A higher preconceptional paternal body mass index influences fertilization rate and preimplantation embryo development

Abstract Background Obesity is a worldwide problem affecting the health of millions of people throughout the life course. Studies reveal that obesity impairs sperm parameters and epigenetics, potentially influencing embryonic development. Objective To investigate the association between preconceptional paternal body mass index (BMI) and embryo morphokinetics using a time‐lapse incubator and in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) outcomes. Materials and methods Participants were recruited from a tertiary hospital in this prospective periconceptional cohort study. A total of 211 men were included: 86 with normal weight (BMI < 25.0), 94 overweight (BMI 25–29.9), and 41 obese (BMI ≥ 30). These men were part of a couple that underwent IVF/ICSI treatment with ejaculated sperm after which 757 embryos were cultured in a time‐lapse incubator. The main outcome parameters consisted of fertilization rate, embryo developmental morphokinetics, embryo quality assessed by a time‐lapse prediction algorithm (KIDScore), and live birth rate. Results A higher paternal BMI was associated with faster development of the preimplantation embryo, especially during the first cleavage divisions (t2: −0.11 h (p = 0.05) and t3: −0.19 h (p = 0.01)). Embryo quality using the KIDScore was not altered. The linear regression analysis, after adjustment for confounders (paternal age, ethnicity, smoking, alcohol use, education, total motile sperm count, and maternal age and BMI), showed an inverse association between paternal BMI and fertilization rate (effect estimate: −0.01 (p = 0.002)), but not with the live birth rate. Discussion and conclusion Our data demonstrate that a higher preconceptional paternal BMI is associated with a reduced fertilization rate in IVF/ICSI treatment. Our findings underline the importance of a healthy paternal weight during the preconception period.


INTRODUCTION
Overweight and obesity are worldwide problems affecting the health of millions of people throughout the life course. 1 The pathophysiologic origin of obesity is complex and results from the interplay between inadequate dietary intake, limited exercise, and genetic predisposition. 2 The prevalence of obesity is also high in the reproductive population, with estimates up to 50%. The influence of obesity on reproductive health is widely studied in women, showing lower oocyte and embryo quality, a longer time to pregnancy, and increased risks of congenital malformations, miscarriages, [3][4][5] preeclampsia, preterm birth, and fetal death. 6 However, the negative effects of obesity in men in the reproductive period are largely neglected and understudied. With the global burden of the male obesity, 7 we hypothesize that periconceptional paternal obesity also affects reproductive outcome.
Studies reveal that obesity impairs sperm concentration, motility, and sperm deoxyribonucleic acid (DNA) quality. 8,9 Obesity is characterized by a systemic and chronic inflammatory state, with adipocytes continuously releasing inflammatory factors and thereby inducing a pro-inflammatory state and excessive oxidative stress that increases sperm DNA damage. 10 The increased exposure to reactive oxidative species due to excessive oxidative stress is also associated with changes in DNA methylation patterns and chromatin constitution during spermatogenesis, with a potential impact on a paternal epigenetic contribution on subsequent embryogenesis. [11][12][13] The influence of paternal obesity on the preimplantation embryo development has scarcely been studied in human with conflicting results. 14 In vitro preimplantation embryo development is of interest since it provides a unique insight into the direct impact of paternal factors through sperm, undisturbed by the effects of the maternal in vivo uterine environment. Since obesity itself is associated with sperm quality parameters and pregnancy chance, we hypothesize that preconception paternal weight is also associated with preimplantation embryo development and fertility treatment outcomes. Maternal health also remains of interest since primordial germ cells up to the oocyte maturation period are exposed to the intrinsic maternal environment. In addition, maternal obesity is known to be associated with decreased oocyte quality. 15 Preimplantation embryo development can be studied using timelapse embryo culture, which uses incubators with a built-in microscope designed for automated time-lapse embryo assessment by acquiring images. This provides a controlled culture environment and captures comprehensive information on embryo development without the need for handling or disturbing the developing embryo. The use of timing of embryo developmental events, also referred to as embryo morphokinetic parameters, has added another dimension to current traditional morphology classification scores.
Because of the epidemic burden of obesity, which also involves men of reproductive age, the main aim of this study is to investigate associations between preconception paternal obesity and developmental morphokinetics of preimplantation embryos and specific in vitro fertilization-intracytoplasmic sperm injection (IVF-ICSI) treatment outcomes.

Study design, population, and patient inclusion
Couples were enrolled in the prospective virtual embryoscope study, which is embedded in the Rotterdam Periconception Cohort (predict study). 16 The predict study is an ongoing prospective tertiary hospitalbased cohort embedded in the outpatient clinic of the Department

In vitro fertilization procedures
Ovarian stimulation, oocyte retrieval, the IVF/ICSI procedures, and assessment of embryo morphology were performed as described previously. 17  Fertility treatment outcomes were retrieved from medical records.

Statistical analysis
Baseline characteristics of men are depicted as median or number with the corresponding interquartile range (IQR) or percentage. All analyses were performed using SPSS package 25

Baseline
After inclusion of a total of 396 preconceptional subfertile couples, patients were excluded because of no paternal participation (n = 52), no available time-lapse data (n = 28), total fertilization failure (n = 12), oocyte donation/vitrification (n = 2), more than 1 year between study intake and fertilization (n = 5) and use of surgically retrieved sperm BMI of men with total fertilization failure was not significantly different from normal fertilization (28.4 kg/m 2 and 26.7 kg/m 2 (p = 0.47)).
Also, after logistic regression analysis we did not find an association between paternal BMI and total fertilization failure (p = 0.20).   (Table S1). Significantly more men were highly educated in the normal weight group (54.7%) compared to the overweight (35.5%) and obese group (22.0%) (p = 0.01).

Embryo morphokinetic parameters
The crude results of the linear mixed models for paternal BMI as con-

Embryo quality and treatment outcomes
Embryo morphokinetic quality and implantation potential was assessed using the KIDScore. In the proportional odds model, paternal BMI was not associated with the KIDScore (beta: −0.01 (p = 0.64)), which remained the same after adjustment for confounders (beta: −0.01 (p = 0.62)) ( Table 2).   In normal weight men of the total study group, the fertilization rate was 0.88, whereas the rate decreased significantly to 0.81 and 0.76 in the overweight and obese groups. A comparable, but not significant decrease in the fertilization rate was also observed after both IVF and ICSI treatment (Table S2). In linear regression analysis, paternal BMI was negatively associated with fertilization rate (beta −0.01 (p = 0.001)), meaning that with every increase in paternal BMI point, the fertilization rate decreased 1%.
After adjustment for both maternal and paternal confounders, the negative association remained significant (beta −0.01 (p = 0.002)) ( Table 2). Results are comparable when also including men from couples that were initially excluded due to total fertilization failure (beta −0.01 (p = 0.005)). The embryo usage rate was not associated with paternal BMI after adjustment for confounders (beta −0.001

Main findings
In this study, we show that a higher paternal BMI is associated with a reduced fertilization rate and faster development of the preimplantation embryo, with stronger effects on the first cleavage divisions as compared to the 6-to 8-cell stage. Paternal BMI was not associated with overall morphokinetic embryonic quality of the day 3 embryo, as determined by the KIDScore embryo evaluation algorithm, the embryo usage rate, embryo implantation, and live birth rate.

Strengths and limitations
Strength of this study is the use of a standardized method to determine BMI by the same two researchers over the complete study period, instead of relying on self-reported data. We adhered to the WHO TA B L E 2 Periconceptional paternal body mass index (BMI) and associations with sperm quality, IVF/ICSI treatment outcomes and pregnancy outcomes Crude Model 1  Female germ cells enter and undergo the first part of meiosis during the fetal development, and resulting oocytes are then exposed to the intrinsic maternal environment, determined by multiple biologic pathways and exposures for many years before meiotic resumption and ovulation. For example, maternal obesity is associated with excessive chronic oxidative stress which can also lead to decreased oocyte quality as evidenced by lower rates of normally fertilized oocytes and decreased ongoing pregnancy rates. 15,22 A limitation is that our study was conducted in a time period that culture until day 3 was routine clinical practice in most hospitals, as well as in ours. Therefore, future research should also investigate the associations between periconceptional parental nutrition and lifestyle factors and the outcome after embryo culture until day 5, that is, blastocyst quality. Since the study is an observational cohort, we adjusted for potential maternal and paternal confounders, however, residual confounding cannot be fully excluded. We also did not correct for other potential important confounders, such as stress and medication.

Interpretation
Our results regarding the impact of BMI on embryo morphokinetics are only supported by previous studies in obese women. These showed a delay in the first cleavage divisions of embryos. 23,24 In addition, Leary et al. showed that embryos from women with overweight and obesity develop faster into a blastocyst. In this study, alterations in glucose and pyruvate metabolism of the embryo were suggested as an underlying cause, as embryo metabolism is determined by the oocyte and the oocyte may directly inherit such impairments from the maternal environment. 25 Before fertilization is carried out, sperm are selected by density gradient centrifugation and washing, removing the seminal fluid. The influence on the embryo development of any nutrients present in seminal fluid is therefore limited.
We show negative associations between paternal body weight and fertilization rate. Reports in the literature are conflicting: studies where less than 300 participants were included, reported no association, 26,27 whereas studies with over 600 participants showed negative associations. 28 While we show no association between paternal BMI and live birth rate, a recent meta-analysis showed that paternal obesity is linked to a decreased live birth rate (OR 0.88). 29 The seven included studies combined included more than 13.000 IVF-ICSI cycles.
Importantly, each individual study (ranging from 170 cycles to 25,000 cycles) found non-significant results, indicating a power problem. It is likely that this is also the case in our study with 221 IVF-ICSI cycles.
Obesity has been shown to negatively influence sperm quality and sperm DNA damage. Different underlying mechanisms, such as (epi)genetic, endocrinological, and environmental effects, are described in the literature linking obesity to sperm quality. 28 Obesity is strongly linked to decreased sperm count via hyperinsulinemia, increased scrotal temperature, and increased oxidative stress. 30 The Necdin (NDN), but higher levels on the H19 gene. 33 A recent study shows that paternal aging can impact on the fertilization rate and day 5 embryo quality and that is preceded by deregulated methylation at imprinted genes in sperm. 34 Interestingly, the methylation status of sperm is dynamic and under environmental pressure. More than 1500 unique genes had altered sperm DNA methylation profiles in six men undergoing Roux-en-Y gastric bypass surgery already 1 week after the surgery, which remained until at least 1 year after surgery. 35 It remains unknown which factors cause obesity related sperm DNA-methylation differences, however some hypotheses are proposed in the current literature. Several studies have demonstrated that the sperm epigenome is responsive to dietary factors and that negative and positive influences are transferred to future generations. [36][37][38] Obesity in general is strongly associated with elevated estrogen levels, both in women and men. Results from animal studies suggest that increased exposure to estrogen, by increased activity of aromatase present in fat tissue, may lead to abnormal methylation patterns in sperm cells providing a possible mechanism how body fat mass can impact DNA methylation. 30,39 Recently, a novel potential epigenetic mechanism was identified. 40,41 Sperm cells carry different types of ribonucleic acid (RNA) and also the epididymal epithelium produces exosome vesicles, which are able to transfer RNA molecules to the passing sperm cells. 42 In mice, such RNAs have been shown to be critical for fertilization and pre-and post-implantation embryo development. 43,44 A study in human sperm cells identified the level of expression of a large number of these human sperm RNAs to be responsive to BMI. 41 From these data, we hypothesize that obesity-related molecular mechanisms, hormonal imbalances, diet, and other obesity-related factors can be involved in the causation of (epigenetic) changes in the sperm quality of obese men. The exact underlying pathophysiologic mechanism in our study remains to be elucidated and future research should focus on investigating the role of underlying pathophysiological mechanisms.
We do not show any significant effect of paternal BMI on the pregnancy rate, fetal heartbeat at 12 weeks and live birth rate. This can be explained by the IVF-ICSI procedure itself, either the ovarian stimulation, culture medium or the fact that with ICSI the embryologist selects the sperm cell, which are additional factors influencing pregnancy and live birth rate and could overrule the epigenetic effects of paternal obesity on sperm.

CONCLUSION
In this study, we show that paternal body weight has a strong negative association with fertilization rate and embryos that develop faster especially in the first cleavage divisions. We found no associations between paternal body weight and pregnancy chance and live birth rate. Explanations for our findings might be the induced alterations in sperm quality, DNA damage, and epigenetic programming caused by chronic exposure to excessive oxidative stress and altered glucose and estrogen levels. Our results demonstrate a paternal impact on the pre-implantation embryo development with potential consequences for the post-implantation embryo. Therefore, more research has to be done to investigate if there is a direct impact of paternal obesity on the reproductive outcomes through mechanisms such as excessive intrinsic oxidative stress. In general, it remains important to advise overweight and obese men to achieve a healthy nutritional state and lose weight prior to treatment to optimize the outcome of a time intensive and expensive fertility treatment.