YTHDF2 regulates MSS51 expression contributing to mitochondria dysfunction of granulosa cells in polycystic ovarian syndrome patients.

RESEARCH QUESTION: Granulosa cells (GCs) dysfunction plays a crucial role in the pathogenesis of polycystic ovary syndrome (PCOS). It is reported that YTH domain-containing family protein 2 (YTHDF2) is upregulated in mural GCs of PCOS patients. What effect does the differential expression of YTHDF2 have in PCOS patients? DESIGN: Mural GCs and cumulus GCs from 15 patients with PCOS and 15 ovulatory controls and 4 cases of pathological sections in each group were collected. Real-time PCR, Western Blot, immunohistochemistry, and immunofluorescence experiments were conducted to detect gene and protein expression. RNA immunoprecipitation assay was performed to evaluate the binding relationship between YTHDF2 and MSS51. Mitochondrial morphology, cellular ATP and ROS levels and glycolysis-related gene expression were detected after YTHDF2 overexpression or MSS51 inhibition. RESULTS: In the present study, we found that YTHDF2 was upregulated in GCs of PCOS patients while MSS51 was downregulated. YTHDF2 protein can bind to MSS51 mRNA and affect MSS51 expression. The reduction of MSS51 expression or the increase in YTHDF2 expression can lead to mitochondrial damage, reduced ATP levels, increased ROS levels and reduced expression of LDHA, PFKP and PKM. CONCLUSIONS: YTHDF2 may regulate the expression of MSS51, affecting the structure and function of mitochondria in GCs and interfering with cellular glycolysis, which may disturb the normal biological processes of GCs and follicle development in PCOS patients.

Potential biomarkers for clinical outcomes of IVF cycles in women with/without PCOS: Searching with metabolomics.

Background: Polycystic ovary syndrome (PCOS) is a heterogeneous endocrinological and metabolic disorder which is the common cause of female infertility. The dysmetabolism displayed in it has not been completely ascertained. Metabonomics may shed light on understanding many small molecule endogenous metabolites and their associated metabolic pathways. Objective: To analyze the different metabolites and related metabolic pathways in follicular fluid and embryo culture fluid of PCOS and non-PCOS groups. Finding markers predictable for clinical outcomes of in vitro fertilization-embryo transfer (IVF-ET) treatment. Population and sample: 60 women who underwent IVF-ET were selected, including 30 with PCOS and 30 with the fallopian tubal issues only. We collected the first tube follicular fluid (FF) of all patients at the time of oocyte pick up and the waste embryo culture medium (ECM) after D3 high-quality embryo transplant. Methods: All samples were performed nontargeted Ultra High Performance Liquid Chromatography-Mass Spectrometry (UHPLC-QE-MS) analysis. Related metabolic pathways were screened by KEGG annotation. To search potential indicators, the logistic regression was made combined with clinical data. Mean outcome measures: Predictive performance of markers of clinical outcomes (pregnancy rate, delivery rate, live birth rate, miscarriage rate) of assisted reproductive technology (ART). Results: Comparing the PCOS group against the non-PCOS group, we found 11 significantly different metabolites in the FF and 56 in the ECM. There are a total of 11 kinds of biomarkers associated with clinical outcomes. Androsterone sulfate, Glycerophosphocholine, and Elaidic carnitine seem robust to predict the abortion rate of the PCOS group, with an AUC of 0.941, 0.933, 0.933, respectively. The glycerol phospholipid metabolic pathway is enriched in both the follicular fluid and embryo culture fluid. Conclusions: The differential metabolites were mainly a variety of lipids. Some of them can predict clinical outcomes to a certain extent.

miR-188-3p-targeted regulation of ATG7 affects cell autophagy in patients with nonobstructive azoospermia.

BACKGROUND: Nonobstructive azoospermia (NOA) is one of the most difficult forms of male infertility to treat, and its pathogenesis is still unclear. miRNAs can regulate autophagy by affecting their target gene expression. Our previous study found that miR-188-3p expression in NOA patients was low. There are potential binding sites between the autophagy gene ATG7 and miR-188-3p. This study aimed to verify the binding site between miR-188-3p and ATG7 and whether miR-188-3p affects autophagy and participates in NOA by regulating ATG7 to influence the autophagy marker genes LC3 and Beclin-1. METHODS: Testicular tissue from 16 NOA patients and 16 patients with normal spermatogenesis and 5 cases in each group of pathological sections were collected. High-throughput sequencing was performed to detect mRNA expression differences. Quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting, immunohistochemical staining and immunofluorescence were used to detect protein localization and expression. Autophagosome changes were detected by electron microscopy. The targeting relationship between miR-188-3p and ATG7 was confirmed by a luciferase assay. RESULTS: ATG7 protein was localized in the cytoplasm of spermatogenic cells at all levels, and the ATG7 gene (p = 0.019) and protein (p = 0.000) were more highly expressed in the NOA group. ATG7 expression after overexpression/inhibition of miR-188-3p was significantly lower (p = 0.029)/higher (p = 0.021) than in the control group. After overexpression of miR-188-3p, the ATG7 3'UTR-WT luciferase activity was impeded (p = 0.004), while the ATG7 3'UTR-MUT luciferase activity showed no significant difference (p = 0.46). LC3 (p = 0.023) and Beclin-1 (p = 0.041) expression in the NOA group was significantly higher. LC3 and Beclin-1 gene expression after miR-188-3p overexpression/inhibition was significantly lower (p = 0.010 and 0.024, respectively) and higher (p = 0.024 and 0.049, respectively). LC3 punctate aggregation in the cytoplasm decreased after overexpression of miR-188-3p, while the LC3 punctate aggregation in the miR-188-3p inhibitor group was higher. The number of autophagosomes in the miR-188-3p mimic group was lower than the number of autophagosomes in the mimic NC group. CONCLUSIONS: LC3 and Beclin-1 were more highly expressed in NOA testes and negatively correlated with the expression of miR-188-3p, suggesting that miR-188-3p may be involved in the process of autophagy in NOA. miR-188-3p may regulate its target gene ATG7 to participate in autophagy anDual luciferase experiment d affect the development of NOA.

Effect of ambient air pollutants on in vitro fertilization-embryo transfer pregnancy outcome in Zhengzhou, China.

With the acceleration of China's urbanization and industrialization, air pollution has become a major environmental problem. Retrospective data analysis of 6564 patients who underwent IVF-ET in the center for reproductive medicine of the First Affiliated Hospital of Zhengzhou University from 2015 to 2020. Different stages were selected from 90 days before oocyte retrieval to 35 days after transfer and divided into five exposure periods. Multivariate logistic regression was used to analyze the relationship between six ambient air pollutants (PM2.5, PM10, NO2, SO2, CO and O3) and the IVF-ET pregnancy outcome. The results showed that air pollutants can significantly affect the IVF pregnancy outcome. The harmful effects of ambient air pollutants are more obvious in the patients aged < 35 years, single embryo transfer and cleavage stage embryo transfer.

Different expression and localization of aquaporin 7 and aquaporin 9 in granulosa cells, oocytes, and embryos of patients with polycystic ovary syndrome and the negatively correlated relationship with insulin regulation.

OBJECTIVE: To investigate the expression of aquaporin 7 (AQP7) and aquaporin 9 (AQP9) in the granulosa cells of patients with polycystic ovary syndrome (PCOS) and healthy women and detect their localization in oocytes at the germinal vesicle (GV), metaphase I (MI), MII, embryo, and blastocyst stages and the in vitro response to insulin stimulation. DESIGN: Randomized, assessor-blinded study. SETTING: Reproductive medical center. PATIENT(S): A total of 40 women (aged 20-38 years) comprising 29 cases of primary infertility and 11 cases of secondary infertility, of whom 17 had an initial diagnosis of PCOS and three received a PCOS diagnosis after an infertility examination. INTERVENTION(S): Controlling different concentrations of insulin and different treatment times in cultures of normal human granulosa cells in vitro. MAIN OUTCOME MEASURE(S): Expression of AQP7 and AQP9 genes and proteins in granulosa cells detected by real-time quantitative polymerase chain reaction, and localization in oocytes at the GV, MI, MII, embryo, and blastocyst stages by Western blot, immunohistochemical, and immunofluorescence assays, and concentrations of insulin in follicular fluid by enzyme-linked immunosorbent assay. RESULT(S): The expression levels of the AQP7 mRNA and protein in the granulosa cells of patients with PCOS were higher than found in healthy controls. We found AQP7 protein expressed in human oocytes at GV, MI, MII, embryo, and blastocyst stages; it was mainly located in the nucleoplasm. In the PCOS group, the expression level of AQP9 mRNA and protein in granulosa cells was lower, and AQP9 protein was expressed in oocytes at the GV, MI, MII, embryo, and blastocyst stages; it was localized on the nuclear membrane. Compared with healthy women, the insulin expression in patients with PCOS was higher. In cultures of normal human granulosa cells in vitro, the expression of AQP7 and AQP9 mRNA and protein decreased with the increase in insulin concentration; expression statistically significantly decreased when the insulin concentration was 100 nmol/L, and after 6 to 24 hours of exposure the lowest expression levels were found at 12 hours. CONCLUSION(S): The different localization and expression of AQP7 and AQP9 between the two groups suggests that they might be involved in oocyte maturation and embryonic development through different regulatory pathways. The expression levels of AQP7 and AQP9 were negatively correlated with insulin regulation, suggesting that insulin might affect the maturation of PCOS follicles by changing AQP7 and AQP9 expression.

Can Time-Lapse Incubation and Monitoring Be Beneficial to Assisted Reproduction Technology Outcomes? A Randomized Controlled Trial Using Day 3 Double Embryo Transfer.

Objective: To determine if the application of time-lapse incubation and monitoring can be beneficial to clinical outcomes in assisted reproductive technology. Methods: A total of 600 patients were equally randomized to three groups, namely, conventional embryo culture and standard morphological selection (CM group), time-lapse culture and standard morphological selection (TLM group), and time-lapse culture and morphokinetic selection (TLA group). Notably, 424 undergoing fresh autologous in vitro fertilization cycles were analyzed, 132 patients in the CM group, 158 in the TLM group, and 134 in the TLA group. Main outcomes included clinical outcomes, embryo development rates, and perinatal outcomes. Results: Clinical pregnancy rates in the time-lapse groups were significantly higher than in the CM group (CM 65.2% vs. TLM 77.2% vs. TLA 81.3%). Implantation rates and live birth rates were significantly higher for the TLA group (59.7 and 70.9%) compared with the CM group (47.7 and 56.1%) but not compared with the TLM group (55.4 and 67.1%). There was no statistical difference in miscarriage and ectopic pregnancy rates among the three groups. Overall, birth weight was significantly higher in the time-lapse groups (CM 2,731.7 ± 644.8 g vs. TLM 3,066.5 ± 595.4 g vs. TLA 2,967.4 ± 590.0 g). The birth height of newborns in the TLM group was significantly longer than that of the CM group and TLA group (CM 48.3± 4.4 cm vs. TLM 49.8± 2.3 cm vs. TLA 48.5± 2.7 cm). Conclusion: Time-lapse incubation and monitoring have a significant benefit on clinical pregnancy rates and on overall birth weights while morphokinetic analysis is not necessary. Clinical Trial Registration: [www.ClinicalTrials.gov], identifier [NCT02974517].

Comparative study on the pregnancy outcomes of in vitro fertilization-embryo transfer between patients with different ovarian responses (a STROBE-compliant article).

There have been few studies on large-sample data of cleavage-stage embryo and blastocyst transfers. We compared the pregnancy outcomes of patients with different ovarian responses after the transfer of different numbers of embryos in different developmental stages.Patients were divided into 3 groups including low response group, medium response group, and high response group according to different ovarian responses. Patients in each group were further divided into 4 subgroups including group A: transfer of 1 D3 embryo, group B: transfer of 2 D3 embryos; group C: transfer of 1 D5 blastocyst; and group D: transfer of 2 D5 blastocysts.In low ovarian responders, the implantation rate, clinical pregnancy rate and live birth rate were significantly lower in the group A than in the groups B and C. In medium ovarian responders, the implantation rate was significantly higher, but the multiple pregnancy rate was significantly lower in the group C than in the group B. The multiple pregnancy rate was significantly higher in the group D than in the group C. In high ovarian responders, the implantation rate was significantly lower, but the multiple pregnancy rate was significantly higher in the group B than in group C.Based on the above results, the single blastocyst transfer is preferable for the patients with different ovarian responses.

Identification and characterization of intestine microRNAs and targets in red swamp crayfish, Procambarus clarkii infected with white spot syndrome virus.

MicroRNAs (miRNAs) are small non-coding endogenous RNA molecules that play important roles in the innate immunity system of invertebrates, especially in the aspect of antivirus. In the present study, high-throughput small RNA Illumina sequencing systems were used to identify differentially expressed miRNAs (DEMs) from the intestines of Procambarus clarkii that were infected with white spot syndrome virus (WSSV). As a result, 39 known and 12 novel miRNAs were identified in both NG and WG small RNA libraries. Seven DEMs were determined to be involved in the antiviral innate immunity in the intestines of P. clarkii. The results of the target gene predictions of the DEMs showed that the putative target genes of these 7 DEMs are related to tight junctions, vascular smooth muscle contraction regulation of the actin cytoskeleton, focal adhesion, RNA transport, mRNA surveillance, viral carcinogenesis, and Salmonella infection. These results provide theoretical insights for future studies on the antiviral immunity of crustaceans.

Reduced microRNA-188-3p expression contributes to apoptosis of spermatogenic cells in patients with azoospermia.

BACKGROUND AND AIMS: Human mutL homologl (MLH1) works coordinately in sequential steps to initiate repair of DNA mismatches, and aberrant MLH1 expression is related to spermatogenetic malfunction. In the present study, MLH1 expression in patients with azoospermia was investigated, and moderating effects of miR-188-3p on MLH1 expression and spermatogenesis were identified. METHODS: Testicular tissues from 16 patients with obstructive azoospermia (OA) and non-obstructive azoospermia (NOA), and tissues of eight healthy patients were collected. Real-time PCR, Western blotting and immunohistochemical staining were used to detect MLH1 expression. Chromatin immunoprecipitation assay and luciferase reporter assay were performed to evaluate histone acetylation level of miR-188-3p and relationships between miR-188-3p and MLH1. RESULTS: Testicular MLH1 expression at mRNA and protein levels was significantly increased, while miR-188-3p expression was lower in patients with OA and NOA than that in controls. Reduced histone acetylation level of miR-188-3p promoter was observed in patients with azoospermia. Overexpression/inhibition of HDAC1, but not HDAC2, contributed to the significant reduction/increase of miR-188-3p expression. miR-188-3p targeted 3' UTR of MLH1 and regulated MLH1 expression. miR-188-3p inhibitor led to elevation of apoptotic level of spermatogenic cells in mice, while this effect was reversed by si-MLH1. CONCLUSION: Down-regulation of miR-188-3p by reducing histone acetylation up-regulated MLH1 expression and contributed to promotion of apoptosis in spermatogenic cells, in patients with azoospermia.

Analysis of Related Causes for No Embryos Transferred and Corresponding Coping Measures in Assisted Reproductive Technology.

OBJECTIVE: To analyze the related causes for no embryos transferred in assisted reproductive technology (ART) in order to provide corresponding coping measures for infertile couples. STUDY DESIGN: The data of 607 couples who underwent ART and had no embryos transferred in our reproductive center between January 2010 and January 2014 were retrospectively analyzed. RESULTS: The cycles of no embryos transferred accounted for 3.99% (607/15,224) of total cycles. Of those, complete fertilization failure, oocyte retrieval failure, and complete abnormal fertilization accounted for 28.3% (172/607), 25.7% (156/607) and 22.24% (135/607), respectively. The incidence of complete abnormal fertilization was higher in IVF than in ICSI (p<0.05). In both IVF and ICSI cycles, the incidences of no embryos transferred were higher in the patients retrieving ≤3 oocytes than in the patients retrieving >3 oocytes (p<0.05). In IVF cycles the incidences of no embryos transferred were higher in the patients with primary infertility than in those with secondary infertility (p<0.05). CONCLUSION: The main causes of no embryos transferred are complete fertilization failure, oocyte retrieval failure, and complete abnormal fertilization. Retrieving adequate number of mature oocytes is the key to success of ART. Patients who experienced complete abnormal fertilization in IVF or the patients with primary infertility who experienced complete fertilization failure or normal fertilization without cleavage should receive ICSI in the next treatment.

Effects of Vitrification on Outcomes of In Vivo-Mature, In Vitro-Mature and Immature Human Oocytes.

BACKGROUND/AIMS: To observe the effects of vitrification on spindle, zona pellucida, embryonic aneuploidy and DNA injury in in vivo-maruted, in vitro-mature and immature human oocytes. METHODS: Between January 2009 and February 2015, 223 immature oocytes from 450 infertile patients, and 31 in vivo-mature oocytes from 3 infertile couples were collected. Of the 223 immature oocytes, 113 were used for in vitro culture before vitrification. Some oocytes were randomly divided into in vivo-mature group (group A, n = 15), in vitro-mature group (group B, n = 88) and immature group (group C, n = 85), and then the oocytes with spindle in these three groups after freezing-thawing were selected to use for Polscope imaging, embryonic aneuploidy screening and embryo development evaluation. Other oocytes were randomly divided into group A (n = 16), group B (n = 25) and group C (n = 25) for detecting DNA injury. RESULTS: After thawing, spindle occurrence rate, spindle Retardance value, and cleavage rate were significantly higher in groups A and B than in group C (all P < 0.05), but there were no statistical differences in fertility rate, high-quality embryo rate, blastulation rate and aneuploidy rate amongst the three groups (all P > 0.05). Zona pellucida density (ZPD) was significantly lower in group A than in groups B and C both before and after vitrification (all P < 0.05). ZPD was significantly higher after thawing than before vitrification (all P < 0.05), but zona pellucida thickness (ZPT) was not significantly changed in all the three groups (all P > 0.05). Rate of comet cells was significantly lower in group A than in groups B and C (all P < 0.01). Comet tail was significantly longer in group C than in groups B and A (all P < 0.05). CONCLUSION: In vivo- and in vitro-mature human oocytes are more suitable to vitrification than immature human oocytes. Spindle Retardance value has more predictive value for embryonic development potential than ZPD and ZPT.

The effects of anticancer drugs TSA and GSK on spermatogenesis in male mice.

OBJECTIVE: The effect of anticancer drugs Trichostation A (TSA) and GSK2126458 (GSK) on genetic recombination of sperm meiosis in mice was investigated, and their clinical feasibility of fertility preservation in cancer patients was also assessed. METHODS: Eighteen Kunming mice were randomly given TSA or GSK at the concentrations of 0, 0.1 and 0.2 umol/L for three months. Immunofluorescence was used to evaluate the genetic recombination of homologous chromosomes and fidelity of chromosome synapsis. Sperm density, motility and viability were also examined to investigate the spermatogenic function. RESULTS: The average number of MLH1 foci in each spermatocyte was greatly higher in TSA (0.1) group than that in control (P<0.05), but no difference with the TSA (0.2) group (P>0.05). The frequency of SC with no MLH1 foci was lower while the frequency of SC with one MLH1 foci was higher in spermatocyte of mice with different doses of TSA compared with controls (P<0.05). The weight of left testis in TSA (0.1) group was significant decreased compared with that in control (P<0.05). However, no significant differences were observed in average number of MLH1, frequency of SC with 0-3 MLH1 foci, spermatocyte percentage of XY chromosomes containing MLH1 foci and percentages of cells containing gaps and splits among groups with or without the treatment of GSK. Furthermore, there were no statistical differences in body weight, testicular weight, sperm density, sperm motility and sperm viability among the three groups. CONCLUSION: TSA increased genetic recombination frequency of spermatocyte meiosis. GSK had no significant effect on genetic recombination frequency of spermatocyte meiosis and spermatogenic function.

Comparison of vitrified outcomes between human early blastocysts and expanded blastocysts.

We compared the vitrified outcomes between early and expanded blastocysts with or without laser drilling. The grade III embryos from the patients undergoing in vitro fertilization-embryo transfer (IVF-ET) in our reproductive center from September 2009 to February 2015 were incubated into early blastocysts and expanded blastocysts. The early blastocysts and expanded blastocysts were, respectively, divided into laser group (vitrification after laser drilling), non-laser group (direct vitrification), and control group (fresh non-vitrified blastocysts). After thawing, the blastular anabiosis rate, expansion rate, hatching rate, and apoptosis were observed in each group and then were compared amongst groups. This study indicated that the blastular expansion rate (all P < 0.01) and hatching rate (all P < 0.01) were significantly lower, but the blastular apoptosis (all P < 0.05) was significantly higher in both laser and non-laser groups than in the control group in the early blastocysts. In the expanded blastocysts, the blastular anabiosis rate was significantly higher in the laser group than in the non-laser group (P < 0.01), and the blastular expansion rate was significantly higher, but the blastular apoptosis was significantly lower in both laser group and control group than in the non-laser group (all P < 0.05). The blastular expansion rate (all P < 0.01) and hatching rate (all P < 0.01) were significantly higher, but the blastular apoptosis (all P < 0.05) was significantly lower in the expanded laser group than in both early laser and early non-laser groups. We conclude that vitrification for laser-drilling expanded blastocysts can achieve the best outcomes.

Role of PAFAH1B1 in human spermatogenesis, fertilization and early embryonic development.

Spermatogenesis, fertilization and subsequent embryonic development are complex processes that require tight regulation. The PAFAH1B1 gene plays important roles in these reproductive events in mice, but its expression and roles in human reproduction have not been investigated. Expression analysis of testicular tissue by reverse transcription quantitative PCR and immunohistochemistry revealed varied expression levels among samples of different spermatogenic abilities (as assessed by the Johnsen score), with protein expression restricted to spermatogonia, spermatocytes and spermatids. Immunofluorescence on spermatozoa showed expression over the acrosome and midpiece regions of ejaculated samples, whereas a high proportion of percutaneous epididymal sperm aspiration-derived spermatozoa showed expression restricted to the midpiece. Analysis for PAFAH1B1 mRNA also revealed different expression levels among unfertilized oocytes, zygotes, cleavage stage embryos and blastocysts, with protein localized at the membrane level in oocytes and zygotes, and gradually distributing within the cytoplasm of cleavage stage embryos and blastocysts. Interestingly, microinjection of PAFAH1B1 siRNA into zygotes significantly (P = 0.024) increased fragmentation formation rates in subsequent embryonic development stages. Altogether, these are the first results to support a role for PAFAH1B1 in human spermatogenesis and early embryonic development.

Impact of oxygen concentrations on fertilization, cleavage, implantation, and pregnancy rates of in vitro generated human embryos.

The aim of the present study was to determine the impact of oxygen concentration during in vitro culture of human oocytes and embryos on fertilization, cleavage, implantation, pregnancy, multiple gestation and abortion rates. Women 20-48 years old presenting for infertility treatment and accounting for 3484 in vitro fertilization/intracytoplasmic sperm injection cycles were included in the study. Oocytes/embryos were randomly allocated to be incubated under three different oxygen tension environments: (1) 20% O2 in air; (2) initially 20% O2 in air, followed on day 2 (2-4 cells stage) by 5% CO2, 5% O2 and 90% N2; and (3) 5% CO2, 5% O2 and 90% N2 throughout. Interestingly, IVF-derived embryos cultured in 5% O2 yielded higher rates of fertilization and implantation as compared to those incubated in 20% O2 (P < 0.05). Conversely, embryos in 20% O2 yielded higher rates of fertilization, high quality embryo and implantation than those in the 20%-5% O2 group (P < 0.05). Moreover, ICSI-derived embryos cultured in 20% O2 resulted in lower rates of cleavage as compared to those from the 20%-5% O2 group (P < 0.05). These results are consistent with in vitro and subsequent in vivo embryo development being more susceptible to O2 tension fluctuations rather than the degree of O2 tension itself during culture.

Embryo developmental potential of microsurgically corrected human three-pronuclear zygotes.

We explored the embryo development potential of human three-pronuclear (3PN) zygotes reduced to two-pronuclear (2PN) zygotes (3 → 2PN zygotes) by micropuncture. In this study, there were three groups, the 3 → 2PN group (338 zygotes), the non-corrected 3PN group (381 zygotes), and the normal 2PN group (359 zygotes). The first cleavage mode (2-cell cleavage or 3-cell cleavage), 6-8 cell embryogenesis rate, high-quality embryogenesis rate and Day 5/Day 6 blastulation rate were compared between the three groups. The success rate of enucleation was 92.9%. The 2-cell cleavage rate was significantly higher in the 3 → 2PN group (74.3%) than in the 3PN group (36.4%) (P < 0.05), but had no statistical difference compared with the 2PN group (86.0%) (P > 0.05). The 6-8 cell embryogenesis rate was significantly higher in the 3 → 2PN group (91.1%) as compared to the 2PN group (85.6%) (P < 0.05), but had no statistical difference compared with the 3PN group (95.0%) (P > 0.05). Total blastulation rate was significantly higher in the 2PN group (58.8%) as compared to the 3PN group (21.5%) (P < 0.01), and in the 3 → 2PN group as compared to the 3PN group (5.6%) (P < 0.01). Also D5 blastulation rate was significantly higher in the 2PN group (53.7%) as compared to the 3 → 2PN group (8.9%) (P < 0.01), and in the 3 → 2PN group as compared to the 3PN group (1.9%) (P < 0.01). In 3 → 2PN zygotes, the first cleavage mode is mainly 2 cells which is significantly higher than that in 3PN zygotes. Compared with 3PN zygotes, the embryo developmental potential of 3 → 2PN zygotes is improved, but still is lower than that in 2PN zygotes.

Expression of CD11c+HLA-DR+dendritic cells and related cytokines in the follicular fluid might be related to pathogenesis of ovarian hyperstimulation syndrome.

OBJECTIVE: To explore the expressions of CD11c+HLA-DR+dentritic cells in the follicular fluid of patients with OHSS and their significances. SUBJECTS: 100 individuals. TREATMENT: embryos were observed. The distribution of dentritic cells in follicular fluid and the levels of IL-10, IL-12, IL-18 and IL-23 in follicular fluid were detected. METHODS: There were ovarian hyperstimulation syndrome (OHSS) group and control group in this study. The OHSS group consisted of 50 patients with OHSS and the control group consisted of 50 patients who underwent in vitro fertilization-embryo transfer (IVF-ET) only due to male factors. The statuses of embryos were compared between the two groups. The distribution of dentritic cells in follicular fluid was determined with flow cytometry, and the levels of IL-10, IL-12, IL-18 and IL-23 in follicular fluid were detected with enzyme-linked immunosorbent assay (ELISA) in all patients. RESULTS: The two-pronuclear (2PN) fertility rate, high-quality embryo rate and available embryo rate were all significantly lower in OHSS group than in control group (all P<0.05). The number of CD11c+HLA-DR+dentritic cells (P<0.05) and the levels of IL-10, IL-12, IL-18 and IL-23 were all significantly higher in OHSS group than in control group (all P<0.01). CONCLUSION: The follicular fluid of the patients with OHSS is in an inflammatory status, the inflammatory status may be involved in OHSS and the microenvironment of follicular fluid may affects oocyte quality and embryo development.

Expression and potential roles of HLA-G in human spermatogenesis and early embryonic development.

As one of the non-classical major histocompatibility complex(MHC)-1 antigens, Human Leukocyte Antigen G (HLA-G), has been suggested as a prognostic marker to identify the embryo developmental potential. In the present study, we investigated the potential roles of HLA-G in human spermatogenesis and early embryonic development. Quantitative real-time PCR analysis revealed that HLA-G's expression was increased with increased Johnsen score in testicular tissues. There was no significant difference in HLA-G mRNA expression between testicular tissues with Johnsen score of 8-9 and normal sperm from ejaculated semen. HLA-G mRNA expression was detected in human zygotes, embryos and blastocysts but not in unfertilized oocytes. In testicular tissues where sperm was obtained by testicular sperm extraction (Johnsen score was 8 to 9), there were no correlations between HLA-G mRNA expression and fertilization, cleavage and high-quality embryo rates. At 48-72 h post-fertilization, HLA-G expression was higher in fast growing embryos. HLA-G specific siRNA injection into zygotes not only slowed down embryonic cleavage rate at 48 h post-fertilization, but also down-regulated the expression of embryo metabolism related gene (SLC2A1) and cell cycle-regulated gene (CCND2). Taken together, our findings suggested that HLA-G plays significant roles in human spermatogenesis and early embryonic development.

Pregnancy outcomes of day 5 embryo transfer in patients at high risk of developing ovarian hyperstimulation syndrome and analysis of factors affecting blastocyst formation.

OBJECTIVES: To investigate the effects of day 5 embryo transfer (D5ET) compared with day 3 embryo transfer (D3ET) in patients at high risk of developing ovarian hyperstimulation syndrome (OHSS); to analyse factors affecting blastocyst formation. METHODS: Patients at high risk of developing OHSS underwent either D3ET or D5ET. RESULTS: A total of 253 patients received D3ET; 263 received D5ET. The number of embryos transferred was lower in the D5ET group than in the D3ET group. There were no between-group differences in pregnancy or live birth rates. Implantation rate was higher, and multifetation rate lower, in the D5ET group compared with the D3ET group. In addition, the incidence of moderate or severe OHSS was lower in the D5ET group than in the D3ET group. The woman's age, gonadotrophin dosage and insemination method were associated with the quality of blastocyst formation. CONCLUSIONS: In patients with a high risk of developing OHSS, compared with D3ET, D5ET decreased the multifetation rate and the incidence of moderate or severe OHSS, but did not affect the pregnancy or live birth rate. Women of a younger age, who have had an appropriate gonadotrophin dose and insemination by in vitro fertilization, are suitable candidates for blastocyst transfer.

Effects of human cumulus cells on in vitro fertilization outcomes and its significance in short-term insemination.

OBJECTIVE: To observe the effects of cumulus cells on in vitro fertilization. STUDY DESIGN: Oocytes were retrieved from 47 patients (> 10/patient) who underwent short-term insemination from August 2009 to June 2010. The oocytes from each patient were divided into a cumulus cell-free group (cumulus cells were removed from the incubation medium 4 hours after coincubation of male and female gametes) with 389 oocytes and a cumulus cell group (cumulus cells were retained with the gametes until fertilization was evaluated 16-18 hours after co-incubation) with 402 oocytes. RESULTS: Polyspermic fertilization was 0.96 +/- 1.14 in the cumulus cell-free group and 0.47 +/- 0.72 in the cumulus cell group with p < 0.05. There were no significant differences in normal fertilization (5.96 g 1.73 vs. 6.55 +/- 3.72), 1PN fertilization (0.06 +/- 0.25 vs. 0.09 +/- 0.28), fertilization failure (1.34 +/- 1.17 vs. 1.45 +/- 1.84), cleavage (6.06 +/- 2.04 vs. 6.51 +/- 3.94), high-quality embryo (3.94 +/- 1.79 vs. 4.74 +/- 3.45) and usable embryo (5.06 +/- 1.86 vs. 5.68 +/- 3.98) between cumulus cell-free group and cumulus cell group, all with p > 0.05. CONCLUSION: In our study short-term insemination (4 hours) causes a statistical increase in polyspermic fertilization. In order to ensure correct oocyte fertilization and reduction of polyspermic fertilization, it is better to retain the cumulus cells for 16-18 hours.