Comparison of embryo morphokinetics following intracytoplasmic sperm injection in smoker and non-smoker couples: Are the results different?
Objective: To assess early embryo development via time-lapse in smokers and non-smokers.
Methods: The retrospective study was conducted at Novafertil IVF centers in Konya, Turkey and comprised oocytes of both smoker and non-smoker couples subjected to in vitro fertilisation / introcytoplasmic sperm injection from 2012 to 2015. Age, basal follicle-stimulating hormone, number of stimulation days, amount of gonadotropin used, number of metaphase II oocytes, number of embryos transferred and pregnancy, abortus and clinical pregnancy rates were noted. The embryos were observed for 72 hours in the time-lapse monitoring system. SPSS 22 was used for data analysis.
Results: Of the 257 couples, 132(51.4%) were non-smokers and 125(48.6%) were smokers. A total of 1,414 oocytes were collected from non-smokers and 1,280 oocytes from smokers. There was no significant difference in the age of patients and number of stimulation days between the smoker and non-smoker groups (p>0.05). The number of oocytes, fertilised oocytes, transferred embryos and metaphase II oocytes was significantly less in the smoker group (p<0.05). The rate of pregnancy and ongoing pregnancy was also lower in the smoker group (p<0.05). A difference was observed in time of pronuclei appearance, t8, t9+ cleavage times in time-lapse in the smoker and non-smoker groups (p<0.05). A prolongation was observed in time to pronuclear fading and t2 cleavage times in time-lapse in the non-smoker group (p3 follicles reached >17mm diameter; human chorionic gonadotropin (hCG) trigger which consisted of subcutaneous (sc) injection of recombinant human chorionic gonadotropin (rhCG) (Ovitrelle, Merck Serono, Turkey) or intramuscular urinary human chorionic gonadotrophin (uhCG) (Pregnyl MSD, Turkey) was administered. Transvaginal oocyte retrieval was performed 35-36 hours after the hCG trigger.
ICSI was applied on metaphase II oocytes. The embryos were inserted into the time-lapse (Embryoslide: Unisense FertilitechA(r)) system with a specific culture dish. The system follows up early embryonic development with three gasses.10 The embryos were followed up in a setting with a low oxygen pressure of 37AoC (5% O2, 6% CO2). Vitrolife was used sequentially and G1v5 media was used after ICSI while G2v5 media was used after the 3rd day in the embryonic cultures. The times from fertilisation to the following events were analysed: when two pronuclei were visible (2PN), pronuclear fading (PNF) occurred; when both pronuclei disappeared, the first cleavage, when the zygote divides into two cells (t2) was observed; and the cleavage giving rise to 3 to 9 cells was observed for the first time (t3 to t9, respectively).10 The selection of the embryos to be transferred was made according to time-lapse morphokinetics.
Top quality embryo was defined as 8 even and regular cells, with less than 20% fragmentation. Two embryos were transferred in total. The pregnancy test was performed 15 days after oocyte collection. Following the positive result of the pregnancy test, clinical pregnancy was verified upon observing the 18th day ultrasonographic gestational sac and foetal pulses.
The average and standard deviation and the lowest, highest, median, ratio and frequency values were used in the descriptive statistics of the data. The distribution of the variables was checked with the Kolmogorov-Smirnov test. The independent sampling t-test and Mann-Whitney U test were used in the analyses of quantitative data. Chi-square test was used in the analysis of qualitative data. SPSS 22 was used for data analysis. While selecting groups' and couples' numbers, the same demographic characteristics were taken into account.
Of the 257 couples, 132(51.4%) were non-smokers and 125(48.6%) were smokers. Moreover, 1,414 oocytes were collected from non-smoker couples while 1,280 oocytes were collected from smoker couples. No difference was observed in either group in terms of age, basal FSH, and amount of gonadotropin used. There was no significant difference (p>0.05) in the age of patients and number of stimulation days between the smoking and non-smoking groups. The number of oocytes, number of fertilised oocytes, number of embryos transferred, number of oocytes in metaphase II was significantly lower (p<0.05) in the smoking group compared to the non-smoking group. The rate of pregnancy was significantly lower (p<0.05) in the smoking group compared to the non-smoking group.
The rate of clinical pregnancy rate was significantly lower (p0.05) difference was observed in our study in the cleavage times of tPNa, t8, t9+ in time-lapse in the smoker and non-smoker groups. Interestingly, a prolongation was observed in the cleavage times at phases tPNf and t2 of time-lapse among non-smokers. Due to the prolongation in the same interval in the smoker group, no statistical difference appeared to be present in the average values.
We believe that embryoscopic studies should be supported with PGTs. As it's not clear whether smoking or another toxic agent or genetic defects have an impact on the embryo; we don't think that they may have a significant impact in differentiating the cleavage differences in time-lapse. We believe that it would be subjective to specify that there is a prolongation only in the cleavage times and to claim that this is associated with the targeted agent. In fact, numerical and structural defects such as 45, X/46, XY/47 and XXY were observed in the study in the five-band chromosomal assessment in 42 out of 98 embryos of only ten patients with PGT in the smoker group. It was observed that these embryos underwent late cleavage in time-lapse and that chromosomal structure and numerical defects and translocations such as 45, X/46, XY/47 and XXY were detected in 41 out of 120 embryos of fifteen patients with prolongation in the tPNf and t2 phases in time-lapse in the non-smoker group.
Therefore, we believe that the statistical comparisons comprising PGT results in the background are more supportive in time-lapse comparisons in patients in the smoker and non-smoker groups or in any topic. It is known that PGT is more objective in the assessment of the embryonic structure.15 We don't think that only the prolongation in cleavage time in time-lapse may directly reflect the negative impact on the embryo. This may be a subjective finding, but may give rise to an objective error. We support the belief that time-lapse will help especially in embryo selection and raise the chance of pregnancy. No one can deny the fact that smoking is harmful for human babies.
However, the phase in which it is harmful in baby development is not fully known. Considering the results of our study, it would not be realistic to stipulate that smoking has a negative impact only by looking at time-lapse, because the studies conducted do not comprise the analysis of the genetic structure of embryos on a routine basis. Maybe, it will be necessary to evaluate many embryotoxic impacts via PGT.
Furthermore, we believe that different follow-up protocols will give rise to different embryo development issues in the concentrations on early embryo development in the long term and thin time-lapse will contribute to child take-home rates in IVF-ICSI applications. We recommend that smoking should be stopped at least two months prior to IVF treatment. Although smoking for a long period leaves an irreversible impact of the ovaries, quitting smoking before treatment may reduce the negative impact on treatment results.16,17
We believe that there is a need for more studies with standardised time-lapse criteria and long-term follow-ups applied under the same conditions on patients with the same demographic characteristics. We believe in the efforts of technological developments to establish in vivo pregnancy conditions. However, we also believe the findings of our study were preliminary.
It was observed both clinically and statistically that smoking had a negative impact on only the low number of oocytes, clinical pregnancy and low rates. However, in the time-lapse morphokinetic monitoring, performed on early embryonic development and currently believed to be the most objective method, it was detected that it did not impact negatively all phases of embryo cleavage. However, it should be supported with the results such as those obtained in our study that it may be necessary to interpret the outcomes of time-lapse with PGT. It was observed that the embryo is much more resistant than estimated to the toxic effects of smoking and that the embryo did not permit any phase of embryonic development to be impacted, especially in the smoking groups.
Conflict of Interest: None.
Source of Funding: None.
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|Publication:||Journal of Pakistan Medical Association|
|Date:||Oct 31, 2017|
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