Genetic testing on products of conception and its relationship with body mass index.

Purpose The study aimed to investigate the relationship between elevated maternal body mass index (BMI) and foetal chromosomal aberrations by performing single-nucleotide polymorphism (SNP) array-based genetic testing on products of conception (POC).

We retrospectively reviewed the data for 1068 assisted reproductive technology (ART)-conceived POC originated from 1068 patients with early spontaneous miscarriage. First, all types of chromosomal abnormalities were defined. Then, the baseline characteristics, including maternal age, BMI, thyroid-stimulating hormone (TSH), gestational age, fertilization method, reasons for fertility treatment, embryo transfer (ET) cycle, stage of embryo development and the embryo morphology grade, were compared between chromosomally normal and abnormal POC groups. Finally, a multivariate logistic regression model was used to analyse various factors affecting the foetal chromosomal abnormality rate.

The SNP array results showed that 45.3% (484/1068) of POC were chromosomally normal and that 54.7% (584/1068) of POC presented chromosomal abnormalities. Of these 584 chromosomally abnormal POC, 388 (66.4%) were trisomy, 42 (7.2%) had a monosomy, 68 (11.6%) were found with segmental aneuploidy, 46 (7.9%) were mosaic, 28 (4.8%) were identified as polyploidy and 12 (2.1%) were euploid samples with uniparental disomy (UPD). Multivariate logistic regression results showed that the risk of miscarrying chromosomally abnormal POC increased 1.424-fold in women with normal BMI compared to women with an elevated BMI (≥ 25 kg/m2) (OR = 1.424, 95% CI = 1.074–1.888, p = 0.014).

Women with an elevated BMI (≥ 25 kg/m2) are more likely to miscarry chromosomally normal POC. Electronic supplementary material The online version of this article (10.1007/s10815-020-01849-9) contains supplementary material, which is available to authorized users.

Overweight/obese, Genetic test, Miscarriage, Products of conception (POC)

Obesity is currently regarded as a worldwide epidemic and is widely reported to be associated with many diseases [1, 2]. The adverse impacts of obesity on child-bearing women are well recognized [3, 4]. For women who conceived naturally, studies have shown that overweight/obese women had a 1.2-fold increased risk of experiencing a spontaneous miscarriage and a 3.5-fold increased risk of recurrent miscarriage compared with their age-matched counterparts [5]. Additionally, for those undergoing assisted conception, the risk ratio changed to 1.33 for overweight/obese women [6], suggesting raised BMI (≥ 25 kg/m2) as a risk factor for miscarriage for those who conceived naturally or by ART treatment [7, 8]. The mechanism for elevated BMI (≥ 25 kg/m2)-related miscarriage is not yet known. Foetal chromosomal aberrations have been widely implicated as a main cause of miscarriage, contributing up to 70% of pregnancy losses [9]. Previous studies performed karyotyping on POC and concluded that women with increased BMI (≥ 25 kg/m2) were more likely to miscarry euploid POC rather than aneuploid POC [4, 6, 10]. However, these studies were all based on the traditional karyotyping technique, which has several limitations. First, large chromosomal abnormalities missed by conventional karyotyping were found in 2–5% of miscarriages [11]. Additionally, traditional karyotyping is hindered by maternal cell contamination (MCC) [12], falsely producing a high rate (29–58%) of normal female karyotype. Furthermore, due to the limited resolution of traditional karyotyping, it was unable to detect submicroscopic deletions and/or duplications, which are also suggested to be related to spontaneous miscarriage [11, 13, 14]. Therefore, genetic testing of POC using traditional karyotyping techniques may underestimate the aneuploidy rate; thus, accurate whole-genome karyotype screening of POC needs to be performed to investigate the role of foetal chromosomal aberrations in elevated BMI-related miscarriage. SNP array is a frequently used whole-genome screening technique [15, 16]. It has many advantages compared to traditional karyotyping techniques. First, it provides a 25–50-fold higher resolution in monitoring mosaicism, aneuploidy and uniparental disomy (UPD). In addition, it can detect submicroscopic deletions and/or duplications typically down to a 50–100-kb level [17, 18]. Notably, recent studies show that it is able to detect MCC [19], allowing the true POC karyotyping results to be reported. The aim of our study is to investigate the relationship between elevated BMI (≥ 25 kg/m2) and foetal chromosomal aberrations using SNP array-based whole-genome screening of POC.

Inclusion and exclusion criteria for the study cohort This study was approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University. Women who both experienced early spontaneous pregnancy loss after ART treatment and had genetic testing on POC between June 2012 and October 2018 were included. Informed consent was signed by couples before the tests. We excluded samples from women older than 40 years. In addition, POC from women (or their spouses) with abnormal chromosomal karyotypes, multiple pregnancies, who underwent donor’s oocyte treatment and/or pre-implantation genetic testing for aneuploidies treatment, or with abnormal thyroid stimulating hormone (TSH) levels (defined as > 4.0 mU/L or < 0.5 mU/L according to the American Thyroid Association [20]), and had recurrent miscarriage histories (defined as ≥ 2 times) were excluded. Ultimately, a total of 1068 POC from 1068 women were included in our analysis. Data collection Information on the following maternal baseline characteristics was extracted from the electronic records: maternal age, BMI, TSH level, gestational age (in weeks), reasons for fertility treatment (tubal factor, polycystic ovary syndrome (PCOS), male factor, uterine factor (uterus malformation or endometriosis), multiple factors), fertilization method (in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI)), embryo transfer (ET) cycle (fresh or frozen-thawed), stage of embryo development (cleavage stage embryo or blastocyst) and embryo morphology grade. BMI was calculated using the formula BMI = weight/height2 (kg/m2) and was classified with the World Health Organization classification cut-offs: BMI ≥ 25 kg/m2 and BMI < 25 kg/m2. PCOS was defined according to the Rotterdam Criteria [21]. At least two of the following characteristics had to be fulfilled after excluding other pathologies (thyroid disease, adrenal hyperplasia or androgen-secreting tumours): (i) chronic anovulation or oligo ovulation, (ii) hyper-androgenism and (iii) polycystic ovaries on ultrasound [21]. The cleavage-stage embryos were graded according to their cleavage rate and appearance (Supplementary Table S1) [22, 23]: Grade 1 and Grade 2 embryos were considered as high quality, usually with 6–10 cells at day 3 post-fertilization. The blastocysts were graded using the system of Gardner and Schoolcraft (Supplementary Table S2) [22, 23]: 3AA, 4AA, 5AA, 6AA, 3AB, 4AB, 5AB, 6AB, 3BA, 4BA, 5BA and 6BA were considered as high quality. Interventions POC were collected from patients who underwent surgical management of a spontaneous miscarriage with dilation and curettage (D&C) under intravenous anaesthesia in our reproductive medical centre [24]. Then, samples were placed in sterile tubes with normal saline and transported to the lab immediately. POC were thoroughly rinsed with physiological saline and examined under a dissecting microscope to separate chorionic villi from maternal tissue according to a standardized pipeline [25]. Genomic DNA was extracted from the villi using QIAamp DNA Mini Kits (Cat. No. 51306, Qiagen, Germany), followed by SNP array analysis using Human CytoSNP-12v.21 arrays (Illumina, San Diego, CA, USA). All procedures were performed based on the manufacturers’ guidelines. GenomeStudio and KaryoStudio v.1.4 software (both from Illumina) were used for data analysis. The log R ratio (LRR) and B allele frequency (BAF) are informative of copy number status [12]. Detected copy number variants (CNVs) were evaluated according to a scientific publication review and several databases: DECIPHER (https://decipher.sanger.ac.uk/), database of Genomic Variants (http://projects.tcag.ca/variation), OMIM (https://omim.org/) and ClinGen (https://clinicalgenome.org/). CNVs ≥ 10 Mb can almost be identified by traditional karyotyping and were defined as partial aneuploidy [26, 27]. CNVs < 10 Mb were defined as chromosomal submicroscopic abnormality. The CNVs were considered as pathogenic if (i) the size of CNVs was ≥ 5 Mb and with no well-established cytogenetic heteromorphisms or (ii) the size of CNVs was < 5 Mb but contained a region with well-established clinical significance [26–28]. Variants of uncertain significance (VOUS) were defined if CNVs did not correspond to any of the clinically significant phenotypes mentioned above. Significant MCC was characterized as levels of MCC exceeding 30% [27]. Statistical analysis All collected data were divided into two groups based on SNP array results of POC: group 1 consisted of chromosomally normal POC and group 2 consisted of chromosomally abnormal POC. Data were tested for normality prior to the use of t test. Otherwise, the non-parametric test (Mann–Whitney U test) was used. Then, Student’s t tests (continuous variables) and χ2 tests (categorical variables) were used to compare the baseline characteristics between the two groups. Normally distributed continuous data are presented as the mean ± standard deviation (SD), and non-normally distributed continuous data are presented as median and interquartile range (IQR). Categorical variables are presented as percentages. Logistic regression analysis was adjusted for baseline differences between the groups and was used to create baseline characteristics’ predictive probability for miscarrying chromosomally abnormal (p) and chromosomally normal (1-p) POC. Potential confounding factors included TSH level, gestational age (both continuous variables), maternal age, BMI, reasons for fertility treatment, fertilization method, ET cycle, stage of embryo development and embryo morphology grade (all nominal categorical variables). The results of logistic regression analysis were reported as odds ratios (ORs) and 95% confidence intervals (CIs). All p values were two-sided, and p < 0.05 was considered statistically significant. All the data were analysed using SPSS statistics 17.0 (SPSS Inc., IBM Corp., Chicago, IL, USA).

Results of the SNP array test A total of 1068 POC were collected from 1068 women. SNP array analysis was successfully performed on all collected POC. No MCC was observed. Additionally, the male-to-female ratio in POC with a normal karyotype was 1.02, which could partially support no MCC. A total of 484 (45.3%) POC were found to be chromosomally normal, and 584 (54.7%) POC were identified with chromosomal abnormalities (Table 1). Of the 584 chromosomally abnormal POC, 430 (73.6%) were found to have aneuploidy, including 388 (66.4%) with trisomy and 42 (7.2%) with monosomy (Table 1). Segmental aneuploidy was found in 68 (11.6% of all chromosomal abnormality) cases (Table 1), including 17 POC with deletions, 33 POC with duplications and 18 POC with complex abnormality (defined when two or more deletions/duplications involved the same chromosome or two or more chromosomes). The segmental aneuploidy POC involved 83 CNVs in total, including 70 CNVs (in 57 POC) with clinical significance and 13 CNVs in 13 POC that were reported to be VOUS. Mosaic was observed in 46 (7.9% of all chromosomal abnormality) POC (Table 1), including 34 mosaic trisomy, 1 mosaic polyploidy and 11 mosaic segmental aneuploidy. Polyploidy was found in 28 (4.8% of all chromosomal abnormality) POC (Table 1), and segmental UPD was identified in 12 (2.1% of all chromosomal abnormality) POC (Table 1). Table 1. | SNP array results | BMI < 25 kg/m2 (n = 758) | BMI ≥ 25 kg/m2 (n = 310) | Total frequency (n = 1068) | | |---|---|---|---|---| | Normal karyotype | 326 (43.0%) | 158 (51.0%) | 484 (45.3%) | | | Aneuploidy | Trisomy | 296 (39.1%) | 92 (29.7%) | 388 (40.3%) | | Monosomy | 29 (3.8%) | 13 (4.2%) | 42 (3.9%) | | | Polyploidy | 21 (2.8%) | 7 (2.3%) | 28 (2.6%) | | | Segmental aneuploidy | 45 (5.9%) | 23 (7.4%) | 68 (6.4%) | | | UPD | 8 (1.1%) | 4 (1.3%) | 12 (1.1%) | | | Mosaic | 33 (4.4%) | 13 (4.2%) | 46 (4.3%) | BMI body mass index, UPD uniparental disomy Participant characteristics The baseline characteristics are presented in Table 2. According to the SNP array results, 484 participants who miscarried normal POC were categorized into group 1, and 584 participants who miscarried chromosomal abnormal POC were categorized into group 2. All spontaneous miscarriages occurred before 14 weeks of pregnancy, and the median gestational age was 9.0 weeks (IQR, 8.6–9.7). Statistical differences between the two groups were observed in terms of maternal age, gestational age, BMI, reasons for fertility treatment, ET cycle and embryo morphology grade (Table 2). Compared to group 1, group 2 had a significantly higher proportion of POC from women older than 35 years (32.7% in group 2 versus 17.6% in group 1, p < 0.001). The mean maternal age in group 2 was also significantly older than that in group 1 (32.0 ± 4.4 versus 30.3 ± 4.2, respectively, p < 0.001) (Table 2). The median gestational age in group 2 was greater than that in group 1 (9.0 versus 8.9, respectively, p < 0.001). Group 2 had a significant less proportion of POC from women with an elevated BMI (≥ 25 kg/m2) compared to that in group 1 (26.0% versus 32.6%, p = 0.018). PCOS as the reason for fertility treatment was more frequently reported in the chromosomally normal POC group (group 1). Tubal factor was more frequently reported in chromosomally abnormal POC group (group 2). Group 2 had a significant higher proportion of POC from a fresh ET cycle compared to that in group 1 (63.0% versus 49.6%, respectively, p < 0.001). High-grade embryo morphology was more frequently reported in group 2 compared to group 1 (78.4% versus 72.1%, respectively, p = 0.017). Table 2. | Parameter | Group 1 (n = 484) | Group 2 (n = 584) | p value | |---|---|---|---| | Maternal age (years)a | 30.3 ± 4.2 | 32.0 ± 4.4 | < 0.001 | | Maternal ageb | < 0.001 | || | < 35 years | 399 (82.4%) | 393 (67.3%) | | | ≥ 35 years | 85 (17.6%) | 191 (32.7%) | | | TSH level (IUI/ml)c | 2.2 (1.6, 3.1) | 2.2 (1.5, 3.0) | 0.126 | | Gestational age (weeks)c | 8.9 (8.4, 9.4) | 9.0 (8.7, 9.9) | < 0.001 | | BMI (kg/m2)b | 0.018 | || | < 25 | 326 (67.4%) | 432 (74.0%) | | | ≥ 25 | 158 (32.6%) | 152 (26.0%) | | | Reasons for fertility treatmentb | ||| | Tubal factor | 157 (32.4%) | 251 (43.0%) | < 0.001 | | Male factor | 115 (23.8%) | 119 (20.4%) | 0.183 | | Uterine factor | 52 (10.7%) | 43 (7.4%) | 0.053 | | PCOS | 111 (22.9%) | 102 (17.5%) | 0.026 | | Multiple factors | 49 (10.1%) | 69 (11.8%) | 0.380 | | Fertilization methodb | 0.070 | || | IVF | 346 (71.5%) | 446 (76.4%) | | | ICSI | 138 (28.5%) | 138 (23.6%) | | | ET cycleb | < 0.001 | || | Fresh | 240 (49.6%) | 368 (63.0%) | | | Frozen-thawed | 244 (50.4%) | 216 (37.0%) | | | Stage of embryo developmentb | 0.170 | || | Cleavage embryo | 351 (72.5%) | 445 (76.2%) | | | Blastocyst | 133 (27.5%) | 139 (23.8%) | | | Embryo morphology gradeb | 0.017 | || | Average grade | 135 (27.9%) | 126 (21.6%) | | | High grade | 349 (72.1%) | 458 (78.4%) | Group 1: Chromosomally normal POC Group 2: Chromosomally abnormal POC BMI body mass index, TSH thyroid stimulating hormone, PCOS polycystic ovary syndrome, IVF in vitro fertilization, ICSI intracytoplasmic sperm injection, ET embryo transfer aMean ± standard deviation bCase number (%) cMedian (interquartile range) Clinical factors related to foetal chromosomal abnormalities After taking several confounders (maternal age, BMI, TSH level, gestational age, reasons for fertility treatment, fertilization method, ET cycle, stage of embryo development and embryo morphology grade) into consideration, multivariate logistic regression analysis showed that maternal age and BMI were independent predictors for foetal chromosomal abnormality (Table 3). Advanced maternal age was found to be a risk factor, and POC from women older than 35 years had a 2.098-fold increased risk of being chromosomally abnormal compared to those from women younger than 35 years (95% CI = 1.544 to 2.850, p < 0.001). In addition, the risk of miscarrying chromosomally abnormal POC increased as BMI decreased (OR = 1.424 with 95% CI = 1.074 to 1.888, p = 0.014). Compared to women with an elevated BMI (≥ 25 kg/m2), women with BMI < 25 kg/m2 had a 1.424-fold increased risk of miscarrying chromosomally abnormal POC. Table 3. | Multivariable analysis | ||| |---|---|---|---| | Adjusted odds ratio (95% CI) | p value | || | Maternal age (years) | < 35 | 1 | – | | ≥ 35 | 2.098 (1.544, 2.850) | < 0.001 | | | TSH level (IUI/ml) | 0.938 (0.846, 1.040) | 0.225 | | | Gestational age (weeks) | 1.029 (0.937, 1.130) | 0.552 | | | BMI (kg/m2) | ≥ 25 | 1 | – | | < 25 | 1.424 (1.074, 1.888) | 0.014 | | | Fertilization method | IVF | 1 | – | | ICSI | 0.739 (0.537, 1.019) | 0.065 | | | Reasons for fertility treatment | Male factor | 1 | – | | Tubal factor | 1.242 (0.856, 1.804) | 0.254 | | | Uterine factor | 0.692 (0.411, 1.164) | 0.165 | | | PCOS | 0.933 (0.614, 1.418) | 0.745 | | | Multiple factors | 1.006 (0.617, 1.640) | 0.981 | | | Stage of embryo development | Cleavage stage embryo | 1 | – | | Blastocyst | 1.078 (0.752, 1.546) | 0.682 | | | Embryo morphology grade | High grade | 1 | – | | Average grade | 0.699 (0.486, 1.004) | 0.052 | | | Embryo transfer cycle | Frozen-thawed | 1 | – | | Fresh | 1.550 (1.198, 2.008) | 0.001 | CI confidence interval, PCOS polycystic ovary syndrome, IVF in vitro fertilization, ICSI intracytoplasmic sperm injection, TSH thyroid stimulating hormone, BMI body mass index

Genetic testing on POC provides valuable information on the cause of miscarriage. This study performed a SNP array-based whole-genome screening of POC and tried to determine the relationship between foetal chromosomal abnormality and elevated BMI-related miscarriage. The results showed a decreased level of chromosomally abnormal POC rate in women with an elevated BMI (≥ 25 kg/m2). After adjusting for possible confounders, multivariate logistic regression analysis indicated that women with an elevated BMI (≥ 25 kg/m2) were more likely to miscarry chromosomally normal POC, suggesting foetal chromosomal abnormality was probably not be the main cause of elevated BMI-related miscarriage. Aneuploidy is the most common chromosomal abnormality type for miscarriage [26]. However, previous studies failed to determine the role of aneuploidy in elevated BMI (≥ 25 kg/m2)-related miscarriage [4, 6, 10, 29]. Although different karyotyping techniques were used in different studies (traditional karyotyping technique in Kroon et al. [6], Landres et al. [10] and Boots et al. [4], hysteroembryoscopy in Bellver et al. [29]), the conclusions were consistent, and they all determined that women with an elevated BMI (≥ 25 kg/m2) had a lower rate of aneuploidy POC compared to women with normal BMI. In this study, we analysed the aneuploidy rate with a higher-resolution karyotyping technique and found that POC from women with an elevated BMI (≥ 25 kg/m2) had a significantly lower aneuploidy rate than those from women with normal BMI (33.9% versus 42.6%, respectively, p = 0.009). Therefore, the results suggested that aneuploidy may not be the main reason for elevated BMI (≥ 25 kg/m2)-related miscarriage. Apart from aneuploidy, other chromosomal abnormality types, such as polyploidy and segmental aneuploidy, were also found to be associated with miscarriage. Increasing data suggested that CNVs detected in euploid miscarriage may aid in detecting a relevant recessive mutation. This may be in a relevant gene or dosage-sensitive genes, where the loss or gain of copies affects the gene function [13]. Our findings detected 83 CNVs in 68 POC, including 34 CNVs in POC from women with an elevated BMI (≥ 25 kg/m2) and 49 CNVs from women with normal BMI. Of these CNVs, 42.2% (35/83 CNVs) were ≥ 10 Mb in size and were considered to be the specific causative for 27 miscarriages. A total of 42.2% CNVs (35/83CNVs) with a reported submicroscopic abnormality (< 10 Mb) in size were identified as clinically significant for 30 miscarriages. Except for segmental aneuploidy, polyploidy was identified in 28 POC, including 7 (25%) from women with an elevated BMI (≥ 25 kg/m2) and 18 (64.3%) from women with normal BMI. However, neither the distribution of polyploidy nor segmental aneuploidy was found to be significantly different among BMI groups. After considering all types of chromosomal abnormalities, our findings indicated that women with an elevated BMI (≥ 25 kg/m2) had fewer chromosomally abnormal POC than women of normal weight. This finding supports the hypothesis that miscarriage in women with an elevated BMI (≥ 25 kg/m2) may be caused by factors other than foetal chromosomal abnormality, such as differences in endometrium receptivity. Several studies analysing oocyte donor cycles have suggested that endometrium receptivity/environment is the main possible pathological mechanism for the increased incidence of BMI-related miscarriage. A Spanish study included 9587 first oocyte donor cycles and observed statistically significant decreases in implantation rate, clinical pregnancy rate and live-birth rate as BMI increased [30]. Furthermore, a large-scale study (22,317 donor/recipient cycles) observed trends towards lower implantation rate and clinical pregnancy rate and a higher rate of pregnancy loss with increasing BMI [31]. A meta-analysis including 16 studies (more than 16,000 pregnancies) concluded that women with a BMI ≥ 25 kg/m2 were more likely to miscarry even after oocyte donation (OR = 1.52, 95% CI = 1.10–2.09, p < 0.05), indicating a negative effect of increased BMI on endometrial receptivity [32]. Other than elevated BMI (≥ 25 kg/m2) and advanced maternal age-related miscarriage, the present study also provided information on other clinical characteristics related to miscarriage. The first one is fresh ET cycle. Several studies compared the pregnancy outcome between fresh and frozen-thawed ET cycles and observed an increased pregnancy rate in women who underwent frozen-thawed ET cycle [33–35]. There are two possible mechanisms suggested by most studies: one is endometrial advancement induced by ovarian stimulation in a fresh ET cycle, resulting in embryo-endometrium asynchrony; the other is that cryopreservation preferentially eliminates nonviable embryos [35]. Our results showed that the risk of foetal chromosomal abnormality increased 1.550-fold in a fresh ET cycle compared to a frozen-thawed cycle (OR = 1.550, 95% CI: 1.198–2.008, p = 0.001), suggesting that cryopreservation may preferentially eliminate chromosomal abnormal embryos. Furthermore, embryo morphological parameters are major predictors for the success of in vitro fertilization treatment [36]. However, it is still controversial whether embryo morphology relates to the embryo chromosomal constitution [37–39]. The present study found a significantly higher rate of chromosomally abnormal POC in high-grade morphology embryo transfer compared to average-grade embryo transfer (78.4% versus 21.6%, p = 0.017), suggesting that chromosomal abnormality might have no detectable effect on embryo morphology. This study had two main advantages. One is that the accuracy of SNP array in detecting chromosomal abnormality provided true information on POC to be analysed. The other is that spontaneous miscarriage is a complicated disease and has been reported to be associated with several women’s baseline characteristics. The present study included many possible predefined factors and used a multivariable logistic regression model to test and adjust confounders, allowing an accurate relationship between elevated BMI (≥ 25 kg/m2) and foetal chromosomal abnormality to be assessed. Unfortunately, as a retrospective study, we failed to collect all the paternal data, such as age, which has also been reported as a possible risk factor for spontaneous miscarriage [40–42]. This is a limitation of our study.

In summary, this project is the largest of its kind to explore the link between foetal chromosomal abnormality and the increased incidence of BMI-related miscarriage. A high-resolution genetic testing technique was used in this study, providing accurate information on BMI-related miscarriage. After adjusting for several confounding factors, multiple logistic regression analysis showed that women with an elevated BMI (≥ 25 kg/m2) were more likely to have a chromosomally normal miscarriage, suggesting that alternative factors predispose this population to miscarriage. Electronic supplementary material Acknowledgments We thank all infertile couples for their positive participation and cooperation. Funding information This study was funded by the National Natural Science Foundation (31271605). Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest. Footnotes Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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