Effect and Underlying Mechanism of Bu-Shen-An-Tai Recipe on Ovarian Apoptosis in Mice with Controlled Ovarian Hyperstimulation Implantation Dysfunction

The effect and underlying mechanism of Bu-Shen-An-Tai recipe on ovarian apoptosis in mice with controlled ovarian hyperstimulation(COH) implantation dysfunction were studied. The COH implantation dysfunction model in mice was established by intraperitoneal injection of 7.5 IU pregnant mare’s serum gonadotrophin(PMSG), followed by 7.5 IU human chorionic gonadotrophin(HCG) 48 h later. Then the female mice were mated with male at a ratio of 2:1 in the same cage at 6:00 p.m. The female mice from normal group were injected intraperitoneally with normal saline and mated at the corresponding time. Day 1 of pregnancy was recorded by examining its vaginal smears at 8:00 a.m. of the next day. Fifty successfully pregnant mice were equally randomly divided into 5 groups: normal control pregnant group(NC), COH implantation dysfunction model group(COH), low dosage of Bu-Shen-An-Tai recipe group(LOW), middle dosage of Bu-Shen-An-Tai recipe group(MID) and high dosage of Bu-Shen-An-Tai recipe group(HIGH). Then from day 1, the mice in different groups were respectively intragastrically given corresponding treatments at 9:00 a.m. for 5 consecutive days. The concentrations of 17β-estradiol(E) and progesterone(P) were determined by radioimmunoassay(RIA). The ultrastructural changes of ovarian tissues were observed by transmission electron microscope(TEM). The histopathological changes of ovarian tissues were observed by HE staining. The number of atretic follicles and pregnant corpus luteum were also recorded. TUNEL was applied to measure apoptotic cells of ovarian tissues. Western blotting was used to detect the protein expression of apoptosis-related factors like Bax, Bcl-2 and cleaved-caspase-3 in ovarian tissue of mice. The results showed that ovarian weight, the concentrations of Eand P, the number of atretic follicles and pregnant corpus luteum, as well as the apoptosis of granulosa cells were significantly increased in the COH group. The ultrastructures of ovarian tissues in the COH group showed that chromatin in granulosa cells was increased, agglutinated, aggregated or crescent-shaped. The focal cavitation and the typical apoptotic bodies could be seen in granulosa cells in the late stage of apoptosis. After the treatment with different doses of Bu-Shen-An-Tai recipe, the ultrastructural changes of ovarian granulosa cells apoptosis were dramatically improved and even disappeared under TEM. Visible mitochondria and mitochondrial cristae were increased and vacuoles were significantly reduced. The lipid dropltes were shown in a circluar or oval shape. The protein expression levels of Bax and cleaved-caspase-3 were decreased, and the expression of Bcl-2 protein was increased after treatment. It was concluded that Bu-Shen-An-Tai recipe can inhibit the apoptosis of ovarian granulosa cells, probably by up-regulating the protein expression of Bcl-2 and down-regulating Bax and cleaved-caspase-3, which contributes to the formation and maintenance of ovarian corpus luteum. It’s helpful to promote the embryonic implantation, to reduce embryo loss and ultimately to improve the success rate of pregnancy.

Endometrial Mucin-1 and Pinopode in Peri-implantation Phase in Ovarian High Responders during Controlled Ovarian Hyperstimulation Cycles

Objective To investigate effects of ovarian high response on endometrial mucin-1 （MUC1） and pinopode in peri-implantation phase in controlled ovarian hyperstimulation （COH） cycles. Methods Ovarian high response was defined as serum E2 〉 15 000 pmol/L on the day of hCG administration in COH cycle using GnRH agonist and recombinant FSH （n=8）. Healthy and fertile women were used as the natural control （n=10）. Endometrial biopsies were performed on the day of LH＋ 7/hCG＋ 7. Pinopode formation was observed by scanning electron microscope. Expression of MUC1 was detected with quantitative Real-time PCR and immunohistochemistry. Results In high response group, the lumen surface was covered with variant pinopodes and microvillous. The expression of MUC1 mRNA in high response group was lower than that in the natural control （P〈0.05）. Immunostaining for MUC1 protein in glandular and luminal epithelium in high response group was lower than that in the natural control （P〈0. 05）. Conclusion Asynchronized pinopode appearance and lower expression of MUC1 during peri-implantation period were the characteristics of endometrium in high response group, Which may provide a clue of decreased endometrial receptivity in the supraphysiological hormone milieu.

Yiqixue Buganshen Recipe(益气血补肝肾方) Regulates the Expression of Integrin ανβ 3 in the Endometrium of Controlled Ovarian Hyperstimulation Mice

Objective： To observe the effect of Yiqixue Buganshen recipe （益气血补肝肾方, YBR） on the expression of integrin e~ v 13 3 in the endometrium of controlled ovarian hyperstimulation mice. Methods： A total of 180 mice were divided into three groups： model group, treatment group and control group. The treatment and model groups were intraperitoneally injected with gonadotropin-releasing hormone analogue for 7 days; pregnant mare serum gonadotropin was also injected on the 7th day. After 48 h, human chorionic gonadotropin was injected. The control group was injected with an equal volume of saline at the same time. From the start of the experiment, the treatment group was intragastrically administered Jinghouzengzhi Recipe （经后增殖方） and Cuhuangti Recipe （促黄体方）. The model group and the control group were intragastrically administered an equal volume of saline. Real-time reverse transcription polymerase chain reaction and Western blotting were used to detect the mRNA and protein expression of integrin α υ β 3 in mouse endometrium. Results： Integrin α υ β 3 was expressed in mouse endometrium in all groups. Integrin α υ β 3 expression increased gradually along with pregnancy, progressing from pregnant day （Pd） 1. Integrin α υ β 3 expression significantly increased on Pd 4, then began to decrease on Pd 6. Integrin α υ β 3 expression in the treatment group was higher than in the model group, and the difference was statistically significant （P〈0.05）. The difference between the treatment group and the control group was not statistically significant （P〉0.05）. Conclusion： YBR improves endometrial receptivity, and may play an important role in embryonic implantation.

Effects of Er'zhi Tiangui Granule (二至天癸颗粒) on Sequential Expressions of Integrin β3 and Its Ligand Osteopontin in Mouse Endometrium during Controlled Ovarian Hyperstimulation

To investigate the effects of ECzhi Tiangui Granule （二至天癸颗粒, ETG） on sequential expressions of integrin β 3 and its ligand osteopontin in the mouse endometrium during controlled ovarian hyperstimulation （COH） and implantation period. Methods： Seventy-five Mature female Kunming mice were randomly divided into 3 groups, a normal control group, a model group, and a treatment group administrated with ETG for 10 days, 25 in each group. After mated with male mice, every 5 mice were sacrified in each group at the 0, 2nd, 4th, 6th, and 8th days to take their endometrium. In-situ hybridization was used to detect the expressions of integrin β 3 and osteopontin in the endometrium. Results： mRNA expressions of integrin β 3 and osteopontin in the endometrium during implantation period showed similar time sequence rules in the treatment group to those in the normal control group; the peak values of them were a little lower in the treatment group than the normal control without significant differences. In the model group, integrin β 3 mRNA expression was higher at the 2nd day, obviously lower at the 4th and 6th days, and insignificantly lower at the 8th day; and osteopontin expression was remarkably lower at the 4th, 6th, and 8th days, compared with the normal control and the treatment groups （P〈0.05, P〈0.01）. Conclusions： COH might influence the sequential expressions of integrin β 3 and its ligand osteopontin, bring forward the integdn β 3 expression peak, impact on the cooperation of integrin β 3 and osteopontin, so as to damage the endometrial receptivity. ETG could regulate the sequential expressions of integrin β 3 and its ligand osteopontin to improve the mouse endometrial receptivity during COH.

Effects of ovarian response prediction index and follicle-oocyte index on pregnancy outcomes:a retrospective cohort study of 12,218 fresh transfer cycles

Objective:To investigate the potential relationships among the ovarian response prediction index(ORPI),follicle-oocyte index(FOI),and clinical pregnancy rate(CPR)in women undergoing their first in vitro fertilization/intracytoplasmic sperm injectionembryo transfer(IVF/ICSI-ET)fresh cycle transfer.Methods:In this retrospective cohort study,we included 12,218 women who underwent their first IVF/ICSI-ET cycle between December 2014 and January 2021.The primary and secondary outcomes of our study were CPR and cumulative live birth rate(CLBR),respectively.The data were divided into three groups according to the ORPI and FOI tertiles.Multivariate logistic regression analyses,stratification analyses,interaction,restricted cubic splines,and receiver operating characteristic(ROC)curves were constructed to identify the relationships among ORPI,FOI,and CPR.Results:A statistically significant increase in CPR was detected from the lowest to the highest tertile group(ORPI:48.12%,54.07%,and 53.47%,P<0.001;FOI:49.99%,52.95%,and 52.71%,P=0.012).A higher CLBR was observed in the high group(ORPI:38.63%,44.62%,and 44.19%,P<0.001;FOI:41.02%,43.78%,and 42.59%,P=0.039).Multivariate logistic regression analysis revealed no statistically significant differences between ORPI,FOI,and neither CPR(odds ratio[OR][95%confidence interval{CI}],0.99[0.97–1.00]vs.[1.02{0.84–1.24}])nor CLBR(OR[95%CI],0.99[0.97–1.01]vs.0.99[0.81–1.20]).No significant association was found among FOI,ORPI,and CPR,even in the subgroups.Restricted cubic spline analyses indicated the existence of a non-linear relationship across the entire range of FOI and ORPI.The ORPI and FOI variables had poor predictive ability(AUC<0.60)for CPR.Conclusions:Both ORPI and FOI are not reliable predictors of clinical pregnancy or live birth outcomes in fresh ETs.Clinicians and researchers should avoid using FOI and ORPI to assess pregnancy outcomes after fresh ET because of their limited relevance and predictive value.

Association of Thr307Ala and Asn680Ser of Follicle-Stimulating Hormone Receptor Gene Polymorphisms with Gonadotropin Administration during Controlled Ovarian Hyperstimulation

Objective:This study is to investigate the effect of different single-nucleotide polymorphisms of follicle-stimulating hormone receptor(FSHR)gene on gonadotropin(Gn)administration dosage during controlled ovarian hyperstimulation(COH)protocol of in vitro fertilization and embryo transfer.Methods:This retrospective study included 184 Chinese infertile women in Center for Reproduction and Genetics of Suzhou Municipal Hospital from June 2012 to 2014.All of the enrolled patients were homogeneous in some basal characteristics,and they all met the eligibility criteria.Blood tests were conducted on day 3 of menstrual cycle or the day of human chorionic gonadotropin administration for hormonal profile analysis and DNA extraction.DNA sequencing was performed for polymorphism analysis.The participants were classified into threonine(Thr)/Thr,Thr/alanine(Ala),and Ala/Ala groups according to genotype at position 307,and asparagine/asparagine(Asn/Asn),Asn/serine(Ser),and Ser/Ser groups according to genotype at position 680.Logistic regression and correlation analyses were performed to identify the effect of these two polymorphisms on Gn consumption.Results:The frequency of Thr307Ala and Asn680Ser distribution was consistent with Hardy-Weinberg equilibrium(P>0.05).No significant difference was found in age,basal hormone levels for different genotype groups.Logistic regression analysis results revealed that patients with Ser680Ser genotype have a higher risk of requiring a high dose of Gn compared with patients with Asn680Asn genotype,while polymorphism of Thr307 Ala has no such effect.Conclusion:This study suggested that FSHR genotype Asn680Ser would be helpful in determining the dosage of Gn in COH;patients with Ser680Ser genotype may require higher dose of Gn.

Pharmacogenetic study of Asn680Ser and-29A>G in FSHR gene in Chinese women undergoing controlled ovarian hyperstimulation

The outcome of controlled ovarian hyperstimulation(COH)is various and unpredictable.According to previous studies,2 single nucleotide polymorphisms,Asn680Ser and-29A/G,have a pharmacogenetic association with ovarian response to COH.However,studies on the Asn680Ser polymorphism have yielded inconsistent conclusions and only a few studies with small sample sizes have been performed on-29A/G.The association of these 2 polymorphisms with ovarian response remains unclear.The present study evaluated the association of Asn680Ser and-29A/G genotypes with COH.A total of 414 Chinese women undergoing in vitro fertilization-embryo transfer were included.Genotypes for these single nucleotide polymorphisms were identified by high-resolution melting-curve analysis.The value of exogenous follicle-stimulating hormone dosage per oocyte divided by the body surface area(Dosage/Oocyte
Surface)was calculated for each patient as an indicator of ovarian response.The results of statistical analyses showed no association between Asn680Ser genotype and ovarian response.As for-29A/G,heterozygote individuals had more oocytes retrieved(P=0.034).Combinatorial analysis of these 2 single nucleotide polymorphisms showed that genotype A/G-Asn/Asn had lower basal-follicle-stimulating hormone and more oocytes retrieved.Analysis of genotype association with ovarian response also revealed this genotype had a significantly higher risk of developing hyper response(OR=7.86;95%CI:1.31-9.43).To some extent,there were associations between the studied polymorphisms and ovarian response;however,the power of this link is weak and has limited value for clinical prediction.

Effects of Transcutaneous Electrical Acupoint Stimulation on Ovarian Responses and Pregnancy Outcomes in Patients Undergoing IVF-ET:A Randomized Controlled Trial

Objective:To evaluate the influence of different transcutaneous electrical acupoint stimulation(TEAS)modes on ovarian responses and pregnancy outcomes in patients with infertility undergoing in vitro fertilization and embryo transfer(IVF-ET).Methods:Two hundred infertility patients undergoing IVF-ET were divided randomly into experimental groups(TEAS groups:E-Ⅰ,E-Ⅱ,E-Ⅲ,and E-Ⅳ,40 cases each group)and a control group(mock TEAS group,40 patients)using the random number method.The patients in the experimental groups received TEAS treatment of 20,30,40 and 50 m A for the E-Ⅰ,E-Ⅱ,E-Ⅲand E-Ⅳgroups,respectively.The control group received a treatment of 5 m A.TEAS was applied at acupoints of Guanyuan(RN 4),Zhongji(RN 3),Sanyinjiao(SP 6),Zigong(EX-CA 1),and Taixi(KI 13),once a day for 30 min each time for a treatment period of 10-13 d.Treatment effect was assessed using the following indicators:endometrial thickness on the 6 th day of gonadotropin treatment(GN6 day),endometrial thickness on the day on chorionic gonadotropin administration(HCG day),number of ovarian follicles on HCG day,number of ova captured,amount of estrogen required for each harvested ova,number of mature ova divided by the total number of ova,percentage of high-quality embryos,and clinical pregnancy.Results:Endometrial thickness in the experimental groups on the HCG day was significantly better than that of the control group after TEAS stimulation(P=0.01).TEAS exhibited a greater impact on the number of ova captured(P=0.003).However,the effect of TEAS stimulation on the high-quality embryo rate and clinical pregnancy in patients was not statistically significant(P>0.05).Conclusions:TEAS is an effective method in improving the ovarian state.When the stimulus intensity was at 40 m A and above,it could be helpful to improve the patient’s endometrial condition and endometrial receptivity and to retrieve more oocytes.(Trial registration No.Chi CTR-TRC-11001780)

Analysis of related factors affecting cumulative live birth rates of the first ovarian hyperstimulation in vitro fertilization or intracytoplasmic sperm injection cycle: a population-based study from 17,978 women in China

Background:More and more scholars have called for the cumulative live birth rate(CLBR)of a complete ovarian stimulation cycle as a key indicator for assisted reproductive technology.This research aims to study the CLBR of the first ovarian hyperstimulation cycles and analyze the related prognosis factors that might affect the CLBR.Methods:Our retrospective study included first in vitro fertilization or intracytoplasmic sperm injection(IVF/ICSI)cycles performed between January 2013 to December 2014.A total of 17,978 couples of first ovarian hyperstimulation IVF/ICSI cycles were included.The study was followed up for 4 years to observe the CLBR.The multivariable logistic regression model was used to analyze the prognosis factor,P value of<0.05 was considered statistically significant.Results:The cumulative pregnancy rate was 58.14%(10,452/17,978),and the CLBR was 49.66%(8928/17,978).The female age was younger in the live birth group when compared with the non-live birth group(30.81±4.05 vs.33.09±5.13,P<0.001).The average duration of infertility was shorter than the non-live birth cohort(4.22±3.11 vs.5.06±4.08,P<0.001).The preliminary gonadotropin used and the total number of gonadotropin used were lower in the live birth group when compared with the non-live birth group(both P<0.001).Meanwhile,the number of oocytes retrieved and transferrable embryos were both significantly higher in the live birth group(15.35±7.98 vs.11.35±7.60,P<0.001;6.66±5.19 vs.3.62±3.51,P<0.001,respectively).Conclusions:The women's age,body mass index,duration of infertility years,infertility factors,controlled ovarian hyperstimulation protocol,the number of acquired oocytes,and number of transferrable embryos are the prognosis factors that significantly affected the CLBR.

Modified super-long down-regulation protocol improves fertilization and pregnancy in patients with poor ovarian responses

Background The successful end-point of in vitro fertilization （IVF） treatment is for a woman to give live birth. This outcome is based on various factors including adequate number of retrieved eggs. Failure to recruit adequate follicles, from which the eggs are retrieved, is called a ＂poor response＂. How to improve the clinical pregnancy rates of poor responders was one of the tough problems for IVF. Methods The study involved 51 patients who responded poorly to high dose gonadotropin treatment in their previous cycles at our reproductive center, between April 2010 and February 2012. The previous cycle （group A） received routine long protocol; the subsequent cycle （group B） received modified super-long down-regulation protocol. The primary outcome of the study was the number of oocytes fertilized. The increase in the pregnancy rate was the secondary outcome. Differences between the groups were assessed by using Student＇s t test and Z2 test where appropriate. Results The patients＇ average age was （36.64±3.85） years. The mean duration of ovarian stimulation cycles of the group A patients was longer than those of the group B patients. The total dose of follicle-stimulating hormone （FSH） was significantly lower in the subsequent cycle. The peak value of serum estradiol on human chorionic gonadotrophin （hCG） day was lower in group A as compared with group B. The number of metaphase II oocytes recovered was significantly higher in group B. The cleavage rate in group A was significantly lower than in group B, 49 patients in group B reached embryo transfer stage, while 46 patients in group A reached this stage. Patients in group B received significantly more embryos per transfer as compared with group A. More pregnancies and more clinical pregnancies with fetal heart activity were achieved in group B. Conclusions This comparative trial shows that poor responder women undergoing repeated assisted reproduction treatment using modified super-long down-regulation protocol achieve more oocytes, leading to higher fertilization rate, compared to women receiving routine long protocol. Our study also showed that clinical pregnancy rate was significantly improved.

Endometrial Collagen Fibril Hyperplasia is Associated with Implantation Failure in Women Undergoing IVF-ET

Objective To analyse the effects of controlled ovarian hyperstimulation （ COH） on the endometrial expression of collagen fibril （CF） during the peri-implantation period in patients undergoing IVF, and its relation to endometrial receptivity （ER） in repeated implantation failure（RIF）. Methods Peripheral blood and endometrial biopsies were obtained from 45 infertile women on days 5, 7 or 9 after oocytes retrieval or ovulation in a stimulated cycle （SC） and natural cycle （NC） respectively. CF was assayed by transmission electron microscope and quantified by modified Masson dyeing. The outcome of subsequent embryo transfer（ET） was observed. Results Levels of both E2 and progesterone were higher in the peripheral blood in SC than in NC. Also the expression of CF in the stroma in each secretory phase was increased significantly in SC （P 〈O.05）. After embryo transferring, expression levels of CF in the pregnancy group dropped between the mid- and late-secretory phase, but no change in the non-pregnancy group. In the same term, all patients undergone endometrial curettage had higher pregnancy rate than those without. Conclusion Imbalance of production and degradation of endometrial CF in the secretory phase resulting from COH may be the cause of defective ER and implantation failure in some RIF patients. Endometrial curettage may improve implantation rate by inducing appropriate CF hyperplasia and degradation.

Reducing the Trigger Dose of Human Chorionic Gonadotrophin Does Not Affect Final Oocyte Maturation and Subsequently Pregnancy Outcome of Frozen-thawed Embryo Transfer

To compare the efficacy of human chorionic gonadotrophin （hCG） at reduced doses of 2 000 IU and 3 000 IU for moderate or high responders with the dose of 5 000 IU in term of inducing final oocyte maturation for IVF/ICSI and the subsequent pregnancy outcome in frozen-thawed embryo transfer （FET）. Methods In the retrospective cohort study, 2 166patients undergoing IVF/ICSI with moderate or high response were recruited and classified into three groups according to the trigger dose of hCG： 2 000 IU （group A, n=722）, 3 000 IU （group B, n=722） and 5 000 IU （group C, n= 722）. The main outcome was the proportion of mature oocytes retrieved, fertilization rates, clinical pregnancy rates, cumulative pregnancy rates and incidence of ovarian hyperstimulation syndrome （OHSS）. Results No evidence of statistically difference was found in the proportion of mature oocytes retrieved （89.92%, 91.40%, 90.20%, respectively） and fertilization rate （79.8%, 80.07%, 80.51%, respectively） among groups A, B and C. Serum E2 level on the day of hCG injection, the number of mature oocytes retrieved and good-quality embryos in group A were significantly higher than those in group B and group C. Clinical pregnancy rates per transfer cycle （45.95%, 43.97% and 44.25%）, ongoing pregnancy rates （43.17%, 40.91% and 42,53%）, implantation rates （30, 74%, 2Z 78% and 29.86%） and cumulative pregnancy rates per patient （58.31%, 53.6% and 54.85%）A reduced hCG dose of 2 000 IUfor moderate or high responders leads