ABSTRACT
Purpose
To ascertain whether ovarian hyperresponse and oocyte vitrification (OV) are associated with an increase in embryo aneuploidy.
Methods
Retrospective study involving 197 629 retrieved oocytes. We studied the euploidy rate (ER), the ER according to the number of retrieved oocytes (ERaRO) and according to the number of metaphase II oocytes (MIIO) (ERaMII), and the quotient “MIIO/retrieved oocytes” or mature oocyte rate (MOR).
Results
The maximal ERaRO corresponded when only one oocyte was recovered (27% ± 44%), and was somewhat lower when two oocytes were retrieved (14% ± 31%). From the 3rd oocyte until the 36th oocyte, the ERaRO remained constant at around 10%. Regarding MIIO, a similar pattern was observed. For a given number of oocytes, usually the ER was higher if MOR was 100%. Moreover, even when considering only cases with a 100% MOR, the ER was significantly higher in cases where there were 1–2 MIIO. Concerning OV, the regression analysis after adjustment showed that vitrified oocytes had a similar rate of aneuploidy to fresh oocytes.
Conclusions
Both ovarian hyperresponse and OV are safe procedures concerning embryo euploidy. The better ER obtained, especially in cases with 1–2 oocytes, opens a new line of research concerning the superior oocyte quality from the point of view of the euploidy of cycles with 1–2 oocytes.
Keywords: aneuploidy, euploidy rate, metaphase II oocytes, oocyte vitrification, ovarian hyperresponse
Abbreviations
- aCGH
comparative genomic hybridization
- AR
aneuploidy rate
- ERaMII
euploidy rate according to the number of metaphase II oocytes
- ERaRO
euploidy rate according to the number of retrieved oocytes
- MIIO
metaphase II oocytes
- MOR
mature oocyte rate
- NGS
next‐generation sequencing
- VO
vitrified oocytes
1. Introduction
The optimal number of oocytes to be recovered in ART is still a matter of debate. Until recently 8–15 oocytes were usually recommended [1, 2, 3, 4]. However later studies reported higher pregnancy rates with 15–20 oocytes, when considering only fresh cycles [5] or > 25 oocytes when cumulative pregnancy rates were included [6]. Nevertheless, there could be some concerns regarding the aneuploidy rate (AR) linked with ovarian hyperresponse or employing high doses of gonadotropins [7, 8]. Some chromosomal abnormalities have been described in mouse oocytes related to the use of high doses of gonadotropins [9, 10, 11]. In a randomized study, it was observed that AR was higher in the conventional stimulation than in the mild stimulation, although the final number of euploid embryos was similar [12]. However in many trials no association was found with AR and gonadotropin dose/ovarian response [13, 14, 15]. Since ovarian response and gonadotropin dose administration are not independent parameters, and both are related to age, it is difficult to ascertain the influence of any of them.
Oocyte vitrification is an increasingly employed very efficient reproductive technique. Previous publications do not report any association between AR and oocyte vitrification. However, the general unselected population was not analyzed, since they studied patients with a high number of vitrified oocytes per woman [16, 17, 18]. Moreover, sample sizes were usually relatively small and no adjustment was made by age or number of oocytes [16, 17, 18]. However, one study performed with donor oocytes reported that although there was no increase in the AR, there was an increase in the nondisjunction rate on chromosome 13 [19].
2. Material and Methods
The population under study consisted of 197 629 retrieved oocytes (157 746 metaphase II oocytes) (MIIO) corresponding to 16 029 ICSI cycles where PGT‐A was performed in our group of 17 clinics between 2011 and 2019. The inclusion criteria were: (1) cycles performed with own oocytes; (2) partner's sperm resulting from ejaculate. The study was approved by our Ethics Investigation Institutional Board (code CEIC E19/02). Of the 16 029 PGT‐A cycles, in 13 410 cases the oocytes were fresh, in 667 vitrified and in 1952 both fresh and vitrified. Main characteristics of the population and main PGT‐A indications are depicted in Table 1. Main indications for oocyte vitrification were social preservation and low response. General results of the same population have already been described [20] as well as those regarding parental age [21].
TABLE 1.
Characteristics of the PGT‐A cycles.
| Total n (%) | Vitrified oocytes n (%) | Fresh oocytes n (%) | p.vitrified vs. fresh | |
|---|---|---|---|---|
| PGT‐A cycles | 16 029 (100%) | 667 (4.16%) | 13 410 (83.66%) | |
| First cycles | 10 950 (68.31%) | 408 (61.17%) | 7912 (59.00%) | |
| Retrieved oocytes | 197 629 (100%) | 8115 (4.11%) | 166 573 (84.29%) | |
| Retrieved oocytes/cycle, Median [25th; 75th] | 10.0 [7.00; 15.0] | 15.1 (7.47%) | 14.2 (8.19%) | < 0.001 |
| MII oocytes | 157 746 (79.82%) | 8005 (5.07%) | 129 241 (81.93%) | |
| MII oocytes/cycle, Median [25th; 75th] | 9.00 [6.00; 12.0] | 14.9 (7.46%) | 11.2 (6.79%) | < 0.001 |
| Patient age, Median [25th; 75th] | 40.0 [38.0; 41.0] | 40.0 [35.0; 42.0] | 40.0 [37.0; 41.0] | < 0.001 |
| Embryos/cycle, Median [25th; 75th] | 6.00 [4.00; 9.00] | 7.00 [5.00; 10.0] | 6.00 [4.00; 9.00] | < 0.001 |
| Day of biopsy | < 0.001 | |||
| Day 3 | 6378 (39.79%) | 427 (64.02%) | 5659 (42.20%) | |
| Blastocyst | 9651 (60.21%) | 240 (35.98%) | 7751 (57.80%) | |
| Main PGT‐A indication | ||||
| Recurrent pregnancy loss | 1531 (9.55%) | 67 (10.04%) | 1291 (9.63%) | |
| Advanced maternal age | 11 445 (71.40%) | 491 (73.61%) | 9400 (70.10%) | |
| Severe male factor | 864 (5.39%) | 30 (4.52%) | 783 (5.84%) | |
| Implantation failure | 1390 (8.67%) | 38 (5.66%) | 1259 (9.39%) | |
Note: PGT‐A cycles including both fresh and vitrified oocytes are not analyzed separately. Data are provided as total number and percentages, unless reported otherwise.
In the beginning of the study period, almost all the embryo biopsies were performed on day 3. The proportion of biopsies performed at the blastocyst stage progressively increased till almost the totality at the end of the study. PGT‐A technique has been already published [20]. Until 2016 the PGT‐A was performed by means of array‐based comparative genomic hybridization (aCGH) and from 2017 with next‐generation sequencing (NGS). In both cases, when a trophectoderm biopsy was performed the threshold to consider a biopsy aneuploid was above 30%, and mosaicism was not reported during this period of time. Therefore, our policy was to consider all embryos with aneuploidy threshold above 30% as non‐transferable embryos. Only euploid embryos with aneuploidy below 30% were candidates for transfer.
Ovarian stimulation was performed according to the standard clinical criteria, which evolved over the course of the study period [14, 20, 22]. The great majority of patients were stimulated with gonadotropins in antagonist down‐regulated cycles. The dose of gonadotropins was based on age, antimüllerian hormone, antral follicle count, body mass index and previous response. Oocyte triggering was performed with hCG and/or triptoreline. Oocyte pick‐up was performed 36 h later, under sedation and all the follicles > 10 mm were aspirated. In cases of oocyte vitrification, the MIIO were vitrified immediately following the Cryotop method, as previously described [23, 24].
Data were obtained from our central electronic database and were reviewed manually if inconsistencies were detected.
2.1. Statistical Analysis
The normality of continuous variables was assessed using the Shapiro–Wilk test. Variables following a normal distribution were presented as mean and standard deviation (which, as measures of variability, may exceed the mean and are not constrained by the theoretical bounds of the variable), while those not normally distributed were presented as median and interquartile range. Differences were analyzed using the parametric Student's t‐test or the non‐parametric Mann–Whitney U test. Comparisons between groups were performed using the Z‐test, which is appropriate and robust in large‐sample settings where the Central Limit Theorem ensures the approximation to normality.
Categorical variables were presented as frequency and percentage. Differences between groups were analyzed using the Chi‐square test or Fisher's exact test when the expected number was less than 5.
Univariate and multivariate analyses were performed using logistic regression models, with the latter adjusted for age, day of biopsy, PGT‐A technique, PGT‐A indication, year when the PGT‐A was performed and number of MIIO.
All analyses were conducted using R software (version 4.3.1): A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria.
We studied the proportion of euploid embryos with respect to the number of oocytes (retrieved oocytes or MIIO) for each given number of oocytes (retrieved oocytes or MIIO). We defined the euploidy rate according to the number of retrieved oocytes (ERaRO) as the quotient “number of euploid embryos/number of retrieved oocytes.” Similarly, we calculated the euploidy rate (ER) according to the number of MIIO (ERaMII). We defined the mature oocyte rate (MOR) as the quotient “MIIO/retrieved oocytes.”
We considered the following subgroups: Day 3 embryos and blastocysts, first cycles, age groups (< 30, 30–34, 35–39, ≥ 40), year of the PGT‐A, as well as the subgroups resulting from their combinations. The distribution according to the number of oocytes per cycle is depicted in Table S1.
In a subgroup of the population (data from the IVI Bilbao clinic) (n = 39954 MIIO) an additional subanalysis was performed taking AMH values into account. For each age group, three subgroups were established based on the AMH tertile (for their age group).
3. Results
3.1. Euploidy Rate According to the Number of Retrieved Oocytes
3.1.1. Global Population
When considering all the embryos and all the cycles, the maximal ERaRO corresponded when only one oocyte was recovered (27% ± 44%), and was somewhat lower when two oocytes were retrieved (14% ± 31%). Afterwards the ERaRO remained unchanged, around 10%, from the 3rd oocyte to when ≥ 36 oocytes were retrieved (Figure 1A). There were significant differences between one oocyte ERaRO and the remaining groups according to the number of retrieved oocytes (p < 0.0001), as well as between the two oocytes ERaRO and the remaining oocyte numbers (excluding one oocyte) (p = 0.0004). However, no significant differences were found for three or greater oocyte numbers.
FIGURE 1.
Euploidy rate per retrieved oocyte and total number of euploid embryos according to the number of retrieved oocytes. (A) In the total population. Significant differences between one oocyte ERaRO and the remaining oocyte groups (Z‐statistic = 9.10, p < 0.0001), as well as between the two oocytes ERaRO and the remaining oocyte numbers (excluding one oocyte) (Z‐statistic = 3.52, p = 0.0004). No significant differences were found for three (Z‐statistic = 0.63, p = 0.527) or greater oocyte numbers. There was a significant correlation between the number of retrieved oocytes and the number of euploid embryos (ρ = 0.4585; p < 0.0001). (B) In blastocysts. Significant differences between the ERaRO with one oocyte and the rest of the oocyte groups (Z‐statistic = 10.07, p = 0.00001), with two oocytes and the other groups (Z‐statistic = 10.07, p < 0.0001), and with three oocytes and the other groups (Z‐statistic = 2.46, p = 0.014). (C) In day 3 embryos. There were no significant differences between one oocyte and the remaining groups (Z‐statistic = 0.02, p = 0.98) nor two and the remaining groups (Z‐statistic = 1.12, p = 0.26), nor three and the remaining groups (Z‐statistic = 1.12, p = 0.26). (D) By age groups. The following p values were obtained: For one oocyte, age 30–34 (Z‐statistic = 3.57, p = 0.0004), age 35–39 (Z‐statistic = 8.06, p < 0.0001), age ≥ 40 (Z‐statistic = 7.96, p < 0.0001). For two oocytes, age 30–34 (Z‐statistic = 2.51, p = 0.01); age 35–39 (Z‐statistic = 4.27, p < 0.0001); age ≥ 40 (Z‐statistic = 3.77, p = 0.0002). For three oocytes, age 30–34 (Z‐statistic = −0.70, p = 0.481); age 35–39 (Z‐statistic = 3.40, p = 0.001); age ≥ 40 (Z‐statistic = 3.27, p = 0.001). Sample size of the age < 30 group was relatively small, precluding the statistical analysis. In (A–C), data are expressed as mean and standard deviation. Standard deviation is shown as colored bars for the number of embryos and as capped spikes for the euploid rate per retrieved oocyte. In (D), data are shown as mean.
When ERaRO analysis was restricted to blastocysts, a similar graph was obtained, but with higher ERaRO. The ERaRO was 42% ± 50% with one oocyte (p < 0.0001), 19% ± 35% with two oocytes (p < 0.0001), and 14% ± 20% with three oocytes (p = 0.014), and then ranging from 11% to 12% in the majority of cases (Figure 1B).
When the data were restricted to day 3 embryos, the ERaRO did not change regardless of the number of oocytes obtained, ranging from 7% to 9% in the majority of cases (Figure 1C).
When only first PGT‐A cycles were considered, very similar data were obtained (Figure 2).
FIGURE 2.
Euploidy rate (ER) per retrieved oocyte and total number of euploid embryos according to the number of retrieved oocytes in the first PGT‐A cycle. Data are expressed as mean and standard deviation. Standard deviation is shown as colored bars for the number of embryos and as capped spikes for the euploid rate per retrieved oocyte. (A) In the total population. Significant differences between 1 retrieved oocyte and the rest (Z‐statistic = 7.66, p < 0.0001), and between two and the rest (Z‐statistic = 3.33, p = 0.0009). No differences with three or more oocytes compared to the rest (Z‐statistic = 1.57, p = 0.12). (B) In blastocysts. Significant differences between 1 retrieved oocyte and the rest (Z‐statistic = 9.01, p < 0.0001) as well as for two and the rest (Z‐statistic = 4.18, p < 0.0001). No differences with three or more oocytes compared to the rest (Z‐statistic = 1.57, p = 0.12). (C) In day 3 embryos. No significant differences. For one oocyte (Z‐statistic = 0.02, p = 0.98), for two oocytes (Z‐statistic = 1.12, p = 0.26) and for three (Z‐statistic = 1.12, p = 0.2631).
3.1.2. Age Group Analysis
Regarding the total population, in all the age groups, the higher ERaRO was observed with one and two oocytes and in the groups ≥ 35 also with three oocytes, remaining more or less steady afterwards (Figure 1D). In the group < 30 years the reduced sample size precluded the statistical analysis.
When blastocyst data were presented according to age groups, the aforementioned pattern persisted (Figure 3A). It has to be highlighted that the ERaRO for 1 retrieved oocyte was 43% in the age group 30–34 and 62% in the age group 35–39. In all age groups, after excluding the data corresponding to cases with 1–2 retrieved oocytes, the ERaRO was steady, although the mean ERaRO changed remarkably with the age group.
FIGURE 3.
Euploidy rate (ER) per retrieved oocytes according to the number of retrieved oocytes, by age groups. Data are shown as mean. (A) In blastocysts. The following p values were obtained in the different age groups. For one oocyte, age 30–34 (Z‐statistic = 1.67, p = 0.09), age 35–39 (Z‐statistic = 8.51, p < 0.001) and age ≥ 40 (Z‐statistic = 6.74, p < 0.001). For two oocytes, age 30–34 (Z‐statistic = 3.06, p = 0.002), age 35–39 (Z‐statistic = 4.21, p < 0.0001) and age ≥ 40 (Z‐statistic = 3.68, p = 0.0002). For three oocytes, age 30–34 (Z‐statistic = −0.17, p = 0.86), age 35–39 (Z‐statistic = 3.77, p < 0.001) and age ≥ 40 (Z‐statistic = 3.58, p < 0.0001). Sample size of the age < 30 group was relatively small, precluding the statistical analysis. (B) In day 3 embryos. There were no significant differences. For one oocyte, age 30–34 (no cases were registered), age 35–39 (Z‐statistic = −0.12, p = 0.90) and age ≥ 40 (Z‐statistic = 0.69, p = 0.49). For two oocytes, age 30–34 (Z‐statistic = −0.81, p = 0.42); age 35–39 (Z‐statistic = 1.30, p = 0.19) and age ≥ 40 (Z‐statistic = 1.97, p = 0.0491). For three oocytes, age 30–34 (Z‐statistic = −0.83, p = 0.408), age 35–39 (Z‐statistic = 0.06, p = 0.95) and age ≥ 40 (Z‐statistic = 1.30, p = 0.19). Sample size of the age < 30 group was relatively small, precluding the statistical analysis.
When data of day 3 embryos were presented according to age groups, the no changing pattern observed in day 3 embryos in general was also present (Figure 3B).
3.2. Euploidy Rate According to the Number of Metaphase II Oocytes
3.2.1. Global Population
When considering all the embryos and all the cycles, the ERaMII when there was only 1 MIIO was the highest (27% ± 50%) (p < 0.0001), whereas with 2 MIIO the ERaMII was among the highest (16% ± 32%) (p < 0.0001) and then remained steady around 11%–14% in the majority of cases (p = 0.127) (Figure 4A).
FIGURE 4.
Euploidy rate per MII oocyte and total number of euploid embryos according to the number of MII oocytes. (A) In the total population. Proportion test between ratios of 1 MIIO versus the rest indicated significant differences (Z‐statistic = 7.82, p < 0.0001), as well as with 2 MIIO and the rest (Z‐statistic = 4.53, p < 0.0001) and then remained steady (Z‐statistic = 1.53, p = 0.127). There was a significant correlation between the number of obtained euploid embryos and the number of MIIO (ρ = 0.4718; p < 0.0001). (B) In blastocysts. Significant differences between 1 MIIO and the rest (Z‐statistic = 8.08, p < 0.001) as well as with two MIIO and the rest (Z‐statistic = 5.39, p < 0.0001), and with three oocytes and the rest (Z‐statistic = 2.46, p = 0.014). There was a correlation between the number of MIIO and the number of euploid blastocysts (ρ = 0.49; p < 0.0001). (C) In day 3 embryos. Euploidy rate per MIIO and total number of euploid day 3 embryos according to the number of MIIO. When there was only 1 MIIO, ERaMII was significantly higher than in the rest (Z‐statistic = 4.27, p = 0.0002), as well as when there were only two 2 MIIO (Z‐statistic = 1.86, p = 0.06), and afterwards there were no significant differences (Z‐statistic = 0.74, p = 0.46). There was a correlation between the number of MIIO and the number of euploid day‐3 embryos (ρ = 0.41; p < 0.0001). (D) By age groups. For one MIIO the following p‐values were obtained: Age 30–34 (Z‐statistic = 1.85, p = 0.06), age 35–39 (Z‐statistic = 7.86, p < 0.0001), age ≥ 40 (Z‐statistic = 5.19, p < 0.0001). For two MII oocytes: Age 30–34 (Z‐statistic = 1.84, p = 0.06), age 35–39 (Z‐statistic = 6.96, p < 0.0001) and age ≥ 40 (Z‐statistic = 4.73, p < 0.0001). For three MIIO: Age 30–34 (Z‐statistic = −0.050, p = 0.96); age 35–39 (Z‐statistic = 5.27, p < 0.0001) and age ≥ 40 (Z‐statistic = 3.77, p = 0.0001). Sample size of the age < 30 group was relatively small, precluding the statistical analysis. In (A–C), data are expressed as mean and standard deviation. Standard deviation is shown as colored bars for the number of embryos and as capped spikes for the euploid rate per retrieved oocyte. In (D), data are shown as mean.
When ERaMII analysis was restricted to blastocysts, the maximal value was obtained in the 1 MIIO (35% ± 49%) (p < 0.001), followed by that corresponding to two MIIO (21% ± 33%) (p < 0.0001), decreasing with three MIIO (16% ± 20%) (p = 0.014) and then remaining steady around 14% in the majority of cases (Figure 4B).
When data analysis was restricted to day 3 embryos, the ERaMII when there was only 1 MIIO (21% ± 50%) was among the highest ERaMII (p = 0.0002), followed by that with 2 MIIO (p = 0.06) and then remained steady around 9%–13% in the majority of cases (p = 0.458) (Figure 4C).
When only first PGT‐A cycles were considered, the observed patterns were very similar (Figure 5).
FIGURE 5.
Euploidy rate (ER) per MII oocyte and total number of euploid embryos according to the number of MII oocytes in the first PGT‐A cycle. Data are expressed as mean and standard deviation. Standard deviation is shown as colored bars for the number of embryos and as capped spikes for the euploid rate per retrieved oocyte. (A) In the total population. Significant differences between 1 MII oocyte and the rest (Z‐statistic = 7.21, p < 0.0001), and between two and the rest (Z‐statistic = 3.33, p = 0.0009) but not with three or more and the rest (Z‐statistic = 0.39, p = 0.69). (B) In blastocysts. Proportion test between ratios of 1 MII oocytes versus the rest indicated significant differences (Z‐statistic = 7.59, p = 0.00001). Significant differences between 1 MII oocyte and the rest (Z‐statistic = 7.59, p < 0.0001), and between two and the rest (Z‐statistic = 4.36, p < 0.0001) but not with three or more and the rest (Z‐statistic = 1.48, p = 0.14). (C) In day 3 embryos. Proportion test between ratios of 1 MII oocytes versus the rest did not suggest the presence of significant differences (Z‐statistic = 1.91, p = 0.057). There were no significant differences between 1 MII oocyte and the rest (Z‐statistic = 1.91, p = 0.057), between two and the rest (Z‐statistic = −0.22, p = 0.83) and for three or more and the rest (Z‐statistic = −0.65, p = 0.51).
3.2.2. Age Group Analysis
In all the age groups (except in < 30 years, where few cases were available) the maximal ERaMII corresponded with cases with only 1 MIIO, decreasing remarkably when there were 2 MIIO, followed by the values corresponding to 3 MIIO and remaining steady afterward, although the mean ERaMII changed remarkably with the age group (Figure 4D).
A very similar pattern was observed in blastocysts and day 3 embryos in all the age groups (Figure 6).
FIGURE 6.
Euploidy rate (ER) per retrieved oocytes according to the number of retrieved oocytes, by age groups. Data are shown as mean. (A) In blastocysts. For one MII oocyte the following p values were obtained: 0.04 (Z‐statistic = 2.03) (age 30–34), < 0.0001 (Z‐statistic = 6.83) (age 35–39), < 0.0001 (Z‐statistic = 6.01) (age ≥ 40). For two MII oocytes the p values were: 0.03 (Z‐statistic = 2.16) (age 30–34), < 0.0001 (Z‐statistic = 4.47) (age 35–39), < 0.0001 (Z‐statistic = 5.92) (age ≥ 40). For three MII oocytes the p values were: 0.81 (Z‐statistic = 0.24) (age 30–34), < 0.0001 (Z‐statistic = 4.70) (age 35–39), 0.005 (Z‐statistic = 2.8) (age ≥ 40). Data regarding the age group < 30 were not shown, because of the small sample size. Sample size of the age < 30 group was relatively small, precluding the statistical analysis. (B) In day 3 embryos. Data regarding the age group < 30 were not shown, because of the small sample size. For one MII oocyte, the following p values were obtained: Age 30–34 (Z‐statistic = 4.27, p = 0.0002), age 35–39 (Z‐statistic = 4.29, p = 0.0002) and age ≥ 40 (Z‐statistic = 5.83, p < 0.0001); for two MII oocytes: Age 30–34 (Z‐statistic = 0.35, p = 0.73); age 35–39 (Z‐statistic = 5.91, p < 0.0001); age ≥ 40 (Z‐statistic = 1.48, p = 0.14); and for three: Age 30–34 (Z‐statistic = −0.414, p = 0.68); age 35–39 (Z‐statistic = 2.60, p = 0.009); age ≥ 40 (Z‐statistic = 3.11, p = 0.002). Data regarding the age group < 30 were not shown, because of the small sample size. Sample size of the age < 30 group was relatively small, precluding the statistical analysis.
3.2.3. Age Group Analysis and AMH Values
The Pearson correlation analysis showed a strong correlation between AMH and the number of MIIO (p < 0.0001). After adjusting for AMH tertiles, in all age groups (except < 30 years, where few cases were available) the pattern of maximum ERaMII with 1–2 oocytes and subsequent stabilization of ERaMII persisted. Similar MII ERs were observed in each age group, regardless of the AMH tertile for that age group (Figure 7).
FIGURE 7.
Euploidy rate (ER) per MII oocyte according to AMH values in each age group. Solid line: First AMH tertile. Dashed line: Second AMH tertile. Dotted line: Third AMH tertile. (A) Age group 30–34. (B) Age group 35–39. (C) Age group ≥ 40. No significant differences.
3.3. Euploidy Rates and Number of Retrieved Oocytes/MIIO According to the Year in Which PGT‐A Was Performed
A very similar pattern was observed in almost all the years considered: higher ER with 1–2 oocytes and a stable rate thereafter (with oocytes retrieved and with MIIO) (Figure 8).
FIGURE 8.
Euploidy rates and number of retrieved oocytes/MII oocytes according to the year in which PGT‐A was performed. (A) Number of retrieved oocytes in blastocysts. (B) Number of retrieved oocytes in day 3 embryos. (C) Number of MII oocytes in blastocysts. (D) Number of MII oocytes in day 3 embryos.
3.4. Total Number of Euploid Embryos and Number of Retrieved Oocytes/MIIO
The number of obtained euploid embryos was correlated with the number of retrieved oocytes, both in the total population (Figure 1A) as well as in blastocysts (Figure 1B) and in day 3 embryos (Figure 1C), as well as in first cycles (Figure 2), as in the different age groups (data not shown). For example, in the overall population, when 10 oocytes were retrieved, the mean number of euploid embryos obtained (n = 0.89) was somewhat less than half that obtained with 20 oocytes (n = 2.16).
Similar results were obtained regarding MIIO (Figures 4A–C and 5).
3.5. Euploid Rate According the Number of MIIO Split by the MOR
When the ER was shown, considering both the number of retrieved oocytes and the number of resulting MIIO, for a given number of retrieved oocytes in the majority of cases the ER was significantly higher when the MOR was higher (corresponding to the rows in Table 2A). For instance, when there were two retrieved oocytes the ER was 16% ± 33% if they produced 2 MIIO (MOR = 100%), versus 6% ± 16% if they produced 1 MIIO (MOR = 50%) (p = 0.014). Similarly in cases with 5 retrieved oocytes the following ERs were obtained: 11% ± 16% (5 MIIO, MOR = 100%), 10% ± 15% (4MIIO), 9% ± 12% (3MIIO), 6% ± 11% (2MIIO), 9% ± 13% (1MIIO) (p = 0.04).
TABLE 2.
Euploidy rate according to the number of oocytes retrieved and number of MII oocytes. (A) In the total population. (B) In blastocysts. (C) In day 3 embryos.
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Note: The columns correspond to the comparison of the euploidy rate obtained by a given number of MII oocytes out of the total number of oocytes from which they were derived. The rows correspond to the comparison of the euploidy rate with respect to the different numbers of MII oocytes resulting from a given number of oocytes retrieved. The diagonal corresponds to the analysis of all cases in which the number of MII oocytes coincides with the number of oocytes recovered.
When we took the complementary approach, and we considered the number of resulting MIIO according to their MOR (corresponding to the columns of Table 2A), in the majority of cases there was a tendency towards an association between higher ER and higher MOR. For instance, when 4MIIO were obtained, if they came from 4 retrieved oocytes (MOR = 100%) the ER was 11% ± 17%, versus 8% ± 7% if they came from 10 oocytes (MOR = 40%) (p = 0.004).
When we considered only cases with 100% MOR (corresponding to the diagonal of Table 2A), the 1 oocyte with 100% MOR had a significantly higher ER (27% ± 44%) than the ER of the remaining groups of oocytes (p = 0.001). Similarly the 100% MOR obtained in the group of two oocytes had an ER (16.33% ± 33%) that was significantly higher than the ER obtained with > 2 oocytes, which were around 11% (p = 0.03).
A differential analysis was performed on blastocysts and day 3 embryos.
In blastocysts a similar pattern was observed to the previously described concerning the higher ER corresponding with 100% MOR in one and two MIIO (p = 0.002) (diagonal of Table 2B). Concerning the number of resulting MIIO, according to their MOR (corresponding to the columns of Table 2B), significant differences were obtained for the following MIIO groups: 1, 4, 9 and 12. Regarding the number of resulting MIIO oocytes, for a given number of retrieved oocytes (corresponding to the rows in Table 2B) significance was achieved in groups of 4, 7, 9, 10, 11 and 15 oocytes.
However, in day 3 embryos the pattern was not so clear (Table 2C).
3.6. Vitrified Oocytes Versus Fresh Oocytes
Among embryos resulting from vitrified oocytes (VO), compared with those resulting from fresh oocytes, there was an increased AR (72.3% vs. 68.2%) (p < 0.001). However, when a regression analysis was performed, adjusting for age, day of biopsy, PGT‐A technique, indication for PGT‐A, year in which PGT‐A was performed and number of MIIO, the VO had an AR similar to that of fresh oocytes (aOR = 0.95; 95% CI = 0.74–1.20, p = 0.7) (Table 3). There were no differences in the trisomy rate in the crude analysis as well as in the adjusted analysis (aOR = 1.08; 95% CI = 0.81–1.41, p = 0.6). Although the crude analysis showed a higher rate of monosomy in OV (44.7% vs. 40.2%, p < 0.001), the aOR showed no differences (aOR = 0.94; 95% CI = 0.86–1.02, p = 0.14). Although the global analysis showed an increased aneuploidy rate in chromosome 17 (Table 3), it does not persist after adjustment. After adjustment the frequency of the aneuploidy of chromosome 19 and 22 was lower in VO (p = 0.046 and 0.023).
TABLE 3.
Aneuploidy rates of vitrified or fresh oocytes by embryo stage.
| Aneuploidy characteristics | Total | Day 3 embryos | Blastocysts | Adjusted OR [95% CI] p (reference fresh oocytes) | ||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Embryos resulting from vitrified oocytes%, n (N = 2600) | Embryos resulting from fresh oocytes%, n (N = 54 537) | p.overall | Embryos resulting from vitrified oocytes%, n (N = 1769) | Embryos resulting from fresh oocytes%, n (N = 25 391) | p.overall | Embryos resulting from vitrified oocytes%, n (N = 831) | Embryos resulting from fresh oocytes%, n (N = 29 146) | p.overall | ||
| Aneuploidies | 72.27 (1827) | 68.16 (36 629) | < 0.001 | 77.5 (1321) | 79.5 (19 694) | 0.195 | 61.6 (506) | 58.6 (16 935) | 0.094 | 0.95 [0.74; 1.20] 0.7 |
| Trisomies | 37.54 (976) | 36.87 (20 107) | 0.502 | 40.7 (720) | 43.1 (10 932) | 0.056 | 30.8 (256) | 31.5 (9175) | 0.708 | 1.08 [0.81; 1.41] 0.6 |
| Monosomies | 44.69 (1162) | 40.23 (21 941) | 0.004 | 49.5 (875) | 50.8 (12 897) | 0.290 | 34.5 (287) | 31.0 (9044) | 0.069 | 0.94 [0.86; 1.02] 0.14 |
| Sex chromosome gain | 1.46 (38) | 1.23 (670) | 0.338 | 1.75 (31) | 1.50 (380) | 0.452 | 0.84 (7) | 0.99 (290) | 0.795 | 1.01 [0.71; 1.40] > 0.9 |
| Sex chromosome loss | 2.92 (76) | 2.09 (1141) | 0.063 | 3.90 (69) | 2.89 (734) | 0.357 | 0.84 (7) | 1.40 (407) | 0.231 | 1.16 [0.90; 1.47] 0.2 |
| q− | 4.19 (109) | 3.03 (1654) | 0.594 | 3.96 (70) | 3.43 (870) | 0.266 | 4.69 (39) | 2.69 (784) | 0.400 | 0.84 [0.59; 1.15] 0.3 |
| q+ | 1.50 (39) | 2.00 (1092) | 0.085 | 1.47 (26) | 1.68 (426) | 0.572 | 1.56 (13) | 2.29 (666) | 0.208 | 1.05 [0.57; 1.77] 0.9 |
| p− | 1.96 (51) | 1.51 (826) | 0.084 | 1.75 (31) | 1.60 (407) | 0.700 | 2.41 (20) | 1.44 (419) | 0.018 | 1.01 [0.65; 1.49] > 0.9 |
| p+ | 1.19 (31) | 1.12 (613) | 0.820 | 1.13 (20) | 0.95 (242) | 0.540 | 1.32 (11) | 1.27 (371) | 1.000 | 0.84 [0.59; 1.15] 0.3 |
| p−/p+/q−/q+ | 8.46 (220) | 7.26 (3961) | 0.862 | 8.03 (142) | 7.16 (1817) | 0.186 | 9.39 (78) | 7.36 (2144) | 0.181 | 1.08 [0.72; 1.55] 0.7 |
| Chromosome 1 | 5.12 (133) | 4.05 (2206) | 0.249 | 6.33 (112) | 5.91 (1499) | 0.497 | 2.53 (21) | 2.43 (707) | 0.943 | 1.05 [0.86; 1.26] 0.6 |
| Chromosome 2 | 5.27 (137) | 4.42 (2408) | 0.082 | 5.43 (96) | 5.77 (1465) | 0.580 | 4.93 (41) | 3.24 (943) | 0.022 | 1.00 [0.83; 1.20] > 0.9 |
| Chromosome 3 | 2.92 (76) | 3.19 (1738) | 0.489 | 3.39 (60) | 4.26 (1081) | 0.090 | 1.93 (16) | 2.25 (657) | 0.608 | 0.76 [0.35; 1.45] 0.4 |
| Chromosome 4 | 4.54 (118) | 4.12 (2245) | 0.313 | 4.92 (87) | 5.08 (1289) | 0.810 | 3.73 (31) | 3.28 (956) | 0.532 | 1.00 [0.82; 1.21] > 0.9 |
| Chromosome 5 | 5.04 (131) | 3.80 (2072) | 0.125 | 5.71 (101) | 5.03 (1275) | 0.223 | 3.61 (30) | 2.74 (797) | 0.158 | 1.14 [0.94; 1.38] 0.2 |
| Chromosome 6 | 3.65 (95) | 3.24 (1764) | 0.264 | 3.84 (68) | 4.17 (1057) | 0.551 | 3.25 (27) | 2.43 (707) | 0.162 | 0.98 [0.78; 1.22] 0.9 |
| Chromosome 7 | 4.19 (109) | 4.09 (2231) | 0.843 | 4.24 (75) | 4.88 (1239) | 0.245 | 4.09 (34) | 3.40 (992) | 0.328 | 0.91 [0.74; 1.11] 0.4 |
| Chromosome 8 | 4.96 (129) | 4.09 (2229) | 0.164 | 5.94 (105) | 5.28 (1338) | 0.254 | 2.89 (24) | 3.06 (891) | 0.859 | 1.09 [0.90; 1.31] 0.4 |
| Chromosome 9 | 6.35 (165) | 5.56 (3028) | 0.094 | 8.03 (142) | 8.03 (2037) | 1.000 | 2.77 (23) | 3.40 (991) | 0.369 | 0.93 [0.79; 1.10] 0.4 |
| Chromosome 10 | 4.88 (127) | 3.94 (2148) | 0.300 | 5.71 (101) | 5.26 (1334) | 0.443 | 3.13 (26) | 2.79 (814) | 0.637 | 1.04 [0.86; 1.26] 0.7 |
| Chromosome 11 | 5.00 (130) | 4.70 (2562) | 0.508 | 6.17 (109) | 6.13 (1554) | 0.988 | 2.53 (21) | 3.46 (1008) | 0.175 | 0.85 [0.70; 1.02] 0.088 |
| Chromosome 12 | 3.66 (95) | 3.35 (1826) | 0.428 | 4.24 (75) | 4.65 (1181) | 0.463 | 2.41 (20) | 2.21 (645) | 0.799 | 0.87 [0.69; 1.07] 0.2 |
| Chromosome 13 | 5.97 (155) | 5.44 (2968) | 0.272 | 6.56 (116) | 6.90 (1750) | 0.625 | 4.69 (39) | 4.18 (1218) | 0.521 | 0.93 [0.78; 1.11] 0.4 |
| Chromosome 14 | 5.66 (147) | 5.15 (2807) | 0.275 | 6.79 (120) | 6.84 (1734) | 0.976 | 3.25 (27) | 3.68 (1073) | 0.574 | 0.93 [0.77; 1.11] 0.4 |
| Chromosome 15 | 10.1 (261) | 9.70 (5287) | 0.578 | 11.2 (197) | 12.3 (3126) | 0.158 | 7.70 (64) | 7.42 (2161) | 0.808 | 0.97 [0.53; 1.65] > 0.9 |
| Chromosome 16 | 13.1 (340) | 12.4 (6768) | 0.328 | 14.5 (256) | 15.7 (3971) | 0.203 | 10.1 (84) | 9.60 (2797) | 0.667 | 0.75 [0.45; 1.17] 0.2 |
| Chromosome 17 | 5.50 (143) | 4.48 (2443) | < 0.001 | 6.11 (108) | 6.30 (1597) | 0.789 | 4.21 (35) | 2.90 (846) | 0.036 | 0.96 [0.80; 1.15] 0.7 |
| Chromosome 18 | 6.35 (165) | 6.10 (3323) | 0.623 | 7.64 (135) | 7.60 (1929) | 0.993 | 3.61 (30) | 4.78 (1394) | 0.139 | 0.86 [0.72; 1.02] 0.084 |
| Chromosome 19 | 2.81 (73) | 3.39 (1850) | 0.118 | 2.54 (45) | 2.48 (628) | 0.920 | 3.37 (28) | 4.19 (1222) | 0.278 | 0.78 [0.60; 0.99] 0.046 |
| Chromosome 20 | 6.69 (174) | 5.51 (3002) | 0.428 | 8.14 (144) | 7.55 (1915) | 0.389 | 3.61 (30) | 3.73 (1087) | 0.935 | 0.99 [0.84; 1.17] > 0.9 |
| Chromosome 21 | 10.9 (283) | 10.8 (5909) | 0.966 | 12.0 (213) | 13.2 (3343) | 0.182 | 8.43 (70) | 8.81 (2566) | 0.754 | 0.87 [0.71; 1.01] 0.059 |
| Chromosome 22 | 12.4 (323) | 13.4 (7274) | 0.191 | 13.4 (237) | 15.7 (3974) | 0.024 | 10.4 (86) | 11.3 (3300) | 0.417 | 0.49 [0.25; 0.86] 0.023 |
| Chromosome XY | 4.50 (117) | 3.35 (1829) | 0.326 | 5.65 (100) | 4.39 (1114) | 0.524 | 2.05 (17) | 2.45 (715) | 0.525 | 1.14 [0.93; 1.38] 0.2 |
4. Discussion
Since the very beginning of IVF there has been a high concern regarding the consequences of eliminating natural barriers that are present in natural conception. Concerning ovarian stimulation it has been a decades‐long discussion regarding the advantages of obtaining not too many oocytes, speculating that oocyte quality would be impaired if too many oocytes were obtained [15]. However, in the last years we have changed from the mild stimulation recommendation [12], to the recommendation of obtaining 8–14 oocytes [25, 26], to 10–15 [27], reporting that the best pregnancy rates were obtained with 15–20 oocytes, when considering only fresh cycles [5] or > 25 oocytes when cumulative pregnancy rates were included [6].
Early mouse models reported an increase in AR in the mouse oocytes related to the use of high doses of gonadotropins [9, 10, 11]. However, the results obtained with mouse models are difficult to extrapolate to humans, since AR in mice is much lower than in humans and the mechanisms that produce aneuploidy in mice do not appear to be the same as those age‐ related in humans [15, 28].
In IVF embryo aneuploidy is a relatively common event [20]. It has been speculated that AR could be related to the artificial processes linked with ART such as ovarian stimulation or ART laboratory. A number of authors have suggested an association between gonadotropin dose and AR [7, 8]. However, it is difficult to disentangle gonadotropin dose from woman's age, which is a well‐known aneuploidy risk factor [20]. Early studies performed by FISH reported lower AR when lower gonadotropin doses were employed [12, 29], but, on the other hand, similar ARs have been reported in natural and stimulated cycles [14]. Moreover in one study comparing 369 natural cycles with 2846 medicated cycles similar ARs were obtained after adjusting for age [15]. In more recent retrospective studies, no association has been reported with gonadotropin dose, after adjusting for a number of clinical parameters [13, 30, 31, 32], although in one of them higher AR was reported in GNRH antagonist protocols [31]. However, certain ovarian conditions could be responsible for a higher AR and at the same time require a higher dose of gonadotropins. In other words, the higher AR could be due to the underlying ovarian conditions rather than to the dose of gonadotropins. Thus, our study does not focus on the effects, if any, of gonadotropin stimulation on AR. We focus on the number of oocytes retrieved, and the age of the woman, which are better indicators of ovarian reserve than the dose of gonadotropins [33, 34].
In a previous study on 12 298 blastocysts similar AR of blastocysts were reported when they were grouped in 3 groups according to the number of retrieved oocytes (< 10, 10–20 and > 20) in the different groups of age [13]. However, the number of oocytes was not analyzed by establishing a separate group for each exact number of oocytes and multivariate regression according to other clinical parameters was not performed [13]. Our work confirms the safety, regarding ER, of obtaining a high number of oocytes. Indeed, when the number of oocytes was > 3 the ER, although very different in the different categories considered, remained stable in all the categories established on the basis of oocytes retrieved/MIIO, number of cycles, day 3 embryos or blastocysts, age groups, and their combinations. Interestingly, the pattern of ER was very similar in the different years in which the PGT‐A was performed. In our opinion, this reflects that the pattern described does not depend on the ovarian stimulation protocol or laboratory method, which have changed over time.
It should be noted that, unlike previous studies, we did not establish artificial cutoffs for the number of oocytes, but considered it as a continuum. This allowed us to ascertain from a mathematical point of view that the ER remained steady irrespective of the number of oocytes, if it was ≥ 3. Consequently the total number of obtained euploid embryos increased constantly with the number of retrieved oocytes or MIIO.
Our results show that an increase in the number of retrieved or MIIO results in a proportional increase in the number of obtained euploid embryos, revealing the beneficial effect of hyperresponse in this regard. It has to be highlighted that our investigation was focused on ER and that no other conditions associated with ovarian hyperresponse were analyzed. Of course when assessing the risks of ovarian hyperresponse, assessing the extent of the ovarian response does not depend only on the number of expected euploid embryos. Although the risk of ovarian hyperstimulation syndrome has been almost eradicated with GnRH antagonists, some complications associated with ovarian hyperresponse still occasionally occur [35, 36, 37].
It has been shown that AMH is a predictor of aneuploidy independent of age and other clinical parameters [38, 39, 40]. In our study, once age was adjusted for, AMH had no influence on ERaMII for a given number of MIIO. This is presumably because AMH and the number of oocytes are not independent variables but are closely related, as evidenced in our correlation analysis. In line with this, our series shows that the higher the number of oocytes obtained/metaphase II, the higher the total number of euploid embryos.
In our study we obtained an unexpected finding. In the majority of the categories considered the ER was higher when there were only 1 or 2 and sometimes also 3 retrieved oocytes or MIIO, both in total and when only blastocysts were considered. It could be speculated that in cases where there were only 1–2 (or 3 oocytes), this could correspond to the follicles that would have been naturally the dominant follicle(s), whereas in cases with > 3, these rescued oocytes could be of impaired chromosomal status. Moreover, in cases with 1–2 or 3 oocytes, follicular vascularization and intrafollicular hormonal status could promote a milieu more favorable for an euploid development of the oocyte.
In a study with human endometrial cells and mouse embryos, it was reported that hyperestrogenemia impaired embryo quality [41]. Our data could also be consistent with the aforementioned experiment [41] since cycles with 1–2–3 oocytes, those with higher ER, should have lower estrogen levels. In a previous study, no effect of estradiol levels on ER has been reported, although the inferior estradiol group considered was ≤ 2000 pg/mL; thus, the influence of lower values cannot be ascertained [13]. Unfortunately, estrogen levels were not available in our population.
We obtained also interesting findings when we related the ER with the MOR. First of all, for a given number of retrieved oocytes in the majority of cases the ER was higher if the MOR was 100%. In our opinion, it could reflect that cases with higher MOR corresponds to better quality oocytes, thus producing more frequently euploid embryos. Secondly, even when considering only cases with a 100% MOR, the ER were significantly higher in cases where there were only 1 or 2 MIIO. These data are consistent with the aforementioned comments regarding the hypothetical better quality of the oocytes that are the only one or two oocytes collected. Finally for each category established on the basis on the number of MIIO, in many cases, the ER was usually better when the MOR was 100%. This fact is consistent with the hypothesis that there is an association between oocyte quality and embryonic euploidy.
The other main topic of our research was the association with VO and AR. Previous studies do not report any association between AR and oocyte vitrification. However, the populations usually included a relatively small number of cases, and had some specific characteristics: performed with patients with a high number of vitrified oocytes per patient [17, 18]. In one of them day 3 embryos were studied [18], in others blastocysts [16], or both [18] and no adjustments were made by age or number of oocytes [16, 17, 18].
In our population an apparent association was observed between VO and increased risk of AR. However, when a regression analysis was performed, adjusting for age, day of biopsy, PGT‐A technique, PGT‐A indication, and number of MIIO, VO had a similar risk of aneuploidy to fresh oocytes. After adjustment, there was also no association with the trisomy rate or monosomy rate. As far as we know, the different chromosomes involved in AR in embryos resulting from VO have been studied only in one report [42]. The authors analyzed 383 blastocysts reporting an increase in the nondisjunction rate on chromosome 13 and a decrease in the nondisjunction rate on chromosome 18 and the sex chromosomes [42]. No adjustments were made. In our analysis, after adjustment, there was no specific chromosome aneuploidy associated with VO. Even in two chromosomes (chromosomes 19 and 22), the frequency was somewhat reduced, where the p‐values (p = 0.046 and 0.023) should probably be attributed to the large number of calculations performed.
In conclusion both ovarian hyperresponse and oocyte vitrification are safe regarding embryo euploidy. More studies are needed to investigate the lower aneuploidy rates in embryos resulting from 1 to 2 oocytes (or 3).
4.1. Limitations
We did not include in our study the cases in which PGT‐A was intended to be performed, but finally it was not carried out (arrested embryos, immature oocytes). Another limitation of our study is the combination of different biopsy types and different PGT‐A analyses along the duration of the study. We overcome these limitations with regression analysis of the different variables incorporated. Finally, we were not able to discriminate uniform aneuploidies from mosaicism, since during the period of the study the option of reporting mosaicism had not been incorporated into the routine clinical practice.
Funding
The author I.M. was supported by Basque Government funding (grant IT456‐22).
Ethics Statement
The study was approved by our Ethics Investigation Institutional Board (code CEIC E19/02).
Conflicts of Interest
The authors declare no conflicts of interest.
Supporting information
Table S1: rmb270003‐sup‐0001‐TableS1.docx.
Matorras R., Pérez‐Fernández S., Santos‐Zorrozua B., et al., “Both Oocyte Hyperresponse and Oocyte Vitrification Are Safe Procedures Regarding Embryo Euploidy: A Real‐World Study Involving Almost 200 000 Oocytes,” Reproductive Medicine and Biology 25, no. 1 (2026): e70003, 10.1002/rmb2.70003.
Data Availability Statement
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Table S1: rmb270003‐sup‐0001‐TableS1.docx.
Data Availability Statement
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.