Evaluation of Serum Anti-Müllerian Hormone (AMH) Values for 28,016 Bulgarian Women: Prognostic Statistical Model of Age Specific AMH Declining

The present study aims to establish a relationship between serum AMH levels and age in a large group of women living in Bulgaria, as well as to establish reference age-specific AMH levels in women that would serve as an initial estimate of ovarian age. A total of 28,016 women on the territory of the Republic of Bulgaria were tested for serum AMH levels with a median age of 37.0 years (interquartile range 32.0 to 41.0). For women aged 20 - 29 years, the Bulgarian population has relatively high median levels of AMH, similar to women of Asian origin. For women aged 30 - 34 years, our results are comparable to those of women living in Western Europe. For women aged 35 - 39 years, our results are comparable to those of women living in the territory of India and Kenya. For women aged 40 - 44 years, our results were lower than those for women from the Western European and Chinese populations, close to the Indian and higher than Korean and Kenya populations, respectively. Our results for women of Bulgarian origin are also comparable to US Latina women at age 30, 35 and 40 ages. On the base on constructed a statistical model to predicting the decline in AMH levels at different ages, we found non-linear structure of AMH decline for the low AMH 3.5) the dependence of the decline of AMH on age was confirmed as linear. In conclusion, we evaluated the serum level of AMH in Bulgarian women and established age-specific AMH percentile reference values based on a large representative sample. We have developed a prognostic statistical model that can facilitate the application of AMH in clinical practice and the prediction of reproductive capacity and population health.

Endocrine and ovarian responses in water buffalo cows immunized against inhibin and subjected to the Ovsynch protocol

The aim of this study was to investigate the feasibility of stimulating ovarian fol icle development in order to improve fertility in water buffalo cows by immunization against inhibin. The experiment was carried out in early summer (May) and included 24 multi-parity crossbred Murrah-Swamp buffaloes that were divided into immunized (n=11) and control (n=13) groups. Each immunized cow was administered with a 2-mL immunogen of mineral oil adjuvant containing 2 mg of recombinant inhibinα-subunit fusion protein. The controls were treated with the adjuvant only. Al animals received Ovsynch protocol treatment, starting on the day of the antigen administration, and they were artiifcial y inseminated upon behavioral estrus. As a result, al of the immunized buffaloes generated antibodies against inhibin during the experimental period and had higher plasma concentrations of fol icle-stimulating hormone (FSH), activin, and estradiol (E2) related to estrous expression. A higher proportion of immunized animals expressed estrus behavior than did the controls (72%vs. 30%, P<0.05). On aver-age, inhibin-immunized buffaloes had signiifcantly more large fol icles (≥9 mm in diameter) than the controls (mean±SEM;1.2±0.1 vs. 0.84±0.1, respectively;P<0.05) and a slightly higher mean total number of fol icles (≥2 mm;11.4±0.7 vs. 9.0±1.1, respectively;P=0.09) and smal (2–4 mm) fol icles (8.81±0.6 vs. 6.84±1.0, respectively;P=0.12). A higher percentage of cows ovulated in the immunized group than in the control group (91%(10/11) vs. 54%(7/13), respectively;P<0.05). Moreover, inhibin-immunized cows had slightly larger corpus luteum (CL) than the controls 9 days after ovulation and signiifcantly higher (P<0.01) post-ovulation peak plasma progesterone (P4) concentrations. Immunization against inhibin also mar-ginal y increased the conception rate 42 days after insemination (45.8%vs. 15.4%;P>0.05). These results demonstrate that immunization against inhibin, coupled with the treatment with the Ovsynch protocol, can constitute a new technique to increase fertility in water buffalo cows.

Clinical Value of Serum Anti-Mullerian Hormone and Inhibin B in Prediction of Ovarian Response in Patients with Polycystic Ovary Syndrome

The present study aimed to investigate the clinical value of serum anti-mullerian hormone（AMH） and inhibin B（INHB） in predicting the ovarian response of patients with polycystic ovary syndrome（PCOS）. A total of 120 PCOS patients were enrolled and divided into three groups in terms of the ovarian response： a low-response group（n=36）, a normal-response group（n=44）, and a high-response group（n=40）. The serum AMH and INHB levels were measured by enzyme-linked immunosorbent assay（ELISA）. The follicle stimulating hormone（FSH）, luteinizing hormone（LH）, and estradiol（E2） levels were determined by chemiluminescence microparticle immunoassay. The correlation of the serum AMH and INHB levels with other indicators was analyzed. A receiver operating characteristic（ROC） curve was established to analyze the prediction of ovarian response by AMH and INHB. The results showed that there were significant differences in age, body mass index（BMI）, FSH, total gonadotropin-releasing hormone（Gn RH）, LH, E2, and antral follicle counts（AFCs） between the groups（P〈0.05）. The serum AMH and INHB levels were increased significantly with the ovarian response of PCOS patients increasing（P〈0.05）. The serum AMH and INHB levels were negatively correlated with the age, BMI, FSH level, Gn, and E2 levels（P〈0.05）. They were positively correlated with the LH levels and AFCs（P〈0.05）. ROC curve analysis of serum AMH and INHB in prediction of a low ovarian response showed that the area under the ROC curve（AUC） value of the serum AMH level was 0.817, with a cut-off value of 1.29 ng/m L. The sensitivity and specificity were 71.2% and 79.6%, respectively. The AUC value of serum INHB was 0.674, with a cut-off value of 38.65 ng/m L, and the sensitivity and specificity were 50.7% and 74.5%, respectively. ROC curve analysis showed when the serum AMH and INHB levels were used to predict a high ovarian response, the AUC value of the serum AMH level was 0.742, with a cut-off value of 2.84 ng/m L, and the sensitivity and specificity were 72.7% and 65.9%, respectively; the AUC value of the serum INHB level was 0.551 with a cut-off of 45.76 ng/m L, and the sensitivity and specificity were 76.3% and 40.2%, respectively. It was suggested the serum AMH and INHB levels have high clinical value in predicting the ovarian response of PCOS patients.

Anti-Müllerian hormone and antral follicle count predict ovarian response in women less than 45 years following GnRH antagonist multiple-dose protocol

Objective:To speculate which of the following parameters:antral follicle count(AFC),anti-Müllerian hormone(AMH),follicle-stimulating hormone(FSH)and age can be used as a predictor of ovarian response to gonadotropin-releasing hormone(GnRH)antagonist stimulation multiple-dose protocol in women under 45 years,and to determine the cutoff value of these parameters and their correlations for predicting low and high ovarian response.Methods:This prospective study included 462 women with the mean age of(29.3±6.5)years.All women were subjected to the GnRH antagonist stimulation multiple-dose protocol.On the second day of the menstrual cycle,ultrasonography was conducted to determine AFC in both ovaries.Peripheral blood samples were collected to evaluate the level of estradiol,FSH,luteinizing hormone,prolactin,thyroid-stimulating hormone,and AMH.The women were divided into three groups:low response(AHH<1 ng/mL,n=173),normal response(AMH=1.0-3.5 ng/mL,n=175),and high response(AMH>3.5 ng/mL,n=114).Results:A significant decrease was found in the age and FSH level in the high response group compared to other groups(P<0.001).Conversely,a significant increase was shown in AMH,estradiol on human chorionic gonadotropin(hCG)day,AFC,mature oocytes,fertilized oocytes,and embryos transferred in the high response group compared to the other two groups(P<0.001).The receiver operating characteristic(ROC)curves demonstrated that AFC and AMH had the highest accuracy,followed by basal FSH level and age in the prediction of low ovarian reserves(P<0.001)with cutoff values of≤4.50 and≤0.95 for AFC and AMH,respectively.Moreover,the ROC analysis showed that AFC had the highest accuracy,followed by AMH level and age in the prediction of high ovarian reserves with a cutoff value of≥14.50,≥3.63,and≤27.50 years,respectively(P<0.01).A significant decrease was observed in women's age,estradiol level,and oocyte fertilization rate in pregnant women compared to non-pregnant women(P<0.001).Additionally,significant negative correlations were found between the AFC,the number of mature oocytes,fertilized oocytes,embryos transferred,and the age of pregnant women(P<0.001).Conclusions:AFC and AMH predict low and high ovarian response to GnRH antagonist stimulation multiple-dose protocol in women under 45 years.

Effects and safety of Ding Kun Dan on IVF/ICSI-ET outcomes in patients with predicted poor ovarian response:A multicenter randomized clinical trial

Objective:To evaluate whether Ding Kun Dan(DKD)can improve the vitro fertilization/intracytoplasmic sperm injection and embryo transfer(IVF/ICSI-ET)outcomes in patients with predicted poor ovarian response(POR)safely and effectively.Methods:Prospective,multicenter,randomized controlled trial;A total of 278 POR patients were randomized in DKD group or immediate treatment group.Both groups received IVF or ICSI as a standard treatment while in the DKD group,DKD was administrated for 3 months before the IVF/ICSI cycle.The primary outcome was the ongoing pregnancy rate.The secondary outcomes include clinical pregnancy rate,biochemical pregnancy rate,total gonadotropin(Gn)dosage and duration,estradiol(E2)and progesterone(P)levels on human chorionic gonadotropin(hCG)trigger day,cycle cancellation rate,number of oocytes retrieved,high-quality embryo rate and any adverse events.Results:Compared to the immediate IVF treatment group,oral administration of DKD for 3 months before IVF led to a significant increase in ongoing pregnancy rate(30.0%v.s.17.6%,P<0.05),biochemical pregnancy rate(39.2%vs.25.2%,P<0.05),clinical pregnancy rate(36.7%vs.22.7%,P<0.05)and high-quality embryo rate(40.8%vs.32.4%,P<0.05),and a significant decrease in Gn duration(P<0.05).However,no significant differences were found in total dosage of Gn,number of retrieved oocytes,cycle cancellation rate,E2 level and P level on hCG trigger days(P>0.05).No serious adverse events occurred during the intervention period in either group.Conclusion:DKD is a safe and effective intervention to improve the IVF/ICSI-ET outcomes in patients with POR.

Association between body mass index and in vitro fertilization/intra-cytoplasmic sperm injection outcomes:An analysis of 15,124 normal ovarian responders in China

Background:High body mass index(BMI)results in decreased fecundity,and women with high BMI have reduced rates of clinical pregnancy and live birth in in vitro fertilization/intra-cytoplasmic sperm injection(IVF/ICSI).Meanwhile,ovarian responses show great heterogeneity in patients with a high BMI.This study aimed to analyze the effects of a high BMI on IVF/ICSI outcomes in the Chinese female with normal ovarian response.Methods:We performed a retrospective cohort study comprising 15,124 patients from the medical record system of the Reproductive Center of Peking University Third Hospital,with 3530(23.3%)in the overweight group and 1380(9.1%)in the obese group,who had a normal ovarian response(5-15 oocytes retrieved)and underwent fresh embryo transfer(ET)cycles from January 2017 to December 2018,followed by linked frozen-thawed embryo transfer(FET)cycles from January 2017 to December 2020.Cumulative live birth rate(CLBR)was used as the primary outcome.Furthermore,a generalized additive model was applied to visually illustrate the curvilinear relationship between BMI and the outcomes.We used a decision tree to identify the specific population where high BMI had the greatest effect on IVF/ICSI outcomes.Results:High BMI was associated with poor IVF/ICSI outcomes,both in cumulative cycles and in separate fresh ET or FET cycles.In cumulative cycles,compared with the normal weight group,obesity was correlated with a lower positive pregnancy test rate(adjusted odds ratio[aOR]:0.809,95%confidence interval[CI]:0.682-0.960),lower clinical pregnancy rate(aOR:0.766,95%CI:0.646-0.907),lower live birth rate(aOR:0.706,95%CI:0.595-0.838),higher cesarean section rate(aOR:2.066,95%CI:1.533-2.785),and higher rate of large for gestational age(aOR:2.273,95%CI:1.547-3.341).In the generalized additive model,we found that CLBR declined with increasing BMI,with 24 kg/m^(2)as an inflection point.In the decision tree,BMI only made a difference in the population aged≤34.5 years,with anti-Mullerian hormone>1.395 ng/mL,and the first time for IVF.Conclusions:High BMI was related to poor IVF/ICSI outcomes in women with a normal ovarian response,and CLBR declined with increasing BMI,partly due to suppressed endometrial receptivity.A high BMI had the most negative effect on young women with anticipated positive prognoses.

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.

Dynamic measurements of serum inhibin B and estradiol: a predictive evaluation of ovarian response to gonadotrophin stimulation in the early stage of IVF treatment

Objective： We dynamically measured serum inhibin B and estradiol in the early stage of hormonal stimulation to predict the ovarian response in in vitro fertilization （IVF） treatment. Methods： A total of 57 patients （〈40 years of age） who underwent the first cycle of long protocol IVF or introcytoplasmic sperm injection （ICSI） treatment were included. Serum inhibin B, estradiol, follicle stimulating hormone （FSH） and luteinizing hormone （LH） levels were measured four times： （1） on Day 3 of the menstrual cycle （basal）; （2） on the day before the first administration ofgonadotrophin （Gn） （Day 0）; （3） on Day 1 of Gn therapy; and （4） on Day 5 of Gn therapy. Comparisons of these measurements with ovarian responses and pregnancy outcomes were made and analyzed statistically. Results： （1） On Day 1 and Day 5 of recombinant FSH （rFSH） stimulation, ovarian response, i.e., numbers of follicles, oocytes, fertilized oocytes, and embryos, had a positive correlation （rs=0.46-0.61, P 0.000） with raised inhibin B and estradiol concentrations, but a negative correlation （rs=-0.67--0.38, P=0.000 or P〈0.01） with total rFSH dose and total days ofrFSH stimulation. （2） No significant variation （P〉0.05） between the pregnant and non-pregnant groups on the basis of mean age or on all hormone concentrations at four times of the IVF cycle was observed. However, all the seven patients aged 〉35 years did not reach pregnancy. Conclusions： （1） Serum inhibin B and estradiol concentrations obtained shortly after Gn therapy may offer an accurate and early prediction of ovarian response; （2） Low levels of serum inhibin B and estradiol obtained shortly after Gn stimulation indicate the need for a longer period of Gn treatment and a higher daily dosage; （3） No obvious pregnancy difference among patients of age 〈35 years was found; however, IVF pregnancy outcome is significantly lower in women of age 〉35 years.

A preliminary observation on efficacy of growth hormone on poor ovarian responders in IVF/ICSI cycles

Objective To determine efficacy of gonadotropin （Gn） co-treated with growth hormone （GH） on poor ovarian response （POR） patients undergoing in vitro fertiliza- tion/intracytoplasmic sperm injection （1VF/ICSI）. Methods A total of 58 IVF/ICSI-embryo transfer （ET） cycles in POR patients were retrospectively identified. The POR criteria were defined by Bologna consensus. The cycles were divided into two groups： Gn co-treated with GH group （GH~ group, 25 cycles） and Gn only group （GH- group, 33 cycles）. Results of lVF/ICSI for these two groups were compared. Results The number of oocyte retrieved and the normal fertilization rate in the two groups were no difference （P〉O.05）. High-quality embryo rate and implantation rate in the GH＋ group were 39.6% and 38.5%, respectively, which were slightly higher than those in the GH group （35.9% and 25.0%）, but no difference was found on these two parameters （P〉0. 05）. Conclusion GH, to some extent, can improve the quality of embryo and the implantation rate. Whether GH co-therapy has a definite role in improving the outcome of POR patients still needs further evaluation.

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.

Increased Oocyte Production after Acupuncture Treatment during Superovulation Process in Mice

Objective To investigate whether acupuncture treatment during superovulation process improves ovarian response and increases egg production. Methods ICR female mice aged 12 -5 weeks were divided into control group, anaesthesia group and acupuncture group. Female mice were injected intraperitoneally with pregnant mare＇s serum gonadotropin （PMSG）, followed by human chorionic gonadotropin （hCG） injection 56 h later. Anaesthesia group and acupuncture group were anaesthetized three times by injecting 10% nembutal solution according to 7.5-10.0μl/g weight. Acupuncture group was treated three times with puncture Sanyinjiao Points（SP6） under anaesthesia. After 17 h of hCG injection, eggs were recovered and ovaries were isolated. Matured eggs were counted, ovarian histology and expression of follicle stimulating hormone receptor（FSH-R） in ovary were analyzed. Results Acupuncture treatment statistically increased the number of ovulated eggs. Histological section showed that some matured follicles left in the ovaries of control and anaesthesia groups after ovulation. On the contrary, all matured follicles ruptured and converted into corpus lutea in Acupuncture group. Expression of FSH-R in ovary was decreased in acupuncture group compared with those of the two others. Conclusion Acupuncture treatment during superovulation process improves ovarian response so as to increase egg production. The positive effect of acupuncture may be associated with regulated FSH-R expression.

Improved Prediction of In Vitro Fertilization Clinical Outcomes by Combining Ultrasound, Serum Hormones and Body Mass Index

Objective To identify efficient predictors of clinical outcomes of in vitro fertilization （IVF）. Methods Two hundred and one women undergoing their first IVF cycle were measured for antral follicle count （AFC） and endometrial thickness via ultrasound. On day 2 or 3 of the menstrual cycle and the day of human chorionic gonadotropin administration, serum levels of follicle-stimulating hormone （FSH）, luteinizing hormone （LH）, estradiol, and progesterone were determined. The difference of ovarian response and clinical pregnancy rates were measured. Results Basal FSH was the single best predictor of ovarian response in women 〈35 years old before the initiation of treatment, with an area under the receiver operating characteristic curve （A UC） of 0. 752. For women ≥ 35 years old, none of parameters was a single good predictor of lVF pregnancy. For women older than 35 years, adding body mass index （BMI） with AFC and basal FSH improved prediction of ovarian response （A UC=0. 859）. Prediction of clinical pregnancy for women ≥ 35 years was improved either by combining only AFC and basal FSH （AUC=0. 757） or adding BMI to the combined AFC and FSH （AUC=0. 722）. Conclusion Combining AFC, basal FSH, and BMI was the best predictor of ovarian response and clinical pregnancy after IVF in women ≥ 35 years.

Anti-Müllerian hormone, antral follicle count and folliclestimulating hormone for predicting the number of oocytes retrieved in IVF/ICSI cycles

Objective To evaluate the efficacy of anti-Müllerian hormone（AMH）, antral follicle count（AFC） and follicle-stimulating hormone（FSH） for predicting the number of oocytes retrieved in in-vitro fertilization/intracytoplasmic sperm injection（IVF/ICSI）cycles.Methods In this retrospective study, a total of 122 infertile women were divided into two groups： group A, 〈35 years（n=71）; group B, ≥35 years（n=51）. AMH and FSH were determined on 2-5 d of the early menstrual cycle. AFC was tested on the second day of the menstrual cycle before the start of stimulation.Results Group B had higher FSH levels compared with group A（8.2±3.5 IU/L vs 6.8±2.4 IU/L, P〈0.05）. However, levels of AMH and AFC in group B were lower than those of group A（AMH： 4.2±3.5 μg/L vs 2.7±2.7 μg/L; AFC： 9.0±3.9 vs 5.4±3.3, P〈0.05）. The number of oocyte retrieved in the two groups was not significantly different（11.5±6.8 vs 9.6±6.9, P〉0.05）. The level of AMH was more strongly correlated with the number of oocytes retrieved than that of AFC or FSH level. The strengths of the correlation degrees were AMH level, AFC, and FSH level in turn（r=0.600, 0.511,-0.369）.Conclusion AMH would be a useful predictor for ovarian response.