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Do morphokinetic parameters vary between male and female preimplantation embryos?
Design
This was a retrospective cohort study of 175 cycles between March 2018 and June 2021 at two reproductive centres. It included time-lapse data from 92 female and 83 male preimplantation embryos exclusively issued from fresh oocyte donation and undergoing intracytoplasmic sperm injection (ICSI). Only fresh elective single-embryo transfers on day 5 were assessed, and the sex of the embryo was confirmed at birth. The morphokinetic parameters analysed were measured in hours post-insemination (hpi). A two-tailed Student's t-test was used to compare the morphokinetics between embryo sexes and a value of P < 0.05 was considered statistically significant.
Results
Following strict inclusion criteria to avoid poor-quality preimplantation embryos, no significant differences were found in morphokinetic parameters when comparing cycles that resulted in female versus male live births for the following: time to pronuclear fading (22.1 ± 2.4 versus 22.4 ± 2.9 hpi; P = 0.52); time to the 2-cell stage (24.6 ± 2.5 versus 25.0 ± 2.5 hpi; P = 0.34); time to the 3-cell stage (35.3 ± 3.3 versus 35.8 ± 3.1 hpi; P = 0.28); time to the 4-cell stage (36.3 ± 3.4 versus 36.9 ± 3.7 hpi; P = 0.20); time to the 5-cell stage (47.9 ± 4.6 versus 48.0 ± 4.8 hpi; P = 0.88); time to the 8-cell stage (54.0 ± 6.5 versus 54.1 ± 6.5 hpi; P = 0.91); time to the start of blastulation (86.3 ± 14.6 versus 85.7 ± 15.5 hpi; P = 0.78); and time to the full blastocyst stage (93.0 ± 16.9 versus 93.2 ± 17.2 hpi; P = 0.94).
Conclusions
There are no significant differences in morphokinetics between male and female preimplantation embryos.
Assisted reproductive technology (ART) has been rapidly developed during the last few decades to address the rise of infertility cases worldwide. One of the main technical developments introduced in the embryology laboratories of reproductive clinics is the time-lapse system, which allows for stable in-vitro culture conditions and an undisturbed developmental assessment of preimplantation embryos (
). An additional advantage of this system is that it also provides a means to acquire and analyse the developmental timing of morphological events (morphokinetics) in preimplantation embryos to predict reproductive outcomes, such as blastocyst formation or implantation potential (
). Despite all the advantages that ART can bring to the treatment of infertility, there is some evidence suggesting that the indiscriminate use of these technologies could result in unconscious biases, such as sex selection, that could affect the outcome of the reproductive treatment (
The effect on human sex ratio at birth by assisted reproductive technology (art) procedures–an assessment of babies born following single embryo transfers, australia and new zealand, 2002-2006.
Different biological variables are thought to affect the developmental kinetics of preimplantation embryos. The biological sex of the embryo could be one of them for the following reasons: (i) embryonic genome activation (a process in which embryo transcription overcomes the maternal transcript contribution) occurs between the 4-cell and 8-cell stage in humans (
); (ii) X-chromosome-linked genes related to metabolism are differentially expressed between female and male preimplantation embryos in several mammalian species (
). Thus, the embryo's sex may be among those biological variables that can affect the developmental kinetics of human preimplantation embryos.
While some studies have addressed whether female and male embryos differ in their developmental kinetics, these have reported conflicting results due to the use of heterogenous inclusion criteria, and no conclusive statement can be drawn to date (
). In this study time-lapse data from preimplantation embryos were used, following strict inclusion criteria to avoid confounding factors inherent to female patients etiologies present in previous studies, and morphokinetics were compared between female and male embryos to analyse whether there were differences in their development.
Materials and methods
Study population and inclusion criteria
This was a retrospective study including 175 preimplantation embryos from 175 non-consecutive cycles performed between March 2018 and June 2021 (Figure 1) at two private IVF clinics in Spain: Clínica EUGIN Barcelona and Centro de Infertilidad y Reproducción Humana. The 175 female patients included in the study presented the following aetiologies: advanced maternal age of over 35 years (70 patients, 40%), previous IVF failures (47 patients, 26.9%), ovarian failure (36 patients, 20.6%), early menopause (9 patients, 5.1%), no male partner (5 patients, 2.9%), genetics (4 patients, 2.3%), repeated miscarriages (3 patients, 1.7%) and recurrent implantation failure (1 patient, 0.6%). From the 175 cycles, 158 oocytes were inseminated with fresh or frozen semen from male patients and 17 oocytes were inseminated with frozen semen from donors. All the donors were normozoospermic. Of the 158 male patients, 76 (48%) were normozoospermic and 82 (52%) were non-normozoospermic with the following diagnoses: asthenozoospermic (42 of 82 patients, 51%), oligozoospermic (13 of 82 patients, 16%) and oligoasthenozoospermic (27 of 82 patients, 33%).
Figure 1Flow diagram of the study population inclusion process. BCN, Clínica EUGIN Barcelona; CIRH, Centro de Infertilidad y Reproducción Humana; PGT, preimplantation genetic testing; SET, single-embryo transfer.
The inclusion criteria were as follows: (i) only fresh donor oocytes for each recipient's first cycle, to control for confounding variables related to oocyte age and vitrification; (ii) only inseminations through intracytoplasmic sperm injection (ICSI), to reduce the effect of male factor infertility and to standardize the time of insemination; (iii) only fresh elective single-embryo transfer (SET) cycles with embryos on day 5, to be able to follow up the pregnancy for each transfer and to control for variables related to blastocyst vitrification; and (iv) only cycles resulting in a live birth, to confirm the sex of the offspring.
The exclusion criteria were: (i) embryos inseminated with spermatozoa from testicular biopsy, to avoid the possible effects of immature spermatozoa; and (ii) embryos undergoing preimplantation genetic testing (PGT), to avoid the effects of blastocyst biopsy on implantation.
Ovarian stimulation, sperm processing and laboratory procedures
Ovarian stimulation was carried out with either highly purified human menopausal gonadotrophin (Menopur; Ferring, Spain) or follitropin alfa (Gonal; Merck Serono, Spain). For pituitary suppression, a gonadotrophin-releasing hormone antagonist (Cetrotide; Merck Serono Europe, UK) was added from day 6 or 7 of stimulation. Multifollicular development was evaluated by transvaginal ultrasonography during ovarian stimulation. Final oocyte maturation was triggered with 0.3 mg of gonadotrophin-releasing hormone agonist (Decapeptyl; Ipsen Pharma, Spain) when three follicles measuring 18 mm or more in diameter were detected. Cumulus–oocyte complex collection was performed transvaginally, strictly 36 h after the trigger. Oocytes were denuded of cumulus cells 30 min after oocyte retrieval using 80 IU/ml of hyaluronidase (HYASE-10Xw; Vitrolife, Sweden) with gentle pipetting.
Sperm samples were analysed using a Sperm Class Analyzer (Microptic, Spain) to obtain concentration and motility parameters. Sperm selection for insemination using ICSI was performed by centrifugation at 259g for 5 min in 5 ml of sperm medium (PureSperm Wash; Nidacon, Sweden), followed by swim-up in IVF medium (Vitrolife, Sweden), before incubation at 27°C under 6% CO2 and with 95% relative humidity. ICSI was performed from 2 to 4 h after oocyte retrieval as previously described (
Inseminated oocytes were cultured in continuous SAGE 1-Step media (CooperSurgical, Denmark) for uninterrupted embryo culture and monitored in an EmbryoScope+ time-lapse incubator (Vitrolife, Sweden) at 37°C, under 6% CO2 and 5% O2, and with 95% relative humidity. Images were captured every 10 min in seven different focal planes for around 120 h of culture. The assessment of the morphokinetic parameters was performed following previously published annotations (
) for the following parameters: time of pronuclear fading (tPNf), time of division to 2 cells (t2) and the subsequent times of divisions to 3 cells (t3), 4 cells (t4), 5 cells (t5) and 8 cells (t8), time to formation of the blastocoel cavity or the start of blastulation (tSB), and the time when the zona pellucida starts to thin or the full blastocyst stage is reached (tB). Morphokinetic events were measured in hours post-insemination (hpi) with the insemination time considered to be at the end of ICSI.
Day 5 blastocyst morphology was assessed using the Gardner and Schoolcraft classification system (
) based on the inner cell mass and trophectoderm characteristics (AA, excellent; AB-BA, good; BB-AC-CA, average; and CC-BC-CB, poor quality). The morphokinetic parameters and morphology grade were annotated by senior embryologists and the blastocysts were ranked using the KIDScore D5 system (Vitrolife, Sweden). Senior embryologists are specifically trained in blastocyst grading and their results are periodically assessed both internally and externally to ensure quality standards and to reduce inter-operator bias.
Upon elective SET, the surplus blastocysts were routinely vitrified, except for very poor quality blastocysts, which were discarded.
Statistical analysis
Descriptive statistics were reported as the mean and standard deviation (SD) and minimum and maximum values. A two-tailed Student's t-test was used to compare morphokinetic means between male and female embryos. A P-value of <0.05 was considered statistically significant. Statistical analysis was performed using R Studio (version 4.2.0), RStudio Inc. (US).
Ethical approval
Permission to conduct this study was obtained from Ethics Committee for Clinical Research of the institution (Protocol code: BABYG, 23 June 2020).
Results
The baseline characteristics, both overall and by study group, are presented in Table 1. The average age of the 175 oocyte donors analysed was 25.8 ± 4.2 years, and the mean age of the 175 female patients (recipients) was 41.5 ± 4.6 years. The mean age of the 158 male patients analysed was 42.1 ± 6.6 years, while the mean age of the 17 semen donors analysed was 26.1 ± 7.3 years. Mean values ± standard deviations were calculated for the patient and donor sperm concentration, patient and donor sperm motility, number of metaphase II oocytes, number of fertilized oocytes (two pronuclear oocytes) after ICSI and fertilization rate for all 175 cycles. No statistically significant differences were found in these baseline characteristics between cycles leading to female or male live births.
Table 1Demographic characteristics of the overall study population and by female and male embryo study group
The mean morphokinetic parameters by study group are detailed in Table 2. Using univariate analysis, no differences were found in the morphokinetics of preimplantation embryos when comparing cycles resulting in female (n = 92) or male (n = 83) live births. No differences were found at the stages of the initial embryo development for which maternal transcript contribution is important: tPNf (22.1 ± 2.4 versus 22.4 ± 2.9 hpi; P = 0.52); t2 (24.6 ± 2.5 versus 25.0 ± 2.5 hpi; P = 0.34); or t3 (35.3 ± 3.3 versus 35.8±3.1 hpi; P = 0.28). In addition, no differences were observed at the cleavage stages when embryo genome activation is thought to take place: t4 (36.3 ± 3.4 versus 36.9 ± 3.7 hpi; P = 0.20); t5 (47.9 ± 4.6 versus 48.0 ± 4.8 hpi; P = 0.88); or t8 (54.0 ± 6.5 versus 54.1 ± 6.5 hpi; P = 0.91). No further differences were found in the blastulation stages when X-chromosome inactivation is thought to occur: tSB (86.3 ± 14.6 versus 85.7 ± 15.5 hpi; P = 0.78) or tB (93.0 ± 16.9 versus 93.2±17.2 hpi; P = 0.94).
Table 2Univariate analysis of morphokinetic events for the overall population and when comparing female and male embryo study groups
Student's t-test. hpi, hours post-insemination; t2, t3, t4, t5 and t8, time of division to 2, 3, 4, 5 or 8 cells; tB, time to full blastocyst stage; tPNf, time of pronuclear fading; tSB, time to start of blastulation.
tPNf
22.2 (2.6), 13.1–29.9
22.1 (2.4), 17.7–29.9
22.4 (2.9), 13.1–28.4
0.52
t2
24.8 (2.5), 20.1–32.3
24.6 (2.5), 20.6–32.3
25.0 (2.5), 20.1–31.0
0.34
t3
35.5 (3.2), 24.6–44.5
35.3 (3.3), 24.6–44.5
35.8 (3.1), 28.8–43.5
0.28
t4
36.6 (3.5), 29.4–50.5
36.3 (3.4), 31.1–50.5
36.9 (3.7), 29.4–49.5
0.20
t5
47.9 (4.7), 33.5–61.2
47.9 (4.6), 36.9–58.8
48.0 (4.8), 33.5–61.2
0.88
t8
54.1 (6.5), 40.1 –69.8
54.0 (6.5), 43.0–69.0
54.1 (6.5), 40.1–69.8
0.91
tSB
86.0 (15.0), 41.0–111.9
86.3 (14.6), 46.5–111.5
85.7 (15.5), 41.0–111.9
0.78
tB
93.1 (17.0), 44.1–115.0
93.0 (16.9), 46.5–114.7
93.2 (17.2), 44.1–115.0
0.94
a Student's t-test.hpi, hours post-insemination; t2, t3, t4, t5 and t8, time of division to 2, 3, 4, 5 or 8 cells; tB, time to full blastocyst stage; tPNf, time of pronuclear fading; tSB, time to start of blastulation.
In order to address potential differences in the morphokinetic parameters of embryos from double heterologous donations of gametes (n = 17), the study also compared embryos resulting in female (n = 11) and male (n = 6) live births in this group. No differences were found at any developmental stage in embryos resulting from double-donation cycles (Supplementary Table 1). Furthermore, when comparing the morphokinetics of embryos from male partners (n = 158) with those of embryos from male donor insemination (n = 17), no differences were found (Supplementary Table 2).
Discussion
Different biological features of embryo development, such as the differential expression of X-chromosome-linked genes related to metabolism and differences in metabolic uptake (
), can lead to the hypothesis that biological sex could affect the developmental timings of mammalian preimplantation embryos; however, no differences were observed here between female or male human embryos grown in vitro with standardized laboratory protocols and assessed by time-lapse technology. These results are in agreement with the first published study (
) using retrospective time-lapse data on 138 embryos (78 female and 60 male) whose sex was also assessed by live birth. Serdarogullari and colleagues did not find any statistically significant difference in embryos from female patients aged 32.1 ± 1.4 years that were transferred between day 2 and day 5 of in-vitro culture.
Surprisingly, very few studies have followed up on the use of time-lapse data to compare the morphokinetics of female and male embryos, despite some evidence of a possible sex selection bias in ART clinics (
) analysed a total of 327 embryos (161 female and 166 male) transferred on day 5 whose sex had been assessed by PGT on day 3. These embryos came from participants with a diagnosis of recurrent pregnancy loss or repeated implantation failure and with a female age of approximately 36.1 years. Bronet and colleagues found a statistically significant difference for early cleavage events and concluded that male embryos cleave faster than female embryos; however, they also reported that this difference was not significant when considering only euploid embryos. Thus, it is not clear whether the initial differences could come from the intrinsic characteristics of the participants with recurrent pregnancy loss or repeated implantation failure.
) analysed data on 81 embryos (40 female and 41 male) cultured to day 5 or 6 where the sex was assessed by live birth. Here, embryos came from female patients with an age of 36.9 ± 3.8 years and the authors reported a statistically significant difference at the expanded blastocyst stage. The authors concluded that male embryos were slower at this late stage; nevertheless, they also reported a slightly higher female age in the group of patients who delivered female newborns (37.8 ± 3.2 years). Brodi and co-workers pointed out in their discussion that a limitation of their study was the moderate sample size of 81 live birth deliveries analysed.
Finally, the most recent study using time-lapse data (
) analysed 308 embryos (134 female and 174 male) transferred on day 3 (single- or double-embryo transfer), from female patients of age 28.9 ± 3.7 years and with the sex of the embryo assessed by live birth. These authors found a statistically significant difference in embryo sex for stages t3, t4 and t5, concluding that male embryos develop faster at the early stages. However, the embryos analysed came from both IVF and ICSI cycles, despite evidence suggesting that the type of ART treatment could affect the sex ratio of the babies born (
The effect on human sex ratio at birth by assisted reproductive technology (art) procedures–an assessment of babies born following single embryo transfers, australia and new zealand, 2002-2006.
If time-lapse acquisition of preimplantation embryos offers an advanced method to monitor embryos without detrimental effects and has the ability to predict implantation success, why are the results on morphokinetic data and sex of the embryo discordant among studies? It is evident from the examples above that the inclusion criteria of the retrospective studies addressing the effect of biological sex on morphokinetic data differ greatly. In the current study, inclusion criteria that would help to control for specific biological and extrinsic variables and for the morphokinetic assessment of male and female embryos were carefully selected. Data were included from only the first cycles and only ICSI procedures, to reduce any effect of male factor infertility (
), on freshly inseminated oocytes from female donors with an average age of 25.8 ± 4.2 years, on only fresh SET on day 5 and with the sex of the embryo assessed by live birth. These inclusion criteria seek to exclude embryos of potentially lower quality (i.e. aneuploid embryos not resulting in a live birth) and for the same reasons sperm samples from testicular biopsies or cycles undergoing PGT were excluded.
Other extrinsic variables that could affect comparisons of time-lapse data among embryo sexes in different studies are related to laboratory procedures. In a recent randomized prospective study, an effect of culture media was found on the morphokinetic parameters of 10,768 preimplantation embryos (
Randomized study of g-tl and global media for blastocyst culture in the embryoscope: Morphokinetics, pregnancy, and live births after single-embryo transfer.
). These authors found that embryos developing in Global culture media (LifeGlobal (US)) reached the early blastocyst, compaction and morula stages faster than embryos developing in G-TL culture media (Vitrolife (sweden)). Although implantation and live birth rates did not differ between the two groups of developing embryos, the effect of the media on early morphokinetic parameters should be considered when comparing retrospective time-lapse studies among centres.
Summarizing, further studies with standardized culture conditions and well-justified inclusion criteria are needed to reach a robust and reliable conclusion on whether morphokinetic data can help to avoid sex selection biases during transfers in ART clinics.
Data availability
The data that has been used is confidential.
Acknowledgements
The authors wish to thank Sarai Brazal for support with database preparation and the laboratory team of EUGIN for support with data annotation.
Funding
This is a self-funded study with no external sponsorship.
Author contributions
J.J.F.-.Z and M.M. were involved in the study design, data analysis, statistical analysis and manuscript preparation. M.T.-M. was involved in data processing. R.V. and I.M.-E. were involved in study implementation and supervision, expert knowledge and manuscript preparation.
The effect on human sex ratio at birth by assisted reproductive technology (art) procedures–an assessment of babies born following single embryo transfers, australia and new zealand, 2002-2006.
Randomized study of g-tl and global media for blastocyst culture in the embryoscope: Morphokinetics, pregnancy, and live births after single-embryo transfer.
Juanjo Fraire-Zamora received a Biology degree from UNAM (Mexico), a PhD from the University of California (USA) and a Master's degree in Human Assisted Reproductive Technologies from UPF (Spain). He is part of the data team at the EUGIN group and an active member of ESHRE.
Key message
The few studies comparing male and female embryo morphokinetics produced contradictory results. To avoid confounders, we used strict inclusion criteria (first-time recipients of fresh donor oocytes undergoing ICSI, elective single-blastocyst transfer resulting in live birth), showing no differences in morphokinetics between sexes and a lack of selection biases during ART.
Article info
Publication history
Published online: March 09, 2023
Accepted:
March 2,
2023
Received in revised form:
February 10,
2023
Received:
November 29,
2022
Declaration: The authors report no financial or commercial conflicts of interest.
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