- Academic Editor
†These authors contributed equally.
Background: There are few studies evaluating the effects of number and quality of transferred blastocysts on birth outcomes in frozen-thawed transfer cycles. Methods: A retrospective study was conducted, encompassing 5493 frozen-thawed blastocyst transfer cycles
from January 2019 to June 2021. The cycles were categorized into five groups
based on the number and quality of transferred blastocysts, as well as
trichotomized based on maternal age brackets. Pregnancy outcomes such as
implantation rate (IR), clinical pregnancy rate (CPR), multiple pregnancy rate
(MPR), abortion rate (AR), live birth rate (LBR), and neonatal characteristics
were compared and statistically analyzed. Results: The data revealed
that maternal age, quality and number of the transferred blastocysts exerted a
demonstrable impact on both pregnancy and birth outcomes. Within the same
blastocyst transfer groups, it was noted that IR, CPR, and LBR exhibited a
progressive decline as a function of advancing maternal age. Amplifying the
number of homogeneously graded blastocysts for transfer did not conspicuously
elevate CPR and LBR; however, it led to a statistically significant escalation in
MPR (p
With the development of assisted reproductive technologies (ART), particularly
in the domains of embryo culture and cryopreservation, the implantation rate (IR)
is increasing along with the multiple pregnancy rate (MPR). Multiple pregnancies
were commonly regarded as the most consequential adverse outcomes correlated with
ART, as they are associated with elevated risks of maternal and neonatal
morbidity. Although, the reduction in the number of transferred embryos has been
posited as a critical strategy for mitigating multiple pregnancies, this strategy
has not been widely used in clinical practice owing to apprehensions regarding
diminished pregnancy rates [1]. Contrary to cleavage-stage embryos, extending
embryo culture to the blastocyst stage allowed for better evaluation of the
implantation potential, yielding a higher IR [2, 3, 4]. Previous studies have
posited that elective single blastocyst transfer could yield comparable clinical
pregnancy rates (CPR) for patients with a good prognosis [1, 5, 6]. Although live
birth rate (LBR) equivalence was not demonstrated, it was thought the additional
complications associated with multiple gestations outweighed the potentially
higher LBR [5]. Additionally, frozen-thawed single blastocyst
transfers have been found to result in enhanced CPR relative to fresh
single blastocyst transfers in ovulatory women
with a good prognosis [6]. In the context of advanced maternal age (
This retrospective study was conducted from January 2019 to June 2021, and
focused exclusively on the first frozen-thawed blastocyst transfer cycle.
Participants with a diagnosis of either congenital or acquired uterine anomalies,
such as uterine malformation, adenomyosis, submucous myoma, uterine fibroids, or
intrauterine adhesions were excluded. The cycles were partitioned into five
groups based on the number and quality of transferred blastocysts: a single
good-quality blastocyst (G), two good-quality blastocysts (GG), a good-quality
blastocyst and a poor-quality blastocyst (GP), a single poor-quality blastocyst
(P), and two poor-quality blastocysts (PP). Subsequent categorization occurred
according to maternal age brackets:
Embryo culture was performed in Quinn’s IVF sequential medium suite
(Quinn’s, SAGE, New York, NY, USA) after
adding 10% human serum substitute (Quinn’s, SAGE, USA). The atmospheric
conditions were precisely regulated to include 5% O
Two primary protocols were employed for endometrial preparation: natural and
artificial cycles. In natural cycles, either administration of
human chorionic gonadotropin (HCG) guided the transfer planning, or a spontaneous
luteinizing hormone (LH) peak was detected, with blastocysts transfers occurring
on the fifth day post-ovulation. In artificial cycles, a daily oral dose of 3.75
mg commenced on days 2–3 of the menstrual cycle, with dose adjustment made in
accordance with the endometrial thickness as gauged by
ultrasound. Upon reaching an endometrial thickness of
Statistical analyses were executed utilizing SPSS version 22.0
(IBM, Armonk, NY, USA). Quantitative variables were presented as means
The present study encompassed a total of 5493 first frozen-thawed blastocyst
transfer cycles. The maternal age, duration of infertility, endometrial thickness
and body-mass index were 20–45 years, 0–25 years, 8–14 mm and 13–42
kg/m
Age (years) | Embryo | Body-mass index (kg/m |
Natural cycle (%) | Endometrial thickness (mm) | Duration of infertility (years) | Primary infertility (%) |
G | 21.3 |
6.7 (163/2416) | 9.5 |
3.5 |
43.0 (1039/2416) | |
GG | 21.7 |
5.4 (15/277) | 9.5 |
3.7 |
47.7 (132/277) | |
GP | 21.5 |
8.4 (39/464) | 9.5 |
3.7 |
45.7 (212/464) | |
PP | 21.1 |
5.6 (18/320) | 9.5 |
3.6 |
47.8 (153/320) | |
P | 21.6 |
8.9 (10/112) | 9.6 |
3.1 |
38.4 (43/112) | |
p-value | 0.03 | 0.38 | 0.94 | 0.13 | 0.18 | |
35–39 | G | 22.0 |
11.6 (94/813) | 9.6 |
4.5 |
17.5 (142/813) |
GG | 21.8 |
13.8 (16/116) | 9.8 |
4.6 |
20.7 (24/116) | |
GP | 22.3 |
9.4 (24/256) | 9.8 |
4.9 |
20.7 (53/256) | |
PP | 22.1 |
12.3 (30/243) | 9.8 |
5.1 |
26.3 (64/243) | |
P | 22.0 |
12.0 (13/108) | 9.5 |
4.0 |
13.9 (15/108) | |
p-value | 0.46 | 0.72 | 0.09 | 0.03 | 0.02 | |
G | 22.6 |
18.2 (22/121) | 9.4 |
5.9 |
11.6 (14/121) | |
GG | 22.2 |
14.8 (4/27) | 9.6 |
3.9 |
11.1 (3/27) | |
GP | 22.6 |
15.2 (12/79) | 9.4 |
5.4 |
11.4 (9/79) | |
PP | 22.2 |
13.0 (13/100) | 9.5 |
6.7 |
13.0 (13/100) | |
P | 22.7 |
19.5 (8/41) | 9.3 |
5.4 |
14.6 (6/41) | |
p-value | 0.72 | 0.81 | 0.82 | 0.09 | 0.98 | |
Total | G | 21.5 |
8.3 (279/3350) | 9.6 |
3.9 |
35.7 (1195/3350) |
GG | 21.8 |
8.3 (35/420) | 9.6 |
4.0 |
37.9 (159/420) | |
GP | 21.9 |
9.4 (75/799) | 9.6 |
4.3 |
34.3 (274/799) | |
PP | 21.7 |
10.7 (71/663) | 9.6 |
4.6 |
34.7 (230/663) | |
P | 21.9 |
11.9 (31/261) | 9.5 |
3.8 |
24.5 (64/261) | |
p-value | 0.12 | 0.57 |
Note: G, a good-quality blastocyst; GG, two good-quality blastocysts; GP, a good-quality blastocyst and a poor-quality blastocyst; PP, two poor-quality blastocysts; P, a poor-quality blastocyst.
Date presented in Table 2 showed that both the quality and number of transferred
blastocysts exerted a palpable impact on pregnancy outcomes. In total age group,
IR (74.5%) was observed to be highest in group G, and MPR was the highest in
group GG (56.8%) followed by groups GP (36.3%) and PP (29.6%). Additionally,
elevated AR was observed in groups P (23.5%) and PP (23.6%). Groups with
good-quality blastocysts (groups G, GG, and GP) had higher CPR and LBR alongside
diminished AR relative to their counterparts devoid of good-quality blastocysts
(groups P and PP). In the groups with good-quality blastocysts, Group G
demonstrated a higher IR (p/odds
ratio (OR)/95% confidence interval (95% CI)
Age (years) | Embryo | IR | CPR | MPR | AR | LBR |
G | 78.4 (1893/2416) |
78.4 (1893/2416) |
2.7 (52/1893) |
13.7 (259/1893) |
67.5 (1630/2416) | |
GG | 65.9 (365/554) |
78.7 (218/277) |
67.4 (147/218) |
11.0 (24/218) |
70.0 (194/277) | |
GP | 56.7 (526/928) |
80.4 (373/464) |
41.0 (153/373) |
11.5 (43/373) |
70.9 (329/464) | |
PP | 45.8 (293/640) |
68.4 (219/320) |
34.2 (75/219) |
20.1 (44/219) |
54.7 (175/320) | |
P | 58.9 (66/112) |
58.9 (66/112) |
3.0 (2/66) |
22.7 (15/66) |
45.5 (51/112) | |
35–39 | G | 66.5 (541/813) |
66.5 (541/813) |
1.8 (10/541) |
23.7 (128/541) |
50.7 (412/813) |
GG | 50.4 (117/232) |
75.0 (87/116) |
35.6 (31/87) |
31.0 (27/87) |
51.7 (60/116) | |
GP | 45.7 (234/512) |
69.9 (179/256) |
31.8 (57/179) |
17.9 (32/179) |
57.4 (147/256) | |
PP | 36.8 (179/486) |
59.3 (144/243) |
26.4 (38/144) |
20.8 (30/144) |
46.9 (114/243) | |
P | 50.9 (55/108) |
50.9 (55/108) |
1.8 (1/55) |
20.0 (11/55) |
40.7 (44/108) | |
G | 52.1 (63/121) |
52.1 (63/121) |
4.8 (3/63) |
36.5 (23/63) |
33.1 (40/121) | |
GG | 38.9 (21/54) |
63.0 (17/27) |
29.4 (5/17) |
47.1 (8/17) |
33.3 (9/27) | |
GP | 42.4 (67/158) |
70.9 (56/79) |
19.6 (11/56) |
46.4 (26/56) |
38.0 (30/79) | |
PP | 20.0 (40/200) |
35.0 (35/100) |
14.3 (5/35) |
57.1 (20/35) |
15.0 (15/100) | |
P | 26.8 (11/41) |
26.8 (11/41) |
0.0 (0/11) |
45.5 (5/11) |
14.6 (6/41) | |
Total | G | 74.5 (2497/3350) |
74.5 (2497/3350) |
2.6 (65/2497) |
16.4 (410/2497) |
62.1 (2082/3350) |
GG | 59.9 (503/840) |
76.7 (322/420) |
56.8 (183/322) |
18.3 (59/322) |
62.6 (263/420) | |
GP | 51.8 (827/1598) |
76.1 (608/799) |
36.3 (221/608) |
16.6 (101/608) |
63.3 (506/799) | |
PP | 38.6 (512/1326) |
60.0 (398/663) |
29.6 (118/398) |
23.6 (94/398) |
45.9 (304/663) | |
P | 50.6 (132/261) |
50.6 (132/261) |
2.3 (3/132) |
23.5 (31/132) |
38.7 (101/261) |
Note: Superscript lowercase letters (a, b, c) demonstrate the differences of different transferred blastocysts in the same age groups, while subscript
uppercase letters (A, B, C) signify differences of the same transferred blastocysts in the different age groups. Completely different letters indicate a significant difference (p
In different age groups, a progressive attenuation in IR, CPR, and LBR was
discernible with advancing maternal age, reaching statistical significance beyond
39 years (p
Logistic regression modeling, delineated in Table 3, incorporated variables that
were both statistically significant according to Table 1 and clinically salient.
This model indicated that the most potent predictors of live birth were maternal
age and transferred blastocysts. The LBR within cohorts of identically graded
blastocyst transfers exhibited a gradual diminution with increasing age. Cohorts
comprising good-quality blastocysts (groups G, GG and GP) consistently
outperformed those bereft of such blastocysts (groups PP and P) in terms of LBR
(p/OR/95% CI
Wals | p | OR (95% CI) | |
Blastocysts | |||
P | 64.60 | 1.00 | |
G | 26.68 | 2.02 (1.55–2.64) | |
GG | 21.58 | 2.17 (1.56–3.01) | |
GP | 37.43 | 2.51 (1.87–3.37) | |
PP | 2.90 | 0.09 | 1.30 (0.96–1.75) |
Endometrial thickness | 0.69 | 0.41 | 1.02 (0.97–1.08) |
Female age | 151.92 | 0.91 (0.90–0.93) | |
Duration of infertility | 1.85 | 0.17 | 0.99 (0.97–1.01) |
Body-mass index | 0.33 | 0.57 | 1.01 (0.99–1.03) |
Type of infertility | 3.22 | 0.07 | 0.89 (0.79–1.01) |
Endometrial preparation | 2.77 | 0.10 | 1.18 (0.97–1.43) |
Note: OR, odds ratio; 95% CI, 95% confidence interval.
Tables 4,5 offered a meticulous portrayal of neonatal attributes for live-born
singletons and twins. Comparative analyses revealed that the singleton group had
a higher average gestational age (p/OR/95% CI
Characteristics | Singleton | Twin | p | OR/MD (95% CI) | |
(n = 2876) | (n = 380) | ||||
Gestational age (weeks) | 38.5 |
35.8 |
2.66 (2.43–2.88) | ||
0.8% (24) | 4.2% (16) | 0.19 (0.10–0.36) | |||
8.2% (236) | 58.7% (223) | 0.06 (0.05–0.08) | |||
91.8% (2640) | 41.3% (157) | 15.89 (12.45–20.28) | |||
Birthweight (g) | 3276.9 |
2458.57 |
818.38 (772.70–864.05) | ||
0.6% (18) | 2.8% (21) | 0.22 (0.12–0.42) | |||
4.6% (133) | 46.3% (352) | 0.06 (0.05–0.07) | |||
95.4% (2743) | 53.7% (408) | 17.79 (14.21–22.28) | |||
Cesarean section | 61.9% (1779) | 94.5% (359) | 0.10 (0.06–0.15) | ||
Sex ratio (male/female) | 1.3 (1613/1263) | 1.2 (413/347) | 0.39 | 1.07 (0.91–1.26) | |
Congenital malformations | 1.4 % (40) | 1.7% (13) | 0.50 | 0.81 (0.43–1.52) |
Note: MD, mean difference.
Characteristics | G | GG | GP | PP | P | p | |
Gestational age (weeks) | S | 38.6 |
38.2 |
38.2 |
38.3 |
38.0 |
|
T | 34.9 |
35.8 |
35.8 |
36.2 |
34.5 |
0.06 | |
S | 7.0% (144/2050) | 14.5% (19/131) | 9.6% (35/364) | 10.8% (25/232) | 13.1% (13/99) | ||
T | 75.0% (24/32) | 61.4% (81/132) | 57.8% (82/142) | 47.2% (34/72) | 100.0% (2/2) | 0.05 | |
Cesarean section | S | 59.4% (1217/2050) | 67.9% (89/131) | 65.7% (239/364) | 68.1% (158/232) | 76.8% (76/99) | |
T | 87.5% (28/32) | 94.7% (125/132) | 95.1% (135/142) | 95.8% (69/72) | 100.0% (2/2) | 0.44 | |
Birthweight (g) | S | 3296.3 |
3223.7 |
3247.8 |
3225.5 |
3173.8 |
0.01 |
T | 2230.5 |
2469.7 |
2499.0 |
2468.9 |
2137.5 |
0.02 | |
Birthweight |
S | 4.1% (84/2050) | 8.4% (11/131) | 4.9% (18/364) | 4.7% (11/232) | 9.1% (9/99) | 0.04 |
T | 64.1% (41/64) | 43.9% (116/264) | 45.8% (130/284) | 42.4% (61/144) | 100.0% (4/4) | 0.01 | |
Sex ratio (male/female) | S | 1.33 (1171/879) | 1.43 (77/54) | 1.19 (198/166) | 1.02 (117/115) | 1.02 (50/49) | 0.20 |
T | 1.06 (33/31) | 1.42 (155/109) | 1.15 (152/132) | 0.92 (69/75) | 4.00 (4/0) | 0.09 | |
Sex ratio (male/female) | Total | 1.34 (1436/1073) | 1.17 (350/298) | 1.00 (240/239) | 0.01 | ||
Congenital malformations | S | 1.3% (27/2050) | 0.0% (0/131) | 2.2% (8/364) | 1.7% (4/232) | 1.0% (1/99) | 0.41 |
T | 4.7% (3/64) | 0.8% (2/264) | 2.1% (6/284) | 1.4% (2/144) | 0.0% (0/4) | 0.20 |
Note: S, singleton; T, twin. The differences of gestational age, cesarean
section rate, birthweight, preterm labor rate and low birthweight rate were
significant (p
Pertaining to the total age group under study, group G exhibited the highest IR,
the lowest MPR, and analogous CPR, AR, and LBR when compared to other cohorts
employing good-quality blastocysts. Likewise, group P had a higher IR, lower MPR,
and similar CPR, AR and LBR in relation to group PP, which is in consonance with
previous studies [11, 12, 13, 14]. In regard to double blastocyst transfer, the MPR of
groups GG, GP and PP were 56.8%, 36.3% and 29.6%, respectively. Importantly,
these trends across diverse age subgroups were consistent with those observed in
the total age group. Consequently, the obtained results indicated that single
blastocyst transfer appeared to be an efficacious strategy for minimizing MPR
while achieving favorable LBR. The logistic regression analysis further
accentuated that maternal age and transferred blastocysts were factors
significantly correlated with LBR. Specifically, within the age group of
Pregnancy is often conceptualized as a nuanced interplay, mediated by localized
secretion of key factors, between a developmentally competent embryo and a
receptive endometrium. Existing research posits that decidualized human
endometrial stromal cells possess the ability to selectively identify
developmentally impaired embryos and respond by inhibiting the secretion of key
implantation mediators such as Interleukin-1
Multiple pregnancy is considered the most significant adverse event associated
with ART and linked to an increased risk of maternal and neonatal morbidity. The
results indicated that single blastocyst transfer had a lower MPR (2.6% vs
39.3%, p/OR/95% CI
In vitro culture has been shown to induce precocious X-chromosome inactivation, and intracytoplasmic sperm injection (ICSI) is implicated in reducing the number of trophectoderm cells in female blastocysts [20]. Sex ratio was significantly higher toward males in the transfer of blastocyst compared to transfer of cleavage stage embryo [20, 21, 22, 23]. Our data further substantiate a significant positive correlation between sex ratio (male/female) and the proportion of good-quality blastocysts transferred. As, the practice of blastocyst culture and selective single good-quality blastocyst transfer gains scholarly endorsement [6, 12, 24], the potential for such strategies to engender imbalances in neonatal sex ratios remains an emergent area requiring further research.
Our empirical analyses confirm the efficacy of single blastocyst transfer as an optimal strategy for significantly attenuating MPR while ensuring favorable pregnancy and birth outcomes. Nonetheless, it should be noted that this strategy may engender a skewed sex ratio among the neonates. Furthermore, the distinct health metrics of monozygotic twins deserve more attention in the context of ART treatments.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
The study was designed by YL and BM. LZ, PY, NL and YL were involved in planning and managing the data collection. YL and BM were involved in the statistical analysis and wrote the manuscript with support. Critical review was from all authors. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.
Ethical approval was granted by the Ethics Committee of Haikou Mary Hospital on the March 8, 2020 (NO. 02/MLYY/2020). Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.
The authors would like to thank all participants and staff at IVF Center, Mali Hospital, Haikou, China and the Hospital of Shenzhen Immigration Inspection, Shenzhen, China.
Hainan Medical and Health Research Project (21A200331).
The authors declare no conflict of interest.
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