cell samples, their carrying status of mutations and chromosome copy number variations (CNVs) were analyzed by SNP-array or NGS, respectively, and along with mutation direct detection by Sanger sequencing or Gap-PCR. The relationship between female age and the number of blastocysts was analyzed, as well as the proportion of embryos carrying mutations and pathogenic CNVs. The detection success rate and accuracy of different molecular diagnostic techniques used in PGT were compared. Amniocentesis prenatal diagnosis was performed in the second trimester after successful intrauterine transfer of embryos. Results:1) A total of 924 embryo samples were successfully performed genetic testing, with a total success rate of 92.9%, and 389 embryos (42.1%) can be transferred according to these results. 2) In detecting deletional α-thalassemia, the success rate of Gap-PCR [84.9% (465/548)] was lower than that of SNP-array [98.7% (81/82)] and NGS [92.5% (431/466)]. However, the success rate of direct mutation detection by Sanger sequencing [98.5% (440/447)] was not significantly different from that by SNP-array [95.6% (110/115)] and NGS [96.1% (319/332)]. There were 38 embryo samples with direct mutation detection results inconsistent with those based on SNP haplotyping. In addition, 4 embryo samples failed SNP haplotyping due to chromosomal recombination. 3) Compared with NGS, SNP-array had a lower success rate [83.7% (165/197)] in detecting CNVs, but it could find out more types of chromosomal abnormalities. 4) A total of 152 embryo transfers were performed, 107 patients got clinical pregnancies, 69 patients completed amniocentesis prenatal diagnosis, and 42 healthy infants were delivered.Conclusion:In considering the detection efficiency, SNP-array is suitable for analyzing embryos which carry multiple pathogenic genes, rare monogenic or deletion mutations, whereas NGS is suitable for detecting common types of mutations. Meanwhile, using Sanger sequencing and Gap-PCR to directly detect the mutations can improve the success rate and accuracy of PGT. Our findings would provide a basis for PGT technicians to select appropriate detection platforms based on the type of mutations and the situation of patients.

Objective:To investigate the effects of different semen sample collection methods on sperm DNA fragmentation index (DFI) test results, then to evaluate the accuracy of the current semen sample collection method in the assessment of male fertility.Methods:In this study, 50 semen sample obtained on the day of oocyte retrieval from patients undergoing in vitro fertilization at the Reproductive Medical Center in Maternity and Child Health Hospital of Guangxi Zhuang Autonomous Region from August 2021 to January 2022 were collected. For each semen, a small amount of samples were collected in three different retention methods for routine semen and sperm DFI testing. Three different ways of retaining samples were as follows: group A, after mixing of the semen, 50 μL sample was directly collected; group B, after density gradient centrifugation, 50 μL sample was collected at the interface between semen and gradient fluid; group C, after density gradient centrifugation, the sperm pellet was upstream, then 50 μL sample was collected from upstream liquid. After semen treatment, routine semen testing and sperm DFI testing were performed. Pearson was used to analyze the correlation between DFI and the percentage of immobile sperm and the percentage of forward sperm movement. Results:The sperm motility rate of group C [(96.83±2.28)%] was significantly higher than that of group A [(57.16±11.28)%, P<0.001] and group B [(22.54±9.35)%, P<0.001], and there was a statistical difference among the three groups. The immotile sperm rate of group B sample was (77.46±9.35)%, which was significantly higher than that of samples from group C [(3.14±2.31)%, P<0.001] and group A [(42.83±11.28)%, P<0.001]. There was also a statistical difference in DFI among the three groups ( P<0.001). The DFI of group B [37.18% (30.41%, 47.80%)] was significantly higher than that of group A [22.00% (14.75%, 29.25%), P<0.001] and group C [0.78% (0.00%, 2.07%), P<0.001]. Pearson analysis results showed that the DFI of group A and group B was positively correlated with the percentage of immobile sperm ( r=0.304, P=0.032; r=0.612, P<0.001), while the DFI of group B was negatively correlated with the percentage of sperm forward movement ( r=-0.517, P<0.001). Conclusion:For the same semen, the DFI of immotile sperm was significantly higher than that of motile sperm. Therefore, due to the interference of immotile sperm, the DFI value by the current sample retention method cannot accurately reflect the DNA status of active sperm participating in fertilization. This suggests that the samples used for DFI testing should be collected from motile sperm collected by gradient centrifugation, upstream or other methods, which can more accurately assess male fertility.

Objective:To evaluate the detection ability and efficiency of single nucleotide polymorphisms array (SNP-array) and next generation sequencing (NGS) in preimplantation genetic testing (PGT).Methods:Totally 188 couples who carried pathogenic gene mutation and requested preimplantation genetic testing for monogenic (PGT-M) treatment were retrospectively analyzed in the Reproductive Center of Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region during January 2020 and August 2022. After ovulation induction, insemination was conducted by intracytoplasmic sperm injection (ICSI) and cultured in vitro, 995 blastocysts were harvested and biopsied. After whole genome amplification (WGA) of the genetic material from embryonic cell samples, their carrying status of mutations and chromosome copy number variations (CNVs) were analyzed by SNP-array or NGS, respectively, and along with mutation direct detection by Sanger sequencing or Gap-PCR. The relationship between female age and the number of blastocysts was analyzed, as well as the proportion of embryos carrying mutations and pathogenic CNVs. The detection success rate and accuracy of different molecular diagnostic techniques used in PGT were compared. Amniocentesis prenatal diagnosis was performed in the second trimester after successful intrauterine transfer of embryos. Results:1) A total of 924 embryo samples were successfully performed genetic testing, with a total success rate of 92.9%, and 389 embryos (42.1%) can be transferred according to these results. 2) In detecting deletional α-thalassemia, the success rate of Gap-PCR [84.9% (465/548)] was lower than that of SNP-array [98.7% (81/82)] and NGS [92.5% (431/466)]. However, the success rate of direct mutation detection by Sanger sequencing [98.5% (440/447)] was not significantly different from that by SNP-array [95.6% (110/115)] and NGS [96.1% (319/332)]. There were 38 embryo samples with direct mutation detection results inconsistent with those based on SNP haplotyping. In addition, 4 embryo samples failed SNP haplotyping due to chromosomal recombination. 3) Compared with NGS, SNP-array had a lower success rate [83.7% (165/197)] in detecting CNVs, but it could find out more types of chromosomal abnormalities. 4) A total of 152 embryo transfers were performed, 107 patients got clinical pregnancies, 69 patients completed amniocentesis prenatal diagnosis, and 42 healthy infants were delivered.Conclusion:In considering the detection efficiency, SNP-array is suitable for analyzing embryos which carry multiple pathogenic genes, rare monogenic or deletion mutations, whereas NGS is suitable for detecting common types of mutations. Meanwhile, using Sanger sequencing and Gap-PCR to directly detect the mutations can improve the success rate and accuracy of PGT. Our findings would provide a basis for PGT technicians to select appropriate detection platforms based on the type of mutations and the situation of patients.

Objective:To investigate the effects of different semen sample collection methods on sperm DNA fragmentation index (DFI) test results, then to evaluate the accuracy of the current semen sample collection method in the assessment of male fertility.Methods:In this study, 50 semen sample obtained on the day of oocyte retrieval from patients undergoing in vitro fertilization at the Reproductive Medical Center in Maternity and Child Health Hospital of Guangxi Zhuang Autonomous Region from August 2021 to January 2022 were collected. For each semen, a small amount of samples were collected in three different retention methods for routine semen and sperm DFI testing. Three different ways of retaining samples were as follows: group A, after mixing of the semen, 50 μL sample was directly collected; group B, after density gradient centrifugation, 50 μL sample was collected at the interface between semen and gradient fluid; group C, after density gradient centrifugation, the sperm pellet was upstream, then 50 μL sample was collected from upstream liquid. After semen treatment, routine semen testing and sperm DFI testing were performed. Pearson was used to analyze the correlation between DFI and the percentage of immobile sperm and the percentage of forward sperm movement. Results:The sperm motility rate of group C [(96.83±2.28)%] was significantly higher than that of group A [(57.16±11.28)%, P<0.001] and group B [(22.54±9.35)%, P<0.001], and there was a statistical difference among the three groups. The immotile sperm rate of group B sample was (77.46±9.35)%, which was significantly higher than that of samples from group C [(3.14±2.31)%, P<0.001] and group A [(42.83±11.28)%, P<0.001]. There was also a statistical difference in DFI among the three groups ( P<0.001). The DFI of group B [37.18% (30.41%, 47.80%)] was significantly higher than that of group A [22.00% (14.75%, 29.25%), P<0.001] and group C [0.78% (0.00%, 2.07%), P<0.001]. Pearson analysis results showed that the DFI of group A and group B was positively correlated with the percentage of immobile sperm ( r=0.304, P=0.032; r=0.612, P<0.001), while the DFI of group B was negatively correlated with the percentage of sperm forward movement ( r=-0.517, P<0.001). Conclusion:For the same semen, the DFI of immotile sperm was significantly higher than that of motile sperm. Therefore, due to the interference of immotile sperm, the DFI value by the current sample retention method cannot accurately reflect the DNA status of active sperm participating in fertilization. This suggests that the samples used for DFI testing should be collected from motile sperm collected by gradient centrifugation, upstream or other methods, which can more accurately assess male fertility.

Objective To evaluate the effect of resveratrol on mitochondrial function in renal tubular epithelial cells of rats with sepsis-induced acute kidney injury.Methods Ninety-six healthy SpragueDawley rats of both sexes,aged 5-7 weeks,weighing 180-220 g,were divided into 4 groups (n =24 each) using a random number table method:sham operation group (Sham group),sepsis group (group Sep),sepsis plus vehicle group (Sep+Ⅴ group) and sepsis plus resveratrol group (Sep+R group).Sepsis was induced by cecal ligation and puncture (CLP).Normal saline 0.5 ml,vehicle 0.5 ml and resveratrol 10 mg/kg were intraperitoneally injected at 6,12 and 18 h after CLP in Sep,Sep+Ⅴ and Sep+R groups,respectively.At 24 h after CLP,serum concentrations of creatinine (Cr) and blood urea nitrogen (BUN) were measured,and kidney tissues were obtained for examination of the pathological changes (using transmission electron microscopy),and the damage to the renal tubules was scored.The renal tubular epithelial cells (RTECs) were isolated from the kidney cortex at 24 h after CLP for determination of mitochondrial transmembrane potential (by flow cytometry with the fluorescent probe JC-1),intracellular ATP content,mitochondrial permeability transition pore (mPTP) opening,lipid peroxide (LPO) content,and lysosomal membrane permeability.Results Compared with group Sham,the serum concentrations of Cr and BUN,renal tubular damage score,mPTP opening,LPO content and lysosomal membrane permeability were significantly increased,and mitochondrial transmembrane potential and ATP content were decreased in Sep and Sep+Ⅴ groups (P＜0.01).Compared with Sep and Sep+Ⅴ groups,the serum concentrations of Cr and BUN,renal tubular damage score,mPTP opening,LPO content and lysosomal membrane permeability were significantly decreased,and mitochondrial transmembrane potential and ATP content were increased in group Sep+R (P＜0.05).Conclusion Resveratrol improves mitochondrial function in renal tubular epithelial cells of rats with sepsis and reduces acute kidney injury,and the mechanism may be related to inhibiting oxidative stress and decreasing the lysosomal membrane permeability.

A viable spermatozoon is a prerequisite for fertilization in intracytoplasmic sperm injection (ICSI). Thus, it is crucial to select viable but immotile spermatozoa on the day of ICSI. We report conflicting results in the identification of viable but immotile spermatozoa between the eosin-nigrosin staining and the laser test, which resulted in confusion for embryologists during assisted reproductive technology (ART). Three patients’ semen samples that showed no motile spermatozoa are described in this report. To identify viable spermatozoa, we used both the eosin-nigrosin test and the laser test for each sample, and repeated the semen analysis twice in each patient. Viable but immotile spermatozoa selected by the laser test were used for ICSI. Viable spermatozoa were detected by both the eosin-nigrosin and laser tests in two patients (case 1, 95.00% vs. 24.21% and 92.68% vs. 22.22%; case 2, 41.18% vs. 23.48% and 39.81% vs. 22.52%), indicating consistent results between the two methods. In the third patient, the eosin-nigrosin test yielded viability rates of 20.75% and 19.14%, while the result of the laser test was 0%. Thus, testicular aspiration was performed to collect viable sperm from this patient. Normal fertilization was achieved after the injection of viable but immotile spermatozoa selected from these patients by the laser test, resulting in the birth of two healthy babies. Our study documents a case where the eosin-nigrosin test showed a limitation in identifying viable but immotile spermatozoa for ART, while the laser test may overcome this limitation. Larger samples may be required to corroborate the clinical value of the laser test.
Fertilization ; Humans ; Parturition ; Reproductive Techniques, Assisted ; Semen ; Semen Analysis ; Sperm Injections, Intracytoplasmic ; Spermatozoa

OBJECTIVE: The aim of this study was to explore the effects of the insemination method on the outcomes of elective blastocyst culture. METHODS: We retrospectively analyzed the outcomes of elective blastocyst culture performed between January 2011 and December 2014. RESULTS: There were 2,003 cycles of conventional in vitro fertilization (IVF) and 336 cycles of intracytoplasmic sperm injection (ICSI), including 25,652 and 4,164 embryos that underwent sequential blastocyst culture, respectively. No significant differences were found in the female patients' age, basal follicle-stimulating hormone level, basal luteinizing hormone level, body mass index, number of oocytes, maturity rate, fertilization rate, or good-quality embryo rate. However, the blastocyst formation rate and embryo utilization rate were significantly higher in the conventional IVF group than in the ICSI group (54.70% vs. 50.94% and 51.09% vs. 47.65%, respectively, p<0.05). The implantation/pregnancy rate (IVF, 50.93%; ICSI, 55.10%), miscarriage rate (IVF, 12.57%; ICSI, 16.29%), and live birth rate (IVF, 42.12%; ICSI, 44.08%) were similar (p>0.05). No cycles were canceled due to the formation of no usable blastocysts. CONCLUSION: Although the fertilization method had no effect on clinical outcomes, the blastocyst formation rate and embryo utilization rate in the ICSI group were significantly lower than those observed in the conventional IVF group. Therefore, more care should be taken when choosing to perform blastocyst culture in ICSI patients.
Abortion, Spontaneous ; Blastocyst* ; Body Mass Index ; Embryonic Structures ; Female ; Fertilization ; Fertilization in Vitro ; Follicle Stimulating Hormone ; Humans ; Insemination* ; Live Birth ; Luteinizing Hormone ; Methods* ; Oocytes ; Pregnancy ; Pregnancy Rate ; Retrospective Studies ; Sperm Injections, Intracytoplasmic