Abstract
Purpose
During a typical IVF cycle, there is unavoidable attrition from oocytes retrieved to blastocysts formed. Some patients will not have blastocysts available to biopsy or embryos for transfer. The purpose of this study was to predict the number of transferable blastocysts available for patients based on their age and number of 2pn zygotes.
Methods
This was a retrospective cohort study of all fresh autologous IVF and ICSI cycles in which PGT-A was planned from 1/2012 to 3/2020. In total, 746 cycles from 571 patients were analyzed. Patient cycles were stratified into two groups: less than four 2pn zygotes (n = 85) and at least four 2pn zygotes (n = 661). Cycles were then stratified by patient age. Cycle outcomes, including number of cleavage-stage embryos, blastocysts, euploid blastocysts, and low level mosaic blastocysts, were determined.
Results
Cleavage-rate was independent of age and number of 2pn zygotes and ranged between 96 and 100%. Blastocyst conversion and euploid blastocyst conversion rates were directly correlated to age, ranging from 52 to 83% for blastocyst conversion and 0–28% for euploid blastocyst conversion. For patients above the age of 40 years with less than four 2pn zygotes, the risk of having no transferable embryos was 99.7%.
Conclusion
While the literature demonstrates higher live birth rates with the use of PGT-A in women of advancing age, this is inconsequential if there is no embryo available to transfer. Women over 40 years with less than four 2pn zygotes should consider transfer of one or more untested embryos either on day 3 or on day 5.
Supplementary Information
The online version contains supplementary material available at 10.1007/s10815-021-02365-0.
Keywords: IVF, PGT-A, Euploidy, Embryo culture, IVF attrition, Blastocyst
Introduction
Preimplantation genetic testing for aneuploidy (PGT-A) has become a frequently utilized ART procedure to test blastocysts for aneuploidy with the goal of maximizing pregnancy rate per embryo transfer, minimizing multiple gestations with single embryo transfer of a euploid blastocyst, and decreasing risk of a miscarriage due to aneuploidy [1–3]. During a typical IVF cycle, there is unavoidable attrition from oocytes retrieved, to embryos obtained, to blastocysts formed such that some patients, particularly those with advanced age or poor ovarian response, will not have blastocysts available to biopsy. While randomized trials have shown improved pregnancy rates per embryo transfer with the use of PGT-A in patients of advancing age, these trials primarily included patients with good ovarian reserve and multiple blastocysts available [1, 4]. In the largest multicenter randomized trial to date, livebirth rates were not significantly different per cycle start in women over the age of 35 years when analyzed per intention to treat [4]. The optimal age group within poor responders who would benefit most from PGT-A has yet to be determined.
In patients with good response to controlled ovarian hyperstimulation, pregnancy and live birth rates are higher after blastocyst transfer compared to cleavage-stage transfer (OR 1.30 and 1.48, respectively), but there is a significantly higher risk of having no embryos to transfer when culturing to blastocyst stage (OR 2.5, 95%CI 1.76 to 3.55) [5]. To our knowledge, there are no published data that provide more granular predictions for which patients will or will not have blastocysts available to biopsy. We hypothesize that patients with advanced age and poor response are less likely to have blastocysts available for biopsy and euploid blastocysts available for transfer. The purpose of this study was to investigate how patient age and number of zygotes affect embryo attrition in order to improve counseling for patients considering PGT-A.
Materials and methods
Cycle selection and outcome measures
This was a retrospective cohort study of all fresh autologous IVF and ICSI cycles in which PGT-A was planned from 1/2012 to 3/2020 at the Center for Infertility and Reproductive Surgery at Brigham and Women’s Hospital, Boston, MA. Only embryos from autologous IVF or ICSI cycles were included. PGT-A was offered to patients with advancing age (> 38 years), a history of recurrent pregnancy loss, those planning embryo biopsy for preimplantation genetic diagnosis (PGT-M or PGT-SR), and any patients requesting PGT-A. Cycles were excluded if (i) embryos were imported from another institution; (ii) cleavage-stage biopsy was performed; (iii) cleavage-stage transfer was performed; (iv) oocytes, two pronuclear (2pn), or cleavage-stage embryos were frozen; and (v) egg donation or in vitro maturation was used. Approval for this study was obtained from the Partners HealthCare Institutional Review Board (Protocol number 2020P001313).
The primary outcome was number of euploid blastocysts. Secondary outcomes were number of 2pn zygotes, cleavage-stage embryos, blastocysts, biopsy-quality blastocysts, and low level mosaic blastocysts. It is the policy at our institution to transfer low level mosaic embryos only when patients cannot or do not want to undergo further stimulated cycles. In these cases, they meet with a genetic counselor to review the plan and potential outcomes for transfer of the specific mosaicism of the embryo. Our associated labs use 25% as the cut-off for low level mosaicism. No patients underwent mosaic embryo transfer during the duration time of this study.
Clinical and laboratory protocols
Standard controlled ovarian hyperstimulation and monitoring protocols were used. Gonadotropin doses were determined based on age, serum antimullerian hormone (AMH) levels, follicle-stimulating hormone (FSH) levels, antral follicular count, body mass index (BMI), and previous response to stimulation. Ovarian stimulation was performed with the use of exogenous gonadotropins (Gonal-F, EMD-Serono, or Follistim, Organon USA; Menopur, Ferring Pharmaceuticals). Pituitary suppression was attained with the use of GnRH antagonist (Cetrotide, EMD-Serono) or GnRH agonist (leuprolide acetate, Abbott Laboratories). Gonadotropin dosage was adjusted according to each patient’s response to stimulation, which was monitored with the use of transvaginal ultrasounds and serial estradiol levels. When at least two follicles reached a mean diameter of 18 mm, final oocyte maturation was triggered with the use of human chorionic gonadotropin (hCG) (Pregnyl, Arganon Pharmaceuticals; Novarel, Ferring Pharmaceuticals), a GnRH agonist (leuprolide acetate, Abbott Laboratories), or both. The dose of hCG was tailored based on serum estradiol levels on the day of trigger and number of follicles. Patients considered to be at high risk for ovarian hyperstimulation syndrome (OHSS) were given a GnRH agonist, 5000 units hCG, or a combination trigger (leuprolide acetate 40 units with 1500 units hCG). Cycles were canceled when patients had no response to maximum dose gonadotropin stimulation. When recommending cycle cancelation, we reviewed with the patient that the cycle may result in no eggs retrieved and we allowed the patient to make the final decision to cancel. Ultrasound-guided oocyte retrieval was typically performed under intravenous general anesthesia 36 h after trigger.
All gametes and embryos were cultured at 37 °C in a dry incubator under an atmosphere of CO2 (5–6%), O2 (5%), and N2 (89–90%). Box incubators were used from 1/2012 to 4/2014 and benchtop incubators were used after 4/2014. Conventional insemination or ICSI was performed 4–6 or 3–5 h after oocyte retrieval, respectively, followed by a fertilization check 16–18 h afterwards. A single step medium (25-μL microdrops, Global Total, IVFOnLine, Guelph, Ontario, Canada under mineral oil) was used to culture 2pn zygotes (one zygote/drop). Embryos were evaluated on day 3 between 66 and 69 h post-insemination, underwent assisted hatching using laser pulses (ZILOS-tk laser; Hamilton Thorne), and then moved to individual fresh drops of equilibrated Global Total medium for culture to day 5/6. Embryo morphology was evaluated on day 5 between 112 and 115 h and scored according to the stage of development and, if at the blastocyst stage, by quality of the inner cell mass (ICM) and trophectoderm (TE). The stages and grades of those blastocysts eligible for biopsy in our program are shown in Supplemental Table I. Expanding blastocysts and any blastocyst with a “C” grade for both the ICM and TE, or a “D” for either the ICM or TE were considered ineligible. Embryos that were ineligible for biopsy or freeze on day 5 were left in culture and re-evaluated on day 6.
The embryos were biopsied on day 5 or day 6 once biopsy criteria were met. Biopsies were performed using standard techniques by embryologists certified to perform the procedure. Briefly, the embryo was immobilized with the use of a holding pipette and four to five trophectoderm cells were aspirated by means of a biopsy pipette with an internal diameter of 20–30 µm. The biopsied specimens were exposed to wash buffer, and the cells were placed in 0.2-mL polymerase chain reaction tubes with 2–3-µL lysis buffer. The specimens were stored at either − 20 °C or − 80 °C (depending on the predetermined genetic testing lab specifications) prior to being sent for analysis. The biopsied blastocysts were frozen by standard vitrification technique. Embryos that were determined by PGT-A to be euploid were eligible for transfer.
Statistical analysis
Cycles were stratified into two groups based on the number of zygotes at the fertilization check: those with less than four 2pn zygotes versus those with at least four 2pn zygotes. This cut-off value was used based on previous literature which used a cut-off of four 2pn zygotes to proceed with culture to blastocysts stage versus cleavage stage [6, 7]. Cycles were then stratified by patient age. Means and standard deviation were generated for continuous variables and frequencies and proportions for categorical variables. Relative risks (RR) and 95% confidence intervals (CI) were generated using Poisson regression for counts, Poisson regression with an offset for ratios, and log binomial regression for dichotomous outcomes. Regression models were adjusted for age, BMI, AMH, FSH, stimulation protocol, and use of ICSI. Generalized estimating equations were used to account for patients contributing more than one cycle. An alpha of 0.05 was considered statistically significant. All statistical analysis was performed with SAS® version 9.4 (Cary, NC, USA).
Results
A total of 746 cycles from 571 patients were analyzed. Demographic characteristics for cycles with patients with less than four 2pn zygotes (n = 85, 75 unique patients) versus at least four 2pn zygotes (n = 661, 514 unique patients) are represented in Table 1. The less than four 2pn zygotes group had a lower mean AMH, higher mean FSH, and higher incidence of diminished ovarian reserve, and were more likely to have been treated with a poor responder stimulation protocol. Two patients had canceled cycles (both for no response to maximum dose gonadotropin stimulation), two patients had no MII oocytes, and ten patients had no 2pn zygotes.
Table 1.
Demographic characteristics of cycles with planned PGT-A
| Less than four 2pn zygotes N = 85 |
At least four 2pn zygotes N = 661 |
|
|---|---|---|
| Producer age | 38.67 (3.96) | 37.00 (4.22) |
| BMI | 23.41 (3.76) | 24.82 (5.47) |
| AMH | 1.74 (1.43) | 4.21 (3.57) |
| Day 3 FSH | 9.12 (4.40) | 7.42 (2.19) |
| Number of previous cycles | 1.02 (1.59) | 0.70 (1.06) |
| Diagnosis | ||
| Diminished ovarian reserve | 17 (20.00) | 47 (7.11) |
| Endometriosis | 6 (7.06) | 11 (1.66) |
| Male factor | 6 (7.06) | 78 (11.80) |
| Uterine factor | 1 (1.18) | 16 (2.42) |
| Ovulatory dysfunction | 1 (1.18) | 50 (7.56) |
| Tubal Factor | 5 (5.88) | 27 (4.08) |
| Unknown factors | 16 (18.82) | 190 (28.74) |
| Other factor | 33 (38.82) | 242 (36.61) |
| Stimulation protocol | ||
| Poor responder | 42 (49.41) | 115 (17.40) |
| Good responder | 43 (50.59) | 546 (82.60) |
| ICSI | 62 (72.94) | 416 (62.93) |
Values represent mean (SD) for continuous or n (%) for categorical
Stimulation protocol: poor responder includes microflare, patch, letrozole, minimal stimulation, and ULDL; good responder includes antagonist and luteal Lupron other than ULDL
As expected, patients with less than four 2pn zygotes had significantly lower numbers of oocytes retrieved, MII oocytes, 2pn zygotes, cleavage-stage embryos, blastocysts, biopsy-quality blastocysts, and euploid blastocysts (Table 2). However, these patients had a 16% lower oocyte maturity rate (61.2% vs. 78.5%; aRR 0.77, 95%CI: 0.70–0.85) and a 23.8% lower fertilization rate (57.3% vs. 81.1%; aRR 0.75, 95%CI: 0.67–0.84) than those with four or more 2pn zygotes. Both groups had similar conversion of 2pn zygotes to cleavage and blastocyst stages (98.7% and 99.6%; aRR 0.99, 95%CI: 0.96–1.21; and 65.5% and 69.5%; aRR, 1.09 95%CI: 0.98–1.02, respectively). Patients with less than four 2pn zygotes had a 9.5-fold increased risk of having zero blastocysts and a 2.6-fold increased risk of having zero biopsy-quality blastocysts (95%CI: 3.97–22.78 and 1.75–3.73, respectively). Patients with less than four 2pn zygotes had significantly lower numbers of euploid embryos available compared to patients with at least four 2pn zygotes (Mean ± SD: 0.33 ± 0.64 vs. 2.19 ± 2.65; aRR 0.23, 95%CI 0.15–0.34). There was no difference in number of low level mosaic blastocysts (0.00 vs. 0.11 ± 0.37) or low level mosaic blastocysts conversion between groups (0.0% vs 0.6%).
Table 2.
Cycle outcomes for patients planned for PGT-A
| Less than four 2pn zygotes N = 85 |
At least four 2pn zygotes N = 661 (ref) |
Unadjusted RR (95%CI) | aRR (95%CI) | |
|---|---|---|---|---|
| # oocytes retrieved | 6.73 (4.53) | 18.67 (9.84) | 0.36 (0.31–0.42) | 0.43 (0.38–0.48) |
| # MII | 4.12 (3.08) | 14.66 (8.08) | 0.28 (0.24–0.33) | 0.33 (0.29–0.38) |
| % MII/oocytes retrieved | 61.2% | 78.5% | 0.78 (0.71–0.85) | 0.77 (0.70–0.85) |
| # 2pn | 2.36 (0.72) | 11.89 (6.89) | 0.20 (0.18–0.22) | 0.25 (0.23–0.27) |
| % 2pn/MII | 57.3% | 81.1%% | 0.71 (0.61–0.82) | 0.75 (0.67–0.84) |
| # cleavage-stage embryos | 2.33 (0.76) | 11.84 (6.90) | 0.20 (0.18–0.21) | 0.24 (0.22–0.27) |
| % cleavage stage/2pn | 98.7% | 99.6% | 0.99 (0.96–1.02) | 0.99 (0.96–1.21) |
| # blastocysts | 1.64 (0.90) | 7.79 (5.49) | 0.21 (0.18–0.24) | 0.27 (0.23–0.31) |
| % blastocysts/2pn | 69.5% | 65.5% | 1.06 (0.95–1.17) | 1.09 (0.98–1.02) |
| # with zero blastocysts | 11 (12.9%) | 9 (1.4%) | 9.50 (3.97–22.78) | n/a |
| # biopsy-quality blastocysts | 1.13 (0.87) | 5.07 (4.34) | 0.22 (0.19–0.27) | 0.29 (0.25–0.36) |
| % bx quality blastocysts/2pn | 47.9% | 42.6% | 1.12 (0.96–1.31) | 1.23 (1.05–1.44) |
| % bx quality blastocysts/total blastocysts | 68.9% | 65.1% | 1.06 (0.94–1.20) | 1.13 (1.00–1.27) |
| # with zero biopsy-quality blastocysts | 25 (29.4%) | 76 (11.5%) | 2.56 (1.75–3.73) | n/a |
| # euploid blastocysts | 0.33 (0.64) | 2.19 (2.65) | 0.15 (0.10–0.23) | 0.23 (0.15–0.34) |
| % euploid blastocysts/2pn | 14.0% | 18.4% | 0.76 (0.49–1.16) | 0.97 (0.63–1.48) |
| % euploid blastocysts/total oocytes retrieved | 4.9% | 11.7% | 0.42 (0.27–0.65) | 0.55 (0.35–0.86) |
| # low level mosaic blastocysts | 0.00 | 0.11 (0.37) | n/a | n/a |
| % low level mosaic blastocysts/2pn | 0.0% | 0.9% | n/a | n/a |
| % low level mosaic blastocysts/total oocytes retrieved | 0.0% | 0.6% | n/a | n/a |
| # euploid and low level mosaic blastocysts | 0.33 (0.64) | 2.30 (2.69) | 0.14 (0.09–0.22) | 0.22 (0.14–0.32) |
| % euploid and low level mosaic blastocysts/2pn | 14.0% | 19.3% | 0.72 (0.47–1.11) | 0.92 (0.60–1.40) |
| % euploid and low level mosaic blastocysts/total oocytes retrieved | 4.9% | 12.3% | 0.40 (0.26–0.62) | 0.52 (0.33–0.81) |
Values represent mean (SD) for continuous or n (%) for categorical
MII, metaphase II oocytes; 2PN, two pronuclear zygotes
Adjusted for age, BMI, AMH, FSH, stimulation protocol, and ICSI
n/a, algorithm does not converge
When cycle outcomes for patients with less than four 2pn zygotes were stratified by age, there was no difference in numbers of oocytes retrieved, MII oocytes, 2pn zygotes, and cleavage-stage embryos among age groups (Table 3). Cleavage-stage conversion (number of day 3 embryos per 2pn zygote) was 100% for all age groups except for age 38–40 years, in which the cleavage rate was 96.1%. As expected, patients < 35 years had a higher blastocyst conversion rate compared to older patients. Blastocyst conversion rates for each age group were 83.3%, 69.6%, 68.8%, 52.0%, and 66.8% (ages < 35, 35–37, 38–40, 41–42, and > 42 respectively). Compared to patients < 35 years, patients aged 41–42 years had a significantly lower number of blastocysts (1.18 vs. 2.00; aRR 0.59, 95%CI: 0.37–0.95) and biopsy-quality blastocysts (0.73 vs. 1.53; aRR 0.50, 95%CI: 0.26–0.98). These patients also had fewer euploid embryos available (0.09 vs 0.67), although the difference was no longer significant in the adjusted model (aRR 0.14, 95%CI: 0.01–1.57). Of women with less than four 2pn zygotes, none of the patients > 42 years had euploid blastocysts. Additionally, none of the patients with less than four 2pn zygotes had low level mosaic blastocysts.
Table 3.
Association of patient age with outcome when less than four 2pn zygotes are available
| Age | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| < 35 N = 15 |
35–37 N = 15 |
38–40 N = 32 |
41–42 N = 11 |
> 42 N = 12 |
||||||
| Mean (± SD) or n (%) | aRR (95%CI) |
Mean (± SD) or n (%) | aRR (95%CI) | Mean (± SD) or n (%) | aRR (95%CI) | Mean (± SD) or n (%) | aRR (95%CI) | Mean (± SD) or n (%) | aRR (95%CI) | |
| # oocytes retrieved | 7.33 (4.30) | ref | 7.00 (5.48) | 1.02 (0.67–1.57) | 6.97 (5.20) | 0.90 (0.65–1.23) | 5.27 (2.20) | 0.83 (0.57–1.21) | 6.33 (3.31) | 0.89 (0.64–1.26) |
| # MII | 3.73 (2.55) | ref | 4.13 (2.77) | 1.15 (0.76–1.74) | 4.53 (3.83) | 1.06 (0.74–1.52) | 3.64 (1.86) | 1.07 (0.69–1.67) | 3.92 (2.97) | 1.09 (0.73–1.61) |
| # 2pn | 2.40 (0.74) | ref | 2.40 (0.74) | 0.95 (0.75–1.22) | 2.31 (0.74) | 0.91 (0.73–1.14) | 2.27 (0.79) | 0.87 (0.67–1.13) | 2.50 (0.67) | 1.03 (0.85–1.25) |
| # cleavage-stage embryos | 2.40 (0.74) | ref | 2.40 (0.74) | 0.96 (0.75–1.23) | 2.22 (0.83) | 0.88 (0.71–1.11) | 2.27 (0.79) | 0.90 (0.69–1.19) | 2.50 (0.67) | 1.02 (0.84–1.24) |
| % cleavage stage/2pn | 100% | ref | 100% | 1.01 (0.98–1.05) | 96.1% | 0.97 (0.90–1.13) | 100% | 1.04 (0.96–1.13) | 100% | 0.99 (0.96–1.02) |
| # blastocysts | 2.00 (0.76) | ref | 1.67 (0.98) | 0.86 (0.59–1.26) | 1.59 (0.87) | 0.85 (0.62–1.16) | 1.18 (0.87) | 0.59 (0.37–0.95) | 1.67 (0.98) | 0.87 (0.62–1.23) |
| % blasts/2pn | 83.3% | ref | 69.6% | 0.90 (0.69–1.17) | 68.8% | 0.94 (0.74–1.18) | 52.0% | 0.68 (0.42–1.10) | 66.8% | 0.85 (0.66–1.09) |
| # biopsy-quality blastocysts | 1.53 (0.74) | ref | 1.20 (1.08) | 0.77 (0.45–1.34) | 1.03 (0.86) | 0.67 (0.42–1.09) | 0.73 (0.79) | 0.50 (0.26–0.98) | 1.17 (0.72) | 0.81 (0.56–1.17) |
| # euploid blastocysts | 0.67 (0.72) | ref | 0.60 (0.99) | 0.99 (0.37–2.60) | 0.25 (0.51) | 0.38 (0.13–1.11) | 0.09 (0.30) | 0.14 (0.01–1.57) | 0.00 (0.00) | n/a |
| % euploid blasts/2pn | 27.9% | ref | 25.0% | 0.98 (0.38–2.50) | 10.8% | 0.42 (0.14–1.22) | 4.0% | 0.15 (0.01–1.72) | 0.0% | n/a |
| # low level mosaic blastocysts | 0.00 | ref | 0.00 | n/a | 0.00 | n/a | 0.00 | n/a | 0.00 | n/a |
| % low level mosaic blasts/2pn | 0.0% | ref | 0.0% | n/a | 0.0% | n/a | 0.0% | n/a | 0.0% | n/a |
| # euploid and low level mosaic blastocysts | 0.67 (0.72) | ref | 0.60 (0.99) | 0.99 (0.37–2.60) | 0.25 (0.51) | 0.38 (0.13–1.11) | 0.09 (0.30) | 0.14 (0.01–1.57) | 0.00 | n/a |
| % euploid and low level mosaic blasts/2pn | 27.9% | ref | 25.0% | 0.98 (0.38–2.50) | 10.8% | 0.42 (0.14–1.22) | 4.0% | 0.15 (0.01–1.72) | 0.0% | n/a |
Mean ± SD are used for continuous variables and percentages are used for proportions
Adjusted for BMI, AMH, FSH, stimulation protocol, and ICSI
n/a, algorithm does not converge
When cycle outcomes for patients with at least four 2pn zygotes were stratified by age, patients with advancing age had lower mean numbers of oocytes retrieved, MII, 2pn zygotes, cleavage-stage embryos, blastocysts, biopsy-quality blastocysts, euploid blastocysts, and low level mosaic blastocysts (Table 4). Cleavage-stage conversion was 100% for all age groups except for age 38–40 years, in which the cleavage rate was 99.3%. As expected, there was an age-related decrease in blastocyst and euploid blastocyst conversion. Blastocyst conversion rates for each age group were 70.8%, 65.4%, 63.3%, 60.1%, and 52.2% (ages < 35, 35–37, 38–40, 41–42, and > 42 respectively). Euploid blastocyst rates were 28.2%, 19.7%, 13.0%, 5.6%, and 1.7% for each respective age group. Low level mosaic blastocyst conversion rates were 1.2%, 1.1%, 0.6%, 0.6%, and 0.0% for each respective age group. Of those with at least four 2pn zygotes, none of the patients > 42 years had low level mosaic blastocysts.
Table 4.
Association of patient age with outcome when at least four 2pn zygotes are available
| Age | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| < 35 N = 197 |
35–37 N = 154 |
38–40 N = 183 |
41–42 N = 98 |
> 42 N = 29 |
||||||
| Mean (± SD) or n (%) | aRR (95%CI) | Mean (± SD) or n (%) | aRR (95%CI) | Mean (± SD) or n (%) | aRR (95%CI) | Mean (± SD) or n (%) | aRR (95%CI) | Mean (± SD) or n (%) | aRR (95%CI) | |
| # oocytes retrieved | 21.20 (10.64) | ref | 18.89 (9.63) | 0.99 (0.88–1.10) | 16.96 (9.11) | 0.90 (0.81–1.00) | 17.05 (9.54) | 0.92 (0.81–1.05) | 16.52 (7.15) | 0.89 (0.75–1.05) |
| # MII | 16.50 (8.66) | ref | 14.99 (8.18) | 0.99 (0.88–1.11) | 13.39 (7.19) | 0.90 (0.81–0.99) | 13.51 (8.27) | 0.92 (0.81–1.06) | 12.24 (5.32) | 0.85 (0.70–1.03) |
| # 2pn | 13.76 (7.59) | ref | 12.14 (6.85) | 0.96 (0.85–1.09) | 10.61 (5.86) | 0.85 (0.77–0.95) | 10.76 (6.94) | 0.88 (0.76–1.02) | 9.72 (4.95) | 0.81 (0.65–1.01) |
| # cleavage-stage embryos | 13.67 (7.59) | ref | 12.14 (6.85) | 0.97 (0.86–1.09) | 10.54 (5.91) | 0.85 (0.77–0.95) | 10.76 (6.94) | 0.88 (0.76–1.02) | 9.72 (4.95) | 0.81 (0.65–1.01) |
| % cleavage stage/2pn | 100% | ref | 100% | 1.00 (1.00–1.01) | 99.3% | 1.00 (0.98–1.01) | 100% | 1.00 (1.00–1.01) | 100% | 1.00 (1.00–1.01) |
| # blastocysts | 9.74 (6.37) | ref | 7.94 (5.16) | 0.88 (0.76–1.01) | 6.72 (4.69) | 0.76 (0.67–0.87) | 6.47 (4.79) | 0.74 (0.62–0.89) | 5.07 (3.20) | 0.59 (0.47–0.76) |
| % blasts/2pn | 70.8% | ref | 65.4% | 0.91 (0.85–0.99) | 63.3% | 0.90 (0.83–0.97) | 60.1% | 0.85 (0.77–0.93) | 52.2% | 0.73 (0.63–0.86) |
| # biopsy-quality blastocysts | 6.91 (5.24) | ref | 5.27 (3.85) | 0.81 (0.69–0.95) | 4.19 (3.61) | 0.66 (0.56–0.77) | 3.58 (3.28) | 0.57 (0.46–0.71) | 2.14 (2.01) | 0.33 (0.23–0.45) |
| # euploid blastocysts | 3.88 (3.34) | ref | 2.39 (2.28) | 0.66 (0.54–0.81) | 1.38 (1.66) | 0.39 (0.31–0.48) | 0.60 (0.97) | 0.17 (0.12–0.24) | 0.17 (0.38) | 0.04 (0.02–0.10) |
| % euploid blasts/2pn | 28.2% | ref | 19.7% | 0.68 (0.59–0.80) | 13.0% | 0.45 (0.38–0.54) | 5.6% | 0.20 (0.14–0.27) | 1.7% | 0.05 (0.02–0.12) |
| # low level mosaic blastocysts | 0.17 (0.51) | ref | 0.13 (0.39) | 0.67 (0.35–1.28) | 0.06 (0.24) | 0.31 (0.15–0.65) | 0.06 (0.24) | 0.27 (0.10–0.71) | 0.00 | n/a |
| % low level mosaic blasts/2pn | 1.2% | ref | 1.1% | 0.69 (0.36–1.31) | 0.6% | 0.36 (0.17–0.75) | 0.6% | 0.30 (0.12–0.78) | 0.0% | n/a |
| # euploid and low level mosaic blastocysts | 4.05 (3.37) | ref | 2.52 (2.28) | 0.66 (0.55–0.80) | 1.44 (1.67) | 0.38 (0.31–0.47) | 0.66 (1.06) | 0.18 (0.13–0.25) | 0.17 (0.38) | 0.04 (0.02–0.09) |
| % euploid and low level mosaic blasts/2pn | 29.4% | ref | 20.8% | 0.68 (0.59–0.79) | 13.6% | 0.45 (0.38–0.53) | 6.1% | 0.20 (0.15–0.28) | 1.7% | 0.05 (0.02–0.12) |
Mean ± SD are used for continuous variables and percentages are used for proportions
Adjusted for BMI, AMH, FSH, stimulation protocol, and ICSI
Anticipated numbers of biopsy-quality blastocysts and euploid blastocysts by age and number of 2pn zygotes are represented in Supplemental Tables II and III. As expected, patients had more euploid embryos available when they had more 2pn zygotes. With advancing age, patients had fewer euploid embryos available and no patients above 44 years had a euploid embryo.
Discussion
In this study, we investigated how patient age and number of 2pn zygotes affect embryo attrition in PGT-A cycles. We found that cleavage-rate was independent of age and number of 2pn zygotes, and that overall, patients can expect 96–100% of 2pn zygotes to undergo cleavage. Blastocyst conversion was directly correlated to age, ranging from a high of 83% in the < 35-year group to a low of 52% in the 41–42-year group. The same was found for euploid blastocyst conversion, ranging from 28% in the < 35-year group to 0% in the > 42-year group, as expected given advancing age is associated with increased aneuploidy rates [8]. In our study population, zero patients above 44 years had euploid embryos. Given that the number of euploid blastocysts available for transfer depends on the number of 2pn zygotes available, we evaluated cycle outcomes for those with less than four 2pn zygotes. In this group, we found that 99.7% of patients over 40 years had no embryo available for transfer.
There are limited data in the literature focused specifically on blastocyst conversion rates. One can extrapolate conversion rates from studies investigating live birth rates following untested blastocyst transfer (48–80%) [9] and euploid blastocyst transfer after testing with PGT-A (61–63%) [4]. However, these studies, as with most evaluating blastocyst transfer outcomes, randomized patients at the blastocyst stage and not at cycle start. Gat et al. investigated euploidy rates by age and AMH and reported blastocyst conversion rates ranging from 44 to 57%, but only included three age categories (≤ 36 years, 37–40 years, ≥ 41 years) and did not stratify by ovarian response [10]. Morin et al. compared cycle outcomes by AMH, which correlated with ovarian response, and noted blastocyst conversion rates of 51–52% but did not stratify by age [11]. To our knowledge, our study is the first to report blastocyst conversion rates by age and number of 2pn zygotes.
It has been well established that euploidy rates are inversely related to age [8]. However, data are conflicting on how ovarian reserve impacts euploidy rates. Some studies report similar aneuploidy rates for age-matched patients with low versus normal ovarian reserve [11, 12], while other studies report higher aneuploidy in patients with diminished ovarian reserve when controlling for age [10, 13, 14]. If we consider response to ovarian hyperstimulation and number of 2pn zygotes as a marker of ovarian reserve, our data are consistent with those studies reporting similar euploidy rates for age-matched patients with low versus normal ovarian reserve. In our study, the euploid conversion rate (number of euploid blastocysts/2pn) for patients age < 35 years with less than four 2pn zygotes was 27.9% vs. 28.9% for patients with at least four 2pn zygotes. Similar euploidy rates were noted for each age category in these groups (less than four 2pn zygotes: 25.0%, 10.8%, 4.0%, and 0; at least four 2pn zygotes: 19.7%, 13.0%, 5.6%, and 1.7%). Our data offers predicted euploidy rates for patients by both age and response to ovarian hyperstimulation, specifically number of 2pn zygotes, which can be used for patient counseling on day 1 of embryo culture.
PGT-A is an effective tool for lowering multiple pregnancy rates through elective single embryo transfer and for increasing pregnancy rates for select patients [1, 4]. However, the desire to pursue single embryo transfer after PGT-A must be balanced with the ability to perform embryo biopsy for genetic testing. Patients with advancing age and diminished ovarian reserve have significantly lower number of blastocysts available to biopsy [13, 15]. Deng et al. showed that PGT-A did not improve live birth rates in women with poor ovarian response and that thirty-one PGT-A cycles were required to prevent one miscarriage in this group [16]. Additionally, patients who underwent PGT-A were significantly less likely to reach embryo transfer compared to those who underwent day 3 or day 5 transfer of an untested embryo (13.7% vs. 70.6%). Our results show the risk of having no euploid or low level mosaic embryos is 99.7% for patients with less than four 2pn zygotes who are above the age of 40 years. Zero patients above the age of 44 had transferable embryos, regardless of number of 2pn zygotes. These data can be used to counsel patients with low response and advancing age to consider transfer of one or more untested day 3 or day 5 embryos.
Many providers who favor transfer of blastocyst embryos over cleavage-stage embryos quote data demonstrating improved outcomes with blastocyst transfer [17–20]. Importantly, these studies were conducted in good prognosis patients (four to six good-quality cleavage-stage embryos, age < 36 years). Additionally, the large randomized trials assessing cycle outcomes with PGT-A included good prognosis patients with multiple good-quality blastocysts and reported outcomes per embryo transfer, not per retrieval [2, 4]. Our study found that over 99% of women above the age of 40 years who respond poorly to stimulation (less than four 2pn zygotes) will have no blastocyst available for transfer. Cleavage rate of 2pn zygotes in this population was 100% compared to blastocyst conversion rates of 52–67%. While pregnancy and live birth rates for patients undergoing day 3 transfer are lower than those for women undergoing day 5 transfer because such patients are generally of poorer prognosis, day 3 transfer affords some chance of a pregnancy and live birth because an embryo transfer is being performed, as opposed to no transfer in cycles with planned blastocyst transfer but no blastocyst conversion. As such, we recommend discussing the option of day 3 embryo transfer with patients who are > 40 years and have less than four 2pn zygotes. Per ASRM guidelines, women aged 41–42 years can transfer up to five untested cleavage stage embryos. When discussing transfer of one or more cleavage stage embryos, providers should review the risks of multiple gestation and possible miscarriage or ectopic pregnancy [21]. The ideal cutoff for the number of 2pn zygotes to culture embryos to blastocyst stage in this age group has yet to be established. At our institution, we use a conservative cutoff of eight 2pn zygotes in women > 40 years to culture to day 5, but then consider fresh day 5 transfer if only low-quality blastocysts are noted on day 5.
Counseling on anticipated embryo attrition is also important for older women with good ovarian response and many 2pn zygotes. In our patient population, the euploidy rate for women above the age of 42 years with at least four 2pn zygotes is 1.7% (no patients in this group had low level mosaic embryos available). With this in mind, a patient would likely need fifty-nine 2pn zygotes to have a euploid embryo. While this may be an over-estimation of the number of zygotes required given the small number of patients we were able to include in this group, we believe it is reasonable given the presumed 85–90% aneuploidy rate in this population based on blastocyst biopsy results [8]. Providers should discuss practice-specific age-related attrition and aneuploidy rates with their patients. We recognize that a recommendation for transfer of untested embryos does not take into account patient wishes, including the desire to optimize time to pregnancy with selective transfer of only euploid embryos. Shared decision-making is crucial, with a discussion of expectations prior to stimulation, and ideally on day 1 at the fertilization check, when the number of zygotes is known. In the setting of multiple failed cycles with low total numbers of 2pn zygotes, it is also important to discuss alternative treatment options with higher success rates, including the use of donor oocytes.
This study was retrospective in nature and limited by small sample sizes when patients were stratified by age and number of 2pn zygotes. Conversion rates are lab-dependent so these findings may not be generalizable. We are fortunate to practice in a state with mandated insurance coverage for infertility, so our patients with mosaic embryos usually choose to cycle again to obtain a euploid embryo. Studies have shown that transfer of mosaic embryos, particularly those with low level mosaicism or single segmental aneuploidy, can lead to live birth [22]. Mosaic embryo results are lab-dependent so our reported rates of low level mosaic embryos may differ from other institutions. Additionally, our reported rates of transferable embryos may be an under-estimate for providers who offer mosaic embryo transfer and use different labs. To best address the question of who should be recommended PGT-A versus fresh embryo transfer, a prospective trial randomizing poor responders to day 3 versus day 5 transfer is critical. Neuhausser et al. are planning such a prospective randomized trial to address this clinical question [23].
Supplementary Information
Below is the link to the electronic supplementary material.
Data availability
The data underlying this article are available in the article and in its online supplementary material.
Code availability
Not applicable.
Declarations
Ethics approval
This retrospective chart review study involving human participants was in accordance with the ethical standards of the institutional and national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Approval for this study was obtained from the Partners HealthCare Institutional Review Board (Protocol number 2020P001313).
Consent to participate and consent for publication
See IRB approval above.
Conflict of interest
The authors declare no competing interests.
Footnotes
Publisher's note
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Contributor Information
C. E. Gordon, Email: cgordon@bwh.harvard.edu
K. W. Keefe, Email: kwkeefe@bwh.harvard.edu
E. S. Ginsburg, Email: eginsburg@bwh.harvard.com
C. Racowsky, Email: cracowsky@bwh.harvard.edu
A. Lanes, Email: alanes@bwh.harvard.edu
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Data Availability Statement
The data underlying this article are available in the article and in its online supplementary material.
Not applicable.