Novel PLCZ1 mutation caused polyspermy during in vitro fertilization.

ABSTRACT: Failure of oocyte activation, including polyspermy and defects in pronuclear (PN) formation, triggers early embryonic developmental arrest. Many studies have shown that phospholipase C zeta 1 ( PLCZ1 ) mutations cause failure of PN formation following intracytoplasmic sperm injection (ICSI); however, whether PLCZ1 mutation is associated with polyspermy during in vitro fertilization (IVF) remains unknown. Whole-exome sequencing (WES) was performed to identify candidate mutations in couples with primary infertility. Sanger sequencing was used to validate the mutations. Multiple PLCZ1 -mutated sperm were injected into human and mouse oocytes to explore whether PN formation was induced. Assisted oocyte activation (AOA) after ICSI was performed to overcome the failure of oocyte activation. We identified three PLCZ1 mutations in three patients who experienced polyspermy during IVF cycles, including a novel missense mutation c.1154C>T, p.R385Q. PN formation failure was observed during the ICSI cycle. However, injection of multiple PLCZ1 -mutated sperm induced PN formation, suggesting that the Ca 2+ oscillations induced by the sperm exceeded the necessary threshold for PN formation. AOA after ICSI enabled normal fertilization, and all patients achieved successful pregnancies. These findings expand the mutational spectrum of PLCZ1 and suggest an important role for PLCZ1 in terms of blocking polyspermy. Furthermore, this study may benefit genetic diagnoses in cases of abnormal fertilization and provide potential appropriate therapeutic measures for these patients with sperm-derived polyspermy.

Cordycepin delays postovulatory aging of oocytes through inhibition of maternal mRNAs degradation via DCP1A polyadenylation suppression.

Postovulatory aging leads to the decline in oocyte quality and subsequent impairment of embryonic development, thereby reducing the success rate of assisted reproductive technology (ART). Potential preventative strategies preventing oocytes from aging and the associated underlying mechanisms warrant investigation. In this study, we identified that cordycepin, a natural nucleoside analogue, promoted the quality of oocytes aging in vitro, as indicated by reduced oocyte fragmentation, improved spindle/chromosomes morphology and mitochondrial function, as well as increased embryonic developmental competence. Proteomic and RNA sequencing analyses revealed that cordycepin inhibited the degradation of several crucial maternal proteins and mRNAs caused by aging. Strikingly, cordycepin was found to suppress the elevation of DCP1A protein by inhibiting polyadenylation during postovulatory aging, consequently impeding the decapping of maternal mRNAs. In humans, the increased degradation of DCP1A and total mRNA during postovulatory aging was also inhibited by cordycepin. Collectively, our findings demonstrate that cordycepin prevents postovulatory aging of mammalian oocytes by inhibition of maternal mRNAs degradation via suppressing polyadenylation of DCP1A mRNA, thereby promoting oocyte developmental competence.

Growth hormone reduces aneuploidy and improves oocytes quality by JAK2-MAPK3/1 pathway in aged mice.

BACKGROUND: The global delay in women's reproductive age has raised concerns about age-related infertility. The decline in oocyte quality is a limiting factor of female fertility, yet there are currently no strategies to preserve oocyte quality in aged women. Here, we investigated the effects of growth hormone (GH) supplementation on aneuploidy of aged oocytes. METHODS: For the in vivo experiments, the aged mice (8-month-old) were intraperitoneally injected with GH daily for 8 weeks. For the in vitro experiments, germinal vesicle oocytes from aged mice were treated with GH during oocyte maturation. The impacts of GH on ovarian reserve before superovulation was evaluated. Oocytes were retrieved to assess oocyte quality, aneuploidy and developmental potential characteristics. Quantitative proteomics analysis was applied to investigate the potential targets of GH in aged oocytes. RESULTS: In this study, we demonstrated that GH supplementation in vivo not only alleviated the decline in oocyte number caused by aging, but also improved the quality and developmental potential of aged oocytes. Strikingly, we discovered that GH supplementation reduced aneuploidy in aged oocytes. Mechanically, in addition to improving mitochondrial function, our proteomic analysis indicated that the MAPK3/1 pathway may be involved in the reduction in aneuploidy of aged oocytes, as confirmed both in vivo and in vitro. In addition, JAK2 may also act as a mediator in how GH regulates MAPK3/1. CONCLUSIONS: In conclusion, our research reveals that GH supplementation protects oocytes against aging-related aneuploidy and enhances the quality of aged oocytes, which has clinical significance for aged women undergoing assisted reproduction technology.

Patients with MMAF induced by novel biallelic CFAP43 mutations have good fertility outcomes after intracytoplasmic sperm injection.

As a specific type of asthenoteratozoospermia, multiple morphological abnormalities of the sperm flagella (MMAF) is characterized by composite abnormalities, including absent, short, coiled, angulation, and irregular-caliber flagella. Mutations in cilia- and flagella-associated protein 43 ( CFAP43 ) are one of the main causative factors of MMAF established to date. To identify whether there are other CFAP43 mutations related to MMAF and to determine the clinical outcomes of assisted reproductive technology for patients with MMAF harboring different mutations, we recruited and screened 30 MMAF-affected Chinese men using a 22-gene next-generation sequencing panel. After systematic analysis, seven mutations in CFAP43 , including five novel mutations and two previously reported mutations, were identified from four families and related to MMAF in an autosomal recessive pattern. Papanicolaou staining, immunofluorescence, and electronic microscopy further clarified the semen characteristics and abnormal sperm morphologies, including disorganized axonemal and peri-axonemal structures, of the CFAP43 -deficient men. The female partners of two patients were pregnant after undergoing assisted reproductive technology through intracytoplasmic sperm injection, and one of them successfully gave birth to a healthy boy. This study significantly expands the mutant spectrum of CFAP43 , and together with the available information regarding male infertility and MMAF, provides new information for the genetic diagnosis and counseling of MMAF in the future.

Comparison of embryo implantation potential between time-lapse incubators and standard incubators: a randomized controlled study.

RESEARCH QUESTION: What are the potential clinical benefits of embryo culture and assessment in a time-lapse incubator compared with a standard incubator using static assessment? DESIGN: This large multicentre, single-blinded, randomized controlled study included 1224 participants randomly assigned (1:1) to the time-lapse or standard incubator group. In all patients one or two embryos were transferred on day 3. The primary outcome was the implantation rate in the first embryo transfer cycle. Secondary outcomes included the cumulative implantation rate, live birth rate in the first embryo transfer cycle and cumulative live birth rate. RESULTS: Among 1224 participants recruited, 1182 underwent embryo transfer. The number of successfully implanted embryos in the first transfer cycle was significantly higher in the time-lapse incubator group (time-lapse group: 52.35%, standard incubator group: 47.11%, P = 0.014). The implantation rate in the first embryo transfer cycle was still significantly higher in the time-lapse group than the standard incubator group after adjusting for age, body mass index, medical centre and embryo status (relative risk 1.11, 95% confidence interval 1.02-1.20, P = 0.020). However, the cumulative implantation rate, live birth rate in the first embryo transfer cycle and cumulative live birth rate were not statistically different between the groups. CONCLUSIONS: The implantation rate in the first embryo transfer cycle was significantly improved in the time-lapse group, but the effect of the time-lapse system on the cumulative implantation rate or cumulative live birth rate was not significant. The embryo assessment method offered by time-lapse systems rather than an undisturbed environment may play an important role in improving the implantation rate in the first embryo transfer cycle. These results are only applicable to young patients.

Early Spontaneous Abortion in Fresh- and Frozen-Embryo Transfers: An Analysis of Over 35,000 Transfer Cycles.

Background: The aim of this study was to explore the risk factors for early spontaneous abortion (ESA) in fresh- and frozen-embryo transfers. Methods: This retrospective cohort study comprised a total of 35,076 patients, including 15,557 women in the fresh-embryo transfer group and 19,519 women in the frozen-embryo transfer group from January 2016 to December 2020. The primary outcome of this study was ESA, which we defined as the termination of embryonic development before 12 weeks of pregnancy (i.e., an early abortion after artificial multi-fetal pregnancy reduction was excluded). Results: In the 35,076 ART transfer cycles, the incidence of ESA was 5.77% (2023/35,076), and the incidence rates for ESA in fresh and frozen cycles were 4.93% (767 of 15,557) and 6.43% (1,256 of 19,519), respectively. Using a multivariate logistic regression analysis model, maternal age, body mass index (BMI), and number of embryos transferred were independent predictors for ESA. In addition, frozen-thawed transfer was a risk factor for ESA as compared with fresh transfer (OR = 1.207; 95% CI, 1.094-1.331; P = 0.000), blastocyst transfer was risk factor for ESA as compared with cleavage transfer (OR =1.373; 95% CI, 1.186-1.591; P = 0.000 in the total group; OR = 1.291; 95% CI, 1.111-1.499; P = 0.001 in the frozen-transfer group), and unexplained infertility was a protective factor for ESA only in the frozen group (OR = 0.746; 95% CI, 0.565-0.984; P = 0.038). Conclusions: Maternal age, BMI, number of embryos transferred, and frozen-thawed transfer were independent risk factors for ESA in assisted reproductive technology treatment cycles.

Impact of growth hormone supplementation on improving oocyte competence in unexplained poor embryonic development patients of various ages.

OBJECTIVES: To evaluate the effectiveness of Growth hormone (GH) co-treatment during in vitro fertilization (IVF) cycles in women of different ages who manifest unexplained poor embryonic development. METHOD: This cohort study included a total of 2647 patients with unexplained poor embryonic development in their previous IVF procedures: 872 women received GH co-treatment and 1775 untreated women served as a control group. Patients were divided into 6 groups according to treatment and stratified by age (<35 years of age, A-GH group and A-control group; 35-40 years, B-GH group and B-control group; and ≥40 years, C-GH group and C-control group). The primary outcome was the oocyte-cleavage rate and the clinical pregnancy rate (CPR). RESULTS: The oocyte-cleavage rates among the three age groups were significantly higher in the GH group compared to the same-aged control group. In both group A and group B, there was no significant difference in clinical pregnancy rate between the GH group and controls. However, in patients ≥40 years of age, the clinical pregnancy rate in the GH group was significantly higher than in the control group (31.8% vs. 13.7%, p = 0.019). In the three age groups, there was no significant difference in the live birth rate between the GH group and controls. In the multivariate logistic regression analysis model, in both group A and group B, the number of cleaved embryos was independent predictors for CPR (OR = 1.464, 95% CI: 1.311-1.634; respectively, OR = 1.336, 95% CI: 1.126-1.586); Besides, in both group B and group C, age was independent predictors for CPR (OR = 0.657, 95%CI: 0.555-0.778; respectively, OR = 0.622, 95%CI: 0.391-0.989). However, only in group C, supplementation GH increased CPR as compared with not supplementation GH (OR = 2.339, 95%CI: 1.182-6.670). CONCLUSIONS: For patients with unexplained poor embryonic development, supplementation with GH increased the oocyte-cleavage rates in all three age groups, and the clinical pregnancy rate gradually improved commensurate with increasing age. There was no difference in the clinical pregnancy rate in group A and group B, but group C improved significantly. Therefore, compared with patients under 40 years of age, patients ≥40 may benefit more from GH supplementation.

Normal pancreatic volume assessment using abdominal computed tomography volumetry.

ABSTRACT: To determine the normal range of pancreatic volume (PV) in Chinese adults using computed tomography (CT) volumetry. To assess the relationships of PV with patient demographics and clinical parameters. To analyze the degree of correlation between PV values determined by manual segmentation and those calculated by formulas.A total of 240 adults (120 women, 120 men) between the ages of 18 and 79 years were reviewed. There were 6 groups of patients, with 40 patients in each decade regarding age. PV was segmented manually on computed tomography images slice by slice for each patient, and 2 formulas were used to calculate PV∗ and PV#.The mean PV was 77.44 ±â€Š19.11 cm3 (range from 28.55-138.15 cm3). PV was significantly correlated with height (r = 0.427, P < .001), weight (r = 0.525, P < .001), body mass index (r = 0.377, P < .001), the width of the first lumbar vertebral body (r = 0.166, P = .01), the transverse abdominal diameter (r = 0.455, P < .001), and the sagittal abdominal diameter (r = 0.456, P < .001). There was a negative correlation between PV and age (r = -0.209, P = .001). The correlation coefficients between PV and PV∗ and PV# were 0.676 and 0.376, respectively, with both P < .001.PV associated with patient demographics and clinical parameters. A more accurate and simpler formula should be used in the future to calculate and monitor changes in PV.

Prolonged interval time between blastocyst biopsy and vitrification compromised the outcomes in preimplantation genetic testing.

This study aimed to evaluate to what extent the different interval times between trophectoderm (TE) biopsy and vitrification influence the clinical outcomes in preimplantation genetic testing (PGT) cycles. Patients who underwent frozen embryo transfer (FET) after PGT between 2015 and 2019 were recruited. In total, 297 cycles with single day 5 euploid blastocyst transfer were included. These cycles were divided into three groups according to the interval times: <1 h group, 1-2 h group, and ≥2 h group. Blastocyst survival, clinical pregnancy, miscarriage, and ongoing pregnancy rates were compared. The results showed that, in PGT-SR cycles, survival rate in the ≥2 h group (96.72%) was significantly lower than in the <1 h group (100%, P = 0.047). The clinical pregnancy rate in the ≥2 h group was 55.93%, significantly lower than in the <1 h group (74.26%, P = 0.017). The ongoing pregnancy rates in the 1-2 h group and the ≥2 h group were 48.28% and 47.46%, respectively, significantly lower than that in the <1 h group (67.33%, P < 0.05). The miscarriage rate in the 1-2 h group was 18.42%, significantly higher than that in the <1 h group (5.33%, P = 0.027). In PGT-A cycles, the clinical pregnancy and ongoing pregnancy rates in the <1 h group were 67.44% and 53.49%, respectively, higher than that in the 1-2 h group (52.94%, 47.06%, P > 0.05) and the ≥2 h group (52.63%, 36.84%, P > 0.05). In conclusion, vitrification of blastocysts beyond 1 h after biopsy significantly influences embryo survival and clinical outcomes and is therefore not recommended.

[An analysis of donor semen quality in Chongqing Human Sperm Bank].

OBJECTIVE: To evaluate the quality of the donor semen in Chongqing Human Sperm Bank and the influence of age on semen parameters. METHODS: We collected semen samples from 899 donors in Chongqing Human Sperm Bank and divided them into five groups according to the age of the semen donors: 22－25, 26－30, 31－35, 36－40, and >40 years old. Using the Makler Counting Chamber, we measured the semen volume, percentage of progressively motile sperm (PMS), total motile sperm, sperm concentration, total sperm count per ejaculate, and percentage of morphologically normal sperm (MNS). Then, we compared the semen parameters obtained with the fifth percentile and median reference values published in the WHO Laboratory Manual for the Examination and Processing of Human Semen-5th Ed (WHO 5th Ed) and among different age groups using the Kruskall-Wallis H test. RESULTS: The semen volume (1.8 ml), sperm concentration (25.0 × 106/ml), total sperm count (100.7 × 106/ejaculate) and MNS (4.3%) in the semen samples of the 899 donors were obviously higher than the fifth percentile values published in the WHO 5th Ed, and so were the first three parameters (4.0 ml, 88.0 × 106/ml, and 333.7 × 106/ejaculate) than the WHO median reference values. PMS (31.0%) and total motile (38.0%) were lower than the WHO fifth percentile values and so was MNS (11.6%) than the WHO median reference value. PMS (55.0%) and total motile sperm (61.0%), however, were coincident with the median reference values of WHO 5th Ed. Statistically significant differences were observed among the 22－25, 26－30, 31－35, 36－40 and >40 years old groups in perm concentration (88.0 ï¼»1.0－270.0ï¼½ vs 96.0 ï¼»5.0－335.0ï¼½ vs 100.0 ï¼»3.0－200.0ï¼½ vs 105 ï¼»15.0－225.0ï¼½ vs 90.0 ï¼»22.0－159.0ï¼½ × 106/ml, P < 0.05), but not in the semen volume, PMS, total sperm motility, total sperm count or MNS (P > 0.05). CONCLUSIONS: The donor semen in Chongqing Human Sperm Bank is generally of high quality. Sperm concentration significantly increases with age but decreases in men aged >40 years.

[Bacterial culture of donor semen: Analysis of results].

OBJECTIVE: To investigate bacterial infection and the distribution of different bacterial species in the donor semen and the influence of different bacterial counts on semen quality. METHODS: Bacterial colonies in the semen samples from 1 126 donors were counted with the Synbiosis Protocol 3 Automatic Colony Counter and the bacterial species with a colony count ≥104 cfu/ml identified with the VITEK2 Compact Automatic Biochemical Analyzer. The Makler Sperm Counting Board was used to examine the semen quality of the semen samples with a colony count = 0 cfu/ml (n = 22, group A), those with a colony count <104 cfu/ml (n = 22, group B) and those with a colony count ≥104 cfu/ml (n = 22, group C). Univariate analysis was employed for comparison of semen quality among different groups. RESULTS: Among the 1 126 donor semen samples cultured, 5 (0.44%) showed mixed bacterial contamination and 993 (88.58%) showed none but with growth of a certain species of bacteria, 2.22% (22/993) with a colony count ≥104 cfu/ml, mainly including Streptococcus bovis, tiny bacilli, Staphylococcus epidermis, and Staphylococcus aureus, among which gram-positive and gram-negative bacteria accounted for 95.45% (21/22) and 4.54% (1/22), respectively. Compared with group A, groups B and C manifested significantly reduced total sperm count (ï¼»567.5 ± 327.6ï¼½ vs ï¼»421.9 ± 155.9ï¼½ and ï¼»389.9 ± 110.6ï¼½ × 106 per ejaculate, P <0.05) and percentage of progressively motile sperm (ï¼»65.0 ± 6.5ï¼½ vs ï¼»61.0 ± 3.5ï¼½ and ï¼»61.6 ± 4.3ï¼½ %, P <0.05). There were no statistically significant differences among the three groups in the semen liquefaction time, semen pH value, total sperm motility or percentage of morphologically normal sperm (P > 0.05). Of the 284 randomly selected semen samples, 34 (11.97%) were found positive for Ureaplasma urealyticum (UU) and no significant difference was observed in the semen quality between the UU-positive and UU-negative samples (P> 0.05). CONCLUSIONS: The bacteria-positive rate is high in the donor semen and the bacterial species are varied, mainly including gram-positive bacteria. Semen quality is reduced with the increased number of bacterial colonies.

Shortened equilibration time can compromise clinical outcomes in human embryo vitrification.

Vitrification is an important way to cryopreserve human embryos and the recommended time of embryo exposure to the vitrification solution is 1 min. However, practically speaking, the duration of embryos exposure to equilibration solution can vary from 5 to 15 min. The purpose of this study was to investigate the effect of different equilibration times on the outcomes of frozen-thawed embryo transfer cycles. The data were collected from our medical records from January 2012 to June 2013 and a total of 517 cycles were included. These cycles were divided into four groups according to the equilibration time: (i) 5-6 min; (ii) 7-8 min; (iii) 9-10 min and (iv) 11-12 min. The results show that there were no differences in terms of survival rate and fully intact embryo rate among the four groups. However, lower clinical pregnancy, embryo implantation and live birth rates were observed in the 5-6 min exposure group (54.6%, 31.9% and 48.2%, respectively) compared with the three other groups. The corresponding rates in the 9-10 min group (73.5%, 47.6% and 64.7%) were the highest. This study indicated that different equilibration times influenced the clinical outcomes of human embryo vitrification and vitrification with shortened equilibration time compromised the clinical outcomes. Appropriate prolongation of the equilibrium time would probably improve the clinical outcomes.

Abnormally cleaving embryos are able to produce live births: a time-lapse study.

PURPOSE: This study investigated the prevalence of abnormally cleaved embryos and determined which types of abnormally cleaved embryos (1-3c, 2-4c, 3-5c, 4-6c), might be suitable for transfer based on live birth data. METHODS: One hundred seventy-one women (whose transferred embryos were confirmed to be either fully implanted or fully unimplanted) provided 1256 embryos, which were analyzed. RESULTS: Of these embryos, 320 embryos were transferred, of these transferred embryos, 291 embryos were normal and 29 embryos were abnormal, which five embryos were not analyzed because each one was presented one abnormal cleavage type. These 24 embryos were divided into four groups. Inclusion criteria were as follows: women under 37 years of age undergoing first fresh in vitro fertilization (IVF) treatment with a basal antral follicle count of 5-15, body mass index (BMI) of 18-25 kg/m(2), number of retrieved oocytes between 5 and 20, and tubal factors as the cause of infertility. Time-lapse imaging analysis software was used to compare temporal parameters of normal cleavage and abnormal cleavage groups (there were four abnormal groups, based on the prevalence of abnormal cleavage embryos). Cleavage times were analyzed before the abnormal cleavage occurred, and time intervals were analyzed after the abnormal cleavage based upon the types of abnormal cleavage. In addition, the time intervals of t4-t3 and t8-t5 were also analyzed; corresponding time parameters were measured in the normal group as well. Implantation rate, clinical pregnancy rate, ongoing pregnancy rate, and live birth rate were also measured in the normally cleaved and abnormally cleaved embryos. The prevalence of abnormal cleavage was 15.92% (200/1256). T8-t5 was the most important parameter in the prediction of potential development (production of a live-born baby) of abnormally cleaving embryos. CONCLUSIONS: Abnormally cleaving embryos were able to produced live births with T8-t5 the best parameter to predict the developmental potential of abnormally cleaving embryos.

[Protective effect of Angelica sinensis polysaccharides on subacute renal damages induced by D-galactose in mice and its mechanism].

To explore the protective effect of Angelica sinensis polysaccharides(ASP) on subacute renal damages induced by D-galactose in mice and its mechanism. Male C57BL/6J mice were randomly divided into 3 groups, with 10 mice in each group. The D-galactose model group was subcutaneously injected with D-galactose (120 mg x kg(-1)), qd x 42; the ASP + D-galactose model group was intraperitoneally injected with ASP since the 8th day of the replication of the D-galactose model, qd x 35; and the normal control group was subcutaneously injected with saline at the same dose and time. On the 2nd day of after the injection, the peripheral blood was collected to measure the content of BUN, Crea, UA, Cys-C; paraffin sections were made to observe the renal histomorphology by HE staining; senescence-associated ß-g-alactosidase (SA-ß-Gal) stain was used to observe the relative optical density (ROD) in renal tissues; transmission electron microscopy was assayed to observe the renal ultrastructure; the renal tissue homogenate was prepared to measure the content of SOD, GSH-PX, MDA; the content of AGEs and 8-OH-dG were measured by ELISA. According to the result, compared with the D-galactose model group, the ASP + D-galactose model group showed obviously decreases in the content of BUN, Crea, UA, Cysc, AGES, 8-OH-dG, the number of hardening renal corpuscle, renal capsular space and renal tubular lumen, ROD of SA-ß-Gal staining positive kidney cells, mesangial cells, basement membrane thickness, podocyte secondary processes fusion and MDA and increases in the number of normal renal corpuscle, ribosome and rough endoplasmic reticulum in podocytes, the activity of SOD and GSH-PX. In Conclusion, A. sinensis polysaccharides can antagonize kidney subacute damages induced by D-galactose in mice. Its protective mechanism may be correlated with the inhibition of the oxidative stress injury.

[Effect of combined administration of Angelica polysaccharide and cytarabine on liver of human leukemia NOD/SCID mouse model].

Leukemia is a type of malignant tumors of hematopoietic system with the abnormal increased immature leukemia cells showing metastasis and invasion ability. Liver is one of the main targets of the leukemia cells spread to, where they may continue to proliferate and differentiate and cause liver function damage, even liver failure. Our previous studies showed that Angelica polysscharides (APS), the main effective components in Angelica sinensis of Chinese traditional medicine, was able to inhibit the proliferation and induced differentiation of the leukemia cells, however, its effect on the liver during the treatment remains elucidated. In the present study, the human leukemia NOD/SCID mouse model were established by implantation human leukemia K562 cells line, then the leukemia mouse were treated with APS, Ara-c or APS + Ara-c respectively by peritoneal injection for 14 days, to explore the effect and mechanism of the chemicals on the mouse liver. Compared to the human leukemia NOD/SCID mouse model group with the treatments of APS, Ara-c and APS + Ara-c, We found that severe liver damage and pathological changes of the liver were able to alleviate: First, the number of white blood cells in the peripheral blood was significantly lower and with less transplanted K562 leukemia cells; Second, liver function damage was alleviated as liver function tests showed that alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin (TBiL) were significantly reduced, while the albumin (Alb) was notably increased; Third, liver antioxidant ability was improved as the activities of the antioxidant enzymes glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) were significantly increased, and the contents of GSH and malonaldehyde (MDA) were decreased significantly in the liver; Fourth, the inflammation of the liver was relieved as the level of IL-1beta and IL-6, the inflammatory cytokines, were decreased significantly in the liver. Fifth, liver index was increased as the pathological observation showed that leukemia cells with diffused infiltration into the liver lobules were significantly reduced and with a remarkable increase of apoptotic positive cell rate by TUNEL test. Furthermore, the APS + Ara-c combined administration showed an even more significant positive effect. In conclusion, the APS, Ara-c therapy reduced the accumulation of leukemia cells within the liver, reduced the liver function damage and levels of inflammatory factors, improved antioxidant capacity of the liver tissue and thus alleviate the pathological changes of the liver. Moreover, the APS + Ara-c combination therapy may have an additive effect.

[Effect of domestic highly purified urinary follicle stimulating hormone on outcomes of in vitro fertilization-embryo transfer in controlled ovarian stimulation].

OBJECTIVE: To investigate the effect of domestic urine-derived high-purity follicle- stimulating hormone (HP-FSH, Lishenbao) on the outcome of in vitro fertilization(IVF) embryo transfer (ET) in controlled ovarian stimulation (COS). METHODS: From 1 September 2010 to 31 March 2011, total of 3178 infertility patients from 14 Reproductive Center with IVF or intracytoplasmic sperm injection (ICSI) indications who accepted first IVF or ICSI cycle were studied retrospectively. Their causes of infertility include all infertility factors except ovulatory dysfunction infertility and uterine factor infertility. The only long luteal phase gonadotropin-releasing hormone agonist (GnRH-a) protocol was included. Patients were divided into 2 groups according to the type of follicle-stimulating hormone (FSH) agents used: 1932 cases in HP-FSH group and 1246 cases in recombinant FSH (rFSH)group. Patients in both groups were combined with human menopausal gonadotropin (hMG) at doses of 150 U when follicle with diameter reached to 14-16 mm. When 3 dominate follicle with diameter reached 18 mm, hCG at dose of 5000 to 10 000 U or recombinant hCG at dose of 250 µg was administered by intramuscular injection. After 34 to 36 hours, oocytes were obtained guided by ultrasound, then IVF-ET were underwent in their Reproductive Center. The primary endpoint was comparison of live birth rate between the two groups. The secondary endpoints were comparisons of clinical pregnancy rate, miscarriage rate, and implantation rate, as well as COS and IVF outcome between the two groups. RESULTS: (1) There were significantly differences in baseline characteristics of the patients between two groups. The mean age was elder(32 ± 4 versus 30 ± 4, P < 0.01) , the infertility duration was longer (5 ± 4 versus 5 ± 3, P < 0.01) , and antral follicle count (AFC) was less (11 ± 5 versus 13 ± 7, P < 0.01) in patients of HP-FSH group compared with those in patients of rFSH group. (2) As compared with rFSH, the total doses of gonadotropin needed was (2348 ± 1011) U in HP-FSH group versus (2022 ± 659) U in rFSH group, the number of oocytes 13 ± 6 in HP-FSH group and 14 ± 7 in rFSH group, the rate of embryo frozen cycle of 66.30% (1281/1932) in HP-FSH group and 74.88% (933/1246) in rFSH group, which all reached statistical difference (P < 0.01). However, there were no significant different implantation rate [30.49% (1111/3644) versus 32.45% (737/2271)] between two groups. The other clinical parameters did not show significant difference, including clinical pregnancy rate per started cycle [41.61% (804/1932) versus 41.97% (523/1246) ] , clinical pregnancy rate per ET cycle[46.58% (804/1726) versus 48.47% (523/1079)], live birth rate per started cycle[34.21% (661/1932) versus 34.19% (426/1246)], live birth rate per ET cycle [38.30% (661/1726) versus 39.48% (426/1079)], miscarriage rate[13.6% (109/804) versus 16.4% (86/523)], and moderate/severe ovarian hyperstimulation syndrome (OHSS) rate [5.80% (112/1932) versus 7.78% (97/1246)](P > 0.05).(3) Treatment cost: the cost of gonadotropins needed for the patients in HP-FSH group was lower than that in rFSH group (4005 ± 1650 versus 6482 ± 2095, P < 0.01). CONCLUSION: In IVF/ICSI treatment cycles, domestic HP-FSH has similar live birth rate and lower financial burden when compared with rFSH.

Effects of cumulus cells removal after 6 h co-incubation of gametes on the outcomes of human IVF.

PURPOSE: To investigate the effects of cumulus cells removal after 6 h co-incubation of gametes on the fertilization, polyspermy, multinucleation and clinical pregnancy rates in human IVF. METHODS: A total of 1,200 IVF-ET cycles undergoing 6 h co-incubation of gametes in 2009 were included in this study. Inclusion criteria were: female age <38 years, first IVF treatment, with bi-ovary and normal ovarian response, e.g., 4 ~ 20 oocytes could be obtained. A 6 h period of co-incubation was applied in all IVF cycles. According to the history of infertility, cumulus cells were mechanically removed either 6 h post-insemination or 20 h post-insemination. For couples with primary infertility, or unexplained infertility, or mild oligospermia or asthenospermia, the cumulus cells were removed at 6 h of insemination for the polar body observation (6 h group, n = 565). Of these, 80 cycles received early rescue ICSI due to fertilization failure or low fertilization rate at 6 h of insemination. For couples with secondary infertility and normal semen analysis, the cumulus cells were removed at 20 h of insemination as routine (20 h group, n = 635). Of these, three cycles received late rescue ICSI due to fertilization failure at 20 h of insemination. Normal fertilization, polyspermy (≥3PN), multinucleation and clinical pregnancy rates were compared between the two groups (rescue ICSI cycles were not included in the comparison in both groups). RESULTS: Significant difference (P < 0.05) was observed between the groups regarding polyspermy rates (7.48% in 6 h group and 9.22% in 20 h group). No difference was observed between the groups regarding normal fertilization rates (2PN rate) (64.89% in 6 h group and 65.74% in 20 h group). No difference was observed between the groups regarding multinucleation and clinical pregnancy rates (11.01% and 65.15% in 6 h group, 10.75% and 66.93% in 20 h group, respectively). The clinical pregnancy rate was 51.43% in cycles receiving early rescue ICSI, while no clinical pregnancy was obtained in cycles receiving late rescue ICSI. CONCLUSION: The present results indicate that cumulus cells removal at 6 h of insemination is a relatively safe operation, which yielded comparable normal fertilization rate, multinucleation and clinical pregnancy rates compared with 20 h group. This protocol may be beneficial for early obsevation of fertilization failure and make early rescue ICSI possible.

The effects of different sperm preparation methods and incubation time on the sperm DNA fragmentation.

PURPOSE: To study the effect of different sperm preparation methods and incubation times post preparation on sperm DNA fragmentation. METHODS: Sperm DNA fragmentation was assessed by sperm chromatin dispersion (SCD) method on motile sperm prepared by gradient centrifugation or swim-up and incubated in IVF medium for up to 24 hours. Data were analyzed to discover the effect of preparation methods and incubation times on sperm DNA Fragmentation Index (DFI). RESULTS: There were no differences in DFI in sperm samples prepared by gradient centrifugation method or swim-up (3.87 ± 2.14 vs. 3.45 ± 1.83, p = 0.544). However, an increase was observed in DFI in samples prepared by swim-up after 6-8 hours compared with by gradient centrifugation (34° vs. 13°, p = 0.04). In the swim-up group, the DFI level at 4 hours was already significantly higher than it was initially. However, following gradient centrifugation, the DFI at 8 hours was significantly higher than the initial DFI level. CONCLUSION: Sperm samples prepared by gradient centrifugation may be more stable, in terms of DNA fragmentation, than samples prepared by swim-up.

IVF/ICSI outcomes between cycles with luteal estradiol (E2) pre-treatment before GnRH antagonist protocol and standard long GnRH agonist protocol: a prospective and randomized study.

OBJECTIVE: To study if luteal E(2) pre-treatment before GnRH antagonist protocol improves IVF/ICSI outcomes compared with standard long GnRH agonist protocol. DESIGN: A prospective, randomized and controlled study. SETTING: ART center of a state public hospital PATIENT(S): Two hundred twenty infertile women underwent IVF/ICSI treatments. INTERVENTION(S): Participants received oral Estradiol Valerate 4 mg/day preceding the IVF cycle from day 21 until day 2 of next cycle before GnRH antagonist protocol (E(2) pre-treatment group n=109) or received standard long GnRH agonist protocol as control group (n=111). MAIN OUTCOME MEASURE(S): Number of oocytes collected, MII oocytes, fertilization, implantation, live birth and early pregnancy rate, and hormone profiles. RESULT(S): E(2) pre-treatment exerted a significant suppressive effect on FSH but not LH secretion compared with basal FSH and LH levels. In E(2) pre-treatment group serum LH level was significantly higher during COH and serum P was also significantly higher on the day of HCG injection compared with control group. Five patients from E(2) pre-treatment group had elevated LH at all time (>or= 10 IU/L) and also a concomitantly high P (>1 ng/mL). Two of the five women achieved pregnancy but had early pregnancy loss. Overall, IVF/ICSI outcomes such as implantation, clinical pregnancy and live birth rates were similar between E(2) pre-treatment and control groups. CONCLUSION(S): Luteal E(2) pre-treatment before GnRH antagonist protocol significantly increases serum LH level and incidence rate of premature LH but no significant effect is observed on implantation, clinical pregnancy, live birth and early pregnancy loss rates compared with long GnRH agonist protocol. However, more studies in large numbers of cycles are needed to confirm that increased serum LH level by E(2) pre-treatment during COH has no negative effect on the IVF/ICSI outcomes.