Tobacco consumption is associated with slow-growing day-6 blastocysts.

A total of 1,103 women were included during the study period. They underwent combined totals of 941 cycles (85.3%) that led to at least one blastocyst at D5 exclusively and 162 (14.7%) that exclusively obtained blastocysts at D6 after prolonged embryo culture. A flowchart ( Fig. 1 ) depicts the number of cycles with at least one expanded blastocyst at D5 compared with cycles with at least one blastocyst electively expanded at D-6. Figure 1 Patient inclusion flowchart. ICSI = intracytoplasmic sperm injection; IVF = in vitro fertilization. Patient inclusion flowchart. ICSI = intracytoplasmic sperm injection; IVF = in vitro fertilization. Both the women’s ages and their BMIs were similar between the D5 and D6 groups, as were the men’s ages and their BMIs ( Table 1 ). The type and cause of infertility, parity, and history of miscarriage were not significantly different between the D5 group and the D6 group. Conversely, a significantly higher mean number of previous ART cycles (ART cycle rank 2.1 ± 1.4 vs. 1.6 ± 1.1; P <.001) and a significantly higher proportion of women who actively smoked ( 18 , 19 , 20 ) (n = 22/162 [13.6%] vs. n = 82/941 [8.7%]; P =.05) were encountered in the D6 group compared with the D5 group. Regarding ovarian reserve markers, the serum antimüllerian hormone (AMH) concentration was significantly lower in the D6 group compared with the D5 group (3.4 ± 2.6 ng/mL vs. 4.2 ± 4.4 ng/mL; P =.02), as was the antral follicle count (AFC; 18.7 ± 11.3 vs. 22.2 ± 12.8; P <.01), whereas the D3 FSH level and the proportion of women with AMH level <1 ng/mL (n = 15 [9.3%] vs. 71 [7.5%]; P=. 45) were similar between the two groups. ICSI was used more frequently in the D6 group for fertilization compared with the D5 group (n = 131/162 [80.9%] vs. n = 661/941 [70.2%]; P <.01), although the prevalence of male-related infertility was similar between the two groups. Regarding the ovarian response to stimulation, despite the use of a significantly higher dose of gonadotropins in the D6 group compared with the D5 group during the ( P <.01), the E 2 levels at triggering were significantly lower in the D6 group compared with the D5 group ( P =.03), and the mean number of oocytes retrieved ( P <.01) and consequently the mean number of blastocysts ( P <.01) in the D6 group were lower than in the D5 group ( Table 2 ). A multivariate analysis was performed to adjust for potential confounding factors ( Table 3 ). The following variables were found to be independently associated with the entire embryo cohort reaching delayed blastocyst expansion at D6 rather than D5: female active smoking during ART (OR 2.33,95% CI 1.16-4.70; P =.02); previous ART cycles (OR 1.40, 95% CI 1.13-1.72; P <.01); and lower AFC (OR 0.97, 95% CI 0.94-0.99; P =.03). Table 3 Risk factor for cohorts of exclusively day-6 embryos: multiple logistic regression model. Variable a OR (95% CI) P value ART rank b 1.40 (1.14-1.72) <.01 Active smoking 2.33 (1.16-4.70) .02 AFC 0.97 (0.94-0.99) .03 Note: AFC = antral follicle count; ART = assisted reproductive technology; CI = confidence interval; OR = odds ratio. a Variables included in the model: female and male age. b ART rank: total number of ART cycles performed per woman, female and male body mass indexes, parity, previous miscarriage, type and cause of infertility, women's smoking habits, ovarian reserve parameters (FSH, antimüllerian hormone, AFC), type of ovarian stimulation protocol, type of triggering, total dose of gonadotropins used, E 2 level at triggering, sperm origin (donor, partner, or frozen from epididymis or testis), technique of fertilization used, and number of metaphase II oocytes. Risk factor for cohorts of exclusively day-6 embryos: multiple logistic regression model. Note: AFC = antral follicle count; ART = assisted reproductive technology; CI = confidence interval; OR = odds ratio. Variables included in the model: female and male age. ART rank: total number of ART cycles performed per woman, female and male body mass indexes, parity, previous miscarriage, type and cause of infertility, women's smoking habits, ovarian reserve parameters (FSH, antimüllerian hormone, AFC), type of ovarian stimulation protocol, type of triggering, total dose of gonadotropins used, E 2 level at triggering, sperm origin (donor, partner, or frozen from epididymis or testis), technique of fertilization used, and number of metaphase II oocytes.

The study was conducted from January 2012 to December 2018 in the ART unit at the reproductive medicine center of our university-based institution. Infertile patients who underwent in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) cycles in a freeze-all strategy ( 15 , 16 ) in our center with a prolonged embryo culture leading to at least one elective blastocyst viable for implantation were considered. Each woman was included only once (first ART cycle performed in our center). The data had been collected and recorded in a registered database. The inclusion criteria were as follows: 1) a requirement for ART with IVF or ICSI; 2) age ≤42 years at the time of the oocyte retrieval; 3) at least one expanded blastocyst electively vitrified at D5 or D6. The exclusion criteria were: 1) having both D5 and D6 blastocysts vitrified; and 2) having been included previously. Two groups were compared: patients with an entire cohort of blastocysts that reached blastocyst stage at D5 (the D5 group) and those with an entire cohort of blastocysts that reached blastocyst stage at D6 (the D6 group). This study was reviewed and approved by the Ethics Committee of Cochin Hospital (research license AAA-2019-08015) on June 27, 2019. Conventional IVF or ICSI was performed based on the presence or absence of male-factor infertility and ovarian response to stimulation as appropriate. In case of non–male-factor infertility, another indication for ICSI was low oocyte yield (≤4 oocytes) ( 17 ). The embryos were cultured individually in 50 μL Global medium droplets (LifeGlobal) under mineral oil (Origio). The dishes were prepared 24 hours in advance and left in the incubator to equilibrate in an atmosphere of 5.5% CO 2 and 5% O 2 at 37°C. Fertilization was assessed 16–18 hours after insemination or injection, and the embryos were maintained in extended culture in tri-gas incubators. Quality assessment after blastocyst culture was performed on the morning of D5 and on D6 if the blastocyst expansion had not occurred by D5. The blastocysts were graded according to the Gardner criteria ( 3 ). At D5, only expanded blastocysts were retained. Nonexpanded blastocysts on D5 (blastocele expansion less than grade B3, including morula stage, B1, and B2) were cultured for an additional day (i.e., until D6), and only expanded blastocysts were retained at D6 (i.e., exceeding or equal to grade B3). The general characteristics of the patients in both of the groups were recorded prospectively during their medical consultations ( Tables 1 and 2 ). All of the data were compiled into a digital database and analyzed with the use of SPSS software. A P value of <.05 was considered to be statistically significant. For univariate statistical analysis, we used the Pearson χ 2 test or Fisher exact test for qualitative variables and the Mann-Whitney test for quantitative variables. To identify factors associated with having an entire cohort of D6 blastocysts, we performed a logistic regression analysis. The factors were tested by means of univariate analysis and added in a multiple logistic regression model. We included all of the available factors that could be linked to our outcome in the multiple logistic regression model. The degree of correlation between variables was tested, and when two variables were highly correlated, we introduced only one of them and suppressed the other in the model, such as for the number of metaphase II oocytes and the number of oocytes retrieved (Spearman correlation 0.930; P <.001; the last parameter was excluded). In case of significant differences, odds ratios (ORs) and their 95% confidence intervals (95% CIs) were calculated. Table 1 Patient characteristics. Characteristic Overall (n = 1,103) Day-5 blastocyst group (n = 941) Day-6 blastocyst group (n = 162) P value Female  Age at retrieval, y 34.7 ± 4.3 34.6 ± 4.4 34.9 ± 3.6 .447 c  BMI, kg/m 2 23.3 ± 4.4 23.3 ± 4.3 23.6 ± 4.8 .582 c  Women actively smoking a 104 (9.4) 82 (8.7) 22 (13.6) .050 d  Parity 0.2 ± 0.5 0.2 ± 0.5 0.2 ± 0.4 .755 c  Previous history of miscarriage 205 (18.6) 172 (18.3) 33 (20.4) .527 d  Ovarian reserve at day 3  AFC 21.7 ± 12.6 22.2 ± 12.8 18.7 ± 11.3 <.001 c  AMH (ng/mL) 4.1 ± 4.2 4.2 ± 4.4 3.4 ± 2.6 .020 c  FSH (IU/L) 6.8 ± 2.2 6.8 ± 2.2 6.9 ± 2.2 .725 c Male  Age, y 38.8 ± 6.9 38.8 ± 6.8 38.7 ± 7.4 .735 c  BMI, kg/m 2 24.7 ± 3.9 24.7 ± 3.9 24.5 ± 4.0 .369 c Couple  Type of infertility .348 d  Primary 790 (71.6) 669 (71.1) 121 (74.7)  Secondary 313 (28.4) 272 (28.9) 41 (25.3)  Cause of infertility .774 d  Tubal factor 101 (9.2) 88 (9.4) 13 (8.0)  Male factor 335 (30.3) 281 (29.8) 54 (33.3)  Endometriosis 292 (26.5) 253 (26.9) 39 (24.1)  Ovulatory factor 73 (6.6) 65 (6.9) 8 (4.9)  Idiopathic 117 (10.6) 97 (10.3) 20 (12.3)  Diminished ovarian reserve 42 (3.8) 34 (3.6) 8 (4.9)  More than one etiology 143 (13.0) 123 (13.1) 20 (12.3) ART cycle rank, b n 1.7 ± 1.1 1.6 ± 1.1 2.1 ± 1.4 <.001 c Note: Data are presented as mean ± standard error of the mean or n (%). AFC = antral follicle count; AMH = antimüllerian hormone; ART = assisted reproductive technology; BMI = body mass index. a Women actively smoking ( 19 , 20 ): defined as a patient who answered “yes” to the following question: “Did you smoke a cigarette during the past 30 days?” ( 18 ). b Cycle rank: total number of ART cycles performed per woman. c Mann-Whitney test. d Pearson chi-square test. Table 2 Ovarian stimulation characteristics. Characteristic Overall (n = 1,103) Day-5 blastocyst group (n = 941) Day-6 blastocyst group (n = 162) P value Stimulation protocol .653 a  Antagonist protocol 912 (82.7) 782 (83.1) 130 (80.2)  Long agonist protocol 74 (6.7) 61 (6.5) 13 (8.1)  Short agonist protocol 117 (10.6) 98 (10.4) 19 (11.7) Total dose of gonadotropins, IU 2,336.8 ± 945.1 2,290.7 ± 929.4 2,604.7 ± 992.6 <.001 b Type of triggering .356 a  hCG 281 (25.5) 235 (25.0) 46 (28.4)  GnRH agonist 822 (74.5) 706 (75.0) 116 (71.6) ART procedure .006 a  IVF 311 (28.2) 280 (29.8) 31 (19.1)  ICSI 792 (71.8) 661 (70.2) 131 (80.9) Sperm origin .218 a  Sperm donation 35 (3.2) 31 (3.3) 4 (2.5)  Frozen testicular or epididymal 53 (4.8) 41 (4.4) 12 (7.4)  Ejaculated sperm from partner 1015 (92.0) 869 (92.3) 146 (90.1) Estradiol at ovulation induction, pg/mL 2,169.1 ± 1,295.9 2,197.6 ± 1,306.4 2,001.9 ± 1,223.8 .034 b No. of retrieved oocytes 11.4 ± 6.8 11.7 ± 6.8 9.3 ± 5.9 <.001 b Mean no. of MII 9.1 ± 5.5 9.4 ± 5.6 7.5 ± 4.8 <.001 b Mean no. of blastocysts 2.8 ± 2.3 3.0 ± 2.4 1.4 ± 1.0 <.001 b No. of expanded blastocysts <.001 a  1 407 (36.9) 279 (29.6) 128 (79.0)  2 255 (23.1) 237 (25.2) 18 (11.1)  ≥3 441 (40.0) 425 (45.2) 16 (9.9) Note: Data are presented as mean ± standard error of the mean or n (%). ART = assisted reproductive technology; ICSI = intracytoplasmic sperm injection; IVF = in vitro fertilization; MII = metaphase II oocytes. a Pearson chi-square test. b Mann-Whitney test. Patient characteristics. Note: Data are presented as mean ± standard error of the mean or n (%). AFC = antral follicle count; AMH = antimüllerian hormone; ART = assisted reproductive technology; BMI = body mass index. Women actively smoking ( 19 , 20 ): defined as a patient who answered “yes” to the following question: “Did you smoke a cigarette during the past 30 days?” ( 18 ). Cycle rank: total number of ART cycles performed per woman. Mann-Whitney test. Pearson chi-square test. Ovarian stimulation characteristics. Note: Data are presented as mean ± standard error of the mean or n (%). ART = assisted reproductive technology; ICSI = intracytoplasmic sperm injection; IVF = in vitro fertilization; MII = metaphase II oocytes. Pearson chi-square test. Mann-Whitney test.

Active smoking in women undergoing ART is associated with an increased time frame to achieve blastocyst expansion after prolonged culture: Patients who smoked exhibited blastocyst expansion at D6 rather than D5. A cohort of exclusively D6 blastocyst was also associated with a higher number of previous ART cycles and lower AFC. The identification of deleterious components impairing embryo development to the blastocyst stage is of considerable relevance for improving ART outcomes. This is particularly so for preventable and reversible factors such as smoking. The present study shows a correlation between women actively smoking and a delay in blastocyst expansion. However, further studies could establish a causative impact by testing the effectiveness of smoking cessation on blastocyst development.

This study demonstrates that, compared with a D5 cohort, obtaining an exclusively D6 blastocyst cohort was associated with active smoking, based on univariate or multivariate analysis. In addition, previous ART cycles and lower AFC were factors significantly associated with D6 cohorts. To our knowledge, this study is the first to investigate the factors associated with the day of blastocyst expansion (i.e., D5 vs. D6), especially a negative impact of active smoking by women. Although there have been studies that investigated the link between ART characteristics and embryo development, most of them evaluated the influence of only one isolated parameter and generally focused on early embryo morphokinetic aspects rather than on features associated with the developmental capacity to the blastocyst stage. Moreover, this study is the first to show a negative impact of active smoking by women on the time to blastocyst expansion, although no previous study compared blast formation at D5 or D6, particularly in the context of cigarette smoking ( 21 , 22 , 23 ). According to a number of authors, the dose of recombinant FSH, the E 2 level on the day of hCG trigger, and the type of GnRH analogue used during ovarian stimulation affect early embryo developmental kinetics ( 24 ,  25 ). Bodri et al. similarly reported a difference between standard IVF and ICSI techniques on late embryo development, with slightly faster embryo development following ICSI ( 26 ). Bellver et al. reported similar morphokinetic patterns in embryos regarding the women’s weight ( 27 ). Another strength of the present study is that we performed a multivariate analysis after adjustment for several confounders to isolate independently associated factors related to the timing of blastocele expansion at D5 versus D6. Strength also lies in the methodologic design, with a large number of patients (1,103 women) included. Thus, our study’s sample size is likely to have minimized statistical errors. In addition, numerous epidemiologic variables were collected prospectively through face-to-face interviews before ART treatment (regarding infertility data and ovarian stimulation characteristics) to avoid memorization bias and to allow multivariate analysis for adjustment on several confounders. Despite the precautions taken, our study may nonetheless be subject to certain shortcomings and biases. One limitation is the self-reporting of smoking (based on personal interviews) ( 19 ). Thus, there could be a risk of underestimation in this regard because we have no other information about the quantity of tobacco consumption ( 28 ). In addition, it cannot be ruled out that other female or male clinical predictive factors are involved. Other lifestyle factors (e.g., alcohol consumption, recreational drug use) could have influenced blastulation time. Unfortunately, those are not exhaustively available in our database. For example, the proportion of male smokers was not known. A male partner who smokes has been associated with detrimental effects on ART outcomes, although the results are still controversial ( 23 , 29 ). Indeed, a meta-analysis did not report a significant effect of active smoking by male partners on ART outcomes ( 30 ). A number of differences persisted between the groups. The ICSI procedure was performed more frequently in the D6 group compared with the D5 group, and a subsequent impact on blastulation cannot be ruled out. However, recent data in the literature suggest that ICSI techniques may be associated with a slightly faster embryonic development compared with standard IVF ( 26 ), although those authors embryonic development to D5 was assessed and not to D6. In our protocols, the ICSI technique is mainly used in case of male-associated infertility or in case a small cohort of oocytes is obtained after stimulation ( 17 ) . Given that the proportion of male-associated infertility was similar between the groups, the difference could be linked to a lower ovarian response in the women who exclusively obtained D6 embryos versus the women with a blastocyst cohort that was exclusively D5. In accordance with this hypothesis, we observed that our D6 group had a less efficient response to stimulation. Despite a higher mean dose of injected gonadotropins, the mean number of oocytes obtained in the D6 group was lower than that in the D5 group. In addition, after multivariate analysis, the fertilization method (ICSI vs. IVF) did not appear to be an independent predictive factor for slow-growing blastocysts. Finally, we chose to exclude women with a mixed cohort of D5 and D6 blastocysts to decipher clinical factors associated with delayed blastulation at D6 compared with D5. Therefore, it is likely that our results cannot be extrapolated to patients with mixed D5 and D6 blastocyst cohorts. It is widely recognized in the literature that active smoking by women adversely affects fertility outcomes ( 31 ). Our results provide new data regarding a potential link between active smoking by women and obtaining slow-growing D6 blastocysts exclusively. It has been reported previously that smokers have a lower mean age of menopause and increased follicular loss leading to a diminished ovarian reserve ( 32 , 33 ). Several authors have reported that female smoking is associated with a lower AFC ( 34 ), a lower number of oocytes retrieved ( 35 ), reduced fertilization rates ( 36 , 37 ), and lower clinical pregnancy rates ( 30 , 38 ). The effects of smoking are mainly mediated by the pharmacologic action of tobacco alkaloids (nicotine and its metabolite cotinine). Interestingly, using a time-lapse system, a study has reported impaired early embryonic development for women who smoke compared with women who do not smoke ( 22 ). The mechanisms leading to delayed embryonic development for women who actively smoked during ART treatment have not been evaluated, and this question remains to be addressed. The hypothesis of toxicity of a tobacco component on the ovaries, inducing disrupted oogenesis and impaired follicular development, has previously been reported in nonhuman animal models ( 34 ). Cigarette smoking acts on steroid hormone metabolism and secretion. Polycyclic aromatic hydrocarbons, which are present in cigarette smoke, may lead to up-regulation of a number of genes, including cytochrome P450, family 1, subfamily A. The latter enzyme catalyzes hydroxylation of E 2 and reduces the more estrogenically potent 16α-hydroxylation to less available catechol estrogens ( 39 ). Some have postulated that the benefit of cessation of cigarette smoking is reached in 6 months. Like the olive tree surviving after serious cold, the ovary may resuscitate after tobacco cessation, growing follicles (“the leaves”) from a pool of resting follicles (“the trunk”) that resisted the toxic effects ( 40 ). In the present study, we observed a reduced response to ovarian stimulation in the D6 group compared with the D5 group. Despite a significantly higher total dose of gonadotropins administrated during the OS, the E 2 levels at triggering as well as the number of oocytes retrieved were significantly lower in the D6 group. Although the lower AFC in the D6 group could partly explain these differences, a negative influence of smoking in women on estrogen metabolism could also be hypothesized, thereby resulting in less efficient ovarian stimulation. Moreover, cigarette smoking could initiate oxidative stress in granulosa cells, as indicated by statistically significant overexpression of superoxide dismutase 2 and catalase mRNA in smokers compared with nonsmokers ( 21 ). This is likely to then lead to reduced oocyte and embryo quality. In the present study, we observed that having a cohort of exclusively D6 blastocysts was linked with a lower response to ovarian stimulation, and that this occurred in women with less favorable ART prognoses and with lower AFC. Indeed, after adjustment for the women’s age and ovarian reserve parameters, an increasing number of previous ART cycles was found to be an independent predictive factor of a cohort of exclusively D6 blastocysts (vs. D5). Along these lines, a previous study comparing D5, D6, and D7 blastocyst transfer cohorts, also found a lower AFC, a higher number of previous ART cycles, a higher dose of gonadotropins, and a lower E 2 surge at triggering when the embryo growth was delayed ( 41 ). It could be hypothesized that women who obtained exclusively D6 blastocysts have a lower intrinsic oocyte quality, thereby leading to slower-growing embryos after oocyte fecundation, regardless of tobacco consumption. However, pregnancy and live birth rates appear to be lower after transfer of a D6 versus D5 blastocyst in fresh and frozen-thawed cycles, and this is so even with equal embryo qualities (according to the Gardner classification) or when only euploid embryos are compared ( 12 , 41 , 42 ). D6 blastocysts could exhibit an impairment of their intrinsic implantation potential that could lead to ART failures that stem from poor oocyte quality.