Use of Cumulative Live Birth Rate per Total Number of Embryos to Calculate the Success of IVF in Consecutive IVF Cycles in Women Aged ≥35 Years

Objective The use of cumulative live birth rate (CLBR) per ovarian stimulation cycle is proving to be an accurate method to calculate the success of IVF; however, the cycle outcome is closely associated with the number of embryos transferred (ET). Our aim was to report CLBR after IVF according to the number of embryos required to achieve a live birth in women aged ≥35 years, considering age, body mass index (BMI), and ethnicity. Methods We conducted a retrospective cohort study including 1344 patients who underwent IVF between January 2013 and June 2016 at the First Affiliated Hospital of Xinjiang Medical University. The cumulative probability of live birth for each couple was estimated using the Kaplan–Meier method, and survival curves were compared according to age, BMI, and ethnicity using the log-rank test. Results CLBR increased rapidly from 1 to 5 ETs, moderately from 6 to 10 ETs, and slowly thereafter. CLBR was significantly different across 4 categories based on BMI as well as across those based on age; low CLBR was significantly associated with the age of ≥42 years and obesity. Conclusion The association between CLBR and number of ET provides realistic and precise information regarding the success of IVF and can be applied to guide clinicians and patients.


Introduction
The emergence of assisted reproductive technology (ART) has brought hope to infertile couples. Since the birth of the world's first "test tube baby," Louise Brown, 40 years ago, 8 million babies have been conceived worldwide through ART [1]. However, IVF success rate remains far below 100%. Therefore, the ratio of probability of success and cost effectiveness is an important indicator of decision making and should be considered as a key point for patients who have a low ratio [2].
Typically, cumulative live birth rate (CLBR) and pregnancy rate are used as key indicators to assess the quality and success of IVF, and these are indispensable for patients to make decisions before starting the treatment [3,4]. CLBR allows for relevant comparison between different reproductive medical centers. Moreover, it may help patients make decisions regarding continuing treatment or remaining childless.
There is a growing tendency in some countries for couples to have children late in their life. This situation is obvious in China for several reasons, such as the two-child policy issued in 2015 [5]. In addition, Chinese women tend to receive education for a prolonged period during their lifespan, which means that they may get married late and consequently postpone childbearing; women with higher education may focus more attention on their career. Apart from these reasons involving women, the high cost of living (burden of feeding a family and offering high-quality education for children) may contribute to late marriages.
According to a recent study, nearly 57% of the couples of all ages in United Kingdom remained childless even after undergoing 6 complete IVF cycles [6]. Female age is the main factor related to delivering a child through ART [7]. That is, women aged ≥35 years would show a lower live birth rate in a single cycle than younger women. Thus, patients older than 35 years of age require multiple cycles to increase the probability of live birth. However, many patients discontinue treatment after 1 or 2 failed treatment cycles at the beginning of ART, although the final prognosis of achieving a live birth may remain promising. Furthermore, the number of embryos transferred (ET) in each cycle varies from 1 to 3, with 3 being the maximum. This reinforces that CLBR per cycle may not be a reliable index for providing informed advice to patients.
The aim of this study was to calculate CLBR per total number of ET required for achieving a live birth. This pragmatic method predicted outcomes considering each embryo as single opportunity to achieve a live birth. This approach is advantageous in that it can provide doctors and patients with a more accurate probability of live birth. Female age, BMI, and ethnicity were considered the key factors affecting the success of IVF.

. . Study Design and Samples.
We performed a retrospective cohort study including 1344 women aged 35-45 years who underwent IVF/ET between January 2013 and June 2016 at the Reproductive Medicine Center, the First Affiliated Hospital of Xinjiang Medical University, Xinjiang, China. The inclusion criteria were as follows: (1) women with a ≥1-year history of infertility and age of ≥35 years; (2) those undergoing the first cycle of IVF/ICSI; and (3) those in whom own oocytes were used and fresh or frozen-thawed embryos were transferred without preimplantation genetic screening for embryo aneuploidies. The exclusion criteria were as follows: (1) patients undergoing IVF cycles without ET for any reason; (2) those unsuitable for treatment because of severe disease; (3) those with unknown clinical outcome; (4) those aged >45 years; and (5) those with congenital uterine dysplasia. This study was approved by the Institutional Review Board (IRB) of the First Affiliated Hospital of Xinjiang Medical University, and all patients provided written informed consent before treatment.
. . Follow-Up. All patients were followed up for at least 2 years or until (1) they decided to discontinue the IVF treatment; (2) all embryos were transferred; (3) they decided to change to ovum donation; (4) had delivery of at least 1 live child surviving over 1 month; or (5) they were still receiving IVF treatment at the end of the study.
. . Statistical Analysis. To summarize the characteristics of patients, continuous variables were expressed as mean (standard deviation, SD) or median (interquartile range, IQR) and categorical variables were expressed as frequency (percentage). Categorical variables were compared using the 2 or Fisher's exact test. Continuous variables were compared using t test, analysis of variance, or Mann-Whitney U test, depending on data distribution.
The cumulative probability of live birth for each couple undergoing IVF/ICSI treatment during the study period was calculated using the Kaplan-Meier method; this was according to the total ET in each cycle of treatments required to achieve a live birth. The probability of cumulative live birth and outcomes were estimated by the Kaplan-Meier method and compared using the log-rank, Breslow, and Tarone-Ware tests according to each categorical group. A two-tailed P< 0.05 was considered statistically significant in all analyses. All analyses were performed using Stata 13 and R version 3.3.1 (http://www.r-project.org/).

Discussion
We evaluated outcomes of IVF treatment according to the total number of ET until a term live birth was achieved in women aged ≥35 years. Although the patients underwent the same number of treatment cycles, the total number of ET varied because the number of ET in each cycle varied from 1 to 3. Therefore, considering each embryo as a single opportunity for achieving a live birth can be a useful method to evaluate the outcomes of IVF/ICSI treatment, and it has the advantage of providing more accurate probability of treatment success to clinicians and patients. The cumulative probability of live birth was nearly 25% with 3 ET but only half of this rate with 6 ET. This indicates that the number of ET is a determinant of pregnancy and live birth achievements.
The number of ET was an essential factor associated with CLBR. This is generally in agreement with other studies showing that the number oocytes retrieved was a contributing factor to live birth rate after IVF, with recent evidence indicating that CLBR increases with the number of oocytes    retrieved [8][9][10][11]. Not all oocytes can completely develop into a mature egg and be successfully fertilized to become an embryo. Therefore, to obtain 5 embryos, approximately 14 metaphase II oocytes are required, with a fertilization rate of 70% such that 50% of them could develop to a blastocyststage embryo [12,13]. Furthermore, patients with polycystic ovary syndrome (PCOS) typically produce more oocytes, but they are often of poor quality and show a low rate of fertilization [14,15]. Therefore, it seems appropriate and reasonable to evaluate the IVF outcomes by considering the number of ET.
Female age is the main factor to achieve a successful live birth through ART. Natural aging may reduce fertility in a patient seeking IVF treatment [16]. Women aged ≥35 years present increased risk of abortion, significantly reduced pregnancy and live birth rates, and increased risk of pregnancy comorbidities and complications [17][18][19][20]. Women aged ≥46 years were excluded from this study because their pregnancy rate tended to be 0% under a natural condition [21], and a recent study suggested that IVF treatment was futile in women aged ≥44 years (CLBR of 3% was never reached irrespective of the number of retrieved oocytes) [11].  Studies indicated that embryo quality is affected by the age of the woman [22][23][24]. Age was found to be a predictor for the number of retrieved oocytes, number of metaphase II oocytes and embryo quality [25]. Declines in oocytes yields and oocytes quality are major reasons for deteriorating IVF outcomes with advanced female age. Embryo quality was largely determined by oocytes and embryo quality determines most of pregnancy and live birth probability. A possible explanation for this is that women's fertility decreases with increasing age due to a combination of a lowered number of embryos for transfer and decline in embryo quality associated with factors including chromosomal aneuploidy, chromosomal errors, fragmentation, and mitochondrial dysfunction [26][27][28].
Our results show that IVF overcomes infertility in women aged ≤41 years. Women at ≥42 years should be informed that IVF treatment cannot completely overcome reduced fertility due to age, which is consistent with reports of a previous study [11]. The added value of the present findings is that, for women aged ≥42 years, discontinuation of further treatment may be recommended after 8 embryos are transferred. Moreover, women of advanced age should be informed of the increased risk of pregnancy and perinatal complications. Women who become pregnant at the age of ≥40 years face increased risks of stroke and heart attack later in their life compared with women who become pregnant at a younger age [29]. Increased risk of neonatal birth defects such as Down syndrome and cerebral palsy is associated with increased age during pregnancy [18,30].
Studies assessing the potential impact of female BMI on IVF outcomes have reported conflicting results. Multiple studies and a meta-analysis indicated that higher BMI was associated with fewer retrieved oocytes, higher cancellation rates, higher miscarriage rate, and lower pregnancy and live birth rates [31][32][33][34][35][36]. Meanwhile, other studies reported no adverse effect of increased BMI on IVF outcomes [37][38][39][40]. This difference in conclusions may be due to disparities of ethnicity, dietary structure, lifestyle, genetics, and environmental factors. Therefore, we categorized BMI into 4 groups and applied the Kaplan-Meier method to analyze CLBR of each group. Obese women showed the lowest CLBR among these 4 groups, and CLBR was limited to 25% after 5 embryos were transferred. Obese women show dysregulated meiotic spindle formation, which may lead to reduced size and quality of oocytes and embryos [41,42]. Thus, obese women should be counseled regarding the detrimental effects of obesity and benefits of weight loss, including improvements in treatment outcomes, as recommended by a recent meta-analysis.
Our study has several strengths. We considered each embryo as a single opportunity for achieving live birth. The plots we produced are easy for clinicians and patients to understand and show the specific probability of treatment success according to patient characteristics. We did not restrict the treatment to 6 cycles as did other studies, [6] allowing us to assess prognosis to a considerable degree. Nonetheless, our study has several limitations. First, the etiology of infertility was not considered. There are numerous reasons for male and female infertility, and some etiologies, such as mild male factor infertility and low ovarian response, are difficult to define and classify. Second, we did not consider embryo quality owing to the lack of standard quantifiable items to evaluate it. Third, we did not account for confounders such as smoking status and alcohol intake. Finally, the retrospective nature of the study cannot exclude all biases.

Conclusions
In summary, the present study provides the expected probability of having a child based on the number of ET. Clinicians can provide accurate probabilities to patients using visual graphics such as the Kaplan-Meier curves. This may assist patients to make difficult decisions regarding whether to pursue the IVF/ICSI treatment. Women aged ≥42 years may not experience any benefit with CLBR after 8 embryos are transferred; thus, they should be discouraged from continuing IVF treatment. Obese women seeking fertility treatment should be counseled on the detrimental effects of obesity and benefits of weight reduction, and they should be informed that weight loss via diet and exercise is associated with improved probability of becoming pregnant, with a trend toward improved live birth rate.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.