Effect of Early Cumulus Cell Removal on the Fertilization and Clinical Outcome in Human <i>in Vitro</i>Fertilization

The short-term insemination technique has been widely applied, although it is not clear whether the mechanical desorption of cumulus cells can affect normal fertilization and birth outcomes. Therefore, the present study aimed to evaluate whether early cumulus cell removal could affect in vitro fertilization outcomes. Based on a history of infertility and semen analysis, 61 patients were identified, and the cumulus cells were mechanically removed at either6 h post-insemination (Group A, 387 mature oocytes) or 18 - 20 h post-insemination as traditional in vitro fertilization (Group B, 424 mature oocytes). There were no significant differences in the rates of fertilization, available embryos, high quality embryos, birth, premature birth, pregnancy, or implantation when we compared the two groups. However, Group A exhibited a significantly higher polyspermy rate, and significantly lower rates of blastocyst formation and high-quality blastocyst formation, compared with Group B. Therefore, the results of present study indicate that early cumulus cell removal (at 6 h post-insemination) may negatively affect the rates of polyspermy, blastocyst formation, and high-quality blastocyst formation.

In vitro maturation of human oocytes maintaining good development potential for rescue intracytoplasmic sperm injection with fresh sperm

BACKGROUND The outcomes of the use of commercial in vitro maturation(IVM)medium to culture immature oocytes obtained from conventional ovulation induction,followed by rescue intracytoplasmic sperm injection(RICSI),are not ideal.It is thus difficult to widely adopt this approach in clinical practice.Therefore,it is necessary to explore methods for improving the clinical outcome of IVM.AIM To study the effect of sperm on the developmental potential of in vitro-matured oocytes in conventional culture.METHODS This was a retrospective study of patients whose immature oocytes were harvested from conventional oocyte stimulation cycles and underwent ICSI at our hospital between June 2018 and August 2020.RICSI was performed using sperm collected on the day of oocyte harvest(old)and sperm collected on the day of RICSI(fresh)and oocytes matured in vitro after 24 h of culture in conventional medium.The rates of in vitro oocyte maturation,normal fertilization,normal cleavage,day-3 top-quality embryos,and useful blastocyst formation were compared between the two groups.RESULTS In total,102 germinal vesicle(GV)-stage immature oocytes were cultured in the old sperm group.In the fresh sperm group,122 GV-stage immature oocytes were collected and cultured in vitro for 24 h.There were no significant differences in the general conditions of males and females between the two groups(P>0.05).The oocyte maturation,normal fertilization,and normal cleavage rates of the old and fresh groups were 51.0%vs 55.7%,61.5%vs 64.7%,and 93.8%vs 93.2%,respectively.None of the rates differed significantly(P>0.05)between the two groups.However,the day-3 top-quality embryo and useful blastocyst rates of the old and fresh sperm groups were 16.6%vs 63.4%;6.67%vs 34.6%,respectively.The day-3 top-quality embryos and useful blastocyst rates of the old sperm group were significantly lower than those of the fresh group(P<0.05).CONCLUSION In vitro maturation with conventional culture medium combined with the use of fresh sperm collected on the day of RICSI is an easy-to-implement strategy for patients whose oocytes are completely or mostly immature.

Presence of Meiotic Spindles Indicates Early Cleavage of Embryos

Objective To assess whether the detection of the meiotic spindle could anticipate the appearance of early cleavage. Methods Oocytes were obtained from stimulated ovaries of consenting patients undergoing oocytes retrieval for ICSI. Spindles were imaged with the Polscope. After ICSI, oocytes with or without spindles were cultured for examination of early cleavage and embryo development. A total of 328 oocytes from 50 cycles were examined with the Polscope and inseminated by ICSI. Results Spindles were imaged in 81.7% of oocytes. After ICSI, more oocytes with spindles （78.4%） fertilized normally than oocytes without spindles （53.3%） （P〈0. 001）. At 25-27 h post ICSI, more fertilized oocytes developed from oocytes with spindles （81.9%） were detected early cleavage than those from oocytes without spindles （28.1%） （P〈0.001）. Significantly more embryos with early cleavage （82.2%） developed to high quality embryos at d 3 compared with the embryos without early cleavage （48.3%） （P=0.001）. The value of rs related to the relationship between spindles and early cleavage was 0.420 （P〈0.001）. Conclusion The existing of the early cleavage may have a predictive value on the opportunity of high quality embryos and the existing of the spindle may have a predictive value in the appearance of early cleavage.

Effects of short gamete co-incubation in human in vitro fertilization

Traditionally oocytes have been exposed to sperm overnight for 16-20 h.This long period of co-incubation, however,has been shown to create problems with high levels of reactive oxygen species(ROS),which may affect embryo viability and cause hardening of the zona pellucida.Recently,several positive and negative effects of reducing the co-incubation time to 90-120 min were reported.The objective of this review was to discuss whether short exposure of oocytes to sperm in vitro might affect the clinical results and several problems arose in short gamete co-incubation.

Analysis of Factors Influencing Pregnancy Rate in Frozenthawed Embryo Transfer

Objective To analyse factors influencing the outcome of frozen-thawed embryo transfer （FET）. Method A retrospective analysis was performed in our center on 129 thawing cycles from March 2001 to April 2003. The related parameters were compared between conceived and non-conceived cycles. Results There were totally 129 clinical pregnancies in these transfers （pregnancy rate： 27.1%）. Frozen-thawed embryos were transferred to natural cycles and CC cycling and hormone replacement treatment had equal success. Groups of IVF and ICSI did not differ significantly in pregnancy rates （P〉0.05）. The pregnancy rates for one, two, three and four pre-embryos transfer were 0, 20.0%,44.1% and 75.0%, respectively （P〈0.05）. There were statistical differences between pregnancy group or non- pregnancy group in the endometrial thickness, CES, CES/No. of embryo. A higher pregnancy rate was observed in embryo transfers which had at least one 4-cell grade I embryo （d 2）（P〈0.01）. Conclusions The most important factors influencing the implantation rate and pregnancy rate of frozen-thawed embryo transfer are age, endometrium thickness, and the number, morphology and growth rate of transferred frozen embryos of women participants.

RNAi-mediated Human Nestin Silence Inhibits Proliferation and Migration of Malignant Melanoma Cells by G1/S Arrest via Akt-GSK3β-Rb Pathway

Human Nestin（hNestin） has been found to express in melanoma, and its expression is positively correlated with the advanced stage of melanoma. However, the precise role of hNestin in the development of melanoma has not been fully understood. The present study aimed to explore the role of hNestin in the proliferation and invasion of melanoma cells. The lentivirus vector carrying a short hairpin RNAs（shRNAs） targeting hNestin（hNestin-sh RNA-LV） was stably infected into human melanoma cells UACC903, which expressed high levels of hNestin. The effects of hNestin knockdown on the proliferation, apoptosis, migration of melanoma cells and the related signaling pathways were investigated by immunofluorence, Western blotting and reverse transcription polymerase chain reaction（RT-PCR）, respectively. The results showed that hNestin was expressed in most melanoma specimens and the melanoma cells studied. Knockdown of hNestin expression significantly inhibited the proliferation of melanoma cells, blocked the formation of cell colony, arrested cell cycle at G1/S stage and suppressed the activation of Akt and GSK3β. hNestin-silent cells also showed a sheet-like appearance with tight cell-cell adhesion, decreased membrane expression of N-cadherin and β-catenin, and attenuated migration. Furthermore, hNestin silence resulted in the inhibition of tumor growth in vivo. Our study indicates that hNestin knockdown suppresses the proliferation of melanoma cells, which might be through affecting Akt-GSK3β-Rb pathway-mediated G1/S arrest, and hNestin silence inhibits the migration by selectively modulating the expression of cell adhesion molecules in the process of epithelial-mesenchymal transition.

Spleen-Yang-deficiency patients with polycystic ovary syndrome have higher levels of visfatin

OBJECTIVE:To study serum visfatin levels in women with polycystic ovary syndrome(PCOS)grouped by Traditional Chinese Medicine(TCM)patterns.To study the correlations of serum visfatin levels with homeostatic model assessment insulin resistance(HOMA-IR),fasting plasma glucose(FPG),fasting insulin(FINS),body mass index(BMI),testosterone(T),total cholesterol(TC),and triglycerides(TG).METHODS:Two hundred and twelve PCOS patients were placed into the following TCM pattern subgroups:Kidney-Yang deficiency(KYD)group,Spleen-Yang deficiency(SYD)group,stagnant Liver-Qi transforming into heat(SLQTH)group,and Kidney-Yin deficiency(KYIND)group.The correlations between serum visfatin levels and HOMA-IR,FPG,FINS,BMI,T,TC,andTG were analyzed.RESULTS:Of all patients with PCOS,there were 82in the KYD group(38.6%),67 in the SYD group(31.6%),37 in the SLQTH group(17.5%),and 26 in the KYIND group(12.3%).Visfatin levels in all PCOS subgroups were higher than those in the control group(P<0.01 or P<0.05).Among these subgroups,the visfatin levels in the SYD group were significantly higher than those in the other three TCM pattern groups(P<0.05).There were no statistical differences among the remaining three pattern groups.The levels of BMI,FINS,HOMA-IR,T,and TG were significantly higher in all subgroups than those in the control group(P<0.05).There were no significant differences in FPG and TC between all PCOS subgroups and the control group(P>0.05).The SYD group had higher levels of FINS and HOMA-IR compared with the KYD,SLQTH,and KYIND groups(P<0.05).In all subgroups,after controlling for BMI,TG,TC,and age,visfatin was positively correlated with FINS(r=0.197,P=0.015)and HOMA-IR(r=0.173,P=0.033),and was not correlated with T.CONCLUSION:KYD and SYD patterns are most common in PCOS patients.Increased visfatin is a common pathophysiologic manifestation in PCOS patients.The SYD group had the highest levels of visfatin,and visfatin was positively correlated with FINS and HOMA-IR.

Effect of Long Protocol on the Outcome of In Vitro Fertilization-embryo Transfer in Patients with Endometriosis Compared with Prolonged Protocol

Objective To investigate the effect of different down-regulation protocol on the in vitro fertilization-embryo transfer （IVF-ET） outcomes in infertile patients with endometriosis （EMs）.Methods A retrospective case control study was performed. Totally 294 infertile patients with EMs were enrolled. And 109 patients （116 cycles） received prolongedprotocol as the control, 185 patients （193 cycles） received long protocol as case group, all followed by standard controlled ovarian hyperstimulation （COH）. Response to gonadotropins, the fertilization rate, the cleavage rate, the implantation rate, the clinical pregnancy rate and the miscarriage rate were measured and analyzed between the two groups. Results A trend toward better ovarian response was observed in long protocol group. Higher fertilization rate, lower total dose of rFSH, shorter duration of stimulation and more endometrial thickness on the day of hCG injection were observed in long protocol group compared with those of prolonged protocol group, and the difference was significant （P〈0.05）. In addition, the clinical pregnancy rate, the cleavage rate and the implantation rate also had an increase trend in long protocol group compared with those of prolonged protocol group, but without significant differences. Conclusion Long protocol regimen before IVF-ET in patients with EMs resulted in a trend toward better ovarian response and higher clinical pregnancy rates than prolonged protocol regimen did.

A Delayed-interval Delivery of An in vitro-fertilized Twin Pregnancy with Premature Rupture of Membranes

This article presents a case of conservative managements of an in vitro-fertilized twin pregnancy with early loss of the first fetus. A 37-year-old woman in the 21st week of a twin pregnancy delivered her one of the fetuses with unfortunate outcome after the premature rupture of membranes. The umbilical cord of the first fetus was ligated at the cervical level and the second surviving fetus was left in utero. In order to save the surviving fetus, conservative managements including combination of steroids, tocolytics, antibiotics and cervical cerclage were performed. The second fetus was delivered vaginally in the 27th week of pregnancy, 4 h after premature rupture of membranes of the remaining amniotic sac, 41 d after the first fetus was born. The child and the mother have been followed up for 4 years and they are healthy. Conservative managements including steroids, tocolytics, antibiotics and cervical cerclage in a closely monitored environment would be given following the delivery of the first fetus. Delayed-interval pregnancy appears to be safe for the mother and the remaining fetuses.

Related Factors of in vitro Fertilization and Embryo Transfer Patients with Complete Fertilization Failure

Objective To find the possible factors predicting fertilization failure of in vitro fertilization-embryo transfer （1VF-ET）. Methods The IVF-ET patients with complete fertilization failure （experimental group, n =32） were analyzed retrospectively. The patients whose oocytes retrieved at the same day and cultured on the same incubators with ≥ 50% fertilization rates were matched as the control （n=56）. Results The infertility duration, superovulation days, the rates of primary case, progesterone （P） level 〉3.12 nmol/L rate and rate of severe abnormal sperm （abnormal sperm rate 〉95%） in experimental group were significantly higher than those in the control （6.4 ±3.1 years, 12.6 ±2.2 d, 56%, 43%, 43% vs 4.6±2.9years, 11.6 ±% 1.3 d, 33%, 23%, 23%, respectively, P〈0.05）. Conclusion We should pay attention to these patients with primary infertility, longer infertility duration and superovulation days （〉6.4 years and 〉12.6 d） and having increased level of P on hCG injection day （〉3.12 nmol/L）, abnormal sperm rate 〉95% at the same time. They should be included in such patients at high risk of fertilization failure.

Expression of Cellular FLICE-like Inhibitory Protein in Granulosa Cells in In Vitro Fertilization Patients with Advanced Endometriosis

Background:This study evaluated the expression of cellular FLICE-like inhibitory protein(cFLIP)in granulosa cells(GCs)obtained from in vitro fertilization-embryo transfer patients with advanced endometriosis.Methods:A total of 267 patients with advanced endometriosis were enrolled in this study.They were divided into clinical pregnancy group(n=114)and nonpregnancy group(n=153).The expressions of cFLIP in mRNA and protein level were measured by real-time polymerase chain reaction(RT-PCR)and Western blotting.The related factors on the clinical pregnancy were analyzed using logistic regression analysis.Coefficients of correlation were calculated using the nonparametric rho-Spearman test.Results:The number of oocytes retrieved,fertilization rate,and cleavage rate were significantly and independently related with clinical pregnancy(P>0.05).RT-PCR and Western blotting analysis showed that the expressions of cFLP in mRNA and protein level were significantly higher in the clinical pregnancy group than in nonpregnancy group(P>0.05).cFLIP had a significantly positive correlation with the number of oocytes retrieved(P>0.05)and no correlation with fertilization rate and cleavage rate(P<0.05).Conclusion:Higher expression of cFLIP increased the pregnancy rate in women with advanced endometriosis.

Application of Ultra-low-dose Incremental Gn Protocol in Controlled Ovarian Hyperstimulation of the Patients with Ovary Hyperreaction

Objective To explore the clinical outcome of ultra-low-dose incremental protocol in the controlled ovarian hyperstimulation （COH） for the ovary hyperreaction patient. Methods A prospective, randomized, controlled study was conducted between June 2006 and February 2008 on 120 women who underwent COH. The patients＇ clinic outcomes were compared between ultra-low-dose incremental protocal （experimental group, n=60） and other ovulation stimulating protocols （control group, n=60）. Results The mean number of large follicles （≥14 mm） on the day of oocytes retrivaling, the mean number of harvested oocytes and the incidence rate of OHSS were significantly lower in experimental group. However, the gonadotropin stimulating time was significantly longer in experimental group. And the rate of leading follicle punctuation was also higher in this group when compared with control group. The clinical pregnant rate, the mean number of gonadotropin ampoules, the cancel rate were similar in the two groups. Conclusion The application of ultra-low-dose incremental protocal in the COH for the ovarian hyperreaction patients is able to effectively reduce the risk of occurrence of OHSS under the precondition of assurance of pregnancy rate. It is a safe and more effective COH program.

Acute Miliary Tuberculosis and Pelvic Tuberculosis after In vitro Fertilization

Nearly one-fourth of the world’s population is infected with Mycobacterium tuberculosis(MTB).Female genital tuberculosis(TB)is a common cause of infertility in both developing and undeveloped countries.Furthermore,assisted reproduction treatments and pregnancy potentially increase the risk of TB infection and reactivation.In this study,we present the case of a 28-year-old infertile female without a history of TB who developed an acute miliary TB and pelvic TB after in vitro fertilization-embryo transfer(IVF-ET).Elevated serum estrogen levels during controlled ovarian hyperstimulation and T-lymphocyte function inhibition during pregnancy are the risk factors for MTB infection and reactivation.In her 7 th week of gestation,the patient developed fever and spontaneously aborted.Her chest computed tomography images revealed classical miliary TB.Uterine curettage tissue and vaginal secretion samples as well as Gene X-pert MTB/rifampicin(RIF)and TB-RNA test results were positive for MTB.Histological examination of the uterine curettage tissue confirmed the diagnosis of endometrial TB.Treatment with isoniazid,RIF,pyrazinamide,amikacin,and levofloxacin was selected based on the patient’s diagnosis,complications,and test results.Currently,the patient is undergoing anti-TB treatment,and her condition is stable.It is important to rule out the presence of TB in infertile patients before performing IVF-ET to avoid TB dissemination during pregnancy.

Syncytin-A Knockout Induces Placental Developmental Abnormalities Partially through Calpain1-Apoptosis-Inducing Factor-Mediated Trophoblast Apoptosis

Objective:Structural abnormalities and dysfunction of the placenta contribute to pregnancy-related complications,such as preeclampsia.Syncytin-A(synA)has been reported to be expressed in the placenta.The contribution of synA to developmental abnormalities and dysfunction of the placenta remains elusive.In this study,we aimed to explore the role of synA in placental development and functions.Methods:SynA-knockout mice were generated using the CRISPR-Cas9 method,and the phenotypes of the placenta and fetus ofsynA-knockout mice were observed.Real-time quantitative polymerase chain reaction(PCR)and routine PCR were employed to detect the genotypes of the offspring.CD31 immunohistochemistry was used to evaluate the vessel density of the placenta,and the protein levels of key molecules were measured by western blotting.Results:SynA knockout caused fetal death.Furthermore,synA-knockout mice showed placental developmental abnormalities,indicated by a thinner labyrinth layer,thicker spongiotrophoblast layer,lower blood vessel density,and significantly higher numbers of apoptotic trophoblasts,when compared with wild-type littermates.Mechanistically,synA ablation induced apoptosis-inducing factor(AIF)cleavage and nuclear localization and promoted placental trophoblast apoptosis.In addition,synA knockout increased the calpain1 protein levels.The calpain1 inhibitor calpeptin blockedsynA knockout-induced AIF cleavage,partially restoring the placental structural abnormalities ofsynA-knockout mice.Conclusions:SynA knockout leads to placental developmental abnormalities by inducing trophoblastic apoptosis via the calpain1-AIF pathway.

Impact of Blood Clot in Oocyte-Corona-Cumulus Complex during in vitro Fertilization and Embryo Transfer

Objective To study the impact of blood clots in the oocyte-corona-cumulus complexes （OCCC） during in vitro fertilization and embryo transfer （IVF-ET）.Methods The OCCCs were harvested from the patients undergoing long protocol ovarian hyperstimulation. The OCCCs with blood clots removed or not, were randomly grouped into A or B. The OCCCs without blood clots were group C （the control）.Results The patient＇s age, infertility duration, the average GN consumption, the average days of superovulation and an average number of harvested oocytes showed no significant difference in the 3 groups. The fertilization rate and 2PN rate in group A were the highest, which were 85.4% and 71.1%, respectively, followed by group C, which were 77.5% and 64.9%, respectively. The lowest fertilization rate and 2PN rate were in group B, 75.8% and 62.2%, respectively. Those in group A were significantly higher than those in groups B and C （P〈0.01）, while there was no significant difference between group B and group C. The implantation rates and pregnancy rates showed no significant difference in the 3 groups after transplantation, even if group A got the highest rate among the 3 groups. Conclusion Removing the blood clots in OCCC can improve the outcome of IVF-ET without increasing the cost and complexity of the operation.

Effects of Different Serum on in vitro Cultured Human Granulosa Cells

Objective To evaluate the effects of different sera on the growth of human granulosa cells （GCs） cultured in vitro. Methods GCs were obtained from women who underwent follicular aspirates during in vitro fertilization （IVF） program. Five groups were divided according to media supple- mented with different types of sera. Group A, 10% heat-inactivated fetal bovine serum （FBS）; group B, 10% heat-inactivated newborn bovine serum; group C, 10% non heat-inactivated FBS; group D, 10% human serum; group E, bovine serum albumin. Morphological characteristics and viability of GCs measured by trypan blue exclusion assay were evaluated after 24 h of incubation.Results GCs cultured in group A and group B showed multiform morphology compared with other groups. GCs cultured in group D and group E were present with cytoplasmic atrophy and less pseudopodium. Moreover, group A and group B showed a similar level in the viability of GCs （P〉0.05）, which displayed no difference between group D and group E as well （P〉0.05）. Group C had a lower level of viability than group A （P〈0.05） but a higher level than group D （P〈0.01）. Conclusion Heat-inactivated sera can improve the growth of GCs. Different types of sera would have different effects on the growth of GCs cultured in vitro. The pre-culture with different types of sera should be performed to get better efficacy.