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Loss of follicles together with decreased oocyte quality and quantity contribute to age-associated ovarian senescence and infertility. Although underlying mechanisms for ovarian senescence are still unknown, mitochondrial dysfunctions have been reported. Here, we showed age-dependent decreases in ovarian Nicotinamide Adenine Dinucleotide (NAD+) levels in mice whereas supplementing aging mice with nicotinamide riboside (NR), an NAD+ precursor, increased ovarian NAD+ content. We found that increases in ovarian NAD+ levels in aging mice led to increased number of ovarian follicles and ovulatory potential as well as increased live birth rate. NR supplementation also reduced levels of reactive oxygen species and decreased spindle anomalies in aging oocytes, together with increased mitochondrial membrane potential (ΔΨm) and decreased mitochondrial clustering. In addition, NR supplementation improved ovarian mitochondrial energy metabolism. Our data suggested that supplementation with NAD+ precursors in vivo and in vitro could be potential therapeutic approaches for treating age-related ovarian infertility.

We investigated the effects of paternal characteristics, including age, body mass index (BMI), and semen parameters on chromosomal aberration-related miscarriages in couples that underwent treatment with assisted reproductive technology (ART). Single nucleotide polymorphism (SNP) array analysis showed chromosomal aberrations in 60.2% (557/925) of miscarried fetuses, including trisomy in 73.1% (407/557) of cases. There were higher chromosomal aberration rates in fetuses for men aged 20-24 years and ≥30 years compared with controls. After adjusting for age and BMI of the female partners, and the BMI and semen parameters of the males, there was no statistically significant effect of paternal age ≥30 years on the risk of chromosomal aberrations-related miscarriages. However, the odds of chromosomal abnormality-related miscarriage were 148% higher for the youngest fathers (age: 20-24 years) than fathers aged 25-29 years [adjusted odds ratio (OR): 2.48, 95% confidence interval (CI): 1.03-5.96; P=0.042]. Furthermore, high male BMI (adjusted OR: 1.56, 95% CI: 1.14-2.14; P=0.005) and low semen volume (adjusted OR: 2.09, 95% CI: 1.06-4.11; P=0.034) were associated with increased risk of chromosomal aberration-related miscarriages. These findings demonstrate that very young paternal age, high BMI, and low semen volume are associated with increased risk of chromosomal aberration-related miscarriages in couples undergoing ART treatment.

ObjectivesTo evaluate the combined impact of male and female BMI on cumulative pregnancy outcomes after the first ovarian stimulation.DesignRetrospective cohort study.SettingUniversity-affiliated reproductive medicine center.PatientsA total of 15,972 couples undergoing their first ovarian stimulations from June 2009 to June 2016 were included. During the follow-up period between June 2009 and June 2018, 14,182 couples underwent a complete ART cycle involving fresh embryo transfer and subsequent frozen embryo transfers (FETs) after their first ovarian stimulations. Patients with a BMI 2 served as the reference group. Patients with a BMI ≥ 24 kg/m2 were considered to be overweight, and those with a BMI ≥28 kg/m2 were considered to be obese.Intervention(s)None.Primary Outcome MeasureThe primary outcome was the cumulative live birth rate (CLBR), which defined as the delivery of at least one live birth in the fresh or in the subsequent FET cycles after the first ovarian stimulation.ResultsIn the analyses of females and males separately, compared with the reference group, overweight and obese females had a reduced CLBR (aOR 0.83, 95% CI 0.7.92 and aOR 0.76, 95% CI 0.64–0.90). Similarly, overweight males had a reduced CLBR (aOR 0.91, 95% CI 0.83–0.99) compared with that of the reference group. In the analyses of couples, those in which the male was in the reference or overweight group and the female was overweight or obese had a significantly lower CLBR than those in which both the male and female had a BMI 2.ConclusionsThe CLBR is negatively impacted by increased BMI in the female and overweight status in the male, both individually and together.

BackgroundIn recent years, infertility has increased in older women with decreased ovarian reserve (DOR). Studies have shown that women with DOR have fewer oocytes, which are poorer in quality, and have an increased risk of adverse pregnancy outcomes. Pre-pregnancy BMI is significantly correlated with many adverse pregnancy outcomes. Therefore, we conducted this study to explore the correlation between body mass index (BMI) and abortion and live birth in older patients with DOR. MethodsThe clinical data of 2052 older women with infertility and DOR admitted to the Reproductive Medicine Center of the First Affiliated Hospital of Zhengzhou University from August 2009 to May 2018 were analysed retrospectively. Patients were divided into underweight (BMI < 18.5 kg/m 2 ; n = 56), normal weight (18.5 kg/m 2 ≤ BMI < 24 kg/m 2 ; n = 1389), overweight (24 kg/m 2 ≤ BMI < 28 kg/m 2 ; n = 527) and obese (BMI ≥ 28 kg/m 2 ; n = 80). We compared the pregnancy outcomes of patients in each group. ResultsLogistic regression analysis showed that being overweight or obese were independent risk factors for miscarriage (P < 0.05) and protection factors for live births (P

Uniparental embryos derived from only the mother (gynogenetic [GG]) or the father (androgenetic [AG]) are unique models for studying genomic imprinting and parental contributions to embryonic development. Human parthenogenetic embryos can be obtained following artificial activation of unfertilized oocytes, but the production of AG embryos by injection of two sperm into one denucleated oocyte leads to an extra centriole, resulting in multipolar spindles, abnormal cell division, and developmental defects. Here, we improved androgenote production by transferring the male pronucleus from one zygote into another haploid androgenote to prevent extra centrioles and successfully generated human diploid AG embryos capable of developing into blastocysts with an identifiable inner cell mass (ICM) and trophectoderm (TE). The GG embryos were also generated. The zygotic genome was successfully activated in both the AG and GG embryos. DNA methylome analysis showed that the GG blastocysts partially retain the oocyte transcription-dependent methylation pattern, whereas the AG blastocyst methylome showed more extensive demethylation. The methylation states of most known imprinted differentially methylated regions (DMRs) were recapitulated in the AG and GG blastocysts. Novel candidate imprinted DMRs were also identified. The production of uniparental human embryos followed by transcriptome and methylome analysis is valuable for identifying parental contributions and epigenome memory transitions during early human development.

Persistent supraphysiological serum estradiol (E2) levels during controlled ovarian hyper-stimulation (COH) have a detrimental effect on endometrial receptivity. In this study, we explored RNA expression and DNA methylation profiles from patients’ endometrium. The patients were divided into two groups: the COH cycle (n=3, hCG+7) group and normal cycle group (n=3, LH+5). Quantitative RT-PCR was used to validate the expression of selected differentially expressed genes (DEGs). Comparing natural and stimulated endometrium transcriptome profiles revealed 640 DEGs, with a > 2-fold change (FC) and p

STUDY QUESTION Are blastocyst culture and cryopreservation in ART associated with chromosomal abnormalities in miscarried products of conception (POC)? SUMMARY ANSWER Frozen blastocyst transfer in women aged 35 years or older and frozen embryo transfer (ET) (including both cleavage-stage embryo and blastocyst) in women aged WHAT IS KNOWN ALREADY Blastocyst culture and embryo cryopreservation have been previously associated with favorable ART treatment outcomes and widely applied in clinical practice. However, the association between these embryo manipulation procedures and embryonic chromosomal abnormalities has not been evaluated to the best of our knowledge. STUDY DESIGN, SIZE, DURATION This retrospective study included a total of 720 patients who underwent IVF/ICSI, and the retained POC were obtained. A single-nucleotide polymorphism (SNP)-based chromosomal microarray analysis (CMA) of all miscarried conceptuses was performed. PARTICIPANTS/MATERIALS, SETTING, METHODS This study was based on the Clinical Reproductive Medicine Management System/Electronic Medical Record Cohort Database (CCRM/EMRCD) at our center. In total, 720 miscarried POCs were collected from patients undergoing ART (including fresh cleavage-stage ET, fresh blastocyst transfer, frozen cleavage-stage ET and frozen blastocyst transfer), and the incidences and profiles of cytogenetic abnormalities in the miscarried conceptuses were measured via SNP-based CMA. MAIN RESULTS AND THE ROLE OF CHANCE The chromosomal abnormality rate in POC varied from 33.7% to 66.7% among the different ET strategies. In the patients aged ≥35 years, frozen blastocyst transfer was significantly associated with a lower incidence of chromosomal aberrations in the POCs (adjusted odds ratio (aOR): 0.171 (95% CI: 0.040–0.738); P = 0.018) than fresh blastocyst transfer. In the patients aged LIMITATIONS, REASONS FOR CAUTION This study was retrospectively designed, and we cannot draw any definite conclusions from our results regarding the adequate safety of embryo cryopreservation in ongoing pregnancy. WIDER IMPLICATIONS OF THE FINDINGS To our knowledge, this is the first study assessing the associations of ET strategies with the probability of miscarriage associated with embryonic chromosomal abnormalities. However, the underlying mechanism of these associations is unknown; this study may promote research concerning ET strategies and promote comprehensive consultations and recommendations for patients. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the Natural Science Foundation of China (Grant No.81571409), Science and Technology Research Project of Henan (Grant No. 172102310009) and Medical Science and Technology Research Project of Henan (Grant No. 201701005). The authors declare no competing interests. TRIAL REGISTRATION NUMBER N/A

Background: Polycystic ovary syndrome (PCOS) patients have a better ovarian reserve and age-related improvement in endocrine disturbances than non-PCOS patients. The effects of age on in vitro fertilization (IVF) treatment outcomes associated with cumulative live birth rate (CLBR) remain unclear.Objectives: To study the effect of age on CLBR after the first ovarian stimulation in IVF in PCOS patients.Method: This retrospective cohort study included 3,502 PCOS patients and 18,596 patients with tubal factor infertility, who underwent their first IVF cycles and subsequent frozen embryo transfer (ET) attempts. The primary outcome was CLBR associated with a single stimulation cycle and secondary outcomes included the implantation rate, clinical pregnancy rate, live birth rate (LBR), large for gestational age (LGA) rate, small for gestational age (SGA) rate, and preterm birth (PTB) rate of fresh ET cycles.Results: PCOS patients over 40 years had a higher implantation rate (27.8 vs. 15.7%, P < 0.05), clinical pregnancy rate (51.4 vs. 26.1%, P < 0.05), LBR (42.3 vs. 18.2%, P < 0.05), and CLBR (50.0 vs. 21.5%, P < 0.05) than non-PCOS patients over 40 years. These rates were comparable between PCOS patients aged 35 to 40 years and those aged over 40 years (P = 0.263, 0.385, and 0.112, respectively). The changes in the implantation rate, clinical pregnancy rate, and CLBR by age were slower for PCOS patients than for non-PCOS patients (all P < 0.05). Among PCOS patients less than 35 years, BMI was negatively associated with CLBR [aOR: 0.961 (0.939–0.985); P < 0.05]; however, among PCOS patients over 35 years, instead of BMI (P = 0.353), age [aOR: 0.891 (0.803–0.990); P < 0.05] and the number of oocytes retrieved [aOR: 1.093 (1.002–1.078); P < 0.05] were significantly associated with CLBR. No significant differences in LGA, LGA, or PTB were detected between PCOS and non-PCOS patients over 35 years (all P > 0.05).Conclusions: The declines in treatment outcomes with age are slower for PCOS patients than for non-PCOS patients. For patients over 40 years, PCOS patients have reproductive advantages over non-PCOS patients. In contrast to younger PCOS patients (

Context: Recently, two patients with primary ovarian insufficiency (POI) delivered healthy babies after in vitro activation (IVA) treatment followed by auto-transplantation of frozen-thawed ovarian tissues. Objective: This study sought to report the first case of live birth after IVA treatment following fresh ovarian tissue grafting in patients with POI, together with monitoring of follicle development and serum hormonal changes. Design: This was a prospective observational cohort study. Setting: We performed IVA treatment in 14 patients with POI with mean age of 29 years, mean duration since last menses of 3.8 years, and average basal FSH level of 94.5 mIU/mL. Interventions: Prior to IVA treatment, all patients received routine hormonal treatments with no follicle development. We removed one ovary from patients with POI and treated them with Akt stimulators. We improved upon early procedures by grafting back fresh tissues using a simplified protocol. Main Outcome Measures: In six of the 14 patients (43%), a total of 15 follicle development waves were detected, and four patients had successful oocyte retrieval to yield six oocytes. For two patients showing no spontaneous follicle growth, human menopausal gonadotropin treatment induced follicle growth at 6–8 months after grafting. After vitro fertilization of oocyte retrieved, four early embryos were derived. Following embryo transfer, one patient became pregnant and delivered a healthy baby boy, with three other embryos under cryopreservation. Conclusion: IVA technology can effectively activate residual follicles in some patients with POI and allow them to conceive their own genetic offspring. IVA may also be useful for treating patients with ovarian dysfunction including aging women and cancer survivors., We performed in vitro activation treatment following fresh ovarian tissue grafting in primary ovarian insufficiency patients, and achieved the first case of live birth with this method.

The quality of postovulatory metaphase II oocytes undergoes a time-dependent deterioration as a result of the aging process. Melatonin is considered to be an anti-aging agent. However, the underlying mechanisms of how melatonin improves the quality of postovulatory aged oocytes remain largely unclear. In this study, by using mouse model, we found that there were elevated reactive oxygen species levels and impaired mitochondrial function demonstrated by reduced mitochondrial membrane potential and increased mitochondrial aggregation in oocytes aged 24 h, accompanied by an increased number of meiotic errors, unregulated autophagy-related proteins and early apoptosis, which led to decreased oocyte quality and disrupted developmental competence. However, all of these events can be largely prevented by supplementing the oocyte culture medium with 10−3 M melatonin. Additionally, we found that the expression of sirtuin family members (SIRT1, 2 and 3) was dramatically reduced in aged oocytes. In addition,in vitrosupplementation with melatonin significantly upregulated the expression of SIRT1 and antioxidant enzyme MnSOD, but this action was not observed for SIRT2 and SIRT3. Furthermore, the protective effect of melatonin on the delay of oocyte aging vanished when the SIRT1 inhibitor EX527 was used to simultaneously treat the oocytes with melatonin. Consistent with this finding, we found that the postovulatory oocyte aging process was markedly attenuated when the oocytes were treated with the SIRT1 activator SRT1720. In conclusion, our data strongly indicate that melatonin delays postovulatory mouse oocyte aging via a SIRT1–MnSOD-dependent pathway, which may provide a molecular mechanism support for the further application of melatonin in the assisted reproductive technology field.

STUDY QUESTION What is the relationship between telomere length in sperm and early embryonic development in in vitro fertilization (IVF)? SUMMARY ANSWER Sperm telomere length (STL) is positively associated with embryo quality in IVF. WHAT IS KNOWN ALREADY Previous studies have shown that STL differs among human males. STUDY DESIGN, SIZE, DURATION In order to determine the associations between STL, fertilization laboratory parameters and clinical pregnancy in IVF, 418 couples were recruited from August 2013 to August 2014. MATERIALS, SETTING, METHODS We collected semen samples and used quantitative PCR technique to detect the mean STL for each patient. These data were compared with the IVF outcomes. MAIN RESULTS AND THE ROLE OF CHANCE The mean STL was positively correlated with the age of patient (rP = 0.100; P = 0.041) and total sperm count/ejaculate (rp = 0.28; P < 0.001). Analysis of the age-adjusted mean STL in relation to the male patient's paternal and maternal ages at the time of his conception showed significant positive relationships between STL and both paternal (r = 0.16; P = 0.003) and maternal (r = 0.19; P < 0.001) ages at the time of conception. In addition, significant correlations were found between STL and good quality embryo (regression coefficient: 1.63; P < 0.001) and transplantable embryo rates (regression coefficient: 1.57; P < 0.001), but clinical pregnancy rates were not affected (odds ratio = 1.00 [95% CI: 0.93-1.07]; P = 0.90). LIMITATIONS, REASONS FOR CAUTION This study showed that STL was positively associated with embryo quality in IVF. Additional studies are needed to confirm these observations. WIDER IMPLICATION OF THE FINDINGS STL has the potential to be used as a marker for the prediction of embryonic quality. STUDY FUNDING/COMPETING INTERESTS This work was supported by the National Natural Science Foundation of China (Grants 31271605 and 31471404), and the National Science Foundation for Young Scientists of China (Grant 31401274), and Science Foundation of First Affiliated Hospital of Zhengzhou University for Yong Scientists. The authors have declared that no competing interests exist.

This study sought to establish archives of genetic copy number variation (CNV) in human embryonic stem cell (hESC) lines that are associated with known diseases. We collected patients' fresh, discarded zygotes from in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) protocols. A total of 208 fresh, tripronuclear, discarded zygotes were also collected in this study from patients on the third day of their treatment cycle, prior to transfer. The blastula-formation rates were 13.51% (26/192) and 26.7% (4/15) while the high-quality blastocyst formation rates were 5.8% (11/192) and 20% (3/15) in the IVF and ICSI groups, respectively. The inner cell mass (ICM) from each embryo was mechanically separated, and then grown on feeder layers consisting of mouse embryonic fibroblasts and human foreskin fibroblasts (a 1:1 mixture). The hESC karyotype was determined by traditional G-banding; analysis of the results for the Zh19P25 and Zh20P24 cell lines showed that both were 46 XY. CNV and loss-of-heterozygosity analysis of hESC gDNA was performed to assess the genetic characteristics associated with molecular diseases using the high-resolution Infinium High-Density HumanCytoSNP-12 DNA chip. Seven CNVs in Zh19P25 and Zh20P24 were deletions, and a region that corresponds to Potocki-Shaffer disease, 11p11.2-11p11.12 in Zh20P24, showed a 2.98-Mb loss. These data together suggest that single-nucleotide polymorphism (SNP) microarray analysis for molecular cytogenetic features can help to distinguish hESC lines with a normal karyotype from tripronuclear zygotes with known, disease-related characteristics.

Objective To determine the association between leukocyte telomere length and the risk of nonobstructive azoospermia (NOA). Design The mean leukocyte telomere length (LTL) among men with NOA, obstructive azoospermia (OA), and normospermic subjects was determined by quantitative polymerase chain reaction (PCR). We used logistic regression to investigate the association between LTL and the risk of NOA after adjustment for age and body mass index (BMI). Partial correlation analysis was also used to evaluate the relationship of clinical parameters with the mean LTL among men with OA and NOA. Setting Reproductive medicine center. Patients(s) A total of 866 men, including 270 normospermic controls, 247 OA and 349 NOA patients. Intervention(s) None. Main Outcome Measure(s) Leukocyte telomere length. Result(s) The mean relative LTL of men with NOA was significantly shorter than that of those with OA and in normospermic controls (odds ratio [OR] 0.81, 95% confidence interval [CI] 0.64–0.98 vs. OR 0.92, 95% CI 0.70–1.24 vs. OR 0.99, 95% CI 0.83–1.22), respectively). Subjects with shorter telomeres (lowest tertile) had a significantly higher risk of NOA than those with longer telomeres (highest tertile). Interestingly, we also found that a low relative LTL was associated with poor efficiency of spermatogenesis using the Johnsen score after testis biopsy and histopathology in azoospermic patients, after adjusting for patient age and BMI. Conclusion(s) This is the first report that short LTL is associated with NOA, shedding light on an important biological pathway involved in the etiology of this form of male factor infertility.

Better pregnancy outcomes can be obtained by human mature oocyte vitrification, but many problems remain to be resolved in human mature oocyte vitrification. Since mature oocyte development possesses its own maturity cycle, there should be the optimal timing for mature oocyte vitrification. The purpose of this study was to observe the effects of frozen timing on the spindle density, the angle between the polar body and spindle, and embryo development of intracytoplasmic sperm injection (ICSI) in vitrified mouse mature oocytes and explore its possible mechanism. Mouse oocytes were randomly divided into three groups according to different frozen timing including Groups A, B, and C in which oocytes were vitrified within 2 h after ovum pick-up, and 3-4 and 5-6 h after ovum pick-up, respectively. Spindle-related parameters were measured, ICSI was performed. The spindle occurrence rate of vitrified-thawed oocytes was 98.4% in Group A, 82.3% in Group B, and 75.8% in Group C, without statistical differences between pre-vitrification and post-thawing and among the three groups (P > 0.05). The angles between the polar body and spindle were larger after thawing than before vitrification (P 0.05). The damage on the spindle is the slightest and embryo quality is the highest in the mouse oocytes vitrified within 2 h after ovum pick-up. The spindle retardance value is more valuable than the spindle occurrence rate in the evaluation of vitrified-thawed oocyte quality, and is positively correlated with embryo quality.

Several studies have reported improved IVF by shortening the time of sperm-oocyte coincubation from 16-18 hours to 1-4 hours. The objective of this study was to examine the advantages and disadvantages of a shortened sperm-oocyte coincubation time in order to assess the effects of this insemination method for clinical IVF practice. Two insemination methods, the shortened method (4 hours) and the standard method (16-18 hours) of coincubation of sperm-oocytes for two groups of patients based on the quality of sperm were compared. Group I, was composed of couples without male factor; Group II, involved couples with mild male factor. Fertilization, good quality embryos, clinical pregnancy, and implantation rates were compared by two different insemination methods. In Group I, fertilization, clinical pregnancy, and implantation rates were not different between the two insemination methods. However, the polyspermy rate was significantly higher (P0.05) in the shortened (7.3%) than in the standard (4.1%) insemination method. In Group II, the fertilization rate was significantly lower (P0.05) using the shortened insemination method (62.6%) compared to the standard insemination method (68.7%). When fertilization failed with the shortened insemination method, the clinical pregnancy and implantation rates were 34.7% and 24.1%, respectively, from the rescue intracytoplasmic sperm injection (ICSI). The live birth rate from the rescue ICSI was 32.0% with normal infants. The duration of sperm-oocyte coincubation does not affect fertilization, embryo quality, clinical pregnancy, and implantation rates. However, fertilization rates will decrease with the shortened insemination method when the sperm parameters are poor. From the results of the present study we suggest that the combination of the shortened sperm-oocyte coincubation and rescue ICSI method may be an efficient method for IVF treatment in order to prevent fertilization failure when sperm parameters were poor as mild male factor.

Whether there are differences in the pregnancy outcomes of blastocysts cryopreserved during different developmental stages remains under debate because the results among studies are inconsistent. We analyzed blastocyst quality and pregnancy outcomes by considering blastocyst euploidy and investigated the differences in the development potential between blastocysts of different developmental stages (frozen-thawed day 5 [D5] and day 6 [D6] cycles) and their relationship with clinical pregnancy outcomes. In total, 1374 D5 and 255 D6 frozen-thawed blastocyst transfer cycles were retrospectively analyzed. Additionally, the chromosome euploidy and clinical pregnancy rates of 237 blastocysts from 50 pre-implantation genetic diagnosis (PGS) cycles were statistically analyzed. The corresponding euploidy rate and pregnancy outcomes of the D5 and D6 blastocyst transfers were also compared. The clinical pregnancy rate (47.2 vs 40.0 %; P = 0.04) and implantation rate (34.2 vs 28.8 %; P = 0.03) of the D5 blastocysts were higher than were those of the D6 blastocysts. However, the clinical pregnancy rate (52.4 vs 52.6 %; P = 0.97) and implantation rate (38.9 vs 35.6 %; P = 0.39) of the high-quality D5 blastocysts did not significantly differ from those of the high-quality D6 blastocysts. Analysis of blastocyst euploidy in 237 blastocysts examined in 50 PGS cycles showed that the euploidy rates of the D5 and D6 blastocysts were both 48.1 % (P = 0.99). The clinical pregnancy rate of the D5 blastocysts (48.5 vs 17.6 %; P = 0.03) was higher than that of the D6 blastocysts. The euploidy rates (55.2 vs 55.3 %; P = 0.99) and clinical pregnancy rates (60.0 vs 42.9 %; P = 0.77) of the high-quality D5 and D6 blastocysts did not differ. The euploidy rate (55.3 vs 41.5 %, P = 0.03) and clinical pregnancy rate (54.5 vs 25.0 %, P = 0.03) of the high-quality blastocysts were higher than were those of the poor-quality blastocysts. The euploidy rates between the D5 and D6 blastocysts did not differ. High-quality D6 blastocysts in frozen-thawed cycles had similar developmental potential and pregnancy outcomes compared to those of high-quality D5 blastocysts. The quality of the blastocysts was an important factor that affected the pregnancy outcomes of the frozen-thawed cycles.

Background Fluorescence in situ hybridization (FISH) is an irreplaceable method in pre-implantation genetic diagnosis. We explored the effects of a modified single cell fixation method on the cell-nuclear area and FISH signal. Methods From January 2006 to March 2008, the blastomeres with marked nuclei from D3 embryos were selected. Cells were fixed with three different methods. The effects of the three methods on the cell-nuclear areas and FISH signals were then analyzed. Results The cell fixation rate was higher in conventional (Group B, 94.85%) and modified (Group C, 95.79%) Tween-20/HCl + methanol/glacial acetic acid methods than in the methanol/glacial acetic acid method (Group A, 86.73%) with p 0.05. The mean cell-nuclear areas in groups A, B, and C were 55.3, 46.2, and 49.5 microm3, respectively, with p 0.05 between Group B and C. There was no significant difference in signal overlap and splitting rates between the three groups. Conclusions Modified Tween-20/HCl + methanol/glacial acetic acid method fails to increase FISH signal overlap and splitting rates. It is simple and its fixation time is short. It can be widely used in clinical practice.

The purpose of this study was to explore the effects of cumulus cells on vitreous cryopreservation of human mature oocytes and clinical pregnancy outcomes. The study was divided into group A (cumulus cells were removed from the oocytes before freezing) containing 24 participants and 193 oocytes and group B (cumulus cells were retained with the oocytes before freezing) containing 26 participants and 240 oocytes. Based on no significant differences in age, duration of infertility, infertile causes, and number of retrieved oocytes between both groups when oocytes were retrieved from infertile women, we found that the survival rate of post thaw oocytes (88% vs 58%), cleavage rate (80% vs 56%), and high-quality embryo rate (75% vs 59%) were significantly higher in group B than in group A. Under the conditions that there were no significant differences between the 2 groups in the general status of the participants undergoing embryo transfer, the embryo implantation rate (37% vs 15%) and the clinical pregnancy rate (50% vs 17%) were significantly higher in group B than in group A, all with Ps.05. We conclude that the retention of cumulus cells can improve the developmental competence of vitrified-thawed human mature oocytes and clinical pregnancy outcomes.

In this study we evaluated the value of short-time insemination and early rescue intra-cytoplasmic sperm injection (ICSI) in preventing the occurrence of complete fertilisation failure for mild or moderate male infertility patients. A total of 866 couples with borderline semen who underwent in vitro fertilisation treatment in 2010 were included. Regular insemination was performed between January and June of 2010 and short-term insemination was performed from July through December 2010, where, as early as 4 h after insemination, oocytes were denuded from cumulus cells and extrusion of the second polar body was evaluated. Of the 4153 mature oocytes with a detectable second polar body 4 h after insemination, 3874 (93.3%) showed signs of fertilisation on Day 1. Where no second polar body was present in any of the retrieved oocytes for a given patient, rescue ICSI was performed immediately. Similar rates of normal fertilisation and percentage of good-quality embryos were obtained between early rescue ICSI and regular ICSI. Clinical pregnancy occurred in 16 of 43 patients (37.2%) receiving early rescue ICSI. Our results showed early rescue ICSI in combination with evaluation of the second polar body 4 h following insemination is an effective method to prevent complete fertilisation failure for patients with mild or moderate male infertility.