Association of body mass index with embryonic aneuploidy

ORIGINAL ARTICLES: ENVIRONMENT AND EPIDEMIOLOGY

Association of body mass index with embryonic aneuploidy Kara N. Goldman, M.D., Brooke Hodes-Wertz, M.D., M.P.H., David H. McCulloh, Ph.D., Julie D. Flom, M.P.H., and Jamie A. Grifo, M.D., Ph.D. New York University Fertility Center, New York University Langone Medical Center, New York, New York

Objective: To determine whether an association exists between body mass index (BMI) and embryo ploidy in patients undergoing in vitro fertilization (IVF) with trophectoderm biopsy and 24-chromosome preimplantation genetic screening (PGS). Design: Retrospective cohort study. Setting: University-based fertility center. Patient(s): 279 women aged 20–45 years with documented height and weight from the day of oocyte retrieval who underwent 24-chromosome PGS between 2010 and 2013. Intervention(s): None. Main Outcome Measure(s): Primary outcomes: number and percentage of euploid embryos. Result(s): Patients were grouped by World Health Organization (WHO) BMI class: underweight (<18.5, n ¼ 11), normal weight (18.5–24.9, n ¼ 196), overweight (25–29.9, n ¼ 50), and obese (R30, n ¼ 22). Groups were similar by age (mean  standard error of the mean: 37.5  1.2 to 39.2  0.9), ovarian reserve, and IVF cycle parameters. There was no difference in the number or percentage of euploid embryos by BMI category (<18.5: 27.6%  8.5; 18.5–24.9: 34.5%  2.2; 25–29.9: 32.1%  4.3; R30: 30.9%  7.3). Age was inversely related to euploidy, but adjusted multivariate regression models failed to demonstrate a statistically significant relationship between BMI and euploidy in underweight (adjusted odds ratio [AOR] 0.44; 95% confidence interval [CI], 0.09–2.10), overweight (AOR 0.90; 95% CI, 0.43–2.00), or obese (AOR 0.74; 95% CI, 0.25–2.20) patients compared with the normal-weight reference group. Conclusion(s): No statistically significant relationship was identified between BMI and euploidy in an otherwise homogenous cohort of patients undergoing IVF with PGS, suggesting that the negative impact of overweight and obesity on IVF and reproductive outcomes may not be related to aneuploidy. (Fertil SterilÒ Use your smartphone 2015;103:744–8. Ó2015 by American Society for Reproductive Medicine.) to scan this QR code Key Words: Aneuploidy, body mass index (BMI), obesity, 24-chromosome preimplantation and connect to the genetic screening Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/goldmank-bmi-embryonic-aneuploidy/

T

he prevalence of obesity has increased to epidemic proportions over the past decades (1, 2). Obesity has become one of the leading causes of preventable death in the United States (3) and contributes to significant morbidity, including cardiovascular and cerebrovascular disease, diabetes, sleep apnea, arthritis, and cancer (4–6). The endocrine and metabolic alterations in obese women contribute to an

increased risk of subfecundity and infertility, pregnancy loss, poor in vitro fertilization (IVF) outcomes, and obstetric complications (7–13). The etiology for impaired reproductive outcomes in obesity remains unclear, and maternal effects may be secondary to abnormalities in the uterine environment, oocyte quality, or embryonic development. A large retrospective analysis of 9,587 oocyte donation cycles with oocytes from

Received July 23, 2014; revised and accepted November 21, 2014; published online January 7, 2015. K.N.G. has nothing to disclose. B.H.-W. has nothing to disclose. D.H.M. has nothing to disclose. J.D.F. has nothing to disclose. J.A.G. has nothing to disclose. Reprint requests: Kara N. Goldman, M.D., New York University Fertility Center, 660 First Avenue, Fifth Floor, New York, New York 10016 (E-mail: [email protected]). Fertility and Sterility® Vol. 103, No. 3, March 2015 0015-0282/$36.00 Copyright ©2015 Published by Elsevier Inc. on behalf of the American Society for Reproductive Medicine http://dx.doi.org/10.1016/j.fertnstert.2014.11.029 744

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normal-weight donors demonstrated a significant impairment in implantation and live-birth rates with increasing recipient body mass index (BMI), suggesting that the uterine environment rather than the oocyte may be responsible for poor reproductive outcomes in obese women (14). However, compelling data suggest that the detrimental effects of obesity are eliminated when donor oocytes are used, suggesting a more important role for the oocyte rather than the maternal environment. A systematic review and metaanalysis of obese recipients of donor oocytes suggests that obesity does not affect IVF outcomes when donor oocytes are used, suggesting that oocyte quality is the most critical factor affecting IVF outcomes (15). VOL. 103 NO. 3 / MARCH 2015

Fertility and Sterility® Analysis of follicular fluid in obese women has demonstrated a correlation between abnormal metabolites in both the follicular fluid and serum of obese women, leading to logical speculation that these metabolic changes could contribute to altered oocyte quality (16, 17). In mouse models, poor pregnancy outcomes in diabetic mice have been found to result from a maternal effect on oocytes and not the diabetic uterine environment, and oocytes from obese mice show abnormal or delayed meiotic maturation and increased follicular apoptosis (18–20). Oocytes from mice fed a high-fat diet demonstrated spindle defects, chromosome misalignments, and a higher incidence of aneuploidy (19). We theorized that the impact of obesity on oocyte quality could involve alterations in normal mitotic checkpoints and abnormalities in cell division thereby leading to aneuploidy. We determined whether an association exists between obesity as defined by BMI and aneuploidy assessed by trophectoderm biopsy and array comparative genomic hybridization (aCGH) in a population of patients undergoing IVF with 24-chromosome preimplantation genetic screening (PGS).

MATERIALS AND METHODS We performed a retrospective cohort study of patients undergoing IVF with blastocyst culture, trophectoderm biopsy, and aCGH for 24-chromosome preimplantation genetic screening at the Fertility Center at New York University Langone Medical Center. Approval was obtained from the institutional review board of the New York University School of Medicine (study no. S13-00389). Patients were included if they underwent IVF with trophectoderm biopsy/aCGH between October 2010 and April 2013, were between ages 20 and 45 years, and had height and weight documented on the day of oocyte retrieval. Patients using donor oocytes were excluded, and only each patient's first cycle of PGS was included. Cycle parameters analyzed included patient age at the time of oocyte retrieval/biopsy, day-2 follicle-stimulating hormone (FSH) and estradiol (E2) levels, the number of oocytes and metaphase-II (MII) oocytes retrieved, the number of two pronuclear (2PN) zygotes, the number of blastocysts biopsied, and the number of aneuploid and euploid embryos in each patient's cohort.

Ovarian Stimulation Before the initiation of treatment, menstrual day-2 serum E2 and FSH levels were assessed. Patients with acceptable parameters (E2 <75 and FSH <13.5) underwent controlled ovarian hyperstimulation using injectable gonadotropins (Follitropin beta; Schering Plough; Serono Pharmaceuticals; or menotropins, Ferring Pharmaceuticals), with LH suppression achieved using either a gonadotropin-releasing hormone agonist (leuprolide acetate; TAP Pharmaceuticals) or antagonist (ganirelix acetate, Organon; cetrorelix, Serono). Oocyte maturation was induced when R2 follicles reached R17 mm in diameter; ultrasound-guided transvaginal oocyte retrieval was performed 34 to 36 hours later. VOL. 103 NO. 3 / MARCH 2015

24-Chromosome Preimplantation Genetic Screening Laser-assisted breaching of the zona pellucida was performed on day 3 (Cronus; Research Instruments). The embryos were assessed on days 5 and 6, and fully differentiated goodquality blastocysts were biopsied. Trophectoderm cells extruding from the expanded blastocyst were gently pulled using suction, and laser-assisted biopsy was performed. The biopsied trophectoderm cells were loaded into polymerase chain reaction tubes and were sent to the reference laboratory for 24-chromosome analysis using aCGH, as previously described elsewhere (21, 22). After the biopsy, blastocysts were vitrified to be replaced in subsequent frozen cycles.

Data Analysis Univariate analyses were performed using analysis of variance (ANOVA) and chi-square analysis, where appropriate. Data were log-transformed before ANOVA to correct for right-skew and heterogeneity of variance for the following variables: number of MII oocytes, number of 2PN embryos, and number of blastocysts biopsied. Chi-square analysis was applied for the number of euploid embryos, and the patients with R4 euploid embryos were consolidated into one group. Multiple logistic regression and Poisson regression were performed adjusting for age and FSH. Analyses were performed using GraphPad (GraphPad Software) and SAS (v9.2; SAS Institute) software. Data are presented in mean  standard error of the mean (SEM) or percentage.

RESULTS For 279 women who underwent their first cycle of IVF with trophectoderm biopsy/aCGH between October 2010 and April 2013, the height and weight measurements were recorded on the day of oocyte retrieval. The patients pursued PGS for multiple indications, with the most common reasons being multiple IVF failures and advanced maternal age. Patients were divided into four groups by World Health Organization (WHO) BMI class: underweight (<18.5, n ¼ 11), normal weight (18.5–24.9, n ¼ 196), overweight (25–29.9, n ¼ 50), and obese (R30, n ¼ 22). There were no statistically significant differences between BMI groups by age, day-2 FSH and estradiol, number of oocytes and MII oocytes retrieved, number of 2PN embryos, or number of blastocysts biopsied (Table 1). Notably, there were no differences between the groups when comparing the primary outcome of number of euploid embryos (BMI <18.5: 2.5  0.9; BMI 18.5–24.9: 2.3  0.2; BMI 25–29.9: 1.8  0.3; BMI R30: 1.5  0.4; P>.05). The patients in the normal BMI group had the highest percentage of euploid embryos, with a mean euploidy rate of 34.5%  2.2%; but there was no statistically significant difference between BMI groups when comparing the percentage of euploid embryos in underweight patients (BMI <18.5: 27.6%  8.5%), overweight patients (BMI 25–29.9: 32.1%  4.3%), and obese patients (BMI R30: 30.9%  7.3%) (P>.05) (Fig. 1). Age was inversely related to euploidy in multivariate regression models, but age-adjusted and FSH-adjusted 745

ORIGINAL ARTICLE: ENVIRONMENT AND EPIDEMIOLOGY

TABLE 1 Patient and cycle characteristics. BMI Characteristic

<18.5 (n [ 11)

18.5–24.9 (n [ 196)

25–29.9 (n [ 50)

‡30 (n [ 22)

37.5  1.2 6.7  0.9 43  5.3 17.8  3.2 13.2 (5–34) 10.7 (4.5–26) 5.3 (1.3–22)

37.6  0.3 6.2  0.2 46 1.7 15.7  0.6 11.3 (3–39.6) 8.5 (2–32) 4.4 (1–19.7)

38.3  0.6 5.6  0.3 43  3.6 15.1  1.1 11 (4–31) 8.5 (3–24) 3.6 (0.8–17)

39.2  0.9 5.3  0.6 50  6.1 15.2  1.3 11.5 (4.8–27.6) 8.5 (3–23) 3.8 (0.9–16.4)

a

Age (y) FSH on day 2 (IU/L)a Estradiol on day 2 (pg/mL)a No. of oocytes retrieveda No. of MII oocytesb No. of 2PN embryosb No. of blastocysts biopsiedb

Note: BMI ¼ body mass index; FSH ¼ follicle-stimulating hormone; MII ¼ metaphase II; 2PN ¼ two pronuclear. a Mean  standard error of the mean; one-way analysis of variance (ANOVA), P>.05. b Mean (95% confidence interval). Data were log-transformed before ANOVA to correct for right-skew and heterogeneity of variance, P>.05. Goldman. Obesity and aneuploidy. Fertil Steril 2015.

models demonstrated no statistically significant association between BMI and euploidy (Fig. 2). Multivariate logistic regression failed to show a statistically significant relationship between BMI and euploidy in underweight (BMI <18.5: adjusted odds ratio [AOR] 0.44; 95% confidence interval [CI], 0.09–2.10), overweight (BMI 25–29.9: AOR 0.9; 95% CI, 0.43–2.00), and obese patients (BMI R30: AOR 0.74; 95% CI, 0.25–2.20) compared with the normal-weight reference group. Adjusted Poisson regression was applied for the variable ‘‘number of euploid embryos,’’ and again no statistically significant relationship was identified between BMI and euploidy in underweight (BMI<18.5: 0.94; 95% CI,

Number of euploid embryos

FIGURE 1 3.5 3.0 2.5 2.0 1.5

DISCUSSION The negative consequences of obesity are identifiable in every organ system, and obesity plays a significant role in reproductive function (12). Obese women experience inferior outcomes in all aspects of reproduction from conception to delivery, and are at a significantly greater risk for infertility, miscarriage, poor response to assisted reproduction, and increased morbidity throughout the pregnancy and the postpartum periods (12, 13). The relationship between obesity and poor reproductive outcomes is clear, but the etiology remains unknown. Compelling data suggest that obesity impairs oocyte quality, with the effect of obesity overcome when donor oocytes are used in obese women (15). Theorizing that oocytes from obese women are subject to alterations in normal mitotic checkpoints and abnormalities in cell division leading to aneuploidy, we sought to determine whether an association exists between obesity as measured by elevated BMI and

1.0 0.5 0.0

2.5

2.3

1.8

18.5-24.9 25-29.9 WHO BMI (kg/m2) class

1.5

40% 30% 20% 10% 0%

FIGURE 2

≥30 Percentage (%) euploid embryos

<18.5

Percentage euploid embryos

0.64–1.40), overweight (BMI 25–29.9: 0.94; 95% CI, 0.75–1.20), and obese patients (BMI R30: 0.76; 95% CI, 0.50–1.10) compared with the reference group.

51

47.1

38.2

40.7

36.8

32.8

33 32.8 25.1

25

18.8

0

27.6

34.5

32.1

30.9 WHO BMI (kg/m2) class

<18.5

18.5-24.9 25-29.9 WHO BMI (kg/m2) class

≥30

* Age is inversely related to euploidy in multivariate regression. Age and FSH-adjusted models demonstrate no significant association between BMI and euploidy.

Number and percentage (%) of euploid embryos by WHO BMI class.

Age-stratified percentage (%) of euploid embryos by WHO BMI class.

Goldman. Obesity and aneuploidy. Fertil Steril 2015.

Goldman. Obesity and aneuploidy. Fertil Steril 2015.

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Fertility and Sterility® aneuploidy as assessed by 24-chromosome embryonic aneuploidy screening. Despite obvious detectable effects on efficiency of reproduction, we found no association between obesity and the number of euploid embryos or the percentage of embryos that were euploid. Our results suggest that the negative impact of being overweight or obese on reproductive outcomes may not be related to embryonic aneuploidy. Age is undisputedly the most important prognostic factor related to embryo ploidy (23), and ovarian reserve has been inconsistently correlated with chromosomal competence (24–26). To account for the universally accepted confounder of age and the potential confounder of ovarian reserve, our models adjusted for age and day-2 FSH. Other aspects of ovarian stimulation and response to medications have not been shown to impact ploidy and therefore were not adjusted for in our regression models. Poor reproductive outcomes have been documented in obese women independent of method of conception (natural, ovulation induction, or in vitro fertilization/intracytoplasmic sperm injection) (8). Data from multiple large trials unequivocally demonstrate lower IVF pregnancy rates in obese women (27). A large retrospective cohort study of 4,609 patients undergoing a first cycle of IVF demonstrated a statistically significantly diminished odds of implantation, clinical pregnancy, and live birth in women with a BMI R30, and the odds of live birth progressively declined with increasing severity of obesity (28). A cohort study of 152,500 ART cycles similarly reported increased rates of cycle cancellation, low response, treatment failure, and pregnancy failure with increasing BMI (10). An analysis of 45,163 embryo transfers identified through the Society for Assisted Reproductive Technologies (SART) database demonstrated that increasing severity of obesity was associated with decreasing odds of achieving a clinical pregnancy using autologous oocytes, but no difference was noted when donor oocytes were used (11). In a systematic review and meta-analysis examining the effect of being overweight or obese on ART outcomes, even BMI R25 was associated with an increased gonadotropin requirement during ovarian stimulation, decreased pregnancy rates, and a higher miscarriage rate (9). The association of obesity with miscarriage in spontaneous conceptions was further delineated in a systematic review of six studies including a cohort of 28,538 women. Analysis of pooled data demonstrated a higher miscarriage rate in obese versus normal-BMI women (7). In an analysis of tissue karyotypes from women with first-trimester miscarriages, overweight and obese women aged <35 years were actually less likely to have aneuploid miscarriages than women in a healthy weight range, suggesting that miscarriages in young overweight/obese women may be due to factors other than aneuploidy (26). However, the women in that study conceived by various modes of conception, ranging from natural conception to all forms of assisted reproduction, thus adding significant confounders. Notably, the only women included were those having a dilation and curettage (D&C) after miscarriage, thus missing the cohort of women with subclinical pregnancy loss due to aneuploidy, those who miscarried spontaneously, those who were treated medically, and women who underwent dilation and curettage without karyotyping (26). VOL. 103 NO. 3 / MARCH 2015

Given the strong association between obesity and miscarriage and the paucity of data on the association between BMI and embryo ploidy status, we sought to explore a plausible connection between obesity, oocyte competence, and embryonic aneuploidy. Using data from patients pursuing 24-chromosome aneuploidy screening represents an innovative means to understand the effects of obesity on aneuploidy, particularly considering that 30% to 60% of all conceptions spontaneously abort within the first 12 weeks of gestation (29). A large proportion of early losses go unrecognized, and significant loss occurs even before the first missed menses (29, 30). Many of these early losses are likely secondary to aneuploidy, but by definition after a subclinical loss it would be impossible to assess the ploidy status of the conceptus. Examining embryo ploidy at the blastocyst stage sheds light on the relationship between BMI and aneuploidy before the expected attrition of aneuploid embryos occurs via subclinical loss or early clinical pregnancy loss. We acknowledge that aneuploidy-related attrition likely also occurs before the blastocyst stage, and this attrition would certainly bias our results. However, in our study there was no difference in fertilization rate or blastocyst formation rate by BMI category, suggesting that the there was no difference in early loss before the blastocyst stage between the BMI groups. Understanding that ovarian stimulation results in statistically significant weight change during an ovarian stimulation cycle (31, 32), we analyzed BMI data from the day of oocyte retrieval to provide the most accurate reflection of the environment to which the oocyte was exposed. The absolute weight change experienced during ovarian stimulation is minimal, ranging from a weight loss of 2.6 kg in obese women to a weight gain of 2.5 kg in underweight women (32). These small absolute changes, coupled with the fact that the height portion of the BMI measurement remains static, suggests that the weight change experienced during ovarian stimulation would likely have negligible effects on a patient's BMI category and therefore would not have affected our results. Limited data exist on the relationship between BMI and aneuploidy, and our data contribute to the growing understanding of the impact of obesity on reproductive outcomes. This study is limited by the relatively small number of overweight and obese patients in our cohort, reflecting an overall healthier population in the center and region that we serve. Our study was also limited by its retrospective nature. The study question would best be addressed by a multicenter, prospective trial, particularly in centers with a higher proportion of obese patients. Selection bias also presents a limitation, as patients seeking PGS in our clinic often have a history of recurrent IVF failure or recurrent pregnancy loss. Our patient population presenting for PGS may be inherently more prone to aneuploidy than the average IVF patient, thereby limiting external validity. To address the possibility that being offered PGS could be related to BMI, potentially contributing to selection bias, we compared the BMI distribution of our study population to patients undergoing IVF at our center from 2003 to 2012. The BMI distribution was nearly identical between our study population and the representative IVF group (mean and 747

ORIGINAL ARTICLE: ENVIRONMENT AND EPIDEMIOLOGY standard deviation: 23.5  4.1 vs. 23.8  4.7, P¼ .3; median 22.7 vs. 22.7), suggesting that our study population is similar to our center's patient population. Our results demonstrated no relationship between BMI and the number of euploid embryos or percentage of euploid embryos in this otherwise homogenous cohort of patients undergoing IVF with 24-chromosome PGS, suggesting that the negative impact of being overweight or obese on IVF and reproductive outcomes may be related to factors other than embryonic aneuploidy. These findings underscore the need for future research in this field.

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