The comparision among euploidy of preimplantation blastocysts in different controlled ovary stimulation(COH) protocols

doi:10.21203/rs.3.rs-3636577/v1

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Research Article

The comparision among euploidy of preimplantation blastocysts in different controlled ovary stimulation(COH) protocols

https://doi.org/10.21203/rs.3.rs-3636577/v1

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Journal Publication

published 06 May, 2024

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Purpose

To compare differences in euploidy rates for blastocysts in preimplantation genetic testing for aneuploidy (PGT-A) cycles after gonadotropin-releasing hormone agonist (GnRH-a) long and short protocols, GnRH-antagonist (GnRH-ant) protocol, progestin-primed ovarian stimulation and mild stimulation protocols, and other ovary stimulation protocols.

Methods

This was a retrospective cohort study from the Assisted Reproductive Medicine Department of Shanghai First Maternity and Infant Hospital. A total of 1657 PGT-A cycles with intracytoplasmic sperm injection after different controlled ovary hyperstimulation protocols were analyzed, and a total of 3154 embryos were biopsied. Differences in euploidy rate per embryo biopsied, embryo euploidy rate per oocyte retrieved and cycle cancellation rate were compared.

Results

For the PGT-A cycles, the euploidy rate per embryo biopsied was lower in the GnRH-ant protocol than in the GnRH-a long protocol (53.26% vs. 58.68%, respectively). Multiple linear regression showed that the GnRH-ant protocol was associated with a lower euploidy rate per embryo biopsied (β = −0.079, P = 0.011). The euploidy rate per embryo biopsied was not affected by total gonadotropin dosage, duration of stimulation and number of oocytes retrieved. The embryo euploidy rate per oocyte retrieved was similar in all protocols, and was negatively correlated with the total number of oocytes retrieved (β = −0.003, P = 0.003).

Conclusion

Compared with the GnRH-a long protocol, the GnRH-ant protocol was associated with a lower euploidy rate per embryo biopsied. The total gonadotropin dosage, duration of stimulation and number of oocytes retrieved did not appear to significantly influence euploidy rates.

Euploidy rate per embryo biopsied

embryo euploidy rate per oocyte retrieved

ovarian stimulation

preimplantation genetic testing for aneuploidy

cycle cancellation rate.

Of guiding significance selecting clinical ovarian stimulation protocols and obtaining higher quality embryos.
provides direction and reference for future research into the safety and application of GnRH antagonists.

In recent years, assisted reproductive technology has helped many infertile couples achieve clinical pregnancies and live births. However, there are still some couples who have difficulty achieving successful outcomes, because of repeated implantation failure, recurrent pregnancy loss and other reasons. One retrospective analysis reported that the rate of reduction in the percentage of euploid blastocysts increases with age^[1]. In addition, embryos undergoing in vitro fertilization (IVF) have a high rate of aneuploidy^[2–3]. The evaluation of embryo quality in IVF/intracytoplasmic sperm injection (ICSI) mainly depends on embryonic morphological indicators, but good embryo morphology does not necessarily exclude abnormal chromosome composition^[4]. The acquisition of euploid embryos is a prerequisite for successful pregnancy for patients with advanced maternal age and recurrent pregnancy loss/repeated implantation failure. Preimplantation genetic testing for aneuploidy (PGT-A) provides a relatively effective and safe option for the above infertile populations, and may help avoid the psychological pressure and economic burden caused by repeated pregnancy failure. Many studies have shown that the application of PGT-A can reduce abortion rates and increase cumulative live birth rates, especially for elderly women over 35 years of age with recurrent pregnancy loss^[5–6].

For controlled ovary hyperstimulation (COH), the most common protocols are the gonadotropin-releasing hormone agonist (GnRH-a) long protocol and GnRH-antagonist (GnRH-ant) protocol. Both prevent a premature rise in luteinizing hormone (LH), and are widely used for pituitary downregulation in the assisted reproductive cycle^[7–8]. Studies have suggested that the GnRH-a long protocol is more beneficial than the GnRH-ant protocol for improving implantation and clinical pregnancy rates, while the incidence of ovarian hyperstimulation syndrome is significantly reduced in women with the GnRH-ant regimen^[9–10]. Progestin-primed ovarian stimulation (PPOS) was first proposed in 2015 by Yanping et al.^[11], and involves the negative feedback pathway of progesterone blocking the LH surge^[12]. In recent years, more PPOS protocols have been applied in clinical practice, especially in patients with polycystic ovary syndrome. In 1990, Garcia et al.^[13] proposed that the GnRH-a short protocol could also be effective at inhibiting pituitary gonadotropin secretion. Later studies suggested that the pregnancy rate and number of oocytes retrieved by PPOS were inferior to those with the long protocol^[14–15]. According to the American Society for Reproductive Medicine, for poor ovarian responders, the mild stimulation protocol and natural/modified natural cycle should be considered^[16], but compared with that with conventional stimulation such as GnRH-a long /GnRH-ant protocol, the number of oocytes retrieved is significantly reduced^[17], and there is no advantage for women with a normal ovarian response^[18].

Although each of these protocols has advantages and disadvantages, the effects of different protocols on embryo euploidy remains controversial. Wang et al.^[19] suggested that the GnRH-ant protocol was associated with higher aneuploidy rates in early aborted tissues and blastocysts compared with the GnRH-a long protocol. Another study concluded that the total number of euploid blastocysts was not significantly different for patients treated with PPOS versus those treated with conventional GnRH antagonists^[20]. However, there are few comparative studies of euploidy rates among the different protocols, including the GnRH-a short and mild stimulation protocols. The purpose of this study was to compare euploidy rates and other clinical outcomes among the GnRH-a long/short protocols, GnRH-ant protocol, PPOS, mild stimulation protocol and other COH protocols.

Study subjects

Data from the Reproductive Medicine Center of Shanghai First Maternal and Infant Hospital affiliated to Tongji University were retrospectively analyzed for patients who underwent PGT-A cycles between July 2019 and July 2022.

The inclusion criteria were: (1) Advanced maternal age (38 years old and above); (2) Recurrent spontaneous abortion (defined as three or more consecutive pregnancy losses/two or more consecutive pregnancy losses including at least one chromosome abnormality in early aborted tissues within 28 weeks of gestation with the same sexual partner); (3) Repeated implantation failure (failure to achieve a clinical pregnancy after the transfer of at least four good-quality embryos within a minimum of three fresh or frozen cycles, and under 40 years of age).

Exclusion criteria include: (1) Severe teratospermia; (2) One of the spouses exhibited a chromosomal abnormality; (3) Required to suspend treatment for various reasons after entering the cycle; (4) Incomplete data.

The study was approved by the Research Ethics Committee of Shanghai First Maternity and Infant Hospital.

Treatment protocols

The study was approved by the Research Ethics Committee of Shanghai First Maternity and Infant Hospital. The COH protocol was personalized according to the patients’ age, body mass index (BMI), basic hormone levels, antral follicle count and previous response to ovarian stimulation, and the gonadotropin dose was adjusted according to follicle growth and hormone levels. All patients in this study were treated with ICSI cycles.

For the GnRH-a long protocol, 1.25 or 1.88 mg GnRH-a (triptorelin acetate, Diphereline, Ipsen Pharma Biotech, France) was administered from the mid-luteal phase of the previous cycle, and continued until ovarian suppression was achieved (with LH < 5 IU/L and blood estradiol < 50 pg/mL, endometrium < 4–5 mm, and no functional cysts). Then recombinant follicle stimulating hormone (rFSH, Puregon, Organon, Dublin, Ireland or Gonal F, Merck Serono S.p.A, Modugno, Italy) or human menopausal gonadotropin (Lizhu Pharmaceutical Trading Co., Zhuhai, China) were administered at 75–300 IU until the day of ovulation trigger.

For the GnRH-ant protocol, 50–225 IU gonadotropin was administered from days 2–5 of the menstrual cycle, followed by 0.25 mg/day GnRH-ant (ganirelix, Orgalutran, Organon, Dublin, Ireland) administered on day 5 or 6 of gonadotropin treatment until the day of ovulation trigger.

For the PPOS protocol, 20 mg/day dydrogesterone (Abbott Biologicals B.V., Netherlands) was administered from the day of ovarian stimulation to the day of ovulation trigger. This prescribed dose has been applied in previous studies to ensure pituitary suppression.

For the GnRH-a short protocol, GnRH-a was administered from day 2 of the menstrual cycle, gonadotropin was administered from day 3 until the day of ovulation trigger.

For the mild stimulation protocol, 50–100 mg/day clomiphene citrate (Fertilan, Medochmie Ltd., China) or 2.5–5 mg/day letrozole (Ferri, Jiangsu Hengrui Pharmaceuticals, Jiangsu, China) was orally administered for 5 consecutive days starting on day 2–3 of the menstrual cycle, followed by 0–225 IU gonadotropin/day until the trigger day.。

Other protocols were the natural cycle/modified natural cycle protocol.

When three leading follicles each reached diameters ≥ 18 mm, 0.1 mg triptorelin (Decapeptyl, Ferring Pharmaceuticals, Netherlands) and 2000 or 5000 IU human chorionic gonadotropin (Lizhu Pharmaceutical Trading Co., China) were administered to trigger final maturation of oocytes. Oocyte retrieval was performed approximately 36 h later.

Embryo culture and Biopsy

On the day of oocyte retrieval, a semen sample was collected by masturbation after 3–7 days of abstinence. Sperm density gradient centrifugation was followed by ICSI. All successfully fertilized embryos were incubated under suitable conditions to blastocysts, which were graded according to Gardner criteria^[21].

Trophectoderm (TE) biopsy was performed on good-quality blastocysts, ,and approximately five cells were aspirated gently and separated from the blastocyst by applying multiple pulses of a non-contact 1.48-µm diode laser (Saturn 5 ActiveTM, Cooper Surgical, Inc., CT, USA) through a zona pellucida opening created by the laser. The biopsied cells were subsequently washed three times in 1× phosphate buffered saline (PBS) (Life Technologies, NY, USA), transferred to a polymerase chain reaction (PCR) tube containing 2.5 µl 1× PBS, and cryopreserved at – 80°C until analysis was performed. Genetic screening was performed using next-generation sequencing-based VeriSeq PGS assay following standard protocols and manufacturer recommendations (Illumina Inc., San Diego, USA). The PGT-A report classified embryos as euploid, aneuploid, mosaic or non-conclusive, and only euploid embryos were transferred. All good-quality blastocysts after TE biopsy were cryopreserved using vitrification.

Statistical analysis

Euploidy rate per embryo biopsied was assessed as the primary outcome, and the embryo euploidy rate per oocyte retrieved and cycle cancellation rate were secondary outcomes. Euploidy rate was defined as the total number of embryos proven to be euploid divided by the total number of embryos that were biopsied. The embryo euploidy rate per oocyte retrieved was defined as the number of euploid embryos divided by the total number of oocytes retrieved. Cycle cancellation rate was defined as the number of cycles in which no euploid embryo was obtained for various reasons divided by the total number of cycles in each protocol.

Data were analyzed using the Statistical Package for Social Sciences version 25.0 (SPSS Inc., Chicago, IL). Continuous independent variables were presented as mean ± standard deviation or mean ± standard error of the mean. For data that did not conform to a normal distribution, the Kruskal Wallis rank sum test was selected. Categorical data were expressed as percentages and calculated using Chi-square tests. The confounding factors were screened by generalized linear regression and binary logistic regression. P < 0.05 was considered statistically significant.

Baseline characteristics

A total of 1657 PGT-A cycles were included in this study, of which 252 were treated with the GnRH-a long protocol, 487 with the GnRH-ant protocol, 343 with the PPOS, 164 with the GnRH-a short protocol, 321 with mild stimulation protocol, and the remaining 90 with other protocols. The baseline characteristics of the patients are shown in Table 1. As shown in the table, there were significant differences among groups in age, BMI, number of previous IVF attempts, infertility years, infertility types, and serum concentrations of anti-Mullerian hormone, FSH and estradiol, whereas no statistical differences were found in serum concentrations of thyroid-stimulating hormone and LH.

Table 1

Demographic characteristics of the cycles
	GnRH-a long (n = 252)	GnRH-ant (n = 487)	PPOS (n = 343)	GnRH-a short (n = 164)	mild stimulation (n = 321)	others (n = 90)	p value
Age (yr)	36.11 ± 0.28	37.31 ± 0.21	38.29 ± 0.27	38.24 ± 0.41	40.31 ± 0.25	39.92 ± 0.47	<0.001*
BMI(kg/m²)	22.13 ± 0.17	21.77 ± 0.12	22.39 ± 0.16	22.28 ± 0.25	22.74 ± 0.16	22.58 ± 0.35	<0.001*
Number of previous IVF attempts	2.33 ± 0.11	2.34 ± 0.08	3.90 ± 0.16	2.87 ± 0.17	4.44 ± 0.20	3.64 ± 0.24	<0.001*
Infertility years	2.23 ± 0.16	2.69 ± 0.13	3.14 ± 0.17	2.65 ± 0.20	3.08 ± 0.19	2.62 ± 0.26	0.024*
Infertility types
Primary	35(13.89%)	109(22.38%)	77(22.45%)	26(15.85%)	61(19.00%)	23(25.56%)	0.03*
Secondary	217(86.11%)	378(77.62%)	266(77.55%)	138(84.15%)	260(81.00%)	67(74.44%)
TSH(mIU/L)	2.08 ± 0.90	2.01 ± 0.57	2.60 ± 0.40	2.08 ± 0.81	2.31 ± 0.25	1.95 ± 0.13	0.527
AMH(ng/ml)	3.30 ± 0.19	3.69 ± 0.58	2.65 ± 0.17	2.32 ± 0.17	1.50 ± 0.13	2.38 ± 0.47	<0.001*
FSH(IU/L)	6.74 ± 0.25	8.34 ± 0.22	8.23 ± 0.23	8.16 ± 0.33	9.78 ± 0.35	8.60 ± 0.56	<0.001*
LH(IU/L)	4.26 ± 0.29	5.14 ± 0.36	4.35 ± 0.17	5.99 ± 0.74	4.28 ± 0.23	4.68 ± 0.59	0.13
E2(pg/ml)	76.35 ± 10.81	26.68 ± 3.28	33.70 ± 5.05	21.55 ± 2.02	22.16 ± 3.19	72.13 ± 11.53	<0.001*
BMI = Body mass index; AMH = Anti-mullerian hormone; TSH = Thyroid stimulating hormone; FSH = Follicle-stimulating hormone; LH = Luteinixing hormone; E2= Estradio; * P < 0.05

Ovary stimulation outcomes

Of the 1657 PGT-A cycles included in the study, 772 cycles were cancelled because no euploid embryos were obtained for transfer, of which 565 cycles did not obtain high-quality blastocysts assessed by laboratory morphological indicators for biopsy. As shown in Table 2, after preliminary analysis, there were statistical differences in total gonadotropin dosage (P < 0.001), duration of stimulation (P < 0.001), number of oocytes retrieved (P < 0.001), metaphase II (MII) oocyte rate (P < 0.001), fertilization rate (P < 0.001), euploidy rates per embryo biopsied (P = 0.017), embryo euploidy rates per oocyte retrieved (P < 0.001) and cycle cancellation rate (P < 0.001) among all groups, while there were no significant differences in cleavage rate (P = 0.216). These results required further regression analysis to exclude confounding effects due to differences in baseline indicators.

Table 2

Characteristics and outcomes of ovarian stimulation
	GnRH-a long	GnRH-ant	PPOS	GnRH-a short	mild stimulation	others	p value
No. of PGT-A cycles	252	487	343	164	321	90
Total dosage of Gn (IU)	2695 ± 62	2043 ± 29	1926 ± 39	1935 ± 67	1120 ± 34	2055 ± 135	<0.001*
Duration of stimulation (days)	11.36 ± 0.15	8.63 ± 0.09	8.37 ± 0.14	8.62 ± 0.20	7.60 ± 0.17	8.52 ± 0.43	<0.001*
Total No. of retrieved oocytes (n)	2682	4358	2460	1079	1155	415
No. of oocytes retrieved (n)	10.64 ± 0.35	8.95 ± 0.30	7.17 ± 0.33	6.58 ± 0.35	3.60 ± 0.18	4.71 ± 0.47	<0.001*
Total No. of MII (n)	2115	3462	2048	890	947	344
No. of MII (n)	8.39 ± 0.29	7.11 ± 0.24	5.97 ± 0.28	5.43 ± 0.29	2.95 ± 0.14	3.70 ± 0.35	<0.001*
MII rate (%)	78.86	79.44	83.25	82.48	81.99	82.89	<0.001*
Total No. of oocytes fertilized (n)	1487	2541	1578	674	698	248
No. of oocytes fertilized (n)	5.90 ± 0.22	5.22 ± 0.18	4.60 ± 0.22	4.11 ± 0.24	2.17 ± 0.12	2.66 ± 0.25	<0.001*
Oocytes fertilized rate (%)	70.31	73.40	77.05	75.73	73.71	72.09	<0.001*
No. of cleavage-stage embryo (n)	1434	2472	1530	646	682	238
Cleavage rate (%)	96.44	97.28	96.96	95.85	97.71	95.97	0.216
Total No. of blastocysts biopsied (n)	650	1203	661	292	263	85
No. of blastocysts biopsied (n)	2.58 ± 0.15	2.47 ± 0.13	1.93 ± 0.12	1.78 ± 0.16	0.82 ± 0.07	0.86 ± 0.15	<0.001*
No. of euploid blastocysts (n)	353	587	370	147	161	48
Euploidy rate per embyo biopsied (%)	58.68 ± 35.23	53.26 ± 37.16	59.88 ± 37.77	57.79 ± 37.36	63.59 ± 41.78	65.33 ± 40.02	0.017*
Euploidy rate per oocyte retrieved (%)	14.69 ± 16.13	13.71 ± 16.33	15.27 ± 19.99	15.04 ± 20.73	15.84 ± 27.64	13.35 ± 24.66	<0.001*
No. of oocytes retrieved	76	199	158	75	209	55
cycle cancellation rate(%)	30.16	40.86	46.06	45.73	65.11	61.11	<0.001*
PGT-A = preimplantation genetic testing for aneuploidy; Gn = gonadotropin; MⅡ= metaphase II oocytes; * P < 0.05

Multiple linear regressions were performed for the MII oocyte, oocytes fertilized and cleavage rates. When confounding factors, such as age and BMI, were excluded, there were no differences in the MII oocyte, oocytes fertilized and cleavage rates among all the COH protocols (Table 3).

Table 3

Multivariate liner regression analysis for MII rate, oocytes fertilized rate and cleavage rate
Independent variable	MII rate (%)*		Oocytes fertilized rate (%)*		Cleavage rate (%)*
Independent variable	β (95% CI )	p-value	β (95% CI )	p-value	β (95% CI )	p-value
GnRH-a long		.		.		.
GnRH-ant	-0.014(-0.046,0.019)	0.407	0.018(-0.026,0.063)	0.421	0.002(-0.016,0.021)	0.798
PPOS	0.008(-0.027,0.043)	0.666	0.038(-0.011,0.086)	0.132	0.005(-0.015,0.025)	0.611
GnRH-a short	0.005(-0.036,0.046)	0.808	0.027(-0.029,0.084)	0.344	-0.006(-0.029,0.017)	0.623
mild stimulation	-0.02(-0.062,0.021)	0.338	-0.025(-0.083,0.033)	0.399	0.017(-0.007,0.041)	0.155
others	-0.013(-0.069,0.044)	0.659	0.017(-0.062,0.096)	0.67	-0.016(-0.049,0.016)	0.329
*Adjustment include the woman’s age,BMI, number of previous IVF attempts, infertility years, infertility types, serum concentration of AMH, FSH, LH, E2, COH protocol, total gonadotropin dosages, durations of stimulation and numbers of oocytes retrieved.

During the ovary stimulation and embryo culture, some cycles failed because of the inability to obtain oocytes, MII oocytes, morphological high-quality blastocysts and/or euploid embryos. Binary logistic regression indicated that the COH protocol had a significant effect on cycle cancellation rate (Table 4). Compared with the GnRH-a long protocol, the GnRH-ant protocol [Odds ratio (OR) = 1.422, P = 0.047], mild stimulation protocol (OR = 1.333, P < 0.001), and other protocols (OR = 2.155, P = 0.005) were associated with higher cycle cancellation rates, but there were no statistically significant differences between the PPOS and GnRH-a short protocol.

Table 4

Binary logistic regression regression analysis for cycle cancellation rate
Independent variable	β	t	p-value	aOR
GnRH-a long
GnRH-ant	0.352	0.178	0.047*	1.422
PPOS	0.287	0.191	0.132	1.333
GnRH-a short	0.393	0.223	0.078	1.482
mild stimulation	0.851	0.201	<0.001*	2.343
others	0.768	0.276	0.005*	2.155
OR adjusted based on woman’s age,BMI, number of previous IVF attempts, infertility years, infertility types, serum concentration of AMH, FSH, LH, E2, COH protocol *P<0.05

Table 5

Multivariate liner regression analysis for embryo euploidy rate per oocyte retrieved
Independent variable	β	t	p-value	95% CI for Exp(B)
Independent variable	β	t	p-value	Lower	Upper
GnRH-a long		.	.	.	.
GnRH-ant	-0.02	0.0173	0.239	-0.054	0.014
PPOS	0	0.0189	0.991	-0.037	0.037
GnRH-a short	-0.009	0.0219	0.669	-0.052	0.034
mild stimulation	-0.008	0.0223	0.725	-0.052	0.036
others	-0.018	0.0305	0.562	-0.077	0.042
Total dosage of Gn	-8.82E-06	1.02E-05	0.387	-2.88E-05	1.12E-05
Duration of stimulation	0	0.0031	0.943	-0.006	0.006
Total No. of oocytes retrieved	-0.003	0.0011	0.003*	-0.005	-0.001
Adjustment include the woman’s age,BMI, number of previous IVF attempts, infertility years, infertility types, serum concentration of AMH, FSH, LH, E2, COH protocol, total gonadotropin dosages, durations of stimulation and numbers of oocytes retrieved. *P<0.05

Table 6

Multivariate liner regression analysis for euploidy rate per embryo biopsied
Independent variable	β	t	p-value	95% CI for Exp(B)
Independent variable	β	t	p-value	Lower	Upper
GnRH-a long		.	.	.	.
GnRH-ant	-0.088	0.0349	0.011*	-0.157	-0.02
PPOS	-0.034	0.0382	0.379	-0.109	0.041
GnRH-a short	-0.048	0.046	0.297	-0.138	0.042
mild stimulation	-0.047	0.0486	0.338	-0.142	0.049
others	-0.019	0.0668	0.777	-0.15	0.112
Total dosage of Gn	0.00	0.00	0.60	0.00	0.00
Duration of stimulation	-0.001	0.007	0.854	-0.015	0.012
Total No. of oocytes retrieved	0.001	0.0021	0.529	-0.003	0.005
Adjustment include the woman’s age,BMI, number of previous IVF attempts, infertility years, infertility types, serum concentration of AMH, FSH, LH, E2, COH protocol, total gonadotropin dosages, durations of stimulation and numbers of oocytes retrieved. *P<0.05

Multiple linear regression showed that there was no statistical difference in the effect of COH protocol on the embryo euploidy rate per oocyte retrieved (Table IV), whereas COH protocol had a statistical difference in the effect of total number of oocytes retrieved. The more oocytes were retrieved, the lower the utilization rate (β = −0.003; P = 0.003).

When the same confounders were included in multiple linear regression (Table V), no correlation was found between baseline data and euploidy rate for most confounders, except age (P < 0.001), and there was no statistical difference in the effects of total gonadotropin dose, duration of stimulation and number of oocytes retrieved on euploidy rate per embryo biopsied. The euploidy rate per embryo biopsied was correlated with the type of COH protocol. The GnRH-ant protocol was associated with a lower euploidy rate per embryo biopsied compared with the GnRH-a long protocol (β = −0.079, P = 0.011), but all the other protocols showed no statistical differences.

In this study of PGT-A cycles, the GnRH-ant protocol was associated with a lower euploidy rate per embryo biopsied compared with the GnRH-a long protocol, whereas the PPOS, GnRH-a short protocol, mild stimulation protocol, and other protocols did not differ statistically. The total gonadotropin dosage, duration of stimulation and number of oocytes retrieved had no significant effect on embryo euploidy per embryo biopsied. The study also showed that the embryo euploidy rate per oocyte retrieved was not statistically different among protocols, but was inversely associated with the total number of oocytes retrieved. Also, all protocols except PPOS were associated with higher cycle cancellation rates compared with the GnRH-a long protocol.

The main reason for poor reproductive outcomes in patients with advanced maternal age, recurrent abortion and repetitive implantation failure is believed to be the high incidence of embryo aneuploidy^[22]. One study showed that a PGT-A diagnosis of uniform aneuploidy provides a 98% predictive value for early lethality and a 86.3% miscarriage rate^[23]. However, the effects of ovarian stimulation on embryonic euploidy during the PGT-A cycle are still being explored. A retrospective analysis study^[19] showed that for patients with early abortion after IVF/ICSI, the GnRH-ant protocol group had a significantly higher aneuploidy rate in early abortion tissues (48.51% vs. 64.19%) compared with the GnRH-a long protocol group. Also, in PGT-A cycles, the aneuploidy rate of blastocysts was significantly higher in the GnRH-ant protocol than that in the GnRH-a long protocol (39.69% versus 52.27%, respectively), consistent with the results obtained in this study. In contrast, it has also been suggested that there is no difference in chromosomal abnormality rates of blastomeres when GnRH agonists or antagonists are used^[24]. The increase in chromosomal abnormalities observed after ovarian stimulation is mainly due to the increased incidence of chromosomal segregation errors during the first embryo cleavage division, resulting in chromosomal chimerism^[25]. It has been suggested that the use of antagonists may present an endocrinological adverse situation in the early stage of the cycle, and the inhibition of endogenous pituitary gonadotropin secretion may be insufficient, leading to early LH and late follicular development^[26]. However, there are still no clear studies to clarify the causal relationship between GnRH-ant and oocyte quality and aneuploid embryos. A systematic review including meta-analysis showed that the embryo euploidy rates were similar in cycles using PPOS or the GnRH-a protocol^[27]. La Marca et al.^[20] and Yang et al.^[28] also reported that there was no significant difference in embryo euploidy between the PPOS and conventional GnRH-ant protocol. However, Pai et al.^[29] found that compared with the traditional GnRH-ant protocol, PPOS may reduce the euploidy rate for elderly patients using IVF. Baart et al.^[30] reported that the mild stimulation protocol reduced the average stimulation days and total amount of gonadotropin used, and the proportion of high-quality and euploid embryos seemed to be higher than that of traditional stimulation programs. At present, studies on the effects of various COH protocols on embryo euploidy are few and widely debated. .However, about the total dosage of Gn, the duration of stimulation and the number of oocytes retrieved, A study involving an analysis of 12,298 trophectoderm biopsies found that the duration of gonadotropin stimulation and number of oocytes retrieved did not affect the embryo euploidy rate^[31]. Similarly, Barash et al.^[32–33] also reported that embryonic euploidy was not affected by the dose of gonadotropin and number of oocytes retrieved, consistent with the results of the present study.

The current study also found that the COH protocol had no significant effect on the embryo euploidy rate per oocyte retrieved, which was negatively correlated with the number of oocytes retrieved per cycle. In 1997, Meniru et al.^[34] found that the proportion of retrieved oocytes producing embryos of a quality suitable for transfer or cryopreservation (the cycle efficiency index) fell significantly with an increasing number of retrieved oocytes. Also, Timeva et al.^[35] showed that the aspiration of less than five oocytes significantly reduced the pregnancy rate. The aspiration of a large number of oocytes (> 15) did not increase the effect of treatment, but increased the risk of ovarian hyperstimulation syndrome. To sum up, the acquisition of a large number of oocytes may not help with clinical outcomes, especially embryo quality, but the limit of embryo number needs more research, and should be adjusted according to the personal conditions of each patient.

Yu et al.^[36] reported that the cycle cancellation rate was lower with the modified GnRH-a protocol than the mild stimulation and GnRH-ant protocols, while Kuan et al.^[37] found that the GnRH-a long and GnRH-ant protocols generally produced similar cycle cancellation rates in women with endometriosis. In the current study, GnRH-ant, mild stimulation and other protocols were associated with higher cycle cancellation rates compared with the GnRH-a long protocol. The GnRH-a long and GnRH-ant protocols had a similar number of oocytes retrieved per cycle (P = 0.075) and equivalent number of good quality embryos sent for biopsy after morphological assessment (P = 0.382), so it was considered that the higher cycle cancellation rate may be related to the lower embryo euploidy rate. The total number of oocytes retrieved per cycle in the mild stimulation and other protocols was significantly lower than that in the GnRH-a long protocol (P < 0.001), and there was no significant difference in the embryo euploidy rate; thus, the low oocyte count may be one of the reasons for the increased cycle cancellation rate.

At present, most studies focus on clinical outcomes, such as pregnancy and live birth rates, among different protocols, and most studies of the euploid rate also focus on pairwise comparison of PPOS and GnRH-a protocols. The advantage of this study is that it included multiple ovary stimulation protocols for simultaneous horizontal comparison, with a large sample size in a single center. Several confounding factors, including age, sex hormone levels, anti-Mullerian hormone and gonadotropin dose, were included. In addition, our study also provides direction and reference for future research into the safety and application of GnRH antagonists.

There are some limitations in this study. First, this is a retrospective cohort study with a large number of groups, and some key parameters may not be included, resulting in inevitable confounding. Second, this study suggested that the GnRH-ant protocol was associated with a lower euploidy rate per embryo biopsied, but the causal relationship could not be determined, and the specific mechanism should be further studied using cytology and molecular biology methods. Finally, this study is a single-center study, and a larger multi-center study is needed to further determine the universality of the current findings.

PGT-A	Preimplantation genetic testing for aneuploidy
Gn	Gonadotropin
GnRH-a	Gonadotropin-releasing hormone agonist
GnRH-ant	Gonadotropin-releasing hormone antagonist
PPOS	Progestin primed ovarian stimulation
ICSI	Intracytoplasmic sperm injection
COH	Controlled ovary hyperstimulation
ART	Assisted reproductive technology
RIF	Repeated implantation failure
RPL	Recurrent pregancy loss
IVF	In vitro fertilization
AMA	Advanced maternal age
LH	Luteinixing hormone
OHSS	Ovarian hyperstimulation syndrome
ASRM	American Society for Reproductive Medicine
BMI	Body mass index
AFC	Antral follicle count
TE	Trophectoderm
PCR	Polymerase chain reaction
NGS	Next-generation sequencing
AMH	Anti-mullerian hormone
TSH	Thyroid stimulating hormone
FSH	Follicle-stimulating hormone
LH	Luteinixing hormone
E2	Estradio
MⅡ	Metaphase II oocytes

Ethics approval and consent to participate

This study obtained the approval of Research Ethics Committee of Shanghai First Maternity and Infant Hospital. Written informed consent was waived due to the retrospective nature, and patients’ data were used anonymously.

Consent for publication

Not applicable.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

Supported by grants from the Natural Science Foundation of Shanghai (grant No. 21ZR1450700).

Authors' contributions

Kunming Li and Yu Wang contributed to the study conception and design. Data collection and analysis were performed by Yu Wang and Junting Xu. The first draft of the manuscript was written by Yu Wang and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Acknowledgements

The authors thank our patients and all participants in the data collection.

LA Marca A, Capuzzo M, Imbrogno MG, Donno V, Spedicato GA, Sacchi S, Minasi MG, Spinella F, Greco P, Fiorentino F, Greco E. d. Minerva Obstet Gynecol. 2021 Feb;73(1):103-110.
E.B. Baart, E. Martini, I. van den Berg, N.S. Macklon, R-J.H. Galjaard, B.C.J.M. Fauser, D. Van Opstal, Preimplantation genetic screening reveals a high incidence of aneuploidy and mosaicism in embryos from young women undergoing IVF, Human Reproduction, Volume 21, Issue 1, January 2006, Pages 223–233,
Macklon NS, Geraedts JP and Fauser BC (2002) Conception to ongoing pregnancy: the ‘black box’ of early pregnancy loss. Hum Reprod Update 8,333–343.
Magli MC, Jones GM, Gras L, Gianaroli L, Korman I and Trounson AO (2000) Chromosome mosaicism in day 3 aneuploid embryos that develop to morphologically normal blastocysts in vitro. Hum Reprod15,1781–1786.
Simopoulou M, Sfakianoudis K, Maziotis E, Tsioulou P, Grigoriadis S, Rapani A, Giannelou P, Asimakopoulou M, Kokkali G, Pantou A, Nikolettos K, Vlahos N, Pantos K. PGT-A: who and when? Α systematic review and network meta-analysis of RCTs. J Assist Reprod Genet. 2021 Aug;38(8):1939-1957.
Bhatt SJ, Marchetto NM, Roy J, Morelli SS, McGovern PG. Pregnancy outcomes following in vitro fertilization frozen embryo transfer (IVF-FET) with or without preimplantation genetic testing for aneuploidy (PGT-A) in women with recurrent pregnancy loss (RPL): a SART-CORS study. Hum Reprod. 2021 Jul 19;36(8):2339-2344.
Huirne JA, Lambalk CB. Gonadotropin-releasing-hormone-receptor antagonists. Lancet. 2001 Nov 24;358(9295):1793-803
European and Middle East Orgalutran Study Group. Comparable clinical outcome using the GnRH antagonist ganirelix or a long protocol of the GnRH agonist triptorelin for the prevention of premature LH surges in women undergoing ovarian stimulation. Hum Reprod. 2001 Apr;16(4):644-51.
Yang R, Guan Y, Perrot V, Ma J, Li R. Comparison of the Long-Acting GnRH Agonist Follicular Protocol with the GnRH Antagonist Protocol in Women Undergoing In Vitro Fertilization: A Systematic Review and Meta-analysis. Adv Ther. 2021 May;38(5):2027-2037.
Xia M, Zheng J. Comparison of clinical outcomes between the depot gonadotrophin-releasing hormone agonist protocol and gonadotrophin-releasing hormone antagonist protocol in normal ovarian responders. BMC Pregnancy Childbirth. 2021 May 11;21(1):372.
Kuang Y, Cheng Q, Fu Y, etal. Medroxyprogesteroneacetate is an effective oral alternative for preventing premature luteinizing hormone surges in women undergoing controlled ovarian hyperstimulation for invitrofertilization[J]. Fertil Steril, 2015,104:62-70.
Filcori M, Butler JP, Crowley WF Jr. Neurodocrine regulation of the corpusluteum in the human. Evidence for pulsatileprogesteronesecretion.[J]. JClin Invest, 1984,73:1638-1647.
Garcia JE, Padilla SL, Bayati J, Baramki TA. Follicular phase gonadotropin-releasing hormone agonist and human gonadotropins: a better alternative for ovulation induction in in vitro fertilization. Fertil Steril. 1990 Feb;53(2):302-5
Sunkara SK, Coomarasamy A, Faris R, Braude P, Khalaf Y. Long gonadotropin-releasing hormone agonist versus short agonist versus antagonist regimens in poor responders undergoing in vitro fertilization: a randomized controlled trial. Fertil Steril. 2014 Jan;101(1):147-53.
Ou J, Xing W, Li Y, Xu Y, Zhou C. Short versus Long Gonadotropin-Releasing Hormone Analogue Suppression Protocols in IVF/ICSI Cycles in Patients of Various Age Ranges. PLoS One. 2015 Jul 24;10(7):e0133887.
Practice Committee of the American Society for Reproductive Medicine. Electronic address: [email protected]. Comparison of pregnancy rates for poor responders using IVF with mild ovarian stimulation versus conventional IVF: a guideline. Fertil Steril. 2018 Jun;109(6):993-999.
Verberg MF, Eijkemans MJ, Macklon NS, Heijnen EM, Baart EB, Hohmann FP, Fauser BC, Broekmans FJ. The clinical significance of the retrieval of a low number of oocytes following mild ovarian stimulation for IVF: a meta-analysis. Hum Reprod Update. 2009 Jan-Feb;15(1):5-12.
Miserez Zaugg C, Fournet Irion N, Cantero-Perez P, Streuli I. Résultats de la fécondation in vitro en cycle naturel ou avec une stimulation hormonale minimale [Results of in vitro fertilization in natural cycle or with mild stimulation]. Rev Med Suisse. 2014 Oct 22;10(447):1969-70, 1972-4, 1976. French.
Wang J, Zhang J, Zhao N, Ma Y, Wang X, Gou X, Ju Y, Zhang H, Chen S, Wang X. The effect of ovarian stimulation on aneuploidy of early aborted tissues and preimplantation blastocysts: comparison of the GnRH agonist long protocol with the GnRH antagonist protocol. J Assist Reprod Genet. 2022 Aug;39(8):1927-1936.
La Marca A, Capuzzo M, Sacchi S, Imbrogno MG, Spinella F, Varricchio MT, Minasi MG, Greco P, Fiorentino F, Greco E. Comparison of euploidy rates of blastocysts in women treated with progestins or GnRH antagonist to prevent the luteinizing hormone surge during ovarian stimulation. Hum Reprod. 2020 Jun 1;35(6):1325-1331.
Gardner DK, Lane M, Stevens J, Schlenker T, Schoolcraft WB. Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer. Fertil Steril. 2000;73:1155–8.
L'Heveder A, Jones BP, Naja R, Serhal P, Nagi JB. Preimplantation Genetic Testing for Aneuploidy: Current Perspectives. Semin Reprod Med. 2021 Mar;39(1-02):1-12.
Capalbo A, Poli M, Jalas C, Forman EJ, Treff NR. On the reproductive capabilities of aneuploid human preimplantation embryos. Am J Hum Genet. 2022 Sep 1;109(9):1572-1581.
Kyrou D, Verpoest W, Staessen C, De Vos A, Haentjens P, Liebaers I, Devroey P. No relationship between the type of pituitary suppression for IVF and chromosomal abnormality rates of blastomeres: an observational study. Fertil Steril. 2011 Feb;95(2):563-7.
Baart EB, Macklon NS, Fauser BJ. Ovarian stimulation and embryo quality. Reprod Biomed Online. 2009;18 Suppl 2:45-50. doi: 10.1016/s1472-6483(10)60448-8.
Lambalk CB, Banga FR, Huirne JA, Toftager M, Pinborg A, Homburg R, van der Veen F, van Wely M. GnRH antagonist versus long agonist protocols in IVF: a systematic review and meta-analysis accounting for patient type. Hum Reprod Update. 2017 Sep 1;23(5):560-579.
Ata B, Capuzzo M, Turkgeldi E, Yildiz S, La Marca A. Progestins for pituitary suppression during ovarian stimulation for ART: a comprehensive and systematic review including meta-analyses. Hum Reprod Update. 2021 Jan 4;27(1):48-66.
Yang L, Luo K, Lu G, Lin G, Gong F. Euploidy rates among preimplantation genetic testing for aneuploidy cycles with oral dydrogesterone primed ovarian stimulation or GnRH antagonist protocol. Reprod Biomed Online. 2022 Oct;45(4):721-726.
Pai AH, Sung YJ, Li CJ, Lin CY, Chang CL. Progestin Primed Ovarian Stimulation (PPOS) protocol yields lower euploidy rate in older patients undergoing IVF. Reprod Biol Endocrinol. 2023 Aug 8;21(1):72.
Baart EB, Martini E, Eijkemans MJ, Van Opstal D, Beckers NG, Verhoeff A, Macklon NS, Fauser BC. Milder ovarian stimulation for in-vitro fertilization reduces aneuploidy in the human preimplantation embryo: a randomized controlled trial. Hum Reprod. 2007 Apr;22(4):980-8.
Irani M, Canon C, Robles A, Maddy B, Gunnala V, Qin X, Zhang C, Xu K, Rosenwaks Z. No effect of ovarian stimulation and oocyte yield on euploidy and live birth rates: an analysis of 12 298 trophectoderm biopsies. Hum Reprod. 2020 May 1;35(5):1082-1089.
Barash OO, Hinckley MD, Rosenbluth EM, Ivani KA, Weckstein LN. High gonadotropin dosage does not affect euploidy and pregnancy rates in IVF PGS cycles with single embryo transfer. Hum Reprod. 2017 Nov 1;32(11):2209-2217.
Wu Q, Li H, Zhu Y, Jiang W, Lu J, Wei D, Yan J, Chen ZJ. Dosage of exogenous gonadotropins is not associated with blastocyst aneuploidy or live-birth rates in PGS cycles in Chinese women. Hum Reprod. 2018 Oct 1;33(10):1875-1882.
Meniru GI, Craft IL. Utilization of retrieved oocytes as an index of the efficiency of superovulation strategies for in-vitro fertilization treatment. Hum Reprod. 1997 Oct;12(10):2129-32.
Timeva T, Milachich T, Antonova I, Arabaji T, Shterev A, Omar HA. Correlation between number of retrieved oocytes and pregnancy rate after in vitro fertilization/intracytoplasmic sperm infection. ScientificWorldJournal. 2006 Jun 21;6:686-90.
Yu R, Jin H, Huang X, Lin J, Wang P. Comparison of modified agonist, mild-stimulation and antagonist protocols for in vitro fertilization in patients with diminished ovarian reserve. J Int Med Res. 2018 Jun;46(6):2327-2337.
Kuan KKW, Omoseni S, Tello JA.Comparing ART outcomes in women with endometriosis after GnRH agonist versus GnRH antagonist ovarian stimulation: a systematic review. Ther Adv Endocrinol Metab. 2023 Jul 4;14:

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The comparision among euploidy of preimplantation blastocysts in different controlled ovary stimulation(COH) protocols

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