Initial serum HCG levels are higher in pregnant women with a male fetus after fresh or frozen single blastocyst transfer: A retrospective cohort study.

OBJECTIVE: Substantial previous studies have almost reached an agreement on the gender effect on maternal serum human chorionic gonadotropin (MsHCG) in and after the late first trimester of pregnancy. However, there is little knowledge of the sex-related difference in MsHCG level at the preliminary stage of pregnancy. The purpose of this study is to reveal this difference in women after fresh or frozen single blastocyst transfer (SBT). MATERIALS AND METHODS: A total of 252 fresh SBT cycles and 1486 frozen-thawed SBT cycles collected between June 1, 2014 and May 30, 2017 were retrospectively analyzed in our center. Patients with MsHCG level ≥5 IU/L on day 11 after transfer, achieving a singleton intrauterine pregnancy and subsequent live birth were included. We compared MsHCG levels between women gave birth to a male neonate and those gave birth to a female one in fresh or frozen SBT cycles, respectively. RESULTS: A total of 136 neonates including 57 females and 79 males were born following fresh SBT. The male-female ratio was 1.39:1. The average MsHCG level of male fetuses was higher than that of female fetuses on day 11 after transfer (549.82 ± 253.24 IU/L versus 439.03 ± 198.41 IU/L, P < 0.05). Correspondingly, a total of 431 infants was born after frozen SBT, containing 188 females and 243 males. The male-female ratio was 1.29:1. Initial MsHCG level remained higher in women with a male neonate than the counterparts with a female neonate (894.43 ± 622.17 IU/L versus 758.05 ± 624.33 IU/L, P < 0.05). It was also found the pregnant women following frozen-thawed SBT exhibited higher initial MsHCG level than those following fresh SBT in whether male-bearing or female-bearing gestations. CONCLUSIONS: MsHCG levels are higher in pregnant women with a male fetus than those with a female one on day 11 after fresh or frozen SBT. A sex-specific response to the stress in the process of in vitro embryo culture was suggested.

[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.

Association of serum autoantibodies with pregnancy outcome of patients undergoing first IVF/ICSI treatment: A prospective cohort study.

The relevance of antiphospholipid (aPL), antinuclear (ANA) or antithyroid (ATA) antibodies in women undergoing in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) are controversial. The present study aims to investigate which autoantibodies are associated with the pregnancy outcome of patients undergoing first IVF/ICSI treatment. A total of 3763 IVF/ICSI patients were recruited from January to December 2015. Forty-five patients positive for aPL presenting adverse outcomes in their first cycle received low-dose aspirin treatment before the second transfer. Logistic regression analyses were performed to assess any association between autoantibodies and IVF/ICSI outcomes. The aCL-IgG was significantly associated with live birth rate (OR 0.58, 95% CI 0.36-0.96, p<0.05) and miscarriage rate (OR 2.04, 95% CI 1.23-3.40, p<0.01). The aCL-IgM was associated with miscarriage rate (OR 2.14, 95% CI 1.29-3.54, p<0.01). The aß2GPI-IgG was associated with implantation rate and clinical pregnancy rate (OR 0.61, 95% CI 0.24-0.96, p<0.05; OR 0.40, 95% CI 0.13-0.87, p<0.05, respectively). After the low-dose aspirin treatment, the live birth rate (37.0% vs. 19.1%, p<0.05) increased significantly in patients with positive for aPL. In contrary, the aß2GPI-IgM, ANA, anti-thyroglobulin (aTG) and anti-thyroperoxidase (aTPO) antibodies had no association with IVF/ICSI outcome. It is suggested that the presence of aCL-IgG, aCL-IgM and aß2GPI-IgG might exert a detrimental effect on IVF/ICSI outcomes. Low-dose aspirin treatment could be useful for patients positive for these antibodies. Therefore, it is suggested that these antibodies should be assessed prior to IVF/ICSI treatment.

Human chorionic gonadotropin potentially affects pregnancy outcome in women with recurrent implantation failure by regulating the homing preference of regulatory T cells.

PROBLEM: Human chorionic gonadotropin (hCG) and regulatory T cells (Tregs) have been suggested to play important roles during the initial stage of pregnancy. However, the clinical relevance and mechanism of the effects of hCG on Treg functions in women with recurrent implantation failure (RIF) remain to be elucidated. METHOD OF STUDY: Thirty-four RIF and twenty-three control women were included in the study. Endometrial and peripheral Tregs were analyzed by immunohistochemistry and flow cytometry, respectively. Tregs were generated from naïve CD4+ T cells by stimulation with anti-CD3/CD28 in the presence or absence of hCG, and the subsets were analyzed by flow cytometry, Western blotting, and qPCR. RESULTS: The percentages of endometrial FOXP3+ Tregs and peripheral CCR4+ FOXP3+ Tregs were significantly lower in the women with RIF than in the healthy controls. In addition, the percentages of CCR4+ FOXP3+ Tregs and TGF-ß-expressing FOXP3+ Tregs were increased following the stimulation of naïve CD4+ T cells with anti-CD3/CD28, and these increases were concomitant with AKT and ERK dephosphorylation. CONCLUSIONS: The results of this study provide novel evidence supporting a role of hCG in regulating the differentiation of peripheral FOXP3+ Tregs. The alterations of circulating Tregs may positively affect the pregnancy outcomes of patients with a history of RIF.

The pro-inflammatory and anti-inflammatory cytokine profile in peripheral blood of women with recurrent implantation failure.

Limited information is available on the balance state of pro- and anti-inflammatory cytokines in patients with recurrent implantation failure (RIF). This study assessed the pro- and anti-inflammatory cytokines in plasma of 34 patients with RIF, compared with those of 25 women with a successful pregnancy in the first IVF/intracytoplasmic sperm injection-embryo transfer (IVF/ICSI-ET) cycle. The IFN-γ, IL-1ß, IL-6 and IL-4 concentrations were higher, whereas the TGF-ß1 concentration was lower in the RIF group compared with the control group. Furthermore, the ratios of pro-inflammatory and anti-inflammatory cytokines IFN-γ/IL-4, IFN-γ/IL-10, IFN-γ/TGF-ß1, IL-6/IL-10, IL-6/TGF-ß1, IL-1ß/TGF-ß1 and TNF-α/TGF-ß1 were higher in the RIF group (all P < 0.01). The results suggested a shift toward a pro-inflammatory state in peripheral blood of the patients with RIF.

Decrease in fertilization and cleavage rates, but not in clinical outcomes for infertile men with AZF microdeletion of the Y chromosome.

This study aimed to explore whether the presence of a Y chromosome azoospermia factor (AZF) microdeletion confers any adverse effect on embryonic development and clinical outcomes after intracytoplasmic sperm injection (ICSI) treatment. Fifty-seven patients with AZF microdeletion were included in the present study and 114 oligozoospermia and azoospermia patients without AZF microdeletion were recruited as controls. Both AZF and control groups were further divided into subgroups based upon the methods of semen collection: the AZF-testicular sperm extraction subgroup (AZF-TESE, n = 14), the AZF-ejaculation subgroup (AZF-EJA, n = 43), the control-TESE subgroup (n = 28) and the control-EJA subgroup (n = 86). Clinical data were analyzed in the two groups and four subgroups respectively. A retrospective case-control study was performed. A significantly lower fertilization rate (69.27 versus 75.70%, P = 0.000) and cleavage rate (89.55 versus 94.39%, P = 0.000) was found in AZF group compared with the control group. Furthermore, in AZF-TESE subgroup, the fertilization rate (67.54 versus 74.25%, P = 0.037) and cleavage rate (88.96 versus 94.79%, P = 0.022) were significantly lower than in the control-TESE subgroup; similarly, the fertilization rate (69.85 versus 75.85%, P = 0.004) and cleavage rate (89.36 versus 94.26%, P = 0.002) in AZF-EJA subgroup were significantly lower than in the control-EJA subgroup; however, the fertilization rate and cleavage rate in AZF-TESE (control-TESE) subgroup was similar to that in the AZF-EJA (control-EJA) subgroup. The other clinical outcomes were comparable between four subgroups (P > 0.05). Therefore, sperm from patients with AZF microdeletion, obtained either by ejaculation or TESE, may have lower fertilization and cleavage rates, but seem to have comparable clinical outcomes to those from patients without AZF microdeletion.

Increased circulating Th1/Th2 ratios but not other lymphocyte subsets during controlled ovarian stimulation are linked to subsequent implantation failure after transfer of in vitro fertilized embryos.

PROBLEM: To investigate alterations of circulating lymphocyte subsets in women undergoing controlled ovarian stimulation (COS) and survey their relations with pregnancy outcome. METHOD OF STUDY: Fifty-one women were included in this study, of which 24 women were successfully pregnant in the index IVF cycle (the successful pregnancy group, sPG) and 27 women failed to get pregnant (the non-pregnancy group, nPG). Peripheral blood was collected at three time points during COS: on the day before the first GnRHa administration (GnRHa day), on the day before the first Gn administration (Gn day), and on the day before HCG administration (HCG day). The intracellular Th1(IFN-γ and TNF-α)/Th2(IL-10) ratios in circulating Th cells after in vitro phorbol-12-myristate-13-acetate stimulation, the NK cytotoxicity, and the percentage of T, B and NK cell subsets were assessed by flow cytometer. A third group consisting of patients with recurrent implantation failure (RIF group, n = 18) was also enrolled, and the intracellular Th1/Th2 ratios were assessed at HCG day. RESULTS: Intergroup comparison: The ratios of IFN-γ/IL-10 and TNF-α/IL-10 at HCG day were significantly higher (P < 0.05) in nPG than those in sPG, while no change at GnRHa day or Gn day. Higher (P < 0.05) ratios of IFN-γ/IL-10 and TNF-α/IL-10 at HCG day were also observed in RIF group compared to those in sPG. No significant change (P > 0.05) was found in other assessed lymphocyte subsets or in the concentrations of progesterone, estradiol, luteinizing hormone, or follicle-stimulating hormone between the sPG and nPG at the three time points. Intragroup comparison: The ratios of IFN-γ/IL-10 and TNF-α/IL-10 at HCG day were significantly (P < 0.05) lower than those at GnRHa day in sPG, but not in nPG. No significant (P > 0.05) difference was found in other assessed parameters or at other time points. CONCLUSIONS: The ratios of Th1/Th2 were fluctuated during COS, and they might affect the pregnancy outcome of IVF. The circulating NK cytotoxicity and percentage of lymphocyte subsets were relatively stable during the COS.

Laser-assisted hatching improves clinical outcomes of vitrified-warmed blastocysts developed from low-grade cleavage-stage embryos: a prospective randomized study.

The aim of this study was to evaluate the effects of quarter zona-pellucida (ZP) opening by laser-assisted hatching (QLAH) on the clinical outcomes following transfer of vitrified-warmed blastocysts developed from low-grade cleavage-stage embryos in patients with all high-grade and fair-grade cleavage-stage embryos transferred without achieving pregnancy. Patients were randomized into two groups: QLAH (n=101) and control (n=102). The implantation and clinical pregnancy rates were significantly higher in the QLAH group compared with the control group (P=0.021 and P=0.034, respectively). The live birth rate of the QLAH group was also higher, although not significantly. When the clinical outcomes according to the day of blastocyst vitrification were compared between the groups, the implantation, clinical pregnancy and live birth rates of the QLAH group were significantly higher (P<0.05) than those of the control group for day 6 blastocysts, but not for day 5 or day 5/day 6 blastocysts. These results suggest that QLAH improves the clinical outcomes of vitrified-warmed blastocysts, especially of day 6 vitrified blastocysts, developed from low-grade cleavage-stage embryos.

Comprehensive analysis of peripheral blood lymphocytes in 76 women with recurrent miscarriage before and after lymphocyte immunotherapy.

PROBLEM: The efficacy of lymphocyte immunotherapy (LIT) for unexplained recurrent miscarriage (uRM) remains indefinite. The objective of the present study is to comprehensively evaluate the alterations in the proportions and functions of peripheral blood lymphocytes in patients with uRM before and after LIT. METHOD OF STUDY: Seventy-six women with uRM were included in the present study. Immunophenotype of peripheral blood lymphocytes (76 patients), NK cytotoxicity (62 patients) and Th1 (IFN-γ and TNF-α)/Th2 (IL-10) ratios (73 patients) were assessed during the luteal phase before LIT and after the third LIT by the flow cytometer. RESULTS: To date, 29 patients had already delivered or been pregnant more than 20 weeks (successful pregnancy group) and 5 patients experienced subsequent abortion (abortion group). The percentages of CD3(+) T cell, CD3(+) CD4(+) T cell and IL-10-producing Th1 cell were significantly increased, whereas the percentages of CD3(+) HLA-DR(+) T cell, CD56(+) CD3(-) NK cell and CD56(dim ) CD16(+) NK cell, the NK cytotoxicity and the ratios of Th1/Th2 were significantly decreased after LIT in the total patients and in the successful pregnant group. The percentages of CD56(+ ) CD3(-) NK cell and IFN-γ-producing cell, and the ratio of IFN-γ/IL-10 were significantly lower after LIT in the successful pregnant group compared to those in the abortion group. CONCLUSIONS: LIT alters the proportions and functions of most peripheral blood lymphocyte subsets. Some of these alterations may be beneficial for pregnancy maintenance, whereas some may be potential markers for predicting subsequent abortion.

Molecular characterization of heat shock protein 70 genes in the liver of three warm freshwater fishes with differential tolerance to microcystin-LR.

Heat shock protein 70 (HSP70) protect cell from oxidative stress by preventing the irreversible loss of vital proteins and facilitating their subsequent regeneration. Silver carp (Hypophthalmichthys molitrix), grass carp (Ctenopharyngodon idellus), and Nile tilapia (Oreochromis nilotica) are three warm freshwater fishes with differential tolerance to microcystin-LR (MC-LR). Full-length cDNAs encoding the HSP70 were cloned from the livers of the three fishes. The HSP70 cDNAs of silver carp, grass carp, and Nile tilapia were 2356, 2348, and 2242 bp in length and contained an open-reading frame of 1950 bp (encoding a polypeptide of 649 amino acids), 1950 bp (649 amino acids), and 1917 bp (638 amino acids), respectively. Like mammalian HSP70, the HSP70 of the three fish was also composed of an ATPase domain from residues 1 to 383 (44 kDa), substrate peptide binding domain from residues 384 to 544 (18 kDa), and a C-terminus domain from residues 545 to 649 (10 kDa). The relatively high conservation of HSP70 sequences among different vertebrates is consistent with their important role in fundamental cellular processes. Using beta-actin as an external control, RT-PCR within the exponential phase was conducted to determine the constitutive and inducible expression level of HSP70 gene among the three fishes (6-12 g) intraperitoneally injected with MC-LR (50 µg kg(-1) body weight). Both constitutive and inducible liver mRNA levels of the fish HSP70 genes showed positive relationships with their tolerance to MC-LR: highest in Nile tilapia, followed by silver carp, and lowest in grass carp. The differential expression pattern of liver HSP70 genes in the three fish indicated a potential role of HSP70 in the detoxification process of MC-LR.

Gene structure, recombinant expression and functional characterization of grass carp leptin.

Leptin is an important hormone for the regulation of food intake, energy expenditure and reproduction in mammals, but information regarding its role in teleosts remains scant. In the present study, the gene structures of grass carp (Ctenopharyngodon idellus) and silver carp (Hypophthalmichthys molitrix) leptins were characterized. Recombinant grass carp leptin (rgc-LEP) was expressed in Escherichia coli and purified, and identified by mass spectrometric analysis. A strong anorexic effect on food intake was observed in grass carp on the first day after intraperitoneal (IP) injection of rgc-LEP, but not during the following days. Body weight of the leptin group (LEP group) and the pair-fed group (PF group) showed no difference throughout the experimental period. The acute and chronic effects on the expression of key genes correlating to food intake, energy expenditure, lipid metabolism and digestion were further characterized by real-time PCR. Accordingly, the mRNA levels of neuropeptide Y (NPY), Stearoyl-CoA desaturase 1 (SCD1) and lipoprotein lipase (LPL) were significantly reduced whereas the mRNA levels of uncoupling protein 2 (UCP2), bile salt-activated lipase (BSAL) and fatty acid elongase (ELO) were significantly elevated on the first day after injection. No effect on the expression of these genes (except LPL) was observed on day 13. In contrast to the down-regulation by exogenous leptin in mammals, the mRNA level of grass carp leptin was elevated 5.76-fold on the first day after rgc-LEP treatment. Our results suggest that leptin has an acute effect on the regulation of food intake, energy expenditure and lipid metabolism in grass carp, but the effect can be rapidly counteracted through mechanisms that are currently unknown.

Transcriptional responses of alpha- and rho-class glutathione S-transferase genes in the liver of three freshwater fishes intraperitoneally injected with microcystin-LR: relationship of inducible expression and tolerance.

Rho-class glutathione S-transferase (GST) is found only in teleost fish with no homologues in mammals. Silver carp (Hypophthalmichthys molitrix), grass carp (Ctenopharyngodon idellus), and Nile tilapia (Oreochromis nilotica) are three warm freshwater fishes with differential tolerance to microcystin-LR (MC-LR): Nile tilapia has a little higher tolerance than silver carp, but both have much higher tolerance than grass carp. Full-length cDNAs encoding the rho-class GST were cloned and sequenced from the livers of the three fishes. The silver carp, grass carp, and Nile tilapia rho-class GST cDNAs were 1078, 1104, and 904 bp in length, respectively, and all contained an open-reading frame (ORF) of 681 bp (encoding a polypeptide of 226 amino acids). Using beta-actin as an external control, semiquantitative RT-PCR was conducted to determine the constitutive and inducible expression level of alpha- and rho-class GST genes among the three fishes (6-12 g) intraperitoneally injected with MC-LR (50 mug kg(-1) body weight). Liver mRNA expression levels of alpha-class GST gene were found to be higher than those of rho-class GST gene in both exposed and control fish of silver carp and grass carp, whereas liver mRNA expression levels of rho-class GST gene were higher than those of alpha -class GST gene in both exposed and control fish of Nile tilapia. Increased liver transcription of GST isoforms was detected at 8-h postinjection of MC-LR in silver carp (alpha- and rho-class GST) and Nile tilapia (rho-class GST), and at 24-h postinjection in silver carp (alpha-class GST) and Nile tilapia (alpha-class GST), but an increase in liver transcription neither of alpha-class GST nor of rho-class GST was detected in grass carp at either 8-h or 24-h postinjection. The inducible expression of the liver GST gene showed a close relationship with their tolerance to MC-LR: high-resistant fish (phytoplanktivorous silver carp and Nile tilapia) had inducible liver expression of either alpha- or rho-class GST, and the high-sensitive fish (herbivorous grass carp) had no inducible liver expression of either one. We suggest that inducible expression (instead of constitutive expression) of the liver GST gene should play an important role in the tolerance to microcystin exposure, and that in addition to alpha-class GST, high-liver expression of rho-class GST gene might have facilitated the evolutionary radiation of teleost fish.