The association between the number of oocytes retrieved and cumulative live birth rate in different female age strata

To evaluate the association between the number of oocytes retrieved and cumulative live birth rate (CLBR) in different female age strata. 17,931 women undergoing their first IVF/ICSI-ET cycle in the Sir Run Run Shaw Hospital of Zhejiang University were grouped by age (A: ≤ 35 years; B: ≥ 36 years) as well as the number of oocytes retrieved (a: ≤ 5; b:6–9; c:10–14; d: ≥ 15). Multivariate regression analysis was performed to assess the OR of CLBR for the variable ‘age’ and ‘number of oocytes retrieved’. The group ≥ 36 years exhibited lower cumulative pregnancy rates (CPRs) and cumulative live birth rates (CLBRs), which are proportional to the number of oocytes retrieved but opposite to increasing age. Multivariate logistic regression analysis revealed that the age and number of oocytes retrieved remain significant independent predictive factors (P < 0.001). Age and number of oocytes retrieved are two independent factors affecting the CLBR. The discrepancy of the minimum number of oocytes retrieved for patients with different ages to achieve ideal CLBR is instructive for clinical practice. The practice of controlling the stimulation dose is feasible for patients ≤ 35 years who can achieve over 60% CLBR once the number of oocytes obtained is more than 6. However, additional stimulation cycles and accumulation of embryos are necessary for elderly group especially those ≥ 38 years old who need more than 14 oocytes to obtain higher live birth rate.

Controlled ovarian stimulation is an important part of assisted reproductive technologies (ARTs) 1,2 .With the substantial advancement of ART in both clinical and laboratory, the number of freeze-thaw embryo transfer cycle have increased along with the pregnancy rate 3,4 .Clinical or ongoing pregnancy rate, embryo implantation rate and live birth rate per transfer have consuetudinary been used to evaluate the successful rate of ART while the cumulative live birth rate (CLBR) appears to be a better one currently.The CLBR, which mainly refers to the first live birth after using all embryos for an integrated cycle (including fresh and subsequent freeze-thaw cycles), varies synchronously with the number of oocytes retrieved 5,6 .Previous studies have manifested that CLBR can even reach as high as 70% once the oocytes retrieved are more than 25 and with no obvious plateau 7 .Despite the benefit of ART in achieving adequate multiple follicles and providing considerable embryos for selection, the optimal number of oocytes need to retrieve and corresponding probability of live birth in elderly women after each ovulation induction therapy have not been determined.
As the two-child policy fully issued in China since 2015, the demand for ART treatment has been growing steadily among women of advanced reproductive age 8 .An additional 90 million couples in China intend to have a second child according to official estimation, of whom 60% are reported to be older than 35 years, and 50% will be at least 40 years old 8 .Female age becomes the main challenge impacting the success rate of ART.Women ≥ 35 years old have been verified to present a significant decline in the pregnancy rates and live birth rates [9][10][11] .As is known to all, ovarian function and oocyte quality are all inextricably linked with female age, with the group ≥ 35 years of age certainly experiencing less chance to achieve high number of oocytes retrieved as well as higher risk of developing aneuploidy oocytes and embryos [12][13][14] .Therefore, challenges that often encountered in clinical practice are as follows: How many oocytes are sufficient to achieve a live birth for women of advanced

Characteristics of basic clinical data.
The baseline data of 17,931 women undergoing their first IVF/ICSI cycle were presented in Table1.Comparisons which mainly between patients ≤ 35 years of age and ≥ 36 years of age revealed significant discrepancies in levels of day3 basal FSH, LH and E2, duration of infertility and infertility factors.The group (age ≥ 36 years of age) had significant longer duration of infertility (5.0 ± 4.0 vs 3.3 ± 2.3, p < 0.001) but with higher basal FSH (8.4 ± 3.4 vs 7.1 ± 2.6, p < 0.001) and E2(43.0 ± 27.4 vs 41.0 ± 28.2, p < 0.001) levels.In addition, the ovarian stimulation characteristics reported in Table 2 showed that stimulation protocol, duration of stimulation or insemination type all varied strikingly in two age groups.The cumulative pregnancy rate and live birth rate..The cumulative pregnancy rate (CPR) and cumulative live birth rate (CLBR) after one complete IVF/ICSI cycle including fresh and subsequent frozenthaw cycles from first oocyte retrieval, which were significantly lower in the aged ≥ 36 years group compared with the aged ≤ 35 years group (CPR: 45.0% vs. 74.9%;OR: 3.639; 95% CI: 3.367-3.932;P < 0.001; CLBR: 34.4% vs. 68.3%;OR: 4.125 95% CI: 3.811-4.466;P < 0.001; respectively), as shown in Table 3.Moreover, statistical analysis was performed in different age strata after grouped according to the number of oocytes retrieved.As expected, the CLBR, taking into account fresh and frozen cycles, increased strikingly parallel with the number of oocytes retrieved in specific age strata but decreased with increasing age for a given number of retrieved oocytes (Fig. 1a,b,c).In addition, we performed ROC curves to offer more precise data regarding the optimal number of oocytes required to achieve a cumulative live birth (CLB) between young (≤ 35 years group) and older participants (≥ 36 years of age) (Fig. 2a,b).

Discussion
Results of this study indicated that the CLBR was proportional to the number of retrieved oocytes, and this steady trend was evident in all female age strata.Notably, female age has a negative effect on the relationship between the number of retrieved oocytes and CLBR, with lower CLBR accompanied by increasing age for a given number of retrieved oocytes.Furthermore, older women exhibited a significantly lower LBR and CLBR in their fresh or frozen embryo transfer cycles compared with younger women.The CLBR, which decreased strikingly from 68.3% in the group ≤ 35 years of age to 34.4% in the group ≥ 36 years of age, can even drop to less than 20% in the group ≥ 40 years of age.
Our research is completely consistent with two recent studies investigating the effect of retrieved oocytes numbers as well as frozen embryo transfer on the CLBR 1,7 .Polyzos et al. confirmed that the CLBR could reach as high as 70% once the number of retrieved oocytes was more than 25 7 .However, according to our analysis, the CLBR closing to 80% is possible as long as the retrieved oocytes numbers were more than 25, which can be explained by the discrepancies that CLBs are mainly from freeze-thaw embryo transfers in our study, accounting for 88.9% in the group ≤ 35 years of age and 87.8% in the group ≥ 36 years of age.Since vitrification was introduced into our reproductive center, 97% of the survival rate in embryo cryopreservation, nearly 60% of the pregnancy rate in freezing-thawing embryo transfer, 35% of the embryo implantation rate and 29% of the live birth rate per embryo were achieved among approximately 6,000 freezing-thawing cycles every year 15 .
In the past decades, researches have been proposed to explore the optimal number of oocytes need to retrieve during controlled ovarian stimulation although verdicts are controversial.Rubio C and Verberg M F, et al. demonstrated that ovarian hyperreactivity may not only increase the risk of chromosomal abnormalities in embryos but reduce the quality of embryos and the rate of high-quality embryos 16,17 .However, other prospective studies concluded that the live birth rate (LBR) is impervious to the ovarian hyperreactivity despite its negative role in fertility rate and the CLBR accompanied by the number of available embryos remains to increase, which can also be reflected in our investigation.Based on our analysis, the fertilization rate and high-quality embryo rate of IVF/ICSI in the group ≤ 35 years of age were significantly lower than those ≥ 36 years of age while the clinical pregnancy rate, embryo implantation rate, live birth rate and CLBR were higher.Consequently, for elderly patients, embryos accumulation by multiple ovulation induction can raise the rate of normal karyotype embryo, which then increase the chance of a live birth accordingly.Notably, the stratification of participants primarily relies on the risk of aneuploidy in our study, which is known to increase significantly beyond 35-36 years.Future researches can further speculate the potential correlation between age, the number of oocytes retrieved and the likelihood of ploidy particularly in the absence of preimplantation genetic testing for aneuploidy (PGT-A), which may also be responsible for the decline in cumulative live birth rate after the age of 35.
As is well known, the number of oocytes retrieved and the age of patients make up the most important independent factors affecting the CLBR.Consistent with other studies, we confirmed the independent property of them after controlling confounding factors including "fertilization rate", "high-quality embryo rate" and "fertilization type".The correlation analysis between the number of oocytes retrieved and the CLBR in each age stratum revealed that the number of oocytes retrieved had potential predictive value in the CLBR.For patients ≤ 35 years of age, the CLBR can reach more than 60% as long as the number of oocytes retrieved is between 6 and 8; but patients aged 36-37 years of age needed 9-13 oocytes to achieve a CLBR aboving 60%.What's worse, for patients aged 38-39 years of age, the CLBR of more than 60% can be accomplished only when the oocytes numbers are greater than 14 while the CLBR in patients ≥ 40 years of age can hardly reach 60% even if the number is more than 14.
The most significant limitation of this study lies in its retrospective design, which is associated with inherent bias in the research itself that affects our outcomes.In addition, the long study period and clinical operational errors may inevitably be confounding factors influencing experimental results.Although we tried our best to minimize selection bias and control for knowable confounders through strict inclusion criteria, other undiscovered confounders cannot be completely eliminated.At the same time, during the period from 2013 to 2017 of the case data collected in this study, our reproduative center had not included AMH test and the antral follicle count as the routine testing program and we can not obtain these values, so we could only use basic endocrine value to represent the difference between ovarian function, which will be further improved and optimized in our subsequent study.In addition, the protocol of ovulation induction in fresh embryo transfer cycles can hardly be reconcile which varied in different groups, the older group mainly adopted the long scheme while the younger tend to receive the short scheme, leading to the discrepancy in the number of oocytes retrieved, and thus inevitably affecting the CLBR.Furthermore, the endometrial preparation pattern of freezing-thawing cycles is also inconsistent in the whole population.However, it is worth clarifying that no matter which preparation has an equivalent effect on the clinical outcome of freeze-thawing embryos 18 .

Conclusion
The age and number of oocytes retrieved are two independent vital factors affecting cumulative live birth rate.The cumulative live birth rate in patients ≤ 35 years old can achieve over 60% as long as the number of oocytes obtained is more than 6, indicating that practice of controlling the stimulation dose to ensure the live birth rate and reduce the occurrence of OHSS is feasible for them.However, the elderly patients, especially those ≥ 38 years old, need to retrieve more than 14 oocytes to obtain higher live birth rate.Therefore, it is of great necessity to receive additional stimulation cycles and accumulation of embryos.

Figure 1 .a
Figure 1. a The association between CLBR and the number of oocytes.b The association between CLBR and the age.c The association between CLBR and the number of oocytes in different female age strata.

Figure 2 .
Figure 2. a ROC curve in women ≤ 35 years of age.b ROC curve in women ≥ 36 years of age.
reproductive age?How many IVF/ICSI cycles are required for the elderly women to increase the probability of live birth?The purpose of this research was to reveal the relationship between the number of oocytes retrieved and the cumulative live birth rate (CLBR) in different female age strata after categorizing patients by age.

Table 1 .
Characteristics of basic clinical data.

years of age ≥ 36 years of age Statistic P value OR (95% CI)
To control possible confounding variables that might have changed over the study period, a multivariate logistic regression analysis was applied to estimate the OR with SE and 95% CI of the CLBR for variable "age" and "number of oocytes Vol.:(0123456789) Scientific Reports | (2023) 13:14516 | https://doi.org/10.1038/s41598-023-41842-7www.nature.com/scientificreports/Multivariate logistic regression analysis for the cumulative live birth rate.. retrieved".As shown in Table 4, after adjustment for relevant confounders including duration of stimulation, duration of infertility, endometrial thickness, insemination type, fertilization rate, and good quality embryo rate, the age and number of oocytes retrieved remained the independent predictive factors (P < 0.001).The OR of CLBR dropped from 3.695 (95%CI: 2.960-4.611) in the aged ≤ 35 years group to 3.164 (95%CI: 2.563-3.906)and 2.230 (95%CI: 1.785-2.786) in the aged 36-37 years and 38-39 years group respectively.In addition, the fertilization rate, good embryo quality rate and insemination method also have prominent effect on CLBR.

Table 2 .
Clinical outcomes of fresh cycle and freeze-thaw cycle.

Table 3 .
Comparions with the cumulative pregnancy and live birth rate. ≤

Table 4 .
Multivariate logistic regression analysis for the cumulative live birth rate.