Characteristics of oocyte fertilization and early embryo development in infertile women with different ovarian reserves in the Changde region, China.

The ovary is a dynamic tissue with many compartments, each possessing distinct and variable biological properties. The functions of the ovary include steroidogenesis and biosynthesis of estrogen, progestin, androgens, and produce mature oocyte to assure fertility. [ 1 , 2 ] During each reproductive cycle consists of folliculogenesis, follicle rupture, luteal formation, and regression. [ 3 ] Ovarian reserve can reflect the fertility potential of women. When the function of ovarian reserve is abnormal, the ability of oocyte production and the quality of oocyte will be seriously affected. [ 4 ] At present, anti-Müllerian hormone (AMH) is widely used as a golden maker for evaluating the ovarian reserve of infertile women before they ondergo treatment. [ 5 ] Studies have shown that higher serum AMH levels are positively associated with a greater number of retrieved oocytes following ovarian stimulation, but these levels are not correlated with oocyte quality or embryo quality. [ 6 , 7 ] A recent study suggested that high serum AMH levels may lead to decreased oocyte developmental competence, which could negatively impact fertilization and early embryonic development in women undergoing in vitro fertilization (IVF) cycles. [ 8 ] Moreover, several studies have failed to find a significant association between serum AMH levels and embryo quality in women undergoing IVF. [ 9 – 11 ] The relationship between serum AMH levels and the quality of oocytes and embryos remains unclear and is currently the subject of ongoing research. Further well-designed clinical studies are needed to establish the true nature of this relationship. So what is the relationship between different ovarian reserve and hormone levels, oocyte quality, embryo quality? Yet, to our best knowledge, variations in AMH levels have been observed across different regions, populations, age groups, and studies, leading to some debate regarding its correlation with fertility. Consequently, establishing geographically based AMH reference intervals is crucial for accurate interpretation of test results. Thus, we conducted a retrospective observational study in order to compare the serum estradiol level on trigger day, number of follicles > 14 mm, fertilization, early embryonic development in women with different ovarian reserve and to characterize the relationship between oocyte fertilization, early embryonic development and the ovarian reserve in the Changde area.

Conceptualization: Donghong Li, Bingru Luo. Formal analysis: Lin Lei, Bingru Luo. Funding acquisition: Donghong Li. Investigation: Lei Liu, Yunhao Wang. Methodology: Yunhao Wang. Project administration: Chao Yang. Software: Bingru Luo. Supervision: Weijing Wang. Validation: Chao Yang. Visualization: Weijing Wang. Writing – original draft: Donghong Li. Writing – review & editing: Donghong Li.

Between January 2018 and July 2022, a total of 325 infertile women who underwent IVF procedure were recruited in the Maternal and Child Health Hospital of Changde City, Department of Reproductive Medicine. The patient’s demographics, baseline characteristics of the cycles, clinical data and parameters related to early embryonic development were extracted from assisted reproduction management information system. The inclusion criteria involve eliminating various factors that can influence embryonic development, with the exception of ovarian reserve. The specific criteria are as follows: infertile women who were undergoing their first fresh IVF cycle; both ovaries were intact, and renal, liver, and hematological parameters were normal; no patient had received surgery or interventional medication within the last 3 months; without malformed uterus, endometriosis III/IV stage or adenomyosis; no male factors; living in Changde area for more than 10 years; had signed written informed consent and was approved by the Ethics Committee of the Maternal and Child Health Hospital of Changde City. The controlled ovarian stimulation treatment included protocols such as long gonadotrophin-releasing hormone agonist protocol, the luteal phase ovarian stimulation protocol, the gonadotrophin-releasing hormone antagonist protocol, and the minimal stimulation protocol. Recombinant follicle-stimulating hormone (FSH; Gonal-F alfa, Merck Serono, Germany) or Urofollitropin (Livzon, Guangdong, China) was initiated on day 2 or 3 of the menstrual cycle. The gonadotrophin starting dose was selected based on age, body mass index (BMI), antral follicle count (AFC), serum AMH level and serum basal FSH levels. The administration typically lasts approximately 8 to 13 days, with the gonadotrophin dose being adjusted dynamically in response to changes in sex hormones levels and AFC. When at least 2 dominant follicles measuring over 18 mm in diameter were observed by ultrasound, a 5000 to 10,000 IU dose of human chorionic gonadotropin (Livzon, Guangdong, China) would be administered by intramuscular injection to trigger the final maturation of the follicles. Oocyte retrieval was carried out under the guidance of transvaginal ultrasound 35 to 36 hours after HCG injection. In conventional IVF cycles, oocytes were inseminated 3 hours after retrieval with a concentration of 1.5 × 10 5 spermatozoa in IVF-plus medium (Vitrolife, Goteborg, Sweden), 18 hours post-insemination, the oocytes were checked for the presence of 2 pronuclei and 2 polar bodies to confirm fertilization. All fertilized oocytes are then placed into G1-plus medium (Vitrolife, Goteborg, Sweden) for culturing. On day 3, the cleavage stage embryos are assessed morphologically using Racowshy scoring system, [ 9 ] with grade I and II embryos being considered good quality. For continued culture, the day 3 embryos are transferred to G2-plus medium (Vitrolife, Goteborg, Sweden), and on day 5 or 6, blastocyst stage embryos are evaluated morphologically and scored using the Gardner grading system, [ 10 ] with a score ≥ BB indicating a top quality blastocyst. All culture conditions are kept at 37°C, with 5% O 2 and 6% CO 2 . All the data obtained in this study was statistically analyzed using SPSS 25.0 (IBM, New York) software. The natural logarithm of serum AMH levels and age were fit to a quadratic regression model. The frequency distribution of AMH test results was assessed for normality, and the reference interval was determined based on the outcomes of the normality test. To demonstrate the multiple of risk factors in individuals with risk factors compared to those without, the odds ratio and its 95% confidence interval were used. After controlling for the influence of age and BMI, the correlation between AMH and biomarkers was analyzed using partial correlation tests. The continuous data are reported as mean ± SD, were analyzed using analyses of the Mann–Whitney test or the independent samples t -test. The categorical data are presented as frequencies and percentages, were assessed using a Chi-square test. P  < .05 was considered statistically significant.

A total of 325 infertile women were recruited in the study. By conducting a power analysis with a significance level of 0.05 and a desired power of 0.80, using SPSS 25.0 software, the calculation shows that a sample size of 325 participants is appropriate. The results of the normality test indicated that the distribution of serum AMH levels across different age groups was normal. Figure 1 illustrates the distribution of the natural logarithm of serum AMH levels plotted against age. The regression equation for the natural logarithm ( y ) of serum AMH levels and age ( x ) is as follows: y  = 6E−05 x 3 − 0.0077 x 2  + 0.2663 x − 2.1663, with an R 2 value of 0.225. The peak serum AMH levels are estimated to occur around 25 years of age, as indicated by the coefficients of the regression equation. Distribution of the natural logarithm of serum AMH levels versus age. The natural logarithm of serum AMH levels for all patients was plotted along the y -axis, while their ages were represented on the x -axis. AMH = anti-Müllerian hormone. Due to the nonuniform distribution of serum AMH levels across different age groups, the reference range is represented by the median value and the 5th to 95th percentiles, encompassing 90% of the data. These are divided as follows: 20 to 24 years, 5.50 (0.62–18.10) ng/mL; 25 to 29 years, 5.13 (0.39–23.00) ng/mL; 30 to 34 years, 4.21 (0.28–20.76) ng/mL; 35 to 39 years, 2.76 (0.15–10.53) ng/mL; 40 to 41 years, 1.30 (0.03–4.01) ng/mL; 45 to 47 years, 0.73 (0.31–1.64) ng/mL (Fig. 2 ). Distribution of serum AMH levels across different age groups. The serum AMH levels of all patients were plotted on the y -axis, with age represented on the x -axis. The data revealed a gradual decline in serum AMH levels as age increased, and AMH levels were significantly lower in individuals aged 45 and older compared to other age groups. AMH = anti-Müllerian hormone. Based on the distribution characteristics of serum AMH levels in different age groups observed in this study, the patients were categorized into 3 groups: low ovarian reserve (LOR, n = 53, AMH  5.5 ng/mL). The population demographics of the women are presented in Table 1 . The average age in LOR was significantly higher than in NOR and HOR ( P  < .05), respectively. And the average age in NOR was significantly higher than in HOR ( P  < .05). The proportion of primary infertility in LOR was significantly lower than in NOR and HOR ( P   .05). The duration of infertility and BMI were not significantly different between the 3 groups ( P  > .05). Comparison of population demographics between different ovarian reserve groups. BMI = body mass index, HOR = high ovarian reserve, LOR = low ovarian reserve, NOR = normal ovarian reserve. Table 2 demonstrates the basal endocrine hormone and AFC between different ovarian reserve groups. The AMH, AFC in LOR were significantly lower than in NOR and HOR ( P  < .001), respectively. And compared with NOR, the AMH, AFC were found to be significantly increased in HOR ( P  < .001). However, the basal FSH in LOR was significantly higher than in NOR and HOR ( P  < .001), respectively. And the basal FSH in NOR was significantly higher than in HOR ( P  < .001). The basal LH in HOR was significantly higher than in LOR and NOR ( P   .05). The basal E 2 was not significantly different between the 3 groups ( P  > .05). Comparison of basal endocrine hormone and antral follicle count between different ovarian reserve groups. AFC = antral follicle count, AMH = anti-Müllerian hormone, FSH = follicle-stimulating hormone, HOR = high ovarian reserve, LH = luteinizing hormone, LOR = low ovarian reserve, NOR = normal ovarian reserve. Table 3 shows that the clinical data during controlled ovarian stimulation between different ovarian reserve groups. The total gonadotrophin in NOR was significantly higher than in LOR and HOR ( P   .05). The serum E 2 level on trigger day, number of follicles > 14 mm in LOR were significantly lower than in NOR and HOR ( P   14 mm were found to be significantly increased in HOR ( P   14 mm were not significantly different between the 3 groups ( P  > .05). Comparison clinical data during controlled ovarian stimulation between different ovarian reserve groups. HOR = high ovarian reserve, LOR = low ovarian reserve, NOR = normal ovarian reserve. Laboratory data between different ovarian reserve groups are presented in Table 4 . The number of oocytes retrieved in LOR was significantly lower than in NOR and HOR ( P  < .001), respectively. And number of oocytes retrieved in NOR was significantly lower than in HOR ( P  < .05). The rate of total fertilization, rate of 2PN fertilization, number of day 3 good quality embryo, number of blastocyst formation and number of top-quality blastocyst in LOR were significantly lower than in NOR and HOR ( P   .05). The rate of day 3 good quality embryo and rate of blastocyst formation were not significantly different between the 3 groups ( P  > .05). Comparison of laboratory data between different ovarian reserve groups. HOR = high ovarian reserve, LOR = low ovarian reserve, NOR = normal ovarian reserve.

Studies have considered that AMH, AFC, basal FSH levels and age factors should be used to evaluate ovarian reserve. [ 11 , 12 ] Of these parameters, AMH levels have been considered good markers of the ovarian reserve during assisted reproductive technology (ART) compared with other predictors. [ 13 – 15 ] Şükür et al [ 5 ] confirmed AMH level was observed to be highly correlated with the AFC, age, and basal FSH level. Therefore, AMH has been widely used as a golden marker for evaluating ovarian reserve before ART treatment. Our investigation uncovered a significant association between the serum AMH levels of infertile women in the Changde region and their age. These levels remain notably high until the age of 30, experience a marked decrease between the ages of 30 and 40, and further descend beyond the age of 45. Moreover, the relationship between serum AMH levels and age reveals a quadratic pattern after undergoing natural logarithm transformation, with the estimated peak occurring at 25 years of age. These findings are consistent with those observed in similar studies. [ 16 , 17 ] Consequently, in this research, patients were categorized into the LOR, NOR, and HOR groups based on their AMH concentrations. The average age and basal FSH levels in the LOR group were notably higher than those in the NOR group, and these parameters in the NOR group were, in turn, significantly higher than in the HOR group. Additionally, the AFC in the LOR group was found to be significantly lower than in the NOR group, whereas a significant increase in AFC was observed in the HOR group compared to the NOR group. It was further confirmed that AMH level exhibited a strong correlated with AFC, age, and basal FSH levels. An intriguing observation from this study is that the prevalence of primary infertility among the LOR was notably lower compared to both the NOR and HOR groups. However, no notable differences were detected the NOR and HOR groups. But, in the Tsai HW’s study, there was no significant difference in the proportion of primary infertility. [ 8 ] This may be due to the number of study samples, regional characteristics and the infertile women in this study who were undergoing their first fresh IVF cycle. Furthermore, our results also showed that the basal LH level of the HOR was significantly higher than that of the other 2 groups, which was consistent with the current domestic research. [ 18 , 19 ] In this study, 29 patients with polycystic ovary syndrome were in the HOR, accounting for 72.50%. As you know, polycystic ovary syndrome patients with hyperandrogenism, androgen will be further converted into estrone in adipose tissue, and the hypothalamus and pituitary will promote the secretion of LH under the continuous action of estrone, resulting in high levels of LH. [ 20 , 21 ] Thus, high levels of LH is a feature of HOR in the study. With the rapid development of ART, controlled ovarian stimulation has been widely used in IVF. Because of the synchronous development of multiple follicles caused by controlled ovarian stimulation, the supraphysiological level of E 2 has a negative impact on endometrial receptivity, oocyte and embryo quality. [ 22 – 24 ] At present, most studies are to analyze the effect of supraphysiological E 2 and other estrogen levels on pregnancy outcome under normal ovarian function conditions, but serum E 2 levels on trigger day are closely related to ovarian reserve and the size and number of follicular during controlled ovarian stimulation. [ 12 , 13 , 20 – 22 ] In our study, we set out to compare the serum estradiol level on the trigger day, the number of follicles > 14 mm, fertilization rates, and early embryonic development among women with varying ovarian reserve. Additionally, we aimed to delineate the relationship between oocyte fertilization, early embryonic development, and different ovarian reserve levels. In this study, the total gonadotrophin in NOR was significantly higher than in LOR and HOR respectively, but the duration of stimulation was not significantly different between the 3 groups. It may be that the HOR reduced the dosage of gonadotrophin to avoid ovarian overstimulation, while the LOR also reduced the dosage of gonadotrophin because of intentional use of low dose. Controlled ovarian stimulation can cause abnormal follicular growth in IVF cycles, resulting in an imbalance in the ratio of E 2 and P levels. [ 9 ] In a retrospective cohort study, suggested that the simultaneous increase of E 2 and P levels or the increase of P levels would have adverse effects on embryo quality and clinical pregnancy rate. [ 11 ] The findings of this study revealed that the serum E 2 levels on the trigger day were significantly lower in the LOR compared to the NOR, and similarly, the NOR had significantly lower levels than the HOR. However, no significant differences in P levels were observed among the 3 groups. Furthermore, the LOR group had a significantly lower number of follicles > 14 mm and retrieved oocytes compared to the NOR group, and similarly, the NOR group had significantly fewer follicles and retrieved oocytes than the HOR group. Consequently, it appears that a higher ovarian reserve correlates with a greater number of follicles > 14 mm and a higher yield of retrieved oocytes. The latest research data show that when the serum E 2 concentration increases, the number of oocytes retrieved and the clinical pregnancy rate will gradually increase, but the fertilization rate will gradually decrease. [ 25 ] However, our study did not show that elevated E 2 level will decrease fertilization rate, This is due to the difference in the subjects between the 2 studies, which were mainly patients with in vitro maturation of follicles, and this study was conducted in patients with IVF. Based on the IVF laboratory data from this study, it is evident that the total fertilization rate and the 2PN fertilization rate in LOR group were significantly lower than those in the NOR and HOR groups. This discrepancy is attributed to the exclusion of male factors as causes of infertility in this study, indicating that the oocyte is the primary factor affecting fertilization. In 2021, Aslih et al [ 26 ] showed that only a fully mature oocyte are recognized and penetrated by sperm, and any abnormalities cytoplasmic and membrane maturation can lead to fertilization failure or abnormalities. Likewise, Silva et al [ 27 ] identified a significant negative correlation between the oocyte maturation rate and the fertilization rate. Drawing on these findings, our study suggests that a LOR may decrease the fertilization rate, as LOR is associated with reduced oocyte maturation, which in turn leads to lower fertilization rates. The number of day 3 good quality embryos and the rate of blastocyst formation in LOR group were significantly lower than those in NOR and HOR groups, respectively. But these indicators showed no significant differences between the NOR and HOR groups. This study demonstrated that LOR had a negative impact on fertilization rates and the number of embryos in women undergoing ART. And compared to NOR, these indicators in HOR did not increase significantly with the increase of ovarian reserve. The reason is that LOR had fewer AFC and number of oocytes retrieval, lower fertilization rate, which further leads to fewer embryos. This study supports the notion that the accumulation of oocytes and embryos via multiple oocyte retrieval times is an effective treatment strategy for women with LOR undergoing ART. However, the rate of day 3 good quality embryo and blastocyst formation were not significantly different across the 3 groups. The results further suggest that ovarian reserve dose not play a predictive role in the in vitro development of embryos. In conclusion, our study reveals that within the Changde region, there is a significant correlation between higher ovarian reserve and increased serum E 2 levels, as well as an increased number of follicles measuring over 14 mm in diameter on the day of ovulation triggering in infertile women. Conversely, the ratio of E 2 to the number of these larger follicles did not show any correlation with the status of ovarian reserve. Moreover, we noted that a decrease in ovarian reserve tended to lead to a lower fertilization rate of oocytes, while the early stages of embryo development appear to be independent of the ovarian reserve level. It is important to recognize that, although our sample size was sufficient for statistical analysis, the relatively small number of participants does limit the scope of our study. Additionally, the reliance on clinical data and parameters related to early embryonic development from a single center represents another limitation. Consequently, the findings of this investigation are preliminary and require validation through a larger, multicenter cohort study to improve the generalizability of our results.