Evidence that obesity alters the quality of oocytes and embryos

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1 Pathophysiology 15 (2008) Review Evidence that obesity alters the quality of oocytes and embryos Rebecca L. Robker Discipline of Obstetrics and Gynaecology, Research Centre for Reproductive Health, School of Paediatrics and Reproductive Health, University of Adelaide, Adelaide, SA 5005, Australia Received 12 February 2008; received in revised form 24 April 2008; accepted 28 April 2008 Abstract Infertility is more common in overweight and obese women, with reproductive impairments occurring at many levels of the hypothalamicovarian-uterine axis. These impairments lead primarily to longer times to conception and decreased pregnancy s and have resulted in increasing numbers of overweight and obese women seeking assisted reproduction technologies, such as in vitro fertilization or IVF. Even after undertaking IVF procedures obese women have decreased pregnancy s compared to mode weight women, suggesting there may be intrinsic differences in the oocytes of these patients. Definitive data is lacking however, and thus the effect of obesity on oocyte quality remains one of the biggest controversies in reproductive medicine. This review summarizes the studies to date which have yielded information about the effects of obesity on human oocyte quality and pre-implantation embryo development. In addition recent results from our laboratory which clearly demonst that diet-induced obesity in mice impairs oocyte developmental competence are discussed Elsevier Ireland Ltd. All rights reserved. Keywords: Obesity; Female fertility; Oocyte; Embryo; Body mass index (BMI); In vitro fertilization (IVF); Assisted reproduction technologies (ART) Contents 1. Introduction Assisted reproduction outcomes in obese women Effects of obesity on oocyte quality and early embryo development Oocyte developmental competence in an obese mouse model Conclusions References Introduction Female fertility is exquisitely sensitive to bodyweight. A critical threshold of body fat is required for girls to enter puberty [1] nature s way of ensuring she can carry a pregnancy to term; however, excessive body fat has a detrimental effect on female fertility [2] and as the incidence of obesity steadily increases, particularly in young women [3], its harmful consequences are becoming increasingly apparent. The effects of obesity are not restricted to a single aspect of female reproduction but are manifest at several levels: altered levels Tel.: ; fax: address: Rebecca.robker@adelaide.edu.au. of hypothalamic gonadotropins, anovulation, altered steroid production, reduced conception s, longer times to conception, increased miscarriage s, increased risk of many pregnancy complications ranging from hypertension to preterm birth, and increased incidence of fetal abnormalities [4 6]. The primary pathologies altered gonadotropin and steroid hormone levels contribute initially to anovulation. Studies have shown however that even in women who are ovulating regularly increased BMI correlates with reduced conception s [7,8], suggesting that obesity affects critical peri-conception events, such as oocyte and/or embryo quality. If pregnancies are detected in obese women, they are still at greater risk of pregnancy loss. Miscarriages in obese women typically occur very early [9 11], with the majority of studies /$ see front matter 2008 Elsevier Ireland Ltd. All rights reserved. doi: /j.pathophys

2 116 R.L. Robker / Pathophysiology 15 (2008) Fig. 1. Schematic of the ovary, the ovulated oocyte and resultant blastocyst stage embryo. Alterations to the ovarian environment, such as during obesity, impact oocyte developmental competence and subsequent embryo quality. In human IVF the oocyte is aspid from preovulatory ovarian follicles and fertilized in vitro. The embryo is typically replaced into the uterus after 2 or 3 days, at the 2-cell or 8-cell stage, respectively. In our animal studies female mice ovulated and mated with males followed by removal of the fertilized oocyte from the oviduct. This zygote was cultured in vitro for 5 days to the blastocyst stage and then the embryonic cells (inner cell mass) and placental precursor cells (trophectoderm) were assessed. describing pregnancy loss by 6 7 weeks gestation as detected by ultrasound. Thus the decreased pregnancy s observed with increasing BMI are primarily due to loss at the pre- or peri-implantation windows, which is often indicative of suboptimal embryo health. These very earliest stages of embryo growth are primarily controlled by the quality of the oocyte, also known as oocyte developmental competence; making it likely that alterations to the oocyte contribute significantly to the reduced conception and pregnancy s that are commonly observed in obese women. Oocyte quality is of course exceedingly difficult, if not impossible, to directly assess in women attempting to conceive naturally; however the process of IVF enables close inspection of oocytes and their fertilization and subsequent development into blastocysts, prior to uterine transfer and implantation Assisted reproduction outcomes in obese women The rising incidence of obesity coupled with its detrimental effects on fertility has led to greater numbers of overweight and obese women utilizing assisted reproduction technologies (ART), such as in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). Both of these increasingly common techniques involve stimulation of ovarian follicle growth with high doses of exogenous gonadotropins and retrieval of oocytes (or eggs) from the preovulatory follicles of the ovary (see Fig. 1). The oocytes are then fertilized in vitro by incubation with sperm, or microinjection of it directly into the egg as is the case with ICSI, and between 2 and 5 days later the resultant embryo is transferred into the recipient uterus. Biochemical pregnancy is assessed by positive hcg levels, usually 14 days following oocyte retrieval, with implantation assessed by ultrasound at 3 6 weeks. The effects of obesity on the fertility of women undergoing ART mirror those of women conceiving naturally [12,5]. One large study [13] found that in the first IVF cycles of 8457 European women, a BMI 27 reduced the chance of a live birth by a striking 33%, irrespective of infertility diagnosis. Similarly, an earlier study [14] found a linear reduction in fecundity (probability of achieving one pregnancy) with increasing BMI such that the pregnancy among very obese women (BMI 35) was half that of mode weight women (BMI ). Ferlitsch et al. [15] found that pregnancy in women undergoing IVF was negatively correlated with body weight, specifically that raising BMI by one unit decreased the odds of pregnancy by Two independent studies [16,17] have demonstd that fat distribution is an additional factor in determining the effects of obesity on pregnancy s. Analysis of pregnancy s of 500 women undergoing artificial donor insemination who were deemed to be healthy normally cycling women, found that waist hip ratio (WHR), rather than BMI, was the best predictor of pregnancy success, even after controlling for age, length of cycle, smoking and reasons for artificial insemination [17]. A smaller study [16] analysed pregnancy s in women undergoing IVF with the authors maintaining that such analysis better controls hormonal stimulation, oocyte maturation and infertility diagnoses. Although the numbers of pregnancies analysed was relatively small, in women with a BMI 25 the clinical pregnancy was 27.3% in those with a WHR of 0.70 to <0.80 but less than half of that (8.7%) in women with a WHR of Together these indicate that abdominal fat distribution in particular results in decreased pregnancy s. However, the negative effects of obesity on fertility outcomes can also be seen using body mass indices other than BMI and WHR. In a study of women with polycystic ovary syndrome (PCOS) undergoing IVF, elevations in any of four different indices of body mass BMI (kg/m 2 ), the National Health and Nutrition Index for women (W/H 1.5 ), ponderal index (W 0.33 /H), and surface area, were correlated with the likelihood of having no ovarian follicle 10 mm on day 7 of stimulation protocol and of having no oocytes retrieved [18]. Women with PCOS are disproportionately overweight, typically with excessive fat distributed abdominally (android) rather than on the lower body (gynoid). It is not known whether obesity influences the onset of overt PCOS in susceptible women or whether the hormonal imbalances that define PCOS (hyperandrogenemia and elevated LH) are responsible for altered metabolism and/or body fat distribution. In light of the higher prevalence of PCOS in obese women, Dokras et al. [19] stratified their observations of IVF outcomes according to PCOS status in the obese and morbidly

3 Table 1 Assisted reproduction outcomes in obese women compared to those with mode body mass index Study (reference) Patients (obese) Obese BMI Gonadotropin dose Cancellation Peak E 2 levels Oocyte # Oocyte maturity Fertilization Embryo # Embryo quality Implantation Lewis 1990 [40] 368 (36) >27.6 Crosignani 1994 [18] 111 (44) >22 a Lashen 1999 [41] 333 (76) >27.9 Wittemer 2000 [24] 398 (48) 28 b Wang 2000 [14] 3586 (421) 30 Fedorcsak 2000 [10] 383 (79) 25 Carrell 2001 [25] 247 (34) >30 c Loveland 2001 [42] 139 (69) >25 c Salha 2001 [27] 100 (50) 26 Nichols 2003 [43] Fedorcsak 2004 [9] 2660 (241) 30 Spandorfer 2004 [44] 920 (148) >27 Van Swieten 2005 [26] 162 (29) >30 c Dechaud 2006 [45] 573 (36) 30 Ku 2006 [46] d c Dokras 2006 [19] 1293 (315) 30 c Metwally 2007 [28] 426 (72) 30 e f e e ( ) increased; ( ) decreased; ( ) no different, in obese compared to mode weight women. a Highest BMI tertile of patient population. b Long-stimulation protocol only. c Trend, not statistically significant. d Asian population. e Patients <35 years old only. f Patients 35 years old only. Pregnancy R.L. Robker / Pathophysiology 15 (2008)

4 118 R.L. Robker / Pathophysiology 15 (2008) obese groups. Of particular note, the clinical pregnancy s were not different between women with or without PCOS [19]. Other studies examining IVF outcomes in women with PCOS compared to those with tubal blockage have also seen no difference in pregnancy and live birth s [20 22]. Cumulatively this indicates that decreased pregnancy s with increasing BMI, such as observed in this [19] and many additional studies (see Table 1), is not simply due to the higher prevalence of PCOS in obese women. Table 1 summarizes the studies to date that have assessed the effect of obesity (reflected as increased BMI) on the outcomes of women undergoing ART; IVF was used in the vast majority of cases although in many instances, especially more recently, ICSI was also employed. Many of the studies, particularly the largest [9,14] clearly demonst an effect of increased BMI on implantation and pregnancy s, a result recently confirmed by a systematic review [23]. The hormonal stimulation regimen and choice of drugs (i.e. clomiphene cit, human menopausal gonadotropin, human chorionic gonadotropin, and/or FSH) and whether or not pituitary downregulation (usually with GnRH antagonist) was employed, is variable between clinics, and almost certainly contributes to the disparity in observed outcomes, as would the differences in BMI measures that constitute obesity. It is interesting to note that although BMI does not influence each outcome in every study, when effects are observed across studies they are always in the same direction, i.e. consistently increased or consistently decreased. The earliest impact of obesity on ART efficacy is its effect on gonadotropin responsiveness (Table 1). As also reflected by a systematic review [23], almost all studies report a need to treat obese women with either higher doses of gonadotropins compared to mode weight women or to treat obese women for longer with the same dose in order to elicit a response. It is possible that this exposure of the ovaries and uterus to higher levels of hormones, particularly FSH, may contribute to the poorer pregnancy outcomes in obese women. However, obese women often do not have an adequate response to gonadotropin treatment, i.e. too few ovarian follicles are present or they do not reach an adequate size in order to proceed with oocyte retrieval; an inadequate response is also reflected by reduced peak levels of estradiol (E 2 ). This failed ovarian stimulation is documented as a cancellation with obese women typically having higher s than normal. It is thought that obese women may have altered absorption and/or metabolism of gonadotropins or that they exhibit resistance. In any respect the difficulty in eliciting a normal gonadotropin response in obese women likely contributes to the subsequent problem of reduced numbers of oocytes. Reduced numbers of oocytes were retrieved from obese women in a number of studies (Table 1). This is not likely due to the presence of PCOS in this population since women with PCOS typically have greater numbers of oocytes retrieved for IVF compared to women without PCOS [19 22]. Inconsistent observations regarding BMI effects on oocyte number likely relate to differences in gonadotropin stimulation regimen. In one study where two protocols were directly compared obese women undergoing a long stimulation protocol (where GnRH analogue is used for ovarian desensitization starting at the end of the luteal phase of the previous cycle) had fewer oocytes than normal while obese women undergoing a short protocol (GnRH analogue is commenced in the early follicular phase) did not [24] Effects of obesity on oocyte quality and early embryo development Oocyte quality (or maturity) has been directly assessed using a graded scoring system in only three studies (Table 1). Wittemer et al. [24] determined whether each of the oocytes were good quality (at metaphase I or metaphase II) or bad quality (at germinal stage, post-mature, or with fractured zona). The ratio of good quality oocytes was not affected by the stimulation protocol however, it was significantly reduced in women with a BMI 25 compared to those with BMI [24]. Subsequently, a sepa study reported that fewer MII oocytes were retrieved, although not statistically significant, from obese women (BMI > 30) compared to those with BMIs of [25]. A more recent and larger study found that although there was no difference in the number of follicles aspid, the number of mature oocytes, by nuclear assessment, was significantly reduced in morbidly obese women [19]. That the maturity of oocytes was reduced in each of the three studies which directly scored it indicates that oocyte quality may be altered in women with obesity. Although oocyte maturity is rarely documented in studies of IVF outcomes, fertilization is easily assessed and almost always deemed to be normal in the oocytes of obese women (Table 1). One exception is a recent study which reports a 45% reduction in fertilization s of oocytes from obese women compared to those of mode weight [26]. Another showed a significantly decreased fertilization of 26.6% in women of BMI 26 compared to 37.1% in women with a BMI of [27]. Fertilization should not however be considered an adequate marker of oocyte quality. Differences in oocytes often do not become apparent until later in development when they manifest as altered embryo quality. Almost no studies have looked at the embryo quality of obese women and this represents a huge knowledge gap in our understanding of the long term and transgenerational consequences of obesity on human health. Occasionally it is reported that there is no difference in the quality (or grade) of embryos transferred to lean versus obese women, but this does not address whether the mean embryo grade of a woman s total embryo pool can be affected by obesity. The two studies to date that have investigated mean embryo grade both found that it was decreased in women with a BMI of >30 (Table 1). Carrell et al. [25] determined embryo score on the day of transfer (72 h after oocyte retrieval) by

5 R.L. Robker / Pathophysiology 15 (2008) subtracting points from the number of blastomeres based on the degree of abnormal blastomere morphology or fragmentation. Embryo quality was significantly reduced in the obese group of women compared to those with BMIs of [25]. In the second study [28] embryos were graded on day 2 following oocyte retrieval using a system of assessment of shape of blastomeres, texture of cytoplasm and degree of fragmentation. In addition, embryo quality was reflected in the number of embryos discarded (i.e. they were poor embryos), the number of embryos frozen (i.e. they were good quality embryos) and the embryo utilization (the number of embryos transferred and frozen in relation to the total number). The results showed that mean embryo grade was significantly poorer in obese patients. In support of this observation more embryos were discarded, fewer were frozen and utilization was lower in obese women. To date this is the most comprehensive analysis of embryo quality in obese women and should be verified by larger studies at other centers. Other studies which did not directly analyse oocyte quality found differences in embryo survival which suggest that oocyte quality may be altered by obesity. Reduced oocyte number is itself associated with decreased embryo quality [10]; however, low oocyte count is even more likely to result in pregnancy loss in obese women than in lean, suggesting alterations in oocyte quality with obesity. Another study [29] of unique design suggests that increased BMI, and concomitant insulin resistance in particular, influences oocyte quality in women. The retrospective study analysed endocrine parameters and IVF outcomes of women with polycystic ovaries who had undergone several failed IVF attempts and also served as oocyte donors. One group of patients (I) had displayed embryos which failed to implant in their own uteri as well as when their embryos were donated to recipients, but became pregnant on the first attempt when given donor oocytes; suggesting these women have poor oocyte quality. The second group of patients (II) displayed embryos which failed to implant in their own uterus but implanted successfully when donated to a recipient. The control group (III) was patients with morphologically normal ovaries who were undergoing IVF using their own oocytes. The groups were not different in their gonadotropin or androgen levels indicating none had overt PCOS as defined by current NIH criteria. The fertilization s of Group I were significantly reduced compared to the other groups, again indicating compromised oocyte quality in this subgroup of patients. Interestingly, Group I also had a higher mean BMI than the other groups and impaired response to an oral glucose tolerance test (OGTT) suggesting that insulin resistance in these women contributes to their reduced oocyte and embryo quality. Unfortunately, there were only four women in this group, but it would be illuminating to undertake similar analyses with larger numbers of women attending different clinics. Cumulatively these observations suggest that alterations in the oocytes and embryos in response to obesity may contribute to the decreased pregnancy s and higher early miscarriage s observed in obese women. However it is not entirely clear whether the reduced pregnancy s seen with obesity are solely due to suboptimal embryos which fail to implant or whether there are also alterations in the endometrium which do not permit implantation. To address this specific question a number of a laboratories investigated whether the BMI of women receiving donor oocytes (from women of normal BMI) exhibited reduced implantation and/or pregnancy s. Two studies found no effect of BMI on implantation, pregnancy or miscarriage s [30,31]. Another compared pairs of women who had received donor oocytes from the same donor but had discordant pregnancy outcomes [32] and found that in women who did not become pregnant BMI was not a predictor. Studies by Bellver et al. [33,34], demonstd a trend towards decreased implantation s in obese women receiving donor oocytes, but the authors also conclude that alterations to the endometrium in obese women are likely subtle compared to those at the ovarian level Oocyte developmental competence in an obese mouse model Our recent studies in a mouse model of diet-induced obesity have clearly demonstd alterations in oocyte developmental competence [35]. Female mice were fed a high-fat diet for 16 weeks during which time they gained significantly more weight and developed hyperinsulinemia and dyslipidemia compared to control mice fed a low-fat diet. The mice were then paired with males and ovulation, steroid production and oocyte developmental competence were assessed. A greater number of the obese females failed to ovulate any oocytes. Interestingly in those that did ovulate there were increased numbers of oocytes compared to controls. Levels of progesterone, which is produced by the ovary, were not different. The oocytes isolated from either obese or control mated females were then cultured in vitro in order to closely monitor their development (see Fig. 1). Fertilization s were not different in oocytes from obese females compared to oocytes from controls. However, the oocytes from obese females exhibited slower development to the 4 8-cell stage that was maintained through to the blastocyst stage [35]. These results demonst that oocytes from an obese individual, even when removed from the systemic environment and cultured in vitro, possess intrinsic differences that influence subsequent embryo development. The cell type distribution of the resultant embryos was also disrupted such that blastocysts from oocytes of obese females had a lower inner cell mass to trophectoderm ratio, indicating that blastomeres were preferentially allocated to placental precursors instead of to embryonic cells. Alterations such as these in other animal models have been shown to result in offspring of smaller size and with compromised health. In obese women even modest weight loss or increased exercise can normalize fertility [36,37]. Similarly, treatment

6 120 R.L. Robker / Pathophysiology 15 (2008) with insulin sensitizers, particularly in women with PCOS, leads to improved pregnancy s, indicating that insulin sensitivity is one of the key metabolic regulators of female fertility. To investigate whether insulin resistance influences the effects of obesity on oocyte developmental competence we next treated obese mice, as above, with one of three insulin sensitizers known to have distinct cellular actions: AICAR, a glucose and lipid-lowering AMP kinase inhibitor; sodium salicylate, an IKK inhibitor that reverses insulin resistance; or rosiglitazone, a PPAR transcription factor agonist. The obese mice were treated with the insulin sensitizers for just 4 days prior to pairing with males and reproductive parameters as above were measured and compared to untreated obese mice as well as mice on a control diet. Rosiglitazone, but not AICAR or sodium salicylate, reversed the obesity-induced defects in oocyte developmental competence [35]. Specifically treatment with rosiglitazone completely normalized embryo on-time development from the 4-cell to blastocyst stage, effectively restoring it to identical s as embryos from oocytes of females on control diet. Rosiglitizone treatment also normalized the alterations in cell allocation, such that embryos from obese females treated with rosiglitazone had identical ratios of inner cell mass and trophectoderm cells as embryos from control females [35]. These improvements were associated with corrected hyperinsulinemia and glucose metabolism, as well as reductions in circulating triglycerides. Rosiglitazone treatment also resulted in altered expression of PPRE-regulated genes in ovarian cells. Thus, acute treatment with a specific insulin sensitizer prior to conception can reverse defects in oocyte developmental competence caused by obesity, demonstrating that oocyte developmental competence is responsive to systemic insulin sensitivity prior to ovulation, while still contained in the preovulatory ovarian follicle. Rosiglitazone was the sole insulin sensitizer used in our study that was able to normalize oocyte developmental competence [35], indicating that its target, the transcription factor PPAR, is a key regulator of metabolic mechanisms controlling oocyte quality. It is not known whether activation of PPAR in these obese, insulin-resistant mice influences ovarian cells and/or the oocyte directly, or whether its effects are mediated through rapid improvements in systemic insulin sensitivity. It is interesting to note however that in humans polymorphisms in the PPAR gene are often associated with metabolic phenotypes (increased BMI and Type II diabetes) as well as altered ovarian function [38]. Neither oocyte developmental competence nor early embryo development has been assessed in women taking rosiglitazone, in fact it is not yet approved for use in pregnancy. However other insulin sensitizers, including a related PPAR agonist troglitazone as well as metformin, are commonly used to normalize ovulation s in insulin resistant women and may also positively influence pregnancy s [39]. Again, whether these effects are mediated through direct ovarian actions or systemic alterations in insulin sensitivity must be determined in order to more completely understand how female fertility is dysregulated during metabolic conditions such as obesity. 2. Conclusions Obese mice exhibit impaired oocyte developmental competence that can be reversed with the insulin sensitizer rosiglitazone. Observations from infertility clinics indicate that obese women may also have altered oocyte developmental competence and sub-optimal early embryo development that is likely to influence the decreased pregnancy s observed in these women. More work is clearly necessary however to determine the nature of any alterations in oocyte quality as well as the cellular mechanisms that have led to their impairment. This information is essential in order to more effectively treat obese IVF patients, but perhaps more importantly to better consul obese patients to lose weight prior to undergoing fertility treatments, so as to increase its effectiveness. There is also the necessity to better educate all overweight women of reproductive age about the dangers of excess body fat on their future fertility as well as the early embryonic development of their children. 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