Abstract

STUDY QUESTION

Is the cytoskeletal and chromosomal organization of failed fertilized oocytes from severely obese patients (BMI ≥ 35 kg/m2) altered compared with that in patients with normal BMI (BMI 18.5–24.9 kg/m2)?

SUMMARY ANSWER

Compared with normal BMI patients, severe obesity was associated with a greater prevalence of spindle anomalies and non-aligned chromosomes in failed fertilized oocytes.

WHAT IS KNOWN AND WHAT THIS PAPER ADDS

Obesity is associated with poor reproductive outcomes, but little is known regarding the underlying mechanisms. To address potential mechanisms, our study compared the cytoskeletal and chromosome organization in failed fertilized oocytes from severely obese and normal BMI patients.

DESIGN

The study population was drawn from IVF patients treated in a hospital-based infertility clinic between February 2010 and July 2011. The prevalence of meiotic spindle and chromosome alignment anomalies in failed fertilized oocytes from patients with severe obesity (i.e. Class II and III; BMI 35.0–50.1 kg/m2) was compared with those from patients with normal BMI (BMI 18.5–24.9 kg/m2). Oocytes were fixed and then labeled for tubulin, actin and chromatin. Spindle number and integrity, as well as chromosome alignment, were assessed using immunofluorescence microscopy and, in some cases, confocal microscopy. Generalized estimating equations were applied, which account for the correlation among oocytes from the same patient to estimate odds ratio (OR), 95% confidence intervals (CIs) and two-sided Wald P-values. Models were adjusted for continuous age at cycle start, cycle type (IVF or ICSI) and polycystic ovarian syndrome (PCOS) a priori.

PARTICIPANTS AND SETTING

University-affiliated infertility clinic. A total of 276 oocytes that failed to fertilize from 137 patients were evaluated: 105 oocytes from severely obese women (n = 47) and 171 oocytes from normal BMI patients (n = 90).

MAIN RESULTS AND THE ROLE OF CHANCE

(i) Significantly more oocytes from the severely obese group exhibited two spindles compared with those from the normal BMI group (58.9 versus 35.1%; OR = 2.68, CI = 1.39–5.15, P-value = 0.003).

(ii) Among oocytes with a single spindle, those from severely obese patients showed a significantly higher prevalence of disarranged spindles with non-aligned chromosomes compared with those from normal BMI patients (28.6 versus 8.6%; OR = 4.58, CI = 1.05–19.86, P-value = 0.04).

BIAS, CONFOUNDING AND OTHER REASONS FOR CAUTION

Inclusion of only failed fertilized oocytes, small sample size, unknown factors such as non-PCOS comorbidity.

GENERALIZABILITY TO OTHER POPULATIONS

For this study, by design, it is unclear whether the findings are generalizable to successfully fertilized oocytes, and whether this oocyte-level influence of obesity is generalizable to infertile women who do not undergo stimulation or, more broadly, to spontaneous conceptions in fertile women.

STUDY FUNDING/COMPETING INTEREST(S)

none.

TRIAL REGISTRATION NUMBER

n/a.

Introduction

The prevalence of obesity in developed countries has been increasing during recent decades, and in 2008 exceeded more than 30% of the population in the USA (Flegal et al., 2010). Believed to be associated with a variety of chronic diseases (Malnick and Knobler, 2006; Flegal et al., 2007), obesity is also correlated with reproductive pathology including anovulation, infertility and obstetrical complications (Rich-Edwards et al., 1994; Pasquali et al., 2003; Dokras et al., 2006; Bhattacharya et al., 2007; Knight et al., 2010). Obese women are more likely to report infertility and reduced fecundity than women with normal BMI (Rich-Edwards et al., 1994; Gesink Law et al., 2007). Obesity has been associated with higher rates of early miscarriages (Fedorcsák et al., 2000; Lashen et al., 2004), as well as poor IVF outcomes (Luke et al., 2011; Shah et al., 2011). While little is known regarding the underlying mechanisms for pregnancy failure, impaired endometrial receptivity or compromised oocyte quality have been proposed (Pasquali et al., 2003; Robker, 2008).

Obesity is associated with alterations in hormone production and metabolism related to folliculogenesis (Yen et al. 1970; Bützow et al., 2000; reviewed by Pasquali and Gambineri, 2006; Brewer and Balen, 2010). Recent studies reported an increase in cytokine production in follicular fluid from obese women (La Vignera et al., 2011; reviewed by Robker et al., 2011) as well as higher levels of C reactive protein, triglycerides and free fatty acids (Robker et al., 2009, 2011). Increased lipid levels may lead to oxidative and endoplasmic reticulum stress, as well as to the production of reactive oxygen species which result in mitochondrial and cellular dysfunctions (reviewed by Schrauwen and Hesselink, 2004; Malhotra and Kaufman, 2007; Robker et al., 2011). As the ovary is thought to have a finite pool of oocytes, any adverse effect of obesity on the follicle and, therefore, the oocyte is of significant concern (Purcell and Moley, 2011).

Low pregnancy rates and increased early pregnancy loss are often indicative of abnormalities in the embryo. These very earliest stages of embryo growth are primarily controlled by the quality of the oocyte (reviewed by Gosden and Lee, 2010). Previous animal studies have demonstrated an association between high BMI and alterations in oocyte development (Minge et al., 2008; Igosheva et al., 2010; Jungheim et al., 2010). In the human, a few retrospective studies investigated the effect of obesity on oocyte quality (assessed by the number of mature oocytes retrieved and the number of oocytes fertilized) and embryo quality (based on mean embryo grade, number of embryos transferred and the number of embryos frozen), but the results have been inconsistent (Dechaud et al., 2006; Metwally et al., 2007; Sneed et al., 2008; Bellver et al., 2010). Moreover, a recent study reported a correlation between gross morphological abnormalities in oocytes and BMI (Depalo et al., 2011).

Development of the haploid oocyte is dependent upon normal formation of the meiotic spindle which, in turn, controls precise chromosomal segregation. Integrity of the meiotic spindle is, therefore, crucial for normal cell cycle progression. Given constraints of working with human oocytes, only discarded material can be evaluated. As fertilized and failed to fertilize oocytes are derived from the same cohort, the failed fertilized oocytes, that are clinically useless and otherwise discarded, can be investigated to define types of abnormalities that may provide insight into quality of the whole oocyte cohort. The goal of the present study was to assess cytoskeletal and chromosome organization of failed fertilized oocytes from severely obese patients compared with patients with normal BMI. The rationale for this study was that by comparing oocytes from these two populations, we may be able to identify oocyte-related deficiencies which, in turn, might explain the poor reproductive performance of the severely obese population.

Material and Methods

Source of oocytes

The study was approved by the Institutional Review Board of Partner's Healthcare. Failed fertilized oocytes were defined as those not exhibiting any pronucleus at the fertilization check (16–18 h post-insemination or ICSI). These otherwise discarded oocytes were obtained from women undergoing IVF (with or without ICSI) between February 2010 and July 2011, honoring patients' signed informed consent for use of their discarded materials for research. After insemination/ICSI, only oocytes exhibiting one polar body (presumed MII according to gross morphological appearance) were analyzed. Oocytes from patients with normal BMI (18.5–24.9 kg/m2) and from severely obese patients (i.e. Class II and III obesity: 35.0–50.1 kg/m2) were included. These cut-points were defined per the World Health Organization BMI classification (http://apps.who.int/bmi/index.jsp?introPage=intro_3.html). BMI was calculated using height and weight measurements taken in the clinic. Exclusion criteria were patients with recurrent abortions or known translocations. In order to avoid the influence of hierarchical clustering as a result of multiple cohorts of oocytes from multiple IVF cycles from a single individual, oocytes from only the first cycle performed during the study period for each patient were included.

Processing of oocytes for immunofluorescence analysis

Failed fertilized oocytes were fixed within 3h post-fertilization check, 24.0 ± 1.1 h (mean ± SD) after retrieval (range 21–27 h), with no difference regarding the mean time from retrieval to fixation between the two groups. Fixation was performed at 37°C using a microtubule stabilizing buffer containing 2% formaldehyde and 0.1% Triton X-100, 1 µmol/I taxol, 10 IU/ml aprotinin and 50% deuterium oxide (Combelles et al., 2002, 2003). Samples were then washed and stored at 4°C in a blocking solution of phosphate-buffered saline (PBS) containing 2% bovine serum albumin, 2% normal donkey serum, 0.1 mol/l glycine and 0.01% Triton X-100 containing 0.2% sodium azide (PBS blocking solution) until processing.

Cytoskeletal analysis of the failed fertilized oocytes was undertaken by assessment of tubulin, actin and chromatin using immunofluorescence microscopy. For labeling, fixed oocytes were incubated overnight at 4°C with a mixture of 5 μg/ml monoclonal anti-α-tubulin and anti-β-tubulin (Sigma Biosciences, USA). Following three 15 min washes in PBS blocking solution, oocytes were exposed for 2 h at 37°C PBS blocking solution Alexa Fluor 488-labeled goat anti-mouse IgG at 2.5 μg/ml (Invitrogen, USA). To visualize filamentous actin (f-actin), rhodamine-conjugated phalloidin (10 units/ml, Invitrogen, USA) was included in the secondary antibody reagent. After further washing, oocytes were incubated with 3 μg/ml of 4(6-diamidino-2-phenylindole) (DAPI, Sigma-Aldrich, St Louis, MO, USA) in PBS blocking solution for 1 h at 37°C. To determine the origin of the spindles (maternal or paternal), oocytes with >1 spindle were further labeled with acetylated α-tubulin, thereby permitting the identification of sperm tails with more certainty than if labeling with tubulin alone. Oocytes were relabeled and incubated overnight at 4°C with monoclonal anti-acetylated α-tubulin (Sigma Biosciences, USA; 2.5 μg/ml). Following three 15 min washes in PBS blocking solution, oocytes were incubated for 3 h with anti-β-tubulin (Sigma Biosciences, USA) at 5 μg/ml in PBS blocking solution, and then exposed to purified Alexa Fluor 488-labeled goat anti-mouse IgG, rhodamine-conjugated phalloidin and DAPI as described above. Oocytes were mounted uncompressed in an anti-fading reagent (Vectashield mounting medium, Vector Labs., USA). Labeled oocytes were visualized using a Nikon inverted microscope ECLIPSE Ti-E, equipped with fluorescence. Images were acquired for each oocyte using the Nikon Digital Sight Qi1 camera system (Nikon, Japan) and the NIS Elements AR image analysis software (Version 3.10, Nikon, Japan). In cases when the number of spindles and/or presence of a sperm tail could not be determined, oocytes were re-evaluated using confocal laser microscopy (Zeiss LSM 510 META, Jena, Germany).

Classification of failed fertilized oocytes

The oocytes were classified in a blinded fashion with the microscopist not knowing from which group the oocytes were derived. Oocytes were classified as those that were either activated or not activated. Activated oocytes were defined as having a network of polymerized tubulin throughout the cytoplasm or pronuclear chromatin structure. Non-activated oocytes were assessed for the absence or presence of at least one spindle. Non-activated oocytes without a spindle were defined as oocytes without polymerized tubulin or a spindle structure. Oocytes with spindles were stratified into those having a single spindle, >1 spindle or an accordion-shaped spindle (i.e. very broad spindle with no poles evident) (Fig. 1a–c). Since the accordion-shaped spindles were so markedly different from the other single spindles, we grouped them in a separate category (‘accordion spindle’ category). Those with a single spindle and having at least one identifiable pole were further assessed for the spindle configuration and chromosome alignment (Fig. 1a). As we used a two-dimensional scope for these analyses, we included only those cases in which the spindle was not rotated on the slide, so the spindle bipolarity and chromosome alignment could be determined. Oocytes were classified for spindle and chromosome configuration as previously described (De Santis et al., 2007):

Figure 1

Three-dimensional confocal reconstructions of microtubule (green) and chromatin (red) in oocytes that failed to fertilize. Representative patterns include: (a) a single normal bipolar spindle, with chromosomes aligned along the equatorial plate; (b) two spindles in the same oocyte; (c) a disarranged broad spindle with no poles (accordion-shaped), together with a decondensing sperm head (arrowhead); and (d) two spindles in the same oocyte with a sperm tail attached to one of the two spindles (arrow). An asterisk denotes a polar body structure in each panel. Scale bar, 10 μm.

Figure 1

Three-dimensional confocal reconstructions of microtubule (green) and chromatin (red) in oocytes that failed to fertilize. Representative patterns include: (a) a single normal bipolar spindle, with chromosomes aligned along the equatorial plate; (b) two spindles in the same oocyte; (c) a disarranged broad spindle with no poles (accordion-shaped), together with a decondensing sperm head (arrowhead); and (d) two spindles in the same oocyte with a sperm tail attached to one of the two spindles (arrow). An asterisk denotes a polar body structure in each panel. Scale bar, 10 μm.

  • Bipolar spindle with chromosomes aligned along the equatorial plate (characteristics of a normal MII oocyte).

  • Bipolar spindle, non-aligned chromosomes.

  • Disarranged spindle, aligned chromosomes.

  • Disarranged spindle, non-aligned chromosomes.

Oocytes that exhibited more than one spindle were further labeled with acetylated α-tubulin in order to assess the origin of the spindles (Schatten et al., 1988; Asch et al., 1995; Rawe and Chemes, 2009). In cases in which a sperm tail (positive for acetylated α-tubulin) was attached to one of the poles (Fig. 1d), the spindle was defined as paternal in origin. Spindles not positive for acetylated α-tubulin were classified as of maternal origin.

Stimulation protocols

Stimulation regimens were as previously described (Shah et al., 2011) and included: down-regulation protocols using gonadotrophin releasing hormone (GnRH) agonists, protocols using GnRH antagonists, and poor responder protocols using low dose GnRH agonist flare or estradiol priming.

Assessment of sperm quality

We used total motile sperm number to assess sperm quality in this study as this test was routinely performed in each IVF cycle during sperm preparation in our laboratory. The percent normal forms could not be studied as evaluations for this parameter were routinely performed before initiation of IVF treatment and so were not tied to a specific cycle. Moreover, these evaluations were done in different external laboratories, with some using Kruger strict criteria whereas others used WHO criteria.

Clinical outcome variables assessed

Clinical pregnancy was documented by the presence of an intrauterine gestational sac by ultrasound.

Statistical analyses

Odds ratios (ORs) and 95% confidence intervals (CIs) and their corresponding two-sided Wald P-values were calculated using generalized estimating equations to account for the correlation among oocytes from the same patient. Models were adjusted for age at cycle start, cycle type (IVF or ICSI) and polycystic ovarian syndrome (PCOS) diagnosis a priori. These covariates were chosen based on previous studies that reported an increase in spindle anomalies associated with older maternal age (Battaglia et al., 1996) and differing reasons for fertilization failure after IVF or ICSI (Rawe et al., 2000). Given the higher prevalence PCOS in obese women, PCOS was also chosen a priori as a possible confounder. The individual addition of total number of oocytes retrieved, total number of mature oocytes, infertility diagnosis, other medical conditions, and medications did not change effect estimates by >10% in the main model and were thus not included in the final models (Greenland, 1989). All analyses were performed using Statistical Analysis Software (SAS®) version 9.1 (SAS Institute, Inc., Cary, NC, USA).

Results

Patients and oocytes

A total of 276 oocytes from 137 patients were evaluated for cytoskeletal parameters: 171 oocytes from 90 women in the normal BMI group were compared with 105 oocytes from 47 patients in the severely obese group. Patient age at cycle start, total number of oocytes retrieved, the number of oocytes that failed to fertilize, the percentage of mature oocytes that failed to fertilize and the number of oocytes that were fixed and labeled were similar between the two groups (Table I). Severely obese patients had a higher prevalence of PCOS (27.7 versus 11.1%) compared with normal BMI patients. Proportions of infertility diagnoses, other than PCOS, usage of ICSI, and total motile sperm were similar between the two patient groups. The prevalences of hypertension (HTN) and non-insulin-dependent diabetes mellitus (NIDDM) among severely obese patients were 10.6% (n = 5/47) and 4.3% (n = 2/47), respectively; no occurrence of these diseases was reported among the normal BMI patients. Thirty four percent (n = 16/47) of the severely obese patients and 10.0% (n = 9/90) of the normal BMI patients had a medical condition other than HTN/NIDDM (such as depression, hyper or hypothyroidism and asthma). Of the 47 severely obese patients, 13 (27.7%) were treated with medications not associated with the IVF treatment, compared with 6 of the 90 (6.7%) patients with normal BMI.

Table I

Demographic characteristics by BMI among 137 women undergoing assisted reproduction at a hospital-based infertility clinic.

Characteristic BMI 18.5–24.9 kg/m2, 90 women BMI ≥ 35 kg/m2, 47 women 
BMI 
 Mean (SD) 22.2 (1.6) 41.8 (4.5) 
 Minimum–maximum 18.6–24.9 35.2–50.1 
Age 
 Mean (SD) 35.0 (4.9) 35.8 (4.9) 
 Minimum–maximum 21.8–43.2 24.8–42.7 
Total number of oocytes retrieved 
 Mean (SD) 15.7 (7.8) 13.5 (7.4) 
 Minimum–maximum 3.0–47.0 3.0–33.0 
Total number of MII retrieved 
 Mean (SD) 13.6 (7.3) 11.3 (6.1) 
 Minimum–maximum 3.0–43.0 3.0–31.0 
Total number of 2PN zygotes 
 Mean (SD) 9.5 (6.0) 7.5 (5.3) 
 Minimum–maximum (1–27) (1–27) 
Total number of oocytes that failed fertilization 
 Mean (SD) 3.0 (3.0) 2.7 (2.1) 
 Minimum–maximum 1.0–21.0 1.0–10.0 
% of MII oocytes that failed fertilization 
 Mean (SD) 23.7 (16.4) 26.6 (18.0) 
 Minimum–maximum 4.2–75.0 7.7–81.8 
Number of oocytes that were fixed and labeled 
 Mean (SD) 1.9 (1.2) 2.2 (1.5) 
 Minimum–maximum 1.0–7.0 1.0–7.0 
PCOS, n (%) 10 (11.1%) 13 (27.7%) 
ICSI, n (%) 35 (38.9%) 24 (51.1%) 
Stimulation protocol 
 Down-regulation 52 (58.4%) 22 (46.8%) 
 Antagonist 23 (25.8%) 9 (19.1%) 
 Poor responders 14 (15.7%) 16 (34.0%) 
 FSH onlya 1 (1.1%)  
FSH dose (IU) 
 Mean (SD) 2938 (1901) 3993 (1874) 
 Minimum–maximum 750–9000 1200–9000 
E2 levels (day of hCG) (pg/ml) 
 Mean (SD) 2208 (927.1) 1912 (951.2) 
 Minimum–maximum 617–4222 659–4220 
Total motile sperm (×106
 Mean (SD) 78.7 (75.6) 80.3 (97.6) 
 Median (Q1–Q3b65 (24–109) 43 (4–117) 
 Minimum–maximum 0–382 0–414 
Characteristic BMI 18.5–24.9 kg/m2, 90 women BMI ≥ 35 kg/m2, 47 women 
BMI 
 Mean (SD) 22.2 (1.6) 41.8 (4.5) 
 Minimum–maximum 18.6–24.9 35.2–50.1 
Age 
 Mean (SD) 35.0 (4.9) 35.8 (4.9) 
 Minimum–maximum 21.8–43.2 24.8–42.7 
Total number of oocytes retrieved 
 Mean (SD) 15.7 (7.8) 13.5 (7.4) 
 Minimum–maximum 3.0–47.0 3.0–33.0 
Total number of MII retrieved 
 Mean (SD) 13.6 (7.3) 11.3 (6.1) 
 Minimum–maximum 3.0–43.0 3.0–31.0 
Total number of 2PN zygotes 
 Mean (SD) 9.5 (6.0) 7.5 (5.3) 
 Minimum–maximum (1–27) (1–27) 
Total number of oocytes that failed fertilization 
 Mean (SD) 3.0 (3.0) 2.7 (2.1) 
 Minimum–maximum 1.0–21.0 1.0–10.0 
% of MII oocytes that failed fertilization 
 Mean (SD) 23.7 (16.4) 26.6 (18.0) 
 Minimum–maximum 4.2–75.0 7.7–81.8 
Number of oocytes that were fixed and labeled 
 Mean (SD) 1.9 (1.2) 2.2 (1.5) 
 Minimum–maximum 1.0–7.0 1.0–7.0 
PCOS, n (%) 10 (11.1%) 13 (27.7%) 
ICSI, n (%) 35 (38.9%) 24 (51.1%) 
Stimulation protocol 
 Down-regulation 52 (58.4%) 22 (46.8%) 
 Antagonist 23 (25.8%) 9 (19.1%) 
 Poor responders 14 (15.7%) 16 (34.0%) 
 FSH onlya 1 (1.1%)  
FSH dose (IU) 
 Mean (SD) 2938 (1901) 3993 (1874) 
 Minimum–maximum 750–9000 1200–9000 
E2 levels (day of hCG) (pg/ml) 
 Mean (SD) 2208 (927.1) 1912 (951.2) 
 Minimum–maximum 617–4222 659–4220 
Total motile sperm (×106
 Mean (SD) 78.7 (75.6) 80.3 (97.6) 
 Median (Q1–Q3b65 (24–109) 43 (4–117) 
 Minimum–maximum 0–382 0–414 

PCOS, polycystic ovarian syndrome.

aThis patient was converted from an intrauterine insemination cycle to IVF.

bAs the number of total motile sperms was very skewed, we present the median and quartiles and not only the mean and SD as for the other parameters.

Eighty-seven patients with normal BMI and 46 women in the severely obese group underwent fresh embryo transfer. A greater proportion of obese patients received poor responder protocols when compared with patients having normal BMI (34.0 versus 15.7%, respectively), and their total dose of FSH was also higher, on average. Despite these more aggressive stimulations, obese patients had lower peak estradiol (E2) levels on average than normal BMI patients (Table I). Clinical pregnancy rates were 39.1% (34/87) and 41.3% (19/46) for the two groups, respectively.

Cytoskeletal analysis of the failed fertilized oocytes

While a greater proportion of oocytes from the normal BMI group (14.0%) were activated relative to the proportion activated from the severely obese group (7.6%), this difference was not statistically significantly different (two-sided Wald P-value = 0.09). Reciprocally, there was no significant difference between the proportion of non-activated oocytes with spindles between the groups (91.2 versus 92.8%; two-sided Wald P-value = 0.88; Table II). The non-activated oocytes with spindles (n = 134 and 90, for the normal BMI and severely obese groups, respectively) were further analyzed for spindle configurations.

Table II

The association between spindle characteristics in oocytes that failed fertilization from women with normal BMI compared with severe obesity among 137 women undergoing assisted reproduction at a hospital-based infertility clinic.

 BMI 18.5–24.9 kg/m2, 90 women, 171 oocytes BMI ≥ 35 kg/m2, 47 women, 105 oocytes OR (95% CI)* P-value* 
Activated 24 (14.0%) 8 (7.6%) 0.46 (0.19–1.12) 0.09 
Non-activated, no spindle 13 (8.8%) 7 (7.2%) 0.93 (0.33–2.57) 0.88 
Non-activated with spindles 134 (91.2%) 90 (92.8%) 1.08 (0.39–3.00) 0.88 
1 spindle 86 (64.1%) 34 (37.8%) 0.33 (0.17–0.63) <0.001 
>1 spindle 47 (35.1%) 53 (58.9%) 2.68 (1.39–5.15) 0.003 
Accordion-shaped spindle 1 (0.8%) 3 (3.3%) – – 
 BMI 18.5–24.9 kg/m2, 90 women, 171 oocytes BMI ≥ 35 kg/m2, 47 women, 105 oocytes OR (95% CI)* P-value* 
Activated 24 (14.0%) 8 (7.6%) 0.46 (0.19–1.12) 0.09 
Non-activated, no spindle 13 (8.8%) 7 (7.2%) 0.93 (0.33–2.57) 0.88 
Non-activated with spindles 134 (91.2%) 90 (92.8%) 1.08 (0.39–3.00) 0.88 
1 spindle 86 (64.1%) 34 (37.8%) 0.33 (0.17–0.63) <0.001 
>1 spindle 47 (35.1%) 53 (58.9%) 2.68 (1.39–5.15) 0.003 
Accordion-shaped spindle 1 (0.8%) 3 (3.3%) – – 

OR, odds ratio; CI, confidence interval.

Percentages in parentheses are the proportion of oocytes examined in each group.

*ORs, 95% CIs and two-sided Wald P-values are from generalized estimating equations to account for the correlation between oocytes from the same patient and are adjusted for continuous age at cycle start, cycle type (IVF or ICSI), and PCOS.

The odds of an oocyte having one spindle were significantly lower for the severely obese group (37.8 versus 64.1%; OR = 0.33, CI = 0.17–0.63, two-sided Wald P-value <0.001; Table II). Conversely, the odds of an oocyte having more than one spindle were greater in this group (58.9 versus 35.1%; OR = 2.68, CI = 1.39–5.15, two-sided Wald P-value = 0.003). The prevalence of oocytes having an accordion-shaped spindle was similarly low for both the normal BMI and severely obese groups [1/90 (0.8%) versus 3/105 (3.3%), respectively].

Spindle number and PCOS

Although the sample size was not sufficiently large to study whether the association between BMI and oocyte characteristics differed depending on PCOS status, we did look descriptively at this stratified analysis. The relationship between BMI and oocyte characteristics appeared to be consistent among patients with and without PCOS: in both populations, a lower proportion of oocytes from obese women had 1 spindle and a higher proportion had >1 spindle (Supplementary data, Table S1).

Spindle number and medications

A higher proportion of patients in the severely obese group used medications unrelated to controlled ovarian stimulation, compared with normal BMI patients (13/47 (27.7%) and 6/90 (6.7%), respectively). Of the 19 patients who used such medications, 4 patients (3 in the severely obese group and 1 in the normal BMI group) took anti-depressants and/or anxiolytic drugs. Previous toxicological studies in animal models reported an association between exposure to diazepam and folliculogenesis disturbances and spindle abnormalities (Van Wemmel et al., 2005). Although the use of such medications was not included in the final model as it was not found to be a potential confounder, a sub-analysis was done excluding patients on anti-depressants and, separately, excluding patients on any medication. Associations were slightly attenuated in the first sub-analysis and slightly strengthened in the second, but overall results remained the same (Supplementary data, Table S2).

Single spindle analysis

The association between BMI and spindle configuration/chromosome alignment was assessed for those oocytes having a single spindle that was non-angled on the slide (n = 58 and 21, for the normal BMI and severely obese groups, respectively; Table III). The odds of an oocyte having a bipolar spindle with aligned chromosomes (Group I) and the odds of an oocyte having a bipolar spindle with non-aligned chromosomes (Group II) did not differ significantly between BMI groups. No oocyte from either BMI group was classified as having a disarranged spindle with aligned chromosomes (Group III). However, the odds of an oocyte with a disarranged spindle and non-aligned chromosomes (Group IV) was significantly greater within the severely obese group compared with the normal BMI group (28.6 versus 8.6%; OR = 4.58, CI = 1.05–19.86, two-sided Wald P-value = 0.04).

Table III

The association between BMI and spindle configurations among oocytes with one, non-angled spindle.

Spindle/Chromosome Classification Group BMI 18.5–24.9 kg/m2, 40 women, 58 oocytes BMI ≥ 35 kg/m2, 16 women, 21 oocytes OR (95% CI)* P-value* 
34 (58.6%) 8 (38.1%) 0.40 (0.13–1.27) 0.12 
II 19 (32.7%) 7 (33.3%) 0.96 (0.25–3.69) 0.96 
III   
IV 5 (8.6%) 6 (28.6%) 4.58 (1.05–19.86) 0.04 
Spindle/Chromosome Classification Group BMI 18.5–24.9 kg/m2, 40 women, 58 oocytes BMI ≥ 35 kg/m2, 16 women, 21 oocytes OR (95% CI)* P-value* 
34 (58.6%) 8 (38.1%) 0.40 (0.13–1.27) 0.12 
II 19 (32.7%) 7 (33.3%) 0.96 (0.25–3.69) 0.96 
III   
IV 5 (8.6%) 6 (28.6%) 4.58 (1.05–19.86) 0.04 

OR, odds ratio; CI, confidence interval.

(I) Bipolar spindle with chromosomes aligned along the equatorial plate; (II) Bipolar spindle, non-aligned chromosomes; (III) Disarranged spindle, aligned chromosomes; (IV) Disarranged spindle, non-aligned chromosomes.

*ORs, 95% CIs and two-sided Wald P-values are from generalized estimating equations to account for the correlation between oocytes from the same patient and are adjusted for continuous age at cycle start, cycle type (IVF or ICSI) and PCOS.

Multiple spindle analysis

A total of 100 oocytes had more than one spindle, of which 88 (44 from the normal BMI and 44 from the severely obese group) were further assessed for the presence of sperm inside the oocyte. Twelve oocytes were not further assessed as they were lost trying to retrieve them from the slide. The prevalence of detection of a sperm tail was similar between the groups (79.5 versus 72.7%, normal versus severely obese, respectively, two-sided Wald P-value = 0.60; Table IV). The sperm tail was attached to a pole of one of the spindles (deemed to be of paternal origin) in a greater percentage of oocytes in the normal BMI group relative to the severely obese group (61.4 and 54.5%, respectively), however these proportions were not significantly different (two-sided Wald P-value = 0.65). All oocytes having more than one spindle had at least one spindle without a sperm tail attached (deemed to be of maternal origin).

Table IV

The association between BMI and origin of the second spindle.

 BMI 18.5–24.9 kg/m2, 26 women, 44 oocytes BMI ≥ 35 kg/m2, 21 women, 44 oocytes OR (95% CI)* P-value* 
Sperm tail present in the oocyte 35 (79.5%) 32 (72.7%) 0.74 (0.24–2.28) 0.60 
Sperm tail attached to a spindle 27 (61.4%) 24 (54.5%) 0.81 (0.33–2.01) 0.65 
 BMI 18.5–24.9 kg/m2, 26 women, 44 oocytes BMI ≥ 35 kg/m2, 21 women, 44 oocytes OR (95% CI)* P-value* 
Sperm tail present in the oocyte 35 (79.5%) 32 (72.7%) 0.74 (0.24–2.28) 0.60 
Sperm tail attached to a spindle 27 (61.4%) 24 (54.5%) 0.81 (0.33–2.01) 0.65 

OR, odds ratio; CI, confidence interval.

Percentages in parentheses are the proportion of oocytes examined in each group.

*ORs, 95% CIs and two-sided Wald P-values are from generalized estimating equations to account for the correlation between oocytes from the same patient and are adjusted for continuous age at cycle start, cycle type (IVF or ICSI) and PCOS.

Discussion

To our knowledge, this is the first analysis that has directly assessed a possible association between severe obesity (Class II and III obesity) and oocyte characteristics in women. Our observations indicate a high prevalence of cytoskeletal abnormalities in failed fertilized oocytes from severely obese patients compared with those from normal BMI patients. The oocytes from the severely obese group exhibited a higher prevalence of two or more spindles and, when only one spindle was present, demonstrated a higher prevalence of disarranged spindles with non-aligned chromosomes. These findings were independent of the effect of potential confounders of patient age at cycle start, ICSI and PCOS.

Studies investigating the relationship between BMI and oocyte quality are difficult to compare due to differing cut-points for BMI groups, and differing definitions of oocyte quality. However, many studies indicate that obesity is associated with decreased oocyte quality (Wittemer et al., 2000; Dokras et al., 2006; Tamer Erel and Senturk, 2009; Depalo et al., 2011). Moreover, in a recent study that included 45 000 assisted reproductive treatment cycles, increasing BMI was associated with decreased clinical pregnancy rates from autologous oocytes, but not from donor oocytes (Luke et al., 2011). These results suggest that oocyte quality, rather than uterine receptivity, might underlie the decreasing fertility associated with increasing BMI.

Further support for an adverse effect of BMI on oocyte quality is derived from direct evidence using a diet-induced obese mouse model and, indirectly, from studies of serum and follicular fluids from IVF patients of varying BMI. The mouse studies have shown that obesity is associated with poor oocyte quality, reduced blastocyst survival rate and abnormal ovarian gene expression (Minge et al., 2008), altered mitochondrial properties in oocytes and zygotes (Igosheva et al. 2010), as well as an increase in prevalence of apoptotic ovarian follicles, smaller oocyte size and delayed resumption of meiosis (Jungheim et al., 2010). The human studies have revealed elevated intra-follicular levels of several compounds (e.g. insulin, triglycerides, lactate, C-reactive protein and androgens) in obese women (Robker et al., 2009), in addition to alterations in adipokines (Butzow et al., 1999; Hill et al., 2007; Purcell and Moley, 2011).

Perhaps of even greater relevance to the findings we present is the documented increase in follicular and serum leptin levels in obese women (Butzow et al., 1999; Hill et al., 2007). Animal studies suggest that elevated leptin levels exert a direct inhibitory effect on ovarian function by perturbation of steroidogenesis, and impairment of folliculogenesis and oocyte maturation (Butzow et al., 1999; Duggal et al., 2000; Swain et al., 2004). Moreover, in vitro studies have shown that leptin interferes with cyclic AMP signaling to inhibit steroidogenesis in human granulosa cells (Lin et al., 2009), and granulosa cells play a critical role in support of normal follicular growth and oocyte development (reviewed by Gilchrist et al., 2004).

Taken together, the above studies support the possibility that the oocyte abnormalities we document here in our severely obese patients are attributable to alterations in the ovarian follicular microenvironment (reviewed by Purcell and Moley, 2011). Further work needs to be performed to investigate this possibility.

Our observation that failed fertilized oocytes from severely obese patients exhibit a higher prevalence of two or more spindles merits further consideration. Completion of normal fertilization is dependent upon tightly synchronized events including sperm binding to, and penetration of the oolemma, followed by extrusion of the second polar body, decondensation of the sperm chromatin and formation of male and female pronuclei (Adenot et al., 1991; Gook et al., 1998). These events are dependent upon the normal completion of ‘cytoplasmic programming’ during meiotic maturation (Swain and Pool, 2008). Failure of any of these stages will result in fertilization failure and, if sperm penetration has occurred, may give rise to formation of a paternal, as well as a maternal spindle (Asch et al., 1995; Gook et al., 1998; Kovacic and Vlaisavljevic, 2000). The high prevalence of spindle abnormalities we observed in the severely obese group therefore raises the concern of impaired oocyte quality and possible cytoplasmic immaturity in oocytes otherwise deemed to be mature (based on the MII nuclear stage) in this population. Such a possibility is consistent with recent findings of a higher number of oocytes with cytoplasmic granularity in patients with BMI > 25 kg/m2 compared with women with normal BMI (Depalo et al., 2011). These authors proposed that oocytes from overweight/obese patients are more likely to exhibit an asynchrony between nuclear and cytoplasmic status.

Oocytes with cytoplasmic immaturity are unlikely to respond to the activation signal provided by the spermatozoa (Rosenbusch, 2000). Such failure may involve a deficient calcium signaling system, defective cell signaling or an abnormal response in the cascade pathway downstream of the sperm signal. Regardless, such an immature cytoplasmic environment is associated with abnormal sperm processing and formation of premature chromosome condensation (PCC; reviewed by Nasr-Esfahani et al., 2007). Although unsuccessful oocyte activation can result from cytoplasmic immaturity, another explanation is absence or deficiency of the ‘sperm associated oocyte activation factors’ (Dozortsev et al., 1995). However, as the number of total motile sperm and the proportion of ICSI cycles were similar between our two patient groups, we postulate that the oocyte cytoplasmic immaturity is most likely responsible for the activation failure among severe obesity patients. A sperm tail was found in >70% of the oocytes with multiple spindles (no significant difference between the groups) and in >50% of the cases, the sperm tail was attached to a spindle. Possible reasons for no sperm present could be the failure of sperm penetration or abnormal maternal meiosis (e.g. division without polar body extrusion). In addition, the presence of a sperm within the oocyte not attached to a spindle may suggest that there was a sperm present and even a paternal spindle, but perhaps the tail became detached.

Both one spindle and multiple spindle phenotypes can reflect meiotic arrest or cell cycle issues that result in fertilization failures. Further evaluation of the failed fertilized oocytes with a single spindle is beyond the scope of this current study, and we can only hypothesize that the reasons for these cases were failure either of the sperm to incorporate into the oocytes or failure of the ooplasm to trigger sperm PCC. Assuming that sperm penetration/incorporation was similar between our two patient groups, we suggest that the greater prevalence of two spindles in the severely obese patients may have resulted from a cytoplasm that favors sperm PCC with formation of a meiotic spindle. As the total number of non-activated oocytes having a spindle did not differ between groups, it is possible that cytoplasmic immaturity may be similarly prevalent in both patient groups, but the exact nature of the immaturity may differ.

As PCOS occurred with a higher prevalence in our severely obese patients compared with the normal BMI group, we adjusted for PCOS diagnosis, allowing us to conclude that the higher prevalence of cytoskeletal abnormalities was attributable to BMI independent of the influence of PCOS (Table II). These results are consistent with previous conclusions that obesity has an effect beyond that of PCOS on IVF outcome (Fedorcsák et al., 2000; Dokras et al., 2006; Shah et al., 2011; Sobaleva and El-Toukhy, 2011). Use of medications for specific medical conditions was also more frequent among severely obese patients. However, excluding those patients who used such medications, in particular anti-depressants, did not affect the results. Nevertheless, it is important to note the very small sample sizes that underlie these observations.

Given that we had access only to discarded oocytes, we were limited to evaluating only those oocytes that failed to fertilize. It is unknown whether our results can be extrapolated to fertilized oocytes within the same cohort retrieved from an individual woman in a single stimulation cycle. In addition, the oocytes studied here were obtained following controlled ovarian stimulation, and it is unknown whether such oocytes retrieved from severely obese patients after ovarian stimulation are similar to those ovulated in natural cycles. Failed fertilized human oocytes available for research are aged oocytes (typically having been in culture for at least 24 h since retrieval) and previous studies have suggested that spindle irregularity may result from in vitro oocyte aging (Eichenlaub-Ritter et al., 1988; Pickering et al., 1988). Pickering et al reported 30% alterations in spindle microtubules in aged oocytes (fixed up to 48 h after retrieval), compared with controls (fresh oocytes) although the sample size was small (30 aged oocytes, 9 fresh controls; Pickering et al., 1988). However, that study did not discriminate between spindle morphology among oocytes that were fixed at 24 h versus 48 h. Eichenlaub-Ritter et al. (1988) found increased irregularity in spindle bipolarity as well as the chromosome alignment in failed fertilized oocytes fixed after 4 versus 2 days of retrieval, again suggesting an effect of oocyte aging in culture. These authors suggest that it may take at least 2 days before predominantly asymmetric spindles are found in human oocytes (Eichenlaub-Ritter et al. 1988). We used a similar fixing time (mean of 24 h) as that of several other authors who evaluated failed fertilized human oocytes (Asch et al., 1995; Kovacic and Vlaisavljevic 2000; Rawe et al., 2000; Rosenbuch, 2000). Moreover, as the fixing time was similar for both BMI groups in our study, we believe that the differences in single spindle morphology we observed between groups were not biased by oocyte aging.

In summary, our observations provide novel insight into a possible cause for the reduced fertility in severely obese patients. The high rates of spindle abnormalities we document suggest that, in these patients, at least a proportion of the ‘mature’ oocytes (based on the presence of a polar body) may have a compromised quality that precludes normal completion of fertilization. Further work is warranted to investigate the mechanism underlying the spindle abnormalities we have observed in this population.

Supplementary data

Supplementary data are available at http://humrep.oxfordjournals.org/.

Authors' roles

R.M. performed the fixation and labeling of oocytes as well as the immunofluorescence scoping and writing the manuscript. C.M.H.C. participated in the study design, performed the immunofluorescence and confocal imaging, and editing of the manuscript. S.A.M. participated in the study design, conducted the data analyses and edited the manuscript. K.F.C. conducted the data analysis and edited the manuscript. J.H.F. edited the manuscript. C.R. was responsible for the experimental design, overseeing completion of the study, editing and finalizing of the manuscript.

Conflict of interest

The authors have no conflict of interest.

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Rankin
J
Inappropriate secretion of follicle-stimulating hormone and luteinizing hormone in polycystic ovarian disease
J Clin Endocrinol Metab
 , 
1970
, vol. 
30
 (pg. 
435
-
442
)