Variations in lipid asset, body mass index (BMI) and
abdominal adipose tissue affect the female reproductive
system compromising ovulatory cycle, oocytes
and embryo quality. Obesity is associated with hyperinsulinemia
and insulin resistance causing an increased
hormonal ovarian production and a reduced synthesis
of sex hormone-binding globulin (SHBG), with subsequent
hyperandrogenism. A peripheral conversion
of ovarian and adrenal androgens determinates abnormal
secretions of gonadotropin-releasing hormone
(GnRH), a reduced peak of luteinizing hormone (LH)
and metabolites of progesterone (
Despite this fact, recent studies, did not report differences
in oocyte maturity in function of BMI variation (
The aim of our study was to evaluate possible correlations between variations in BMI, triglycerides, HDL-c, LDL-c, and total cholesterol levels and ovarian response (in terms of oocytes and embryos retrieved, oocytes and embryo quality, fertilization rate and percentage of miscarriages and pregnancies) in women undergoing intracytoplasmic sperm injection (ICSI) cycles.
We conducted a retrospective observational study on 114 couples undergoing ICSI who referred to our Human Reproductive Physiopathology Centre between April 2017 and March 2018. Each couple, before entering the study, subscribed an informed consent, and anonymity was preserved. The study protocol conformed to the ethical guidelines of the Helsinki Declaration (as revised in Tokyo 2004) and was approved by the Local Research Ethics Committee. The enrolled couples underwent ICSI for idiopathic causes after a history of primary or secondary infertility from almost one year, and met the following criteria.
Inclusion criteria: i. Female age between 18-42 years, ii. Primary or secondary infertility for not less than 12 months, iii. ICSI techniques, iv. Negative results for Hepatitis B Australia Antigen (HbsAg), Hepatitis C Virus (HCV), Human Immunodeficiency Virus (HIV) for the male and the female, v. No exposure to toxic agents reported by the couples, vi. Female BMI<35, vii. Normal semen parameters, and viii. Follicle stimulating hormone (FSH) <15 IU/mL.
Exclusion criteria: i. Female age <18 or >42 years, ii. Primary or secondary infertility for less than 12 months, iii. Assisted reproductive techniques (ART) different from ICSI, iv. Positive results for HbsAg, HCV, HIV for the male and the female, v. Exposure to toxic agents reported by the couples, vii. Female BMI >35, vii. severe oligozoospermia, severe asthenozoospermia, and/or azoospermia, viii. FSH >15 IU/mL.
Following obtaining the signed informed consent, FSH, LH, estrogen (E2) and prolactin (PRL) blood test were done on the 3rd day of menstrual cycle. PRL samples were collected three to four hours after waking up in the morning by taking three samples with 20 minutes intervals after correcting the position of the vein catheter. Moreover, blood tests were performed in order to determinate plasma concentrations of total cholesterol, LDL-c, HDL-c and triglycerides. The blood samples were obtained before starting ovarian stimulation by using separated lipoproteins, by electrophoresis or precipitation method. Finally, weight and height of the patients were recorded. According to the BMI, the samples were divided into the following groups: group A: patients with BMI of 18.5-24.9 kg/m2 (45.6%); group B: patients with BMI of 25-29.9 kg/m2 (32.4%); and group C: patients with BMI greater than 30 kg/m2 (22%). Woman who participated in the present study, had been treated with gonadotropin-releasing hormone (GnRH) agonist long protocol using leuprorelin acetate or triptorelin acetate and recombinant FSH was injected 14 days later. E2 level dosage and ultrasound control were performed three times a week after starting recombinant FSH therapy to monitor the follicular growth. When the E2 level and the follicular dimensions were adequate, human chorionic gonadotropin (hCG) triggering was performed using chorionic gonadotropin. All the pick-ups were performed 36- 38 hours after the hCG triggering. Retrieved oocytes were inseminated by ICSI and thus, the zygotes were observed at their maturation by biologist. Embryos were transferred 48-72 hours after the pick-ups. Variable considered in our study were as follows: number of retrieved oocytes and obtained embryos as well as oocytes’ and embryo quality using respectively “Pronuclear Scoring System” and “Day 3 Scoring”.
The pronuclear scoring system was used with the aim of detecting the pronuclear stage. The evaluation began about 16-20 hours after ICSI. Oocytes were examined on the heated stage of an inverted microscope equipped with Hoffman modulation contrast (200 magnification). Normally fertilized oocytes presented two clearly distinct pronuclei (2PN) and two polar bodies. This pattern correlated with increased embryo competence. The presence of one pronucleus (1PN) might be a result of errors in the fertilization process due to the asynchrony in formation/fusion of pronucleus. The formation of three different pronuclei might be due to an altered fertilization process determining a triploid zygote. The “Day 3 Scoring” system evaluates the morphological appearance of embryo on day 3. Embryos were assessed using a scoring system graded 1 to 5 according to morphology, which took into account cell number, evenness of cell division and degree of fragmentation. In the following lines, the characteristics of each grade are explained. Grade 1: 8 cells, <10% fragmentation, good cell-cell contact, absence of multinucleated blastomers, grade 2: 8 cells, 10-20% fragmentation or lacking good cell-cell contact, absence of multinucleated blastomers, grade 3: 6-7 cells or 8 cells with 20% fragmentation or uneven blastomer size, absence of multinucleated blastomers, grade 4: >8 cells or 4-6 cells or 8 cells with >20% fragmentation or uneven blastomer size or multinucleated blastomers, grade 5: <4 cells or grossly fragmented or with half of the blastomers being multinucleated.
Moreover, our study evaluated also the fertilization and rate and percentage of miscarriages as well as clinical pregnancies detected first by β-hCG blood test after 14 days from embryo transfer and then by transvaginal ultrasound visualization.
The primary endpoint was the establishment of a correlation between variations of BMI, triglycerides, HDL- c, LDL-c, and total cholesterol and ovarian response (in terms of oocytes and embryos retrieved, oocytes and embryo quality, fertilization rate and percentage of miscarriages and pregnancies) in ICSI cycles. The Shapiro-Wilk test was implemented to test the normally distribution of the variables. The level of significance of normality test for all variables was alfa=0.05. Analysis of variance (ANOVA) was applied to compare normally distributed variables. Variables with free distributions were analyzed using Kruskal-Wallis method. Normally distributed continuous data were presented as mean ± SD, while categorical data were presented as number (n) and percentage (%). Non-normally distributed data were presented as median (range).
We demonstrated that normal weight patients (group
A) and overweight patients (group B) had mean HDL-c
levels, with parameters included within the normal range.
Significantly high values were found for obese patients
(group C) with a mean value of 45.3 mg/dl (SD: 21.68,
P=0.0032) for HDL-c which was close to the inferior normal
range and the mean value for triglycerides was 101.5
mg/dl (SD: 23.7, P=0.00001,
Levels of blood lipids
|Variables (mg/dl)||Group A||Group B||Group C|
|Mean ± SD||P value||Mean ± SD||P value||Mean ± SD||P value|
|Total cholesterol||183 ± 33.43||0.23||178 ± 23.89||0.12||165 ± 21.79||0.071|
|LDL||110 ± 31.14||0.14||101 ± 25.71||0.13||101 ± 23.89||0.16|
|HDL||64.8 ± 17.49||0.16||60.4 ± 10.34||0.13||45.3 ± 21.68||0.0032|
|Triglycerides||68.1 ± 19.30||0.08||62.9 ± 21.08||0.24||101.5 ± 23.7||0.00001|
P<0.05 were considered significant. LDL; Low-density lipoprotein and HDL; High-density lipoprotein.
Number of retrived oocytes, fertilized oocytes and embryo obtained from each group
|Variables||Group A||Group B||Group C|
|Mean ± SD||P value||Mean ± SD||P value||Mean ± SD||P value|
|Retrived oocytes||7.88 ± 5.99||0.32||6.25 ± 4.15||0.13||3.0 ± 3.16||0.00001|
|Fertilized oocytes||2.97 ± 1.36||0.22||2.19 ± 1.32||0.003||1.25 ± 1.50||0.00001|
|Total embryos||1.81 ± 1.15||0.25||1.94 ± 1.06||0.71||1.00 ± 1.15||0.0036|
P value are significant if <0.05.
Number and percentage of oocytes in stage of maturation and zygotes pronuclear number
|Oocytes/Zigotes||Median||Group A (%)||Group B (%)||Group C (%)|
|M1||18 (20-3)||11.5 (20/173)||14 (18/127)||7.5 (3/40)|
|M2||95 (136-34)||78.6 (136/173)||75 (95/127)||85 (34/40)|
|GV||12 (13-3)||7 (12/173)||10 (13/127)||7.5 (3/40)|
|DEG||1 (5-0)||2.9 (5/173)||1 (1/127)||0 (0/40)|
|PN1||5 (7-0)||4.1 (5/122)||7.7 (7/90)||0 (0/25)|
|PN2||83 (20-117)||95.9 (117/122)||92.3 (83/90)||80 (20/25)|
|PN3||0 (5-0)||0 (0/122)||0 (0/90)||25 (5/25)|
Median (range) is used to indicate the middle value of zygotes in a determinate stage of maturation and with a determinate pronuclear number. Percentage is used to indicate the number of zygotes in a determinate stage of maturation and the percentage of zygote with a determinate pronuclear number in each group. M1; Immature retrieved oocytes in metaphase I, M2; Mature retrieved oocytes in metaphase II, GV; Germinal vesicular, DEG; Degenerated oocytes, and PN1-PN2-PN3; Zygotes with 1 pronucleus, 2 pronuclei or 3 pronuclei.
Number and percentage of embryo descripted by day 3 scoring system
|Embryos||Median||Group A (%)||Group B (%)||Group C (%)|
|G1||16 (42-0)||39.6 (42/106)||22.5 (16/71)||0 (0/18)|
|G2||25 (46-6)||43.4 (46/106)||35.0 (25/71)||33.3 (6/18)|
|G3||14 (16-5)||15.1 (16/106)||20.0 (14/71)||27.7 (5/18)|
|G4||2 (5-0)||1.9 (2/106)||7.0 (5/71)||0 (0/18)|
|G5||0 (2-0)||0 (0/106)||2.8 (2/71)||0 (0/18)|
|DEG||7 (9-0)||0 (0/106)||12.7 (9/71)||40 (7/18)|
Median (range) is used to indicate the middle value of embryo in a determinate stage of maturation percentage is used to indicate the stage of maturation of embryo catalogued by day 3 scoring system in each group. G1; Grade 1 embryo according day 3 scoring system, G2; Grade 2 embryo according day 3 scoring system, G3; Grade 3 embryo according day 3 scoring system, G4; Grade 4 embryo according day 3 scoring system, G5; Grade 5 embryo according day 3 scoring system, and DEG; Degenerated embryo.
Number and percentage of positive βhCG, biochemical miscarriages, clinical miscarriages and clinical pregnancies
|Embryos||Median||Group A (%)||Group B (%)||Group C (%)|
|Positive βhCG||3 (17-2)||16 (17/106)||4.2 (3/71)||11.1 (2/18)|
|Biochemical miscarriages||0 (3-0)||1.8 (3/106)||0 (0/71)||0 (0/18)|
|Clinical miscarriages||2 (2-2)||1.8 (2/106)||2.8 (2/71)||11.1 (2/18)|
|Clinical pregnancies||1 (12-0)||1.9 (12/106)||1.4 (1/71)||0 (0/18)|
βhCG; Beta human chorionic gonadotropin. Median (range) is used to indicate the middle value of positive βhCG, biochemical miscarriages, clinical miscarriages and clinical pregnancies. Percentage is used to indicate the percentage of positive βhCG, biochemical miscarriages, clinical miscarriages and clinical pregnancies for each group.
The present study aimed to investigate the relationship
between obesity and lipid variations and to determinate
their association with ovarian response in terms
of oocyte maturation, fertilization rate, embryo quality
and pregnancy rate in women undergoing ICSI. Our
literature survey demonstrated that obesity is strongly
associated with metabolic syndrome (
Considering the effect of obesity on embryo quality,
Metwally et al. (
Finally, results of our study reported a rate of 19.1%
for β-hCG positivity, with greater success among normal-
weight, compared to overweight and obese patients.
The pregnancy rate was 85.7% (12/14 pregnancies) in
group A, and 14.3% (2/14 pregnancies) in group B, but
no conception in group C. Consistent with our findings,
as far as the pregnancy and live birth rate is concerned,
Luke et al. (
Our study demonstrated that an excess of adipose tissue in women undergoing ICSI was not directly related with altered values of lipoprotein taken in consideration in our study. Overweight and obese patients (BMI: 25-34) showed poor fertilization and pregnancy rates despite the not altered levels of lipoprotein. Strengths of our work were the accurate collection of data on oocyte and embryo quality, as well as the careful processing of collected values. However, as our study was conducted in a small population, further research should be done to better understand the pathogenic mechanisms underlying poor reproductive outcomes in obese and overweight women. Finally, we believe that young women of reproductive age should be appropriately advised about the negative effects of obesity and insulin resistance on fertility, in order to perform some lifestyle modification.