Effects of female body mass index on embryo development and ART outcomes

WANG Ming-kun, HUANG Yuan-hua, MA Yan-lin

Hainan Provincial Key Laboratory for Human Reproductive Medicine and Genetic Research, Hainan Provincial Clinical Research Center for Thalassemia, Key Laboratory of Reproductive Health Diseases Research and Translation (Hainan Medical University), Ministry of Education,Department of Reproductive Medicine, the First Affiliated Hospital of Hainan Medical University, Hainan Medical University, Haikou 570100, China

Keywords:

ABSTRACT Objective: To investigate the effects of female body mass index on embryo development and assisted reproductive technology outcomes, aiming to provide better treatment for patients with different body mass index and provide reference for clinical treatment.Methods: The study retrospectively collected data of 3783 patients who received their first fresh embryo transfer and were ovulated by a long protocol at the Reproductive Medicine Center of the First Affiliated Hospital of Hainan Medical University from January 2015 to October 2021.Patients were divided into four groups based on body mass index (BMI): low weight group,normal weight group, overweight group and obese group.The normal weight group was used as a control to compare the basic information, assisted reproductive technology process,embryo development and assisted reproductive technology outcomes between different groups.Results: Analyzing patients' basic information, we found that the duration of infertility was significantly longer in obese women (P = 0.007).Basal hormone levels in the overweight and obese groups were lower than those in the normal group (P＜0.05).Basal Follicle-stimulating hormone (FSH), basal Luteinizing hormone (LH), basal Estradiol (E2), basal Progesterone (P),and anti-Mullerian hormone (AMH) in the obese group were lower than the normal weight group (P＜0.05), and the number of antral follicle counting (AFC) was reduced in the obese group (P=0.011).The overweight group only showed a decrease in E2 and P levels (P＜0.05).During the ART, there was a significant difference in Gonadotropin (Gn) dosage among the four groups, with the obese group was the most, followed by the overweight group, and the low weight group was the least (P＜0.001).Gn days were increased in the obese group (P＜0.001).LH, E2, and P on trigger day were all lower in the overweight and obese groups than in the normal weight group (P＜0.05).Comparing the embryo development process, we found that the blastocysts of the obese group showed delayed development at the stages of pronuclei disappearance, four-cell and blastocyst formation (P ＜ 0.05).The ART outcomes were worse in the obese group, the clinical pregnancy rate (P=0.044) and live birth rate (P=0.036) were reduced in the obese group.After logistic regression, obesity was found to be a risk factor for clinical pregnancy (OR=0.683, 95% CI: 0.479-0.973, P=0.035) and live birth (OR=0.662,95%CI: 0.459-0.954, P=0.027).Female age was a risk factor for biochemical pregnancy,clinical pregnancy and live birth (P＜0.05).Conclusion: Female obesity prolongs the duration of infertility, causes endocrine disorders, increases Gn dosage and days, and leads to poorer assisted reproductive technology outcomes.Female obesity delays the blastocyst development process and presents as a risk factor for clinical pregnancy and live birth.

1.Introduction

With the development of society and the improvement of living standards, the incidence of overweight and obesity is increasing worldwide.Dietary habits, lifestyle, genetic characteristics, and social factors interact with each other to form the basis of this chronic disease.The degree of obesity is usually measured by body mass index (BMI).The current Chinese standard defines BMI＜ 18.5 kg/m2 as low weight, BMI 18.5～23.9 kg/m2 as normal weight, BMI 24～27.9 kg/m2 as overweight, and BMI≥28 kg/m2 as obesity[1].

Abnormal body mass index usually leads to the occurrence of multi-system diseases, such as hypertension, coronary heart disease,diabetes, etc.Among them, the relationship between obesity and reproductive function has attracted more and more attention.Obesity can affect reproductive function from the aspects of endocrine,lipotoxicity, oxidative stress, inflammatory response and gene expression, and increase the risk of infertility[2,3].At present, a large number of studies have explored the relationship between body mass index and the outcome of Assisted reproductive technology(ART), but due to different sample sizes, grouping criteria, treatment regimens, operation techniques and population characteristics of different studies, there are still controversies.Some studies suggest that body mass index is not a predictor of assisted reproductive outcomes[4-6].Most studies have found that obesity impairs egg quality, affects embryo development, reduces clinical pregnancy rate and live birth rate, and has genetic effects on offspring health[7-12].Time-lapse (TL) technology has been widely used in clinical practice as an emerging technology for embryo culture and selection.TL consists of three parts: an incubator, a microscope, and a software program[13], By providing a stable culture environment and an intelligent image collection and evaluation system, the dynamic monitoring of embryos can accurately record the embryo development process and accurately observe the abnormal development events of embryos, which is helpful to select embryos with higher developmental potential to transfer[14,15].

This study retrospectively analyzed the clinical data of patients receiving ART in the Reproductive Medicine Center of the First Affiliated Hospital of Hainan Medical College.TL technique was used to collect embryo dynamics data from fertilized egg to blastocyst, aiming to explore the effect of female body mass index on embryo development and ART outcomes, so as to provide better ART treatment for patients with different body mass index, to provide reference for clinical diagnosis and treatment.

2.Materials and Methods

2.1 Subjects

A retrospective analysis was conducted on female patients who underwent ART in the Reproductive Medicine Center of the First Affiliated Hospital of Hainan Medical College from January 2015 to October 2021.A total of 3783 patients were enrolled and divided into four groups according to the WHO Asia-Pacific BMI criteria: underweight group (BMI ＜ 18.5 kg/m2), normal weight group (18.5 BMI ＜ 24 kg/m2), overweight group (24 BMI ＜ 28 kg/m2) and obesity group (BMI 28 kg/m2).The normal weight group served as the control group.The basic information, assisted pregnancy process, embryo development process and assisted pregnancy outcome of each group were compared.This study has been approved by the Ethics Committee of Hainan Medical University (HYLL-2023-298).

2.1.1 Inclusion Criteria

All the patients received fresh embryo transfer for the first time and were stimulated by long protocol.They were 20～40 years old and met the conditions of assisted pregnancy.The causes of infertility include tubal factors and male factors.Male body mass index ranged from 18.5～24 kg/m2.

2.1.2 Exclusion Criteria

Patients with polycystic ovary syndrome, clinically significant thyroid dysfunction, and diabetes mellitus with poor glycemic control were excluded.Patients with hypertension, immune system diseases, abnormal uterus, endometriosis (grade Ⅲ,Ⅳ), multiple uterine fibroids, cervical insufficiency, intrauterine adhesions, acute genitourinary infection, adverse pregnancy history, chromosome abnormalities, egg and sperm donation were excluded.Patients with missing basic information and failed follow-up were also excluded.

2.2 Methods

2.2.1 Ovulation protocol

All patients received long protocol ovulation induction, and the dose was adjusted according to the patient’s age, basic endocrine and ovarian reserve.When at least one follicle diameter 20 mm or at least two follicles diameter 18 mm or at least three follicles diameter 17 mm, 10,000 IU of Human chorionic gonadotropin(HCG) was injected intramusitoneally.Oocyte retrieval by transvaginal ultrasound was performed 34 to 36 h later.

2.2.2 Fertilization and embryo culture

In vitro fertilization (IVF) is a process in which eggs and sperm are placed in the same culture dish and co-cultured for about 16～18 h, followed by microscopic observation of fertilization.Intracytoplasmic sperm injection (ICSI) is a procedure in which sperm is injected into the cytoplasm of oocytes at metaphase of meiosis and then transferred to a culture dish for observation.Normal fertilization was defined as Two pronucleus (2PN) in the zygote.

2.2.3 Time-lapse imaging technology

All embryos were cultured in an Embryo ScoPe Plus (Vitrolife,Sweden) incubator at 37 ℃, 6% CO2and 5% O2.TL was photographed at 11 focal planes every 10 min from the beginning of fertilization to record embryo development, accurately recording the development process from fertilized egg to blastocyst.The collected images and data were entered into the Embryo Viewer (Vitrolife,Sweden) database.

2.2.4 Embryo transfer and patients follow-up

On day 3 after oocyte retrieval, the number of embryos transferred was determined according to the patient’s condition.In routine cases, 1～2 cleavage stage embryos/blastocysts can be transferred.The remaining embryos were cryopreserved with the consent of the patients.Luteal support was performed from the day of oocyte retrieval.Biochemical pregnancy was defined as positive urine HCG and serum β-HCG 25 U/L 14 d after embryo transfer.Clinical pregnancy was defined as the presence of a gestational sac on transvaginal ultrasound 35 d after embryo transfer.Patients with clinical pregnancy were referred to the department of obstetrics after 12 weeks of gestation and followed up by telephone until the assisted pregnancy outcome was obtained.

2.3 Statistics

SPSS26.0 statistical software was used to analyze the data.The quantitative data conforming to normal distribution were expressed as mean ± standard deviation (±s ).One-way ANOVA test was used for multiple group comparison, and Bonferroni method was used for pairwise comparison if there was significant difference.The quantitative data that did not meet the normal distribution were expressed as the median (interquartile range) M (IQR), and the non-parametric test (Kruskal-Wallis) was used for multiple group comparison.If there were significant differences, the Kruskal-Wallis one-way ANOVA test was used for pairwise comparison.Qualitative data were expressed as percentage (%), and chi-square test was used for comparison between groups.Binary Logistic regression was used to analyze the correlation between body mass index and the outcome of assisted reproductive technology.P＜ 0.05 was considered statistically significant.

3.Results

3.1 Comparison of basic information

The duration of infertility in the obese group was significantly longer than that in the normal weight group (F=4.020, P=0.007).The basal LH (F=4.445,P=0.004) and basal FSH (F=9.168,P＜0.001) in the obese group were lower.The basal E2 (H=65.346, P＜ 0.001) and basal P (H=19.537,P＜ 0.001) in the overweight and obese groups were lower.AMH (H=13.750, P=0.003) and AFC (F=3.702,P=0.011) in obese group were lower than those in normal weight group.There was no significant difference in age, infertility type and basal T among the four groups (P＞ 0.05).Table 1.

Tab 1Comparison of patients’ basic information

3.2 Comparison of assisted reproductive process

There was a difference in Gn dosage, the highest dose in the obese group, followed by the overweight group and the lowest dose in the low weight group (F=65.138, P ＜ 0.001).The Gn days in the obese group were longer (F=9.418,P＜ 0.001).The trigger day LH(H=9.382,P=0.025), E2(F=38.918, P ＜ 0.001) and P (F=11.131,P＜ 0.05) in overweight and obese groups were lower than those in normal weight group.There were no significant differences in fertilization mode, endometrial thickness, number of oocytes retrieved, normal fertilization rate, transferable embryo rate, highquality embryo rate, blastocyst formation rate and number of transferred embryos among the four groups (P＞ 0.05).Table 2.

Tab 2 Comparison of patients’ ART processes

3.3 Comparison of embryonic development processes

Analysis of the dynamic data of blastocysts showed that the development of blastocysts in the obese group was delayed at the tPNf, t4 and tB stages.The tPNf stage was delayed by 0.4 h(F=4.520,P=0.004), t4 (F= 3.580,P=0.013) and tB (F=4.236,P=0.004).P=0.005).There was no significant difference in other embryo dynamics data among the four groups (P ＞ 0.05).Table 3.

Tab3 Comparison of patients’ embryo dynamics data (h)

3.4 Comparison of assisted reproductive outcomes

The clinical pregnancy rate (χ2=8.122, P=0.044) and live birth rate (χ2=8.548,P=0.036) in the obese group were lower than those in the normal group.The biochemical pregnancy rate in the obese group was lower, but there was no statistical significance (P ＞ 0.05).There were no significant differences in biochemical pregnancy rate,clinical pregnancy rate and live birth rate between the low weight group, the overweight group and the normal weight group (P ＞0.05).Table 4.

Tab 4 Comparison of patients’ ART outcomes (%)

3.5 Logistic regression of assisted pregnancy outcomes

Age was a risk factor for biochemical pregnancy (OR=0.902,95%CI: 0.881-0.923,P＜ 0.001), clinical pregnancy (OR=0.901,95%CI: 0.881-0.922, P ＜ 0.001) and live birth (OR=0.892, 95%CI:0.872-0.913, P ＜ 0.001).Obesity was a risk factor for clinical pregnancy (OR=0.683, 95%CI: 0.479-0.973,P=0.035) and live birth (OR=0.662, 95%CI: 0.459-0.954, P=0.027).There was no correlation between underweight, overweight and the outcome of assisted reproductive technology (P＞ 0.05).Tables 5 and 6.

4.Discussion

In recent years, the relationship between body mass index and fem-ale fertility has attracted increasing attention.Studies have found that abnormal body mass index can lead to female infertility by affecting endocrine level, oocyte quality and endometrial receptivity [2,16-22].With the continuous development of ART technology, more and more infertility patients try to get pregnant through ART.At present,there have been a lot of studies discuss the influence of female body mass index on the outcome of assisted pregnancy, but it is still controversial due to various reasons[1,4,7].In order to reduce the bias and improve the accuracy of the results, we only included women who underwent the long protocol for the first time of fresh embryo transfer, considering that most studies did not distinguish the ovarian stimulation protocol, fresh embryo transfer/frozen embryo transfer and transfer history.

Tab 5 Logistic regression analysis variable assignment

Tab 6 Logistic regression of ART outcomes

4.1 The effect of female body mass index on infertility risk

Abnormal body mass index increases the risk of infertility in women[2], obesity has the most significant effect[23].For each 1 kg/m2 increase in BMI in underweight women, the risk of infertility decreased by 15%.For each 1 kg/ m2increase in BMI in obese women, infertility risk increased by 18%[2].A large retrospective study of 10252 transplant cycles found that obese women were infertile for an average of half a year longer, whereas the effect of low body weight was not significant[1].This is similar to the conclusion of the present study, which found that the age of obese women was not statistically different from that of normal women,but the duration of infertility was prolonged by about 0.57 years,indicating that obesity prolongs the time to pregnancy and aggravates the risk of infertility.Studies have shown that inflammatory response is associated with difficulty in preparing for pregnancy in obese women[3].Fatty acids are closely related to inflammatory response, and trophoblast cells exposed to IL-6 are prone to fatty acid accumulation and increased lipotoxicity[24].There is also a correlation between obesity and the expression of inflammatory genes.Protein phosphatase 1 gene has an up-regulation trend in obese women, which can interfere with the internal environment of the body and cause metabolic abnormalities[25].Inflammatory pathways play an important role in oocyte maturation and embryo implantation[19], it may explain why obese women are less likely to conceive.

4.2 The effect of female body mass index on basal endocrine and ovary

Leptin is a protein secreted by adipose tissue and has an inverse relationship with body mass index[3].Leptin levels in the blood and follicular fluid of obese women are increased to varying degrees[19,26], excessive leptin can inhibit Gonadotropin-releasing hormone (GnRH) secretion, reduce FSH and LH levels, and reduce P and E2 production[17,27].The present study found that the basal endocrine level decreased with the increase of BMI.The levels of FSH, LH, P and E2in the obese group were lower than those in the normal weight group.The results showed that obesity has an inhibitory effect on central endocrine axis and ovarian secretory function, which might be related to leptin content[17].There are less data on women who are underweight.Some studies suggest that there is no significant difference in the endocrine level between low weight women and normal weight women, and the effect of low body mass index on reproductive endocrinology may be limited [1,9].Studies have found that obese women have decreased ovarian function, decreased number of oocytes retrieved, reduced oocyte diameter, and abnormal morphology of cumulus complex[18,28-30].Existing studies generally agree that the dosage of exogenous Gn is proportional to body mass index[7,31,32].That is because obese women have more fat content in the body and a large amount of aromatase causes the rapid conversion of androgens into estrogens,which inhibits the secretion of GnRH and Gn through the negative feedback of the hypothalamic-pituitary-ovarian axis[29].Increased body surface area in obese patients can affect pharmacokinetics and make exogenous Gn less available[17].The results of this study are consistent with previous studies, which found that the basal follicle number was lower in overweight and obese patients, and higher doses of exogenous Gn were required to obtain similar follicle numbers as the normal group.

Obesity increases the content of reactive oxygen species through oxidative stress, stimulates the mitochondria and endoplasmic reticulum of oocytes, increases the apoptosis of the corolla colliculus complex, and hinders the development and maturation of follicles[19].Obesity also changes the content of fatty acids in follicular fluid.n-3 polyunsaturated fatty acids, as a component of neuronal membranes, are related to membrane signal transduction, ion channels and receptor functions[16].In obese women, the levels of n-3 polyunsaturated fatty acids are significantly reduced, resulting in reduced granulosa cell viability and exacerbating oocyte maturation disorders[16].

4.3 Effect of female body mass index on embryonic development

Embryos from obese women have lower developmental potential,manifested by abnormalities in meiosis and mitochondrial dynamics[19].It is concluded that the maternal metabolic environment has a significant effect on blastocyst formation, and the blastocyst formation rate of normal weight women is higher than that of overweight and obese women, while there is no significant difference in the rate of blastocyst formation between women with metabolic syndrome and obese women, suggesting that metabolic disorders may be one of the reasons for the reduction of blastocyst formation rate of obese women[33].High levels of fatty acids and adipokines have adverse effects on embryonic development[34],arachidonic acid can activate the oxidative stress pathway, increase the content of prostaglandins and thromboxanes, and delay the process of embryonic development[34].In addition, early embryo development is mainly regulated by the oocyte genome[35], the expression of genetic genes in obese women is unstable, which may interfere with the process of embryo development and affect embryo quality[36].

4.4 Effect of female body mass index on the outcome of assisted reproductive technology

With the continuous development of ART, the influencing factors of assisted reproductive outcomes have become a research hotspot.At present, a large number of studies have discussed the effect of body mass index on the ART outcome, but due to different grouping criteria, ovulation induction protocol, study subject characteristics, and outcome indicators, there are still controversies.Some studies suggest that body mass index is not a predictor of assisted reproductive outcomes[4-6,37].Studies have also shown that overweight and obesity in women reduce the clinical pregnancy rate and live birth rate, and increase the miscarriage rate[7-12,38,39].There was an inverted U-shaped association between BMI and live birth rate.As BMI increased, the live birth rate increased in underweight women and decreased in obese women, while there was no significant change in the live birth rate among women with BMI between 18.5 and 30.4 kg/m2[40].A large retrospective study included 10,252 patients who underwent frozen embryo transfer for the first time and only transplanted high-quality embryos.The results showed that implantation rate, clinical pregnancy rate and live birth rate in the low-weight group were slightly lower than those in the normal-weight group; implantation rate, pregnancy rate and live birth rate were significantly lower in the obese group, and abortion rate was significantly increased[39].Similar results were obtained in this study: the clinical pregnancy rate and live birth rate of the obese group were significantly reduced, and the pregnancy rate and live birth rate of the low weight group and the overweight group were slightly lower than those of the normal weight group, but there were no statistical significance.

4.5 Outlook

This study strictly formulated the inclusion and exclusion criteria,and all patients underwent fresh embryo transfer for the first time and underwent the long protocol, in order to reduce the occurrence of bias and improve the accuracy of the results.ART provides a new perspective on the effects of BMI, but several limitations should be considered.First, there are differences between endocrine and physiological levels of exogenous Gn applied to ovarian stimulation in women undergoing ART.Second, the medium does not fully mimic the in vivo environment and may attenuate the effects of abnormal BMI on oocytes and embryos.This study focuses on the relationship between pre-pregnancy BMI and ART outcomes.The relationship between the change of BMI and ART outcomes still need to be further explored.Considering the limitations of retrospective studies, prospective studies with large samples are needed to further investigate the effects of abnormal body mass index on embryo development and assisted reproductive outcomes.

Declaration of Conflict of Interest:

All the authors declare no conflicts of interest.

Authors’ contribution

Wang Mingkun: design the article, collect and analyze the data,write the paper;

Huang Yuanhua: Responsible for the quality control of articles

Ma Yanlin: In charge of reviewing articles.