Abstract
Background
Chromosomal microarray analysis (CMA) has been widely applied to explore the genetic etiology in recurrent pregnancy loss (RPL). However, the reproductive prognosis in RPL couples with different types of chromosomally abnormal miscarriage remains unclear.
Objectives
The main purpose of this study was to evaluate the reproductive prognosis among RPL couples after genetic testing in products of conception (POCs) by CMA.
Study design
In this retrospective study, 1101 RPL couples referred for genetic testing in POCs by CMA. A total of 830 couples who met the inclusion criteria were followed up for at least 24 months after the index miscarriage. The rates of live birth and adverse pregnancy events in subsequent pregnancy and cumulative pregnancies were examined.
Results
For couples with three or more miscarriage, compared with those with chromosomally normal miscarriage, a significantly higher subsequent live birth rate was found in couples with chromosomally abnormal miscarriage (66.9% vs 71.6%, P = .040). However, differences in cumulative live birth rate among couples with chromosomally abnormal miscarriage and normal miscarriage were nonsignificant (82.7% vs 80.2%, P = .131). Women with advanced maternal age showed a significant decrease in the live birth rate (P < 0.01) and an increase in the miscarriage rate (P < 0.01) than those aged < 35 years old, regardless of whether the miscarriage was chromosomally normal or abnormal. RPL couples with chromosomally normal miscarriage showed a significant decrease in live birth rates in subsequent pregnancy and cumulative pregnancies, when they had experienced a large number of previous miscarriages; however, no significant difference was observed in those with chromosomally abnormal miscarriage.
Conclusion
For women with three or more previous miscarriages, RPL couples with chromosomally normal miscarriage manifested a poorer reproductive prognosis than those with chromosomally abnormal miscarriage in subsequent pregnancy, while the cumulative live birth rate was similar. Advanced maternal age was a predictor of adverse pregnancy events, regardless of embryonic chromosomal results. Furthermore, among RPL women with large numbers of previous miscarriages, the supportive care and counselling regarding individual risk is necessary for those with chromosomally normal miscarriage.
Supplementary Information
The online version contains supplementary material available at 10.1007/s10815-023-02971-0.
Keywords: Recurrent pregnancy loss, Chromosomal microarray analysis, Reproductive outcome, Live birth rate
Introduction
Clinically recognized pregnancy loss is the most common complication of conception, with an incidence of about 15–25% [1]. The American Society for Reproductive Medicine (ASRM) defined recurrent pregnancy loss (RPL) as two or more clinical pregnancy losses [2], experienced by 1–2% of women trying to conceive [3]. The sequelae of RPL are physical and psychological. Women who experience recurrent pregnancy loss may suffer from high levels of anxiety, depression, and post-traumatic stress disorder [4].Well-established risk factors for RPL have been reported, including genetic, maternal, environmental and lifestyle factors [5]. Among them, chromosomal abnormalities in product-of-conceptions (POCs) contribute to the majority of the cases, accounting for about 30%-57% of RPL couples [6].
Chromosomal microarray analysis (CMA) can simultaneously detect aneuploidy, polyploidy, uniparental disomy (UPD) and copy number variations (CNVs) at the whole genome level and has replaced the application of conventional G-banding karyotype analysis in paediatric and prenatal diagnosis [7]. Furthermore, guidelines recommended genetic analysis of POCs in couples with recurrent miscarriages [8, 9]. Previous studies using karyotype technology to study reproductive outcomes have found that RPL couples who experience chromosomally abnormal miscarriage may have a better prognosis than those who suffer from chromosomally normal miscarriage [10, 11]. However, the information of reproductive outcomes among RPL couples with various types of chromosomally abnormal miscarriage is still limited. Previous studies have only included a few cases of couples with embryonic chromosomally numerical abnormality and have not investigated the reproductive outcomes of couples with pathogenic CNVs [5, 12]. Besides, the association between the times of previous miscarriages and the reproductive prognosis of couples with different types of chromosomally abnormal miscarriage is unclear [13, 14]. Hence, there is limited knowledge on the reproductive outcome in RPL couples with various types of chromosomally abnormal miscarriage.
The main purpose of this study was to assess the reproductive outcomes between RPL couples with various types of chromosomally abnormal miscarriage. Furthermore, among those, we studied the reproductive outcomes according to maternal age and the number of previous miscarriages.
Materials and methods
Study population
This retrospective cohort study enrolled RPL couples who underwent CMA testing of POCs after two or more miscarriages before 13 weeks of gestation between October 2012 and February 2021 at Women's Hospital of Nanjing Medical University. Other potential causes of miscarriage were also investigated, such as anatomic abnormalities (examined by hysterosalpingogram or hysteroscopy), parental chromosomal abnormalities (examined by karyotype analysis or fluorescence in situ hybridization), thyroid dysfunction (examined by blood tests), luteal phase defect (examined by basal body temperature and progesterone levels during the luteal phase), hyperprolactinemia (examined by blood tests), immune system dysfunction (examined by β2-glycoprotein I– dependent anticardiolipin antibodies and lupus anticoagulant). Informed consent for genetic testing was obtained from all couples before CMA testing. Medical records were reviewed systematically to obtain maternal characteristics and CMA results of POCs from the index miscarriage. Previous clinical miscarriages were confirmed by ultrasonic examination of the gestational sac, laboratory test for positive Human chorionic gonadotropin (HCG) and POCs after curettage. The exclusion criteria for the participants were as follows:
Abnormal anatomical structure detected by ultrasound.
Parental chromosomal abnormality.
Endocrine disorder (e.g., hyperthyroidism, hypothyroidism, luteal phase defect, polycystic ovary syndrome or hyperprolactinemia).
Immunological dysfunction (e.g., antiphospholipid syndrome or systemic lupus erythematosus).
Mode of conception: preimplantation genetic testing (PGT), oocyte donation or sperm donation.
This study was approved by the Medical Ethics Committee of Nanjing Maternal and Child Health Hospital.
Chromosomal microarray analysis
Genomic DNA was extracted from aborted chorionic villi samples with the protocol of QIAamp DNA Mini Kit (Qiagen, Germany). Human CytoSNP-12 array (Illumina, USA) and CytoScan750K array (Affymetrix, USA) were applied for CMA detection of POCs in accordance with standard experimental methods [15]. POCs with maternal cell contamination (MCC) detected by CMA were further examined by quantitative fluorescent polymerase chain reaction (QF-PCR) to determine the ratio of embryonic villi to maternal deciduae. A level of MCC exceeding 30% was defined as significant MCC, and cases with significant MCC were excluded [15]. According to the guidelines developed by the American College of Medical Genetics and Genomics (ACMG) and the Institute for Clinical Genome Resources (ClinGen) [16], CNVs were divided into the following five categories: pathogenic CNVs, likely pathogenic CNVs, variant of uncertain clinical significance (VOUS), likely benign CNVs and benign CNVs. Likely benign and benign CNVs were not reported. Based on the size of CNVs, CNVs < 10 Mb were defined as chromosomal microdeletion/duplication and CNVs ≥ 10 Mb were defined as partial aneuploidy.
Reproductive outcomes
All eligible patients were followed up for at least two years after undergoing CMA for genetic testing in POCs. Patients with VOUS in the index miscarriage were excluded from the follow-up study due to ambiguous pathogenicity. Patients conceived by PGT either before or after the CMA testing were also excluded. The index miscarriage was the miscarriage for which CMA was performed. The cumulative miscarriage rate was the probability of having at least one miscarriage during the follow-up period. The reproductive outcomes of the first pregnancy after the index miscarriage (subsequent outcome) and all the following pregnancies during the follow-up period (cumulative outcomes) were obtained by telephone interview or medical records. The primary outcome was live birth, defined as delivery of one or more viable neonate at or beyond 28 weeks gestation. The secondary outcome was adverse pregnancy events, which were subdivided into clinical miscarriage, biochemical miscarriage and ectopic pregnancy.
Statistical analysis
Maternal characteristics were compared between groups using the Fisher’s exact test for categorical variables, and Kruskal–Wallis test for continuous variables. The comparison between reproductive outcomes was performed by binary logistic regression using the unadjusted model and the adjusted model, respectively. In the adjusted model, the maternal age of the index miscarriage was adjusted. In addition, we performed sensitivity analysis to examine the robustness of our results. We repeated binary logistic regression analysis adjusted for maternal age, gestational age of the index miscarriage and previous reproductive history simultaneously. The association between the number of previous miscarriages and the reproductive outcomes was performed by binary logistic regression analysis adjusted for maternal age. Two-tailed P value < 0.050 was defined as statistically significant difference.
Results
Frequency and distribution of embryonic chromosomal abnormalities
A total of 1101 RPL couples referred for genetic testing in POCs by CMA. Nine cases (9/1101, 0.8%) with significant MCC were excluded. Chromosomal abnormality was identified in 647 (647/1092, 59.2%) cases (Table 1). Normal result was identified in 438 (438/1092, 40.1%) cases, and VOUS was identified in seven (7/1092, 0.6%) cases.
Table 1.
Distribution and frequency of embryonic chromosomal abnormalities detected in the index miscarriage of RPL couples
| Chromosomal abnormality | RPL (n, %) |
|---|---|
| Single aneuploidy | 450 (41.2) |
| Autosomal trisomy | 363 (33.2) |
| Autosomal monosomy | 5 (0.5) |
| Monosomy X | 80 (7.3) |
| Sex chromosome trisomy | 2 (0.2) |
| Multiple aneuploidy | 26 (2.4) |
| Polyploidy | 89 (8.2) |
| Triploidy | 69 (6.3) |
| Hypotriploidy | 8 (0.7) |
| Hypertriploidy | 9 (0.8) |
| Tetraploidy | 3 (0.3) |
| Partial aneuploidy (large CNVs) | 55 (5.0) |
| Terminal deletion/duplication | 13 (1.2) |
| Interstitial deletion/duplication | 3 (0.3) |
| Terminal deletion + terminal duplication (suggestive of unbalanced translocation) | 18 (1.6) |
| Multiple deletions and/or duplications | 21 (1.9) |
| Pathogenic microdeletion/duplication | 17 (1.6) |
| Terminal microdeletion/duplication | 5 (0.5) |
| Interstitial microdeletion/duplication | 11 (1.0) |
| Multiple microdeletions and/or duplications | 1 (0.1) |
| UPD (whole-genome) | 10 (0.9) |
| Total | 647 (59.2) |
Data are listed as n (%). CNVs Copy number variations; UPD Uniparental disomy; RPL Recurrent pregnancy loss
The majority of chromosomal abnormalities in POCs are numerical abnormalities, comprising 476 (476/1092, 43.6%) cases with aneuploidy and 89 cases with polyploidy (89/1092, 8.2%). Out of the 476 women who had aneuploid miscarriages, 450 women had single aneuploidy in the products of conception (POCs). The remaining 26 women had multiple aneuploid abnormalities in POCs. Among single aneuploid cases, autosomal trisomy accounted for 80.7% (363/450), followed by monosomy X at 17.8% (80/450). Autosomal monosomy (5/450, 1.1%) and sex chromosome trisomy (2/450, 0.4%) were relatively rare. Single aneuploidy was detected on all chromosomes except chromosome 1 and 19. Among 89 cases with polyploidy, 86 cases were triploidy (86/89, 96.6%) and three cases were tetraploidy (3/89, 3.4%). In addition, ten cases of whole-genome UPD (10/1092, 0.9%) were detected.
A total of 72 cases with pathogenic CNVs were detected, including 55 cases (55/1092, 5.0%) with partial aneuploidy (Table S1) and 17 cases (17/1092, 1.6%) with microdeletion/duplication (Table S2). Among cases with partial aneuploidy, we detected 13 cases (13/55, 23.6%) with terminal deletion/duplication, three cases (3/55, 5.5%) with interstitial deletion/duplication, 21 cases (21/55, 38.2%) with multiple deletions and/or duplications and 18 cases (18/55, 32.7%) with terminal deletion accompanied with terminal duplication. After parental chromosomal analysis, eight couples were identified as balanced translocation carriers (six paternal inherited and two maternal inherited). Of the 17 cases with pathogenic microdeletion/duplication, five cases (5/17, 29.4%) were identified as terminal microdeletion/duplication, 11 cases (11/17, 64.7%) were identified as interstitial microdeletion/duplication and one case (1/17, 5.9%) was identified as multiple microdeletions and/or duplications. Among the 16 couples with parental chromosomal results, 13 cases were considered to be de novo and the remaining three were considered to be maternal inherited.
Baseline characteristics of couples with reproductive outcomes
Among 1092 cases with CMA results, seven cases with VOUS and 255 couples with other potential causes of miscarriage such as anatomic abnormalities, parental chromosomal abnormalities, thyroid dysfunction, luteal phase defect, hyperprolactinemia, immune system dysfunction were excluded. Of the 830 cases (76.0%) that met the inclusion criteria, 493 had abnormal results and 337 had normal results. Sixty-nine couples were lost to follow-up. Among couples with chromosomally abnormal miscarriage, 450 women were successfully followed up. Among those with chromosomally normal miscarriage, 311 women were successfully followed up. Among the 761 couples successfully followed up, 627 conceived at least once, while 73 chose not to conceive and 61 failed to conceive during the follow-up period. The 627 couples who conceived at least once after CMA testing were eventually included in our study. The mean maternal age of the 627 couples was 30.1 years (range 21.0 to 45.0) and the mean number of previous miscarriages was 2.4 (range 2.0 to 7.0). The mean gestational age was 9.8 weeks (range 6.0 to 13.0). The flow chart of follow-up after the index miscarriage in RPL couples was shown in Fig. 1.
Fig. 1.
Flow chart of follow-up after the index miscarriage in couples with recurrent pregnancy loss
The baseline characteristics of couples with reproductive outcomes were shown in Table 2. The follow-up period ranged from 24 to 120 months, with a mean duration of 43 months. No significant difference was observed in the mean follow-up duration of subgroups.
Table 2.
Baseline characteristics of RPL couples who conceived during the follow-up period
| Characteristics | Normal group (n = 257) |
Abnormal group (n = 370) | P value | |||
|---|---|---|---|---|---|---|
| Aneuploidy (n = 246) |
Polyploidy (n = 64) |
CNVs (n = 52) |
UPD (n = 8) |
|||
| Maternal age ± SD | 29.5 ± 4.3 | 31.1 ± 4.1 | 29.7 ± 3.8 | 29.0 ± 3.6 | 31.3 ± 3.9 | < 0.001a |
| No. of previous abortions ± SD | 2.5 ± 0.8 | 2.3 ± 0.7 | 2.5 ± 0.9 | 2.4 ± 0.7 | 2.3 ± 0.4 | 0.546a |
| Gestational age ± SD | 9.8 ± 1.6 | 9.8 ± 1.5 | 9.7 ± 1.4 | 9.8 ± 1.5 | 10.0 ± 1.2 | 0.052a |
| Live birth ≥ 1 | 20 (7.8) | 24 (9.8) | 4 (6.3) | 3 (5.8) | 1 (12.5) | 0.781b |
| Biochemical miscarriage ≥ 1 | 36 (14.0) | 28 (11.4) | 5 (7.8) | 5 (9.6) | 1 (12.5) | 0.656b |
| Ectopic pregnancy ≥ 1 | 8 (3.1) | 5 (2.0) | 1 (1.6) | 0 (0) | 1 (12.5) | 0.259b |
Data were present as average ± SD or number (percentage). Two-tailed p value < .050 was defined as statistically significant difference. RPL Recurrent pregnancy loss; CNVs Copy number variations; UPD Uniparental disomy
a: P value for Kruskal–Wallis test
b: P value for Fisher’s exact test
The baseline characteristics of couples with reproductive outcomes were compared among five subgroups based on CMA results in POCs. The significant difference was only found in maternal age of the index miscarriage (P < 0.001). Women with chromosomally normal miscarriage were significantly younger than those with aneuploid miscarriage (P < 0.001) and pathogenic CNVs (P = 0.040). Differences in gestational age, number of previous miscarriages, or incidence of live birth and adverse pregnancy events before the index miscarriage among subgroups were nonsignificant. In addition, the clinical information of RPL couples aged 35 and under was displayed in Table S3. For the clinical information of those women, the significant difference was observed in maternal age of the index miscarriage (P < 0.001). Women with chromosomally normal miscarriage were significantly younger than those with aneuploid miscarriage (P < 0.001). No significant differences were found in gestational age, number of previous miscarriages, or incidence of live birth and adverse pregnancy events before the index miscarriage among women aged 35 and under.
Subsequent reproductive outcomes
Among 627 couples conceived during the follow-up period, 437 (69.7%) had a subsequent live birth. Adjusted for maternal age, couples with chromosomally abnormal miscarriage presented a significantly higher live birth rate (71.6% vs 66.9%, P = 0.040) and a significantly lower spontaneous miscarriage rate (22.7% vs 27.2%, P = 0.047) than those with chromosomally normal miscarriage. No significant difference was observed in the incidence of biochemical miscarriage or ectopic miscarriage for the subsequent pregnancy between the two groups. Details were presented in Table 3.
Table 3.
Subsequent reproductive outcomes during the follow-up period in RPL couples
| Outcome | Normal group (n = 257) |
Abnormal group (n = 370) | ||||
|---|---|---|---|---|---|---|
| Total (n = 370) |
Aneuploidy (n = 246) |
Polyploidy (n = 64) |
CNVs (n = 52) |
UPD (n = 8) |
||
| Live birth | 172 (66.9) | 265 (71.6) | 174 (70.7) | 46 (71.9) | 39 (75.0) | 6 (75.0) |
| P value | Ref | 0.209 | 0.357 | 0.449 | 0.256 | 0.634 |
| OR (95% CI) | Ref | 1.25 (0.88–1.76) | 1.19 (0.82–1.74) | 1.26 (0.69–2.31) | 1.48 (0.75–2.93) | 1.48 (0.29–7.50) |
| Adjusted P value | Ref | 0.040 | 0.041 | 0.425 | 0.383 | 0.445 |
| Adjusted OR (95% CI) | Ref | 1.46 (1.02–2.11) | 1.52 (1.02–2.28) | 1.29 (0.69–2.42) | 1.37 (0.68–2.77) | 1.94 (0.35–10.62) |
| Spontaneous abortion | 70 (27.2) | 84 (22.7) | 56 (22.8) | 16 (25.0) | 11 (21.2) | 1 (12.5) |
| P value | Ref | 0.195 | 0.248 | 0.718 | 0.365 | 0.372 |
| OR (95% CI) | Ref | 0.79 (0.54–1.13) | 0.79 (0.53–1.18) | 0.89 (0.48–1.67) | 0.72 (0.35–1.47) | 0.38 (0.05–3.16) |
| Adjusted P value | Ref | 0.047 | 0.032 | 0.712 | 0.518 | 0.259 |
| Adjusted OR (95% CI) | Ref | 0.68 (0.46–0.99) | 0.63 (0.41–0.96) | 0.89 (0.46–1.70) | 0.78 (0.37–1.64) | 0.28 (0.03–2.53) |
| Biochemical miscarriage | 13 (5.1) | 17 (4.6) | 13 (5.3) | 1 (1.6) | 2 (3.8) | 1 (12.5) |
| P value | Ref | 0.789 | 0.909 | 0.248 | 0.712 | 0.373 |
| OR (95% CI) | Ref | 0.90 (0.43–1.90) | 1.05 (0.48–2.31) | 0.30 (0.04–2.32) | 0.75 (0.16–3.43) | 2.68 (0.31–23.44) |
| Adjusted P value | Ref | 0.661 | 0.905 | 0.247 | 0.756 | 0.425 |
| Adjusted OR (95% CI) | Ref | 0.85 (0.40–1.78) | 0.95 (0.43–2.12) | 0.30 (0.04–2.32) | 0.79 (0.17–3.60) | 2.43 (0.27–21.52) |
| Ectopic pregnancy | 2 (0.8) | 4 (1.1) | 3 (1.2) | 1 (1.6) | 0 (0) | 0 (0) |
| P value | Ref | 0.703 | 0.621 | 0.567 | 0.998 | 0.999 |
| OR (95% CI) | Ref | 1.39 (0.25–7.67) | 1.57 (0.26–9.50) | 2.02 (0.18–22.67) | - | - |
| Adjusted P value | Ref | 0.746 | 0.678 | 0.568 | 0.998 | 0.999 |
| Adjusted OR (95% CI) | Ref | 1.33 (0.24–7.36) | 1.47 (0.24–9.04) | 2.02 (0.18–22.66) | - | - |
Data were present as n (%). RPL Recurrent pregnancy loss; CNVs Copy number variations; UPD Uniparental disomy; Ref Reference. P value and OR (95% CI) were calculated by binary logistic regression analysis. Adjusted P value and OR (95% CI) were calculated by binary logistic regression analysis adjusted for maternal age of the index miscarriage. Statistically significant differences were marked in bold
In terms of subgroups, we found that couples with aneuploid miscarriage had a significantly higher live birth rate (70.7% vs 66.9%, P = 0.041) and a significantly lower subsequent spontaneous miscarriage rate (22.8% vs 27.2%, P = 0.032) than those with chromosomally normal miscarriage after adjusting maternal age. However, no significant differences were observed in rates of live birth or adverse pregnancy events between any other subgroups of chromosomally abnormal groups (polyploidy, CNVs, UPD) and normal group. Besides, biochemical miscarriage and ectopic miscarriage rates for subsequent pregnancy were similar across all subgroups. These findings remained consistent after adjusting for maternal age, gestational age of the index miscarriage and previous reproductive history in the sensitivity analysis.
Cumulative pregnancies outcomes
Compared with those with chromosomally normal miscarriage, the cumulative miscarriage rate was significantly lower in couples with chromosomally abnormal miscarriage (35.4% vs 29.2%, P = 0.019). However, the cumulative live birth rate did not differ significantly between couples with chromosomally abnormal and normal miscarriage (82.7% vs 80.2%, P = 0.131). Besides, no significant difference was found in the frequency of biochemical miscarriage (4.9% vs 7.0%, P = 0.194) and ectopic pregnancy (1.9% vs 2.3%, P = 0.657). Details are shown in Table 4.
Table 4.
Cumulative reproductive outcomes during the follow-up period in couples with recurrent pregnancy loss
| Outcome | Normal group (n = 257) |
Abnormal group (n = 370) | ||||
|---|---|---|---|---|---|---|
| Total (n = 370) |
Aneuploidy (n = 246) |
Polyploidy (n = 64) |
CNVs (n = 52) |
UPD (n = 8) |
||
| Live birth | 206 (80.2) | 306 (82.7) | 207 (84.1) | 52 (81.3) | 41 (78.8) | 6 (75.0) |
| P value | Ref | 0.418 | 0.244 | 0.844 | 0.830 | 0.721 |
| OR (95% CI) | Ref | 1.18 (0.79–1.78) | 1.31 (0.83–2.08) | 1.07 (0.53–2.16) | 0.92 (0.44–1.92) | 0.74 (0.15–3.79) |
| Adjusted P value | Ref | 0.131 | 0.027 | 0.874 | 0.542 | 0.931 |
| Adjusted OR (95% CI) | Ref | 1.39 (0.91–2.14) | 1.73 (1.06–2.83) | 1.06 (0.51–2.20) | 0.79 (0.37–1.70) | 0.93 (0.16–5.25) |
| Spontaneous abortion | 91 (35.4) | 108 (29.2) | 73 (29.7) | 20 (31.3) | 13 (25.0) | 2 (25.0) |
| P value | Ref | 0.100 | 0.171 | 0.532 | 0.150 | 0.547 |
| OR (95% CI) | Ref | 0.75 (0.54–1.06) | 0.81 (0.56–1.18) | 0.87 (0.49–1.57) | 0.64 (0.33–1.26) | 0.64 (0.13–3.24) |
| Adjusted P value | Ref | 0.019 | 0.019 | 0.506 | 0.218 | 0.392 |
| Adjusted OR (95% CI) | Ref | 0.65 (0.46–0.93) | 0.62 (0.42–0.93) | 0.82 (0.45–1.49) | 0.65 (0.32–1.30) | 0.51 (0.09–2.71) |
| Biochemical miscarriage | 18 (7.0) | 18 (4.9) | 14 (5.7) | 1 (1.6) | 2 (3.8) | 1 (12.5) |
| P value | Ref | 0.260 | 0.547 | 0.133 | 0.406 | 0.559 |
| OR (95% CI) | Ref | 0.68 (0.35–1.33) | 0.80 (0.39–1.65) | 0.21 (0.03–1.61) | 0.53 (0.12–2.36) | 1.90 (0.22–16.27) |
| Adjusted P value | Ref | 0.194 | 0.406 | 0.132 | 0.437 | 0.620 |
| Adjusted OR (95% CI) | Ref | 0.64 (0.32–1.26) | 0.73 (0.35–1.52) | 0.21 (0.03–1.60) | 0.55 (0.12–2.47) | 1.73 (0.20–14.99) |
| Ectopic pregnancy | 6 (2.3) | 7 (1.9) | 4 (1.6) | 2 (3.1) | 1 (1.9) | 0 (0) |
| P value | Ref | 0.702 | 0.571 | 0.718 | 0.856 | 0.999 |
| OR (95% CI) | Ref | 0.81 (0.27–2.43) | 0.69 (0.19–2.48) | 1.35 (0.27–6.85) | 0.82 (0.10–6.96) | - |
| Adjusted P value | Ref | 0.657 | 0.509 | 0.719 | 0.877 | 0.999 |
| Adjusted OR (95% CI) | Ref | 0.78 (0.26–2.36) | 0.65 (0.18–2.36) | 1.35 (0.27–6.84) | 0.85 (0.10–7.19) | - |
Data were present as n (%). RPL Recurrent pregnancy loss; CNVs Copy number variations; UPD Uniparental disomy; Ref Reference. P value and OR (95% CI) were calculated by binary logistic regression analysis. Adjusted P value and OR (95% CI) were calculated by binary logistic regression analysis adjusted for maternal age of the index miscarriage. Statistically significant differences were marked in bold
Adjusted for maternal age, couples with aneuploid miscarriage had a higher live birth rate (84.1% vs 80.2%, P = 0.027) and a lower spontaneous miscarriage rate (29.7% vs 35.4%, P = 0.019) than those with chromosomally normal miscarriage. However, the rates of live birth and spontaneous miscarriage did not differ between any remaining subgroups of couples with chromosomally abnormal and normal miscarriage. Besides, the rates of biochemical miscarriage and ectopic miscarriage were similar among couples with chromosomally normal and abnormal miscarriage. In the sensitivity analysis adjusted for maternal age, gestational age of the index miscarriage and previous reproductive history, these statistically significant findings were consistent with the results. The live birth rates in subsequent pregnancy and cumulative pregnancies are displayed in Figure S1.
Reproductive outcomes of couples with single aneuploid miscarriage
Among 246 couples with single aneuploid miscarriage who conceived at least once during the follow-up period, trisomy 16 was the most frequent aneuploidy (22.4%, 55/246), followed by sex chromosome abnormality (15.4%, 38/246) and trisomy 22 (12.6%, 31/246) (Fig. 2a). The subsequent live birth rates for couples with trisomy 16, sex chromosome abnormalities and trisomy 22 were 72.7%, 78.9% and 61.3%, respectively (Fig. 2b), and the cumulative live birth rates of these couples were 81.8%, 92.1% and 77.4%, respectively (Fig. 2c).
Fig. 2.
Embryonic genetic findings and reproductive outcomes in couples with various single aneuploid miscarriage. a Distribution of embryonic single aneuploidy in 450 couples with reproductive outcomes. b Subsequent reproductive outcome in couples with single aneuploid miscarriage. c Cumulative live birth rate in couples with single aneuploid miscarriage
The subsequent live birth rates for couples with single aneuploid miscarriage involving chromosomes 3, 7, 17 and 18 reached to 100% in this study. Similarly, the cumulative live birth rate for couples with single aneuploid miscarriage involving chromosomes 3, 5, 6, 7, 17 and 18 also represented a live birth rate of 100%. However, the cumulative live birth rates for couples with single aneuploid miscarriage involving chromosomes 11 and 14 were relatively low, at 50.0% and 57.1%, respectively.
Reproductive outcomes according to maternal age
Among couples with chromosomally normal miscarriage, the cumulative live birth rates of women aged < 35 years and ≥ 35 years were 83.7% and 61.9%, respectively. Among couples with chromosomally abnormal miscarriage, the cumulative live birth rates of women aged < 35 years and ≥ 35 years with chromosomally abnormal miscarriage were 87.5% and 60.0%, respectively (Figure S2).
Among couples with chromosomally normal miscarriage, the cumulative miscarriage rates in women aged < 35 years and ≥ 35 years were 30.2% and 61.9%, respectively. Among those with chromosomally abnormal miscarriage, the cumulative miscarriage rates in women aged < 35 years and ≥ 35 years with chromosomally abnormal miscarriage were 24.9% and 49.2%, respectively (Table S4).
Among couples with advanced maternal age, the live birth rate significantly decreased (P < 0.01) and the miscarriage rate increased (P < 0.01) than those aged < 35 years old, regardless of the embryonic CMA results.
Reproductive outcomes according to the number of previous miscarriages
For women with chromosomally normal miscarriage, the subsequent live birth rates of women with 2, 3 or ≥ 4 previous miscarriages were 75.5%, 56.4% and 43.7%, respectively. For women with chromosomally abnormal miscarriage, the subsequent live birth rates of women with 2, 3 or ≥ 4 previous miscarriages were 73.6%, 70.5% and 63.0%, respectively (Table S5). No significant difference was observed in the live birth rate of women with 2 previous miscarriages between chromosomally abnormal and normal groups (P = 0.729). We found that for couples with chromosomally normal miscarriage, the subsequent live birth rate significantly decreased as the number of previous miscarriages increased, suggesting that women may have a better prognosis if they have fewer miscarriages. However, for women with chromosomally abnormal miscarriage, there was no significant difference in subsequent live birth rate according to the number of previous miscarriages, indicating that this relationship does not seem to apply to women with chromosomally abnormal miscarriage. These findings were consistent in cumulative pregnancies.
The number of previous miscarriages was not associated with either subsequent spontaneous miscarriage rate or cumulative miscarriage rate during the follow-up period (Table S6).
Discussion
We conducted a retrospective study to evaluate the reproductive outcomes of RPL couples with various types of embryonic results. Overall, the subsequent live birth rate in couples with chromosomally abnormal miscarriage was higher than those with chromosomally normal miscarriage. However, no significant difference was observed in the cumulative live birth rate in RPL couples with chromosomally abnormal and normal miscarriage. Furthermore, among couples with chromosomally normal miscarriage, the live birth rate in couples with large number of previous miscarriages was significantly low. However, this finding was not available in those with chromosomally abnormal miscarriage.
In this retrospective cohort study, we evaluated the frequency and distribution of embryonic chromosomal abnormalities among RPL couples. Then, we followed up all the eligible couples to assess the reproductive prognosis among RPL couples with various types of embryonic chromosomal abnormalities. We found that RPL couples with chromosomally normal miscarriage showed a poorer reproductive prognosis than those with chromosomally abnormal miscarriage for the subsequent pregnancy. However, the cumulative live birth rate was similar among RPL couples with chromosomally normal and abnormal miscarriage. These findings provided valuable information for counselling RPL couples on their clinical outcomes in future pregnancies according to embryonic chromosomal results.
It is not clear whether there is a significant difference in reproductive prognosis between couples with chromosomally abnormal and normal miscarriage. Carp et al. reported that, after an chromosomally abnormal miscarriage, there was a higher subsequent live birth rate compared to that after a chromosomally normal miscarriage [11], but that difference was nonsignificant. However, we observed that couples with chromosomally normal miscarriage (66.9%) presented a significantly lower chance of subsequent live birth compared with those with chromosomally abnormal miscarriage (71.6%). Moreover, compared with those with chromosomally abnormal miscarriage, couples suffered from chromosomally normal miscarriage presented a higher subsequent miscarriage rate. These findings demonstrated that chromosomally normal miscarriage may be a predictive factor of adverse pregnancy events for the subsequent pregnancy. Possible reasons are some unknown factors that play a role in the recurrence of miscarriage. Nowadays, studies have focused on immunologic mechanisms, spermatozoal, embryonic, and endometrial characteristics to investigate the potential causes for pregnancy loss in unexplained RPL [17–19].
Currently, the reproductive prognosis among RPL couples with various types of chromosomally abnormal miscarriage is still unclear. Ogasawara et al. [10] reported that patients with chromosomally normal miscarriage showed a significantly higher spontaneous miscarriage rate in subsequent pregnancy. Our results showed that couples with aneuploid miscarriage presented a lower risk of spontaneous miscarriage compared with those with chromosomally normal miscarriage. In addition, for women with three or more previous miscarriages, we observed that couples with aneuploid miscarriage showed a higher subsequent live birth rate than those with chromosomally normal miscarriage. For couples with chromosomally normal miscarriage, women may have a better prognosis if they have fewer miscarriages. However, this relationship does not seem to apply to women with chromosomally abnormal miscarriage. Of note, the remaining subgroups of chromosomally abnormal miscarriage (polyploidy, CNVs and UPD) were not predictive of better reproductive prognosis than chromosomally normal miscarriage. Hence, it indicated that couples with aneuploid miscarriage may have a better prognosis than those with chromosomally normal miscarriage in the subsequent pregnancy.
In contrast to the previous study by Xia Z. et al. [20], which reported a similar reproductive prognosis for couples with various types of embryonic chromosomal results after a sporadic miscarriage, the current study focused on reproductive outcomes after recurrent miscarriages. We found that the prognosis in the subsequent pregnancy was worse for couples with chromosomally normal miscarriage than for those with chromosomally abnormal miscarriage. The two groups had comparable cumulative live birth rates, but the cumulative miscarriage rates were higher for couples with chromosomally normal miscarriage than for those with chromosomally abnormal miscarriage. Therefore, couples with chromosomally normal miscarriage may face more miscarriages before achieving a live birth. In summary, CMA is more useful for couples who experienced recurrent miscarriages than for those who had a sporadic miscarriage.
Our study paid particular attention to reproductive outcomes of RPL couples with single aneuploid miscarriage. For all couples with single aneuploid miscarriage, the long-term prognosis would be favorable. However, they were more prone to subsequent spontaneous miscarriage if they had miscarried with trisomy 5 or 6, possibly due to the small sample size of these cases in our cohort. Previous studies have reported that trisomy 16 is the most frequent single aneuploidy [15, 21]. The subsequent live birth rate of couples with trisomy 16 in POCs of the index miscarriage was 72.7%, and the cumulative live birth rate of them reached to 81.8%. Thus, this study could provide valuable assessment and guidance for RPL with single aneuploid miscarriage.
It is well known that maternal age and the number of previous miscarriages are independent risk factors for RPL [22]. In our research, women aged ≥ 35 years old showed a lower live birth rate and a higher spontaneous miscarriage rate, regardless of embryonic results. Among couples with various numbers of previous miscarriages, we did not observe a significant difference in spontaneous miscarriage rates for subsequent pregnancy or cumulative pregnancies. However, among couples with chromosomally normal miscarriage, there was a significant decrease in live birth rates in subsequent pregnancy and cumulative pregnancies when a large number of previous miscarriages had occurred. Nevertheless, this finding was not available in couples with chromosomally abnormal miscarriage. The reason for this may be recurrent unknown factors would reemerge in the following pregnancies of couples with chromosomally normal miscarriage. The prognosis may vary for women who suffered RPL due to random chance or an undiagnosed pathological condition. The former may have a favorable prognosis, while the latter may have a poorer prognosis that cannot be improved by the current diagnostic protocols.
This study demonstrates the relationship between the number of previous spontaneous abortions and the live birth rate. For RPL couples with chromosomally normal miscarriage, individualized treatment and psychological counselling seems to be more urgent.
This was a large cohort study to evaluate the reproductive outcomes of RPL couples. Based on our findings, we provided systematic and accurate genetic counselling for RPL couples regarding their reproductive prognosis and risk assessment. However, this study also had some limitations. Due to the low incidence of specific types of embryonic abnormalities, some subgroups of embryonic abnormalities included a few cases. A few couples (8.3%, 69/830) with RPL met criteria were lost to follow-up and we failed to obtain the reproductive outcomes in them.
Conclusions
In conclusion, this study illuminated that among couples with three or more previous miscarriages, RPL couples with chromosomally normal miscarriage presented a poorer reproductive prognosis than those with chromosomally abnormal miscarriage in subsequent pregnancy. However, the cumulative live birth rates were similar between couples with chromosomally abnormal and normal miscarriage. Advanced maternal age was a predictor of adverse pregnancy events, and independent of types of embryonic chromosomal abnormalities. Furthermore, a large number of previous miscarriages indicated poor reproductive prognosis in RPL couples with chromosomally normal miscarriage, but not in those with chromosomally abnormal miscarriage.
Supplementary Information
Below is the link to the electronic supplementary material.
Figure S1 The live birth rate of RPL couples during the follow-up period (JPG 40 KB)
Figure S2 The live birth rate according to maternal age of the index miscarriage (JPG 30 KB)
Acknowledgements
The study was supported by the National Natural Science Foundation of China (81801445 and 81701427). We greatly appreciate all the participants for their cooperation in this study.
Funding
The study was supported by the National Natural Science Foundation of China (81801445; 81701427).
Declarations
Ethical approval
The research Ethics Committee of Nanjing Maternity and Child Health Care Hospital approved the study (2021KY-004) in accordance with the Helsinki Declaration of 1975, as revised in 2000.
Patient consent
Informed consent was obtained from all the study participants at the time of providing samples.
Conflict of interest
The authors declare that they have no conflict of interest.
Date sharing
All the data generated or analyzed in this study are included in this published article.
Footnotes
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Yiming Li, Ran Zhou, and Zhengyi Xia contributed equally to this work.
Contributor Information
Zhengfeng Xu, Email: zhengfengxu@njmu.edu.cn.
Yan Wang, Email: wangyan@njmu.edu.cn.
References
- 1.Practice Committee of the American Society for Reproductive Medicine. Evaluation and treatment of recurrent pregnancy loss: a committee opinion. Fertil Steril. 2012;98(5):1103–11. 10.1016/j.fertnstert.2012.06.048. [DOI] [PubMed]
- 2.Practice Committee of the American Society for Reproductive Medicine. Definitions of infertility and recurrent pregnancy loss: a committee opinion. Fertil Steril. 2020;113(3):533–5. 10.1016/j.fertnstert.2019.11.025. [DOI] [PubMed]
- 3.Bender Atik R, Christiansen OB, Elson J, Kolte AM, Lewis S, Middeldorp S, Nelen W, Peramo B, Quenby S, Vermeulen N, Goddijn M. ESHRE guideline: recurrent pregnancy loss. Hum Reprod Open. 2018;2018(2):hoy0004. doi: 10.1093/hropen/hoy004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Quenby S, Gallos ID, Dhillon-Smith RK, Podesek M, Stephenson MD, Fisher J, Brosens JJ, Brewin J, Ramhorst R, Lucas ES, McCoy RC, Anderson R, Daher S, Regan L, Al-Memar M, Bourne T, MacIntyre DA, Rai R, Christiansen OB, Sugiura-Ogasawara M, Odendaal J, Devall AJ, Bennett PR, Petrou S, Coomarasamy A. Miscarriage matters: the epidemiological, physical, psychological, and economic costs of early pregnancy loss. Lancet. 2021;397(10285):1658–1667. doi: 10.1016/S0140-6736(21)00682-6. [DOI] [PubMed] [Google Scholar]
- 5.Linehan LA, San Lazaro Campillo I, Hennessy M, Flannery C, O'Donoghue K. Reproductive outcomes following recurrent first-trimester miscarriage: a retrospective cohort study. Hum Reprod Open. 2022;2022(4):hoac045. doi: 10.1093/hropen/hoac045. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Garrido-Gimenez C, Alijotas-Reig J. Recurrent miscarriage: causes, evaluation and management. Postgrad Med J. 2015;91(1073):151–162. doi: 10.1136/postgradmedj-2014-132672. [DOI] [PubMed] [Google Scholar]
- 7.Sahoo T, Dzidic N, Strecker MN, Commander S, Travis MK, Doherty C, Tyson RW, Mendoza AE, Stephenson M, Dise CA, Benito CW, Ziadie MS, Hovanes K. Comprehensive genetic analysis of pregnancy loss by chromosomal microarrays: outcomes, benefits, and challenges. Genet Med. 2017;19(1):83–89. doi: 10.1038/gim.2016.69. [DOI] [PubMed] [Google Scholar]
- 8.American College of Obstetricians and Gynecologists’ Committee on Practice Bulletins—Gynecology. ACOG practice bulletin no. 200: early pregnancy loss. Obstet Gynecol. 2018;132(5):e197–e207. 10.1097/AOG.0000000000002899. [DOI] [PubMed]
- 9.Huchon C, Deffieux X, Beucher G, Capmas P, Carcopino X, Costedoat-Chalumeau N, Delabaere A, Gallot V, Iraola E, Lavoue V, Legendre G, Lejeune-Saada V, Leveque J, Nedellec S, Nizard J, Quibel T, Subtil D, Vialard F, Lemery D. Pregnancy loss: French clinical practice guidelines. Eur J Obstet Gynecol Reprod Biol. 2016;201:18–26. doi: 10.1016/j.ejogrb.2016.02.015. [DOI] [PubMed] [Google Scholar]
- 10.Ogasawara M, Aoki K, Okada S, Suzumori K. Embryonic karyotype of abortuses in relation to the number of previous miscarriages. Fertil Steril. 2000;73(2):300–304. doi: 10.1016/S0015-0282(99)00495-1. [DOI] [PubMed] [Google Scholar]
- 11.Carp H, Toder V, Aviram A, Daniely M, Mashiach S, Barkai G. Karyotype of the abortus in recurrent miscarriage. Fertil Steril. 2001;75(4):678–682. doi: 10.1016/S0015-0282(00)01801-X. [DOI] [PubMed] [Google Scholar]
- 12.Murugappan G, Leonard SA, Newman H, Shahine L, Lathi RB. Karyotype of first clinical miscarriage and prognosis of subsequent pregnancy outcome. Reprod Biomed Online. 2021;42(6):1196–1202. doi: 10.1016/j.rbmo.2021.03.021. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Sugiura-Ogasawara M, Ozaki Y, Kitaori T, Suzumori N, Obayashi S, Suzuki S. Live birth rate according to maternal age and previous number of recurrent miscarriages. Am J Reprod Immunol. 2009;62(5):314–319. doi: 10.1111/j.1600-0897.2009.00741.x. [DOI] [PubMed] [Google Scholar]
- 14.Maconochie N, Doyle P, Prior S, Simmons R. Risk factors for first trimester miscarriage–results from a UK-population-based case-control study. BJOG. 2007;114(2):170–186. doi: 10.1111/j.1471-0528.2006.01193.x. [DOI] [PubMed] [Google Scholar]
- 15.Wang Y, Li Y, Chen Y, Zhou R, Sang Z, Meng L, Tan J, Qiao F, Bao Q, Luo D, Peng C, Wang Y, Luo C, Hu P, Xu Z. Systematic analysis of copy-number variations associated with early pregnancy loss. Ultrasound Obstet Gynecol. 2020;55(1):96–104. doi: 10.1002/uog.20412. [DOI] [PubMed] [Google Scholar]
- 16.Riggs ER, Andersen EF, Cherry AM, Kantarci S, Kearney H, Patel A, Raca G, Ritter DI, South ST, Thorland EC, Pineda-Alvarez D, Aradhya S, Martin CL. Technical standards for the interpretation and reporting of constitutional copy-number variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics (ACMG) and the Clinical Genome Resource (ClinGen) Genet Med. 2020;22(2):245–257. doi: 10.1038/s41436-019-0686-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Wang X, Shi W, Zhao S, Gong D, Li S, Hu C, Chen Z, Li Y, Yan J. Whole exome sequencing in unexplained recurrent miscarriage families identified novel pathogenic genetic causes of euploid miscarriage. Human reproduction (Oxford, England) 2023;38(5):1003–1018. doi: 10.1093/humrep/dead039. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Liao J, Li Y, Li X, Su X, Peng J, Xiao N, Fan X, Chen H, Lu G, Lin G, Cheng L, Gong F. Blood CD4(+)CD25(+) T regulatory cells constitute a potential predictive marker of subsequent miscarriage in unexplained recurrent pregnancy loss. Int Immunopharmacol. 2022;110:108960. doi: 10.1016/j.intimp.2022.108960. [DOI] [PubMed] [Google Scholar]
- 19.Craciunas L, Chu J, Pickering O, Mohiyiddeen L, Coomarasamy A. The metabolomic profile of endometrial receptivity in recurrent miscarriage. Minerva Obstet Gynecol. 2022. 10.23736/S2724-606X.22.05151-X. [DOI] [PubMed]
- 20.Xia Z, Zhou R, Li Y, Meng L, Huang M, Tan J, Qiao F, Zhu H, Hu P, Zhu Q, Xu Z, Wang Y. Reproductive outcomes in couples with sporadic miscarriage after embryonic chromosomal microarray analysis. Ann Med. 2023;55(1):837–48. doi: 10.1080/07853890.2023.2183986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Finley J, Hay S, Oldzej J, Meredith MM, Dzidic N, Slim R, Aradhya S, Hovanes K, Sahoo T. The genomic basis of sporadic and recurrent pregnancy loss: a comprehensive in-depth analysis of 24,900 miscarriages. Reprod Biomed Online. 2022;45(1):125–134. doi: 10.1016/j.rbmo.2022.03.014. [DOI] [PubMed] [Google Scholar]
- 22.Dimitriadis E, Menkhorst E, Saito S, Kutteh WH, Brosens JJ. Recurrent pregnancy loss. Nat Rev Dis Primers. 2020;6(1):98. doi: 10.1038/s41572-020-00228-z. [DOI] [PubMed] [Google Scholar]
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Supplementary Materials
Figure S1 The live birth rate of RPL couples during the follow-up period (JPG 40 KB)
Figure S2 The live birth rate according to maternal age of the index miscarriage (JPG 30 KB)