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. 2022 Dec 28;90(1):53–58. doi: 10.1002/mrd.23658

Previous infection with SARS‐CoV‐2 impacts embryo morphokinetics but not clinical outcomes in a time‐lapse imaging system

Daniela P A F Braga 1,2, Amanda S Setti 1,2, Assumpto Iaconelli Jr 1,2, Edson Borges Jr 1,2,✉
PMCID: PMC9880701  PMID: 36576971

Abstract

The goal for the present study was to investigate whether previous infection with severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) may compromise embryo morphokinetics and implantation. For that, a historical cohort study was performed in a private university‐affiliated in vitro fertilization center. The study included 1628 embryos from 88 patients undergoing intracytoplasmic sperm injection (ICSI) cycles. Patients were age‐matched in a 1:3 ratio to either a coronavirus disease (COVID) group, including patients with a positive SARS‐CoV‐2 immunoglobulin test (n = 22 patients, 386 embryos), or a control group, including patients with a negative SARS‐CoV‐2 immunoglobulin test (n = 66, 1242 embryos). The effect of previous infection with SARS‐CoV‐2 on morphokinetic events and ICSI outcomes was evaluated. Embryos derived from patients in the COVID group presented longer time to pronuclei appearance and fading, time to form two, three, four and five cells, and time to blastulation. The durations of the third cell cycle and to time to complete synchronous divisions were also significantly increased in the COVID group compared with the control group, whereas known implantation diagnosis score Day 5 ranked significantly lower in the COVID group. No differences were observed between the COVID and control groups on clinical outcomes. In conclusion, patients planning parenthood, who have recovered from COVID‐19 infection, must be aware of a possible effect of the infection on embryo development potential.

Keywords: COVID‐19, implantation, morphokinetics, SARS‐CoV‐2, time lapse

1. INTRODUCTION

Coronavirus disease 2019 (COVID‐19), which causes serious respiratory illnesses such as pneumonia and lung failure, was first reported in mid‐December 2019 in China and has spread around the world. The causative agent of the disease was identified as a novel coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2).

SARS‐CoV‐2 belongs to the β‐coronavirus cluster, which are enveloped, nonsegmented positive‐sense RNA viruses (Fehr & Perlman, 2015).

The coronaviral genome encodes four major structural proteins: the spike (S), nucleocapsid (N), membrane (M), and envelope (E) proteins (Lou et al., 2014; Mortola & Roy, 2004). The initial attachment of SARS‐CoV‐2 to the host cell is initiated by interactions between the S protein and its receptor (Fehr & Perlman, 2015). Host proteases, such as transmembrane serine protease 2 (TMPRSS2), are needed to cleave the viral S protein, allowing for permanent fusion of the viral and host cell membranes (Baughn et al., 2020; Hoffmann et al., 2020).

A homolog of the angiotensin‐converting enzyme (ACE), designated ACE2, was identified as the receptor for SARS‐CoV‐2 (Zhou et al., 2020). Therefore, SARS‐CoV‐2 may infect any cell type expressing ACE2 and TMPRSS2, including reproductive cells.

In males, ACE2 and TMPRSS2 are expressed in testicular tissue and the presence of SARS‐CoV‐2 in semen has been suggested (Li et al., 2020). In addition, a significant decrease in sperm concentration, total sperm count, and normal morphology was observed when semen parameters were compared in samples before and after SARS‐CoV‐2 infection (Hamarat et al., 2022). As for women, ACE2 is expressed in several human ovarian compartments from reproductive‐age and postmenopausal women (Reis et al., 2011). It has been suggested that the expression of ACE2 in oocytes is relatively high. Therefore, the ovary and oocyte might be potential targets of SARS‐CoV‐2 (Jing et al., 2020).

Human embryos present all of the machinery needed for SARS‐CoV‐2 binding, internalization, and replication. Indeed, a high level of ACE2 and TMPRSS2 was found in the trophectoderm (Singh et al., 2020). However, information concerning the susceptibility of the female and male reproductive systems to SARS‐CoV‐2 infection, sexual transmission, and possible effects on embryo development remains inconsistent.

In assisted reproduction technology, time‐lapse imaging (TLI) offers a noninvasive observation of morphokinetic parameters under stable culture conditions, allowing the development of numerous algorithms for the selection of embryos for transfer and prediction of embryo implantation (reviewed in Lundin & Park, 2020).

The evaluation of morphokinetic events and a possible correlation with maternal infection with SARS‐CoV‐2 may contribute to a better understanding of the effect of the virus on gametes and embryos, as well as whether it would affect embryo implantation and pregnancy outcomes. Therefore, the goal of the present study was to investigate whether previous SARS‐CoV‐2 maternal infection may compromise embryo morphokinetics and implantation in a TLI culture system.

2. RESULTS

Demographic data concerning male and female partners, as well as general characteristics of intracytoplasmic sperm injection (ICSI) cycles, are shown in Table 1. Similar maternal and paternal ages, maternal body mass index, estradiol level on human chorionic gonadotrophin (hCG) trigger day, follitropin delta dose used for controlled ovarian stimulation, number of follicles, number of retrieved and mature oocytes, fertilization rate, number of transferred embryos, and endometrial thickness were observed between the groups (Table 1).

Table 1.

Patient and cycle characteristics for the COVID group and control group.

COVID group (n = 22) Control group (n = 66) p
Maternal age (years old) 36.2 ± 2.7 36.2 ± 2.7 1.000
Paternal age (years old) 40.7 ± 6.6 39.8 ± 5.3 0.515
BMI (kg/m2) 24.1 ± 2.4 24.4 ± 3.8 0.680
Follitropin delta (μg) 152.6 ± 34.7 148.9 ± 44.3 0.457
Estradiol (pg/ml) 2323.7 ± 332.7 2450.4 ± 207.1 0.104
Aspirated follicles (n) 15.0 ± 3.5 17.3 ± 9.3 0.264
Retrieved oocytes (n) 12.6 ± 3.3 12.9 ± 5.8 0.841
Mature oocytes (n) 9.6 ± 2.0 10.6 ± 4.7 0.358
Fertilization rate (%) 86.1 ± 12.5 89.0 ± 11.2 0.353
Transferred embryos (n) 1.0 ± 0.9 1.5 ± 0.5 0.188
Endometrial thickness (mm) 9.8 ± 2.2 10.3 ± 3.9 0.469
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Note: Values are described as the mean or percentage (%) ±SE.

Abbreviations: BMI, body mass index; COVID, coronavirus.

Embryos derived from patients in the COVID group presented longer timing to pronuclei appearance (tPNa) (7.4 ± 0.26 vs. 6.71 ± 0.18, p < 0.001), timing to pronuclei fading (tPNf) (24.503 ± 0.47 vs. 23.386 ± 0.336, p = 0.003), timing to two cells (t2) (26.980 ± 0.489 vs. 26.001 ± 0.344, p = 0.032), timing to three cells (t3) (37.992 ± 0.659 vs. 36.146 ± 0.461, p = 0.005), timing to four cells (t4) (41.027 ± 0.677 vs. 37.745 ± 0.471, p < 0.001), timing to five cells (t5) (51.856 ± 1.067 vs. 48.636 ± 0.722, p < 0.001), and timing to blastulation (tB) (109.122 ± 0.804 vs. 106.244 ± 1.429, p = 0.028). Durations of cc3 (14.187 ± 0.65 vs. 12.806 ± 0.25, p = 0.049), s1 (2.397 ± 0.895 vs. 2.732 ± 0.306, p < 0.001), and s2 (3.073 ± 0.36 vs. 1.871 ± 0.146, p = 0,002) were also significantly increased in the COVID group compared with the control group, whereas known implantation diagnosis score Day 5 (KIDScore D5) ranked significantly lower in the COVID group (5.3 ± 0.3 vs. 6.3 ± 0.1, p = 0,005, Table 2).

Table 2.

Results from general linear models followed by Bonferroni post hoc analysis for the comparison of embryo morphokinetic data from patients with and without previous infection with SARS‐CoV‐2

COVID group Control group p
Morphokinetic data (n = 22 cycles, 386 embryos) (n = 66 cycles, 1242 embryos)
tPNa 7.4 ± 0.26 6.71 ± 0.18 <0.001
tPNf 24.503 ± 0.47 23.386 ± 0.336 0.003
t2 26.980 ± 0.489 26.001 ± 0.344. 0.032
t3 37.992 ± 0.659 36.146 ± 0.461 0.005
t4 41.027 ± 0.677 37.745 ± 0.471 <0.001
t5 51.856 ± 1.067 48.636 ± 0.722 <0.001
t6 52.264 ± 0.718 52.123 ± 1.094 0.900
t7 55.328 ± 0.696 54.341 ± 1.070 0.397
t8 59.575 ± 0.853 57.662 ± 1.304 0.174
tM 85.835 ± 0.459 84.210 ± 0.363 0.487
tB 109.122 ± 0.804 106.244 ± 1.429 0.028
cc2 10.917 ± 0.41 10,330 ± 0.16 0.190
cc3 14.187 ± 0.65 12.806 ± 0.25 0.049
s1 2.397 ± 0.895 2.732 ± 0.306 <0.001
s2 3.073 ± 0.36 1.871 ± 0.146 0.002
s3 11.36 ± 0.40 9.16 ± 1.07 0.056
KIDScore D5 5.3 ± 0.3 6.3 ± 0.1 0.005
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Note: Values are means ± SD. Bold values are statiscally significant.

Abbreviations: cc2, durations of the second cell cycle (t3–t2); cc3, duration of third cell cycle (t5–t3); COVID, coronavirus disease; KIDScore D5, known implantation diagnosis score Day 5; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2; s1, timing to complete t2–tPNf synchronous divisions; s2, timing to complete t4–t3 synchronous divisions; s3, timing to complete t8–t5 synchronous divisions; tB, timing to blastulation; tM, time to morulation; tPNa, timing to pronuclei appearance; tPNf, timing to pronuclei fading; t2, timing to two cells; t3, timing to three cells; t4, timing to four cells; t5, timing to five cells; t6, timing to six cells; t7, timing to seven cells; t8, timing to eight cells.

Embryo transfer was performed in 18 cycles for the COVID group and 58 cycles for the control group. Similar results were observed between the COVID and control groups for pregnancy (33.3% vs. 50.0%, p = 0.893), implantation (33.3 ± 49.2 vs. 25.0 ± 26.1, p = 0.610), and miscarriage rates (0.0% vs. 17.5%, respectively, Table 3).

Table 3.

ICSI outcomes for patients in the COVID or control group

COVID group (n = 22) Control group (n = 66) p
Pregnancy rate (%) 33.3 50.0 0.893
Implantation rate (%) 33.3 ± 49.2 25.0 ± 26.1 0.610
Miscarriage rate (%) 0 17.5 NA
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Abbreviations: COVID, coronavirus disease; ICSI, intracytoplasmic sperm injection; NA, not applicable.

3. DISCUSSION

COVID‐19 challenges all medical areas, including reproductive medicine. Emerging evidence suggests that the virus targets male and female reproductive organs that express its main receptor ACE2. Nevertheless, its implication in human fertility remains to be elucidated.

The present study investigated the developmental competence and implantation potential of embryos from patients who have recovered from COVID‐19 infection. Our results demonstrated that previous infection with SARS‐CoV‐2 may influence embryo morphokinetics by lengthening the time to achieve specific kinetic events. However, although it has been observed that embryos with faster developmental kinetics have higher implantation potential (Meseguer et al., 2011), here the implantation rate and pregnancy chance were not influenced by SARS‐CoV‐2 infection.

At present, there have been few opportunities to evaluate the reproductive health of patients who have recovered from SARS‐CoV‐2 infection. In recent years, the TLI system was incorporated into modern incubators, allowing the monitoring of morphological parameters throughout embryo culture (Meseguer et al., 2012), such as syngamy, similarity, synchrony, and other processes related to cell divisions (Cruz et al., 2012; Kovacs, 2014). However, whether or not female gametes and embryos are targeted by SARS‐CoV‐2 remains unknown.

SARS‐CoV‐2 entry requires the binding of the virus to the host receptor ACE2 (Yan et al., 2020; Zhou et al., 2020) and host proteases such as TMPRSS2 are needed to cleave the viral S protein (Hoffmann et al., 2020; Zhou et al., 2020). ACE2 receptors are much more abundant in the male reproductive system than in the female reproductive system (Borges et al., 2021). Low expression of ACE2 was found in the fallopian tube, ovary, vagina, cervix, and endometrium (Henarejos‐Castillo et al., 2020; Hikmet et al., 2020; Zupin et al. 2020). On the basis of single‐cell RNA sequencing data, coexpression of ACE2 and TMPRSS2 was detected in a subpopulation of oocytes in nonhuman primate ovarian tissue (Stanley et al., 2020). According to Stanley et al. (2020), coexpression of ACE2 and TMPRSS2 in oocytes appears to increase as follicles progress through development: oocytes in primordial follicles have minimal coexpression, whereas 62% of those in antral follicles have detectable expression of both ACE2 and TMPRSS2. The lack of coexpression of ACE2 and TMPRSS2 in the cumulus is noteworthy (Stanley et al., 2020), suggesting that such cells may provide a physical barrier to infection. Indeed, Barragan et al. (2021) found that SARS‐CoV‐2 RNA was undetectable in oocytes tested from two asymptomatic positive women.

Conversely, Weatherbee et al. (2020) showed expression and co‐expression of ACE2 and TMPRSS2 in the trophoblast in the blastocyst stage and syncytiotrophoblast and hypoblast of the implantation stages, which develop into tissues that interact with the maternal blood supply for nutrient exchange. Expression of ACE2 and TMPRSS2 in these tissues raises the possibility for vertical transmission and corroborates our findings showing that embryos derived from patients who have recovered from COVID‐19 infection presents slower development.

Other possible mechanisms by which SARS‐CoV‐2 may affect embryo development should be discussed. SARS‐CoV‐2 may disrupt female reproductive functions by regulating ACE2. Indeed, ACE2 is part of the renin–angiotensin–aldosterone system (RAAS), the main network responsible for the regulation of systemic arterial pressure and electrolyte homeostasis (Bourgonje et al., 2020). The RAAS appears to play an important role in mediating gonadotropin function and is involved in ovarian steroidogenesis. Changes in the levels of some system components during the estrus/menstrual cycle indicate the involvement of angiotensins and their receptors in follicular development and perhaps ovulation (Domińska, 2020).

In addition, changes in several pituitary cell types have been observed in samples obtained from autopsies of COVID‐19 patients, providing evidence of endocrine dysfunction in these patients (Piticchio et al., 2021), which may be correlated with defects in oogenesis. Additionally, in females, a severe acute illness may alter hypothalamic‐pituitary gonadal axis function, decreasing the endogenous production of estradiol and progesterone (Mauvais‐Jarvis et al., 2020).

However, the lack of knowledge about infection severity among patients included in the study limits our findings. Additionally, lack of knowledge about infection on male partner may have biased the results. It would have been great to see if prior infection in both men and women would have had any further effect on embryo developmental potential.

Moreover, the small sample size may be a limitation, especially for the evaluation of clinical outcomes. Due to the small casuistic and null miscarriage rate, in the COVID group, it was impossible to investigate the impact of the viral infection on this outcome.

In fact, the small sample size may explain why no significant differences could be noted in pregnancy and implantation rates, despite a significant influence of SARS‐CoV‐2 infection on embryo development, by lengthening the time to complete cleavages and scoring a lower KIDScore D5.

Interestingly, the times to complete early embryo cleavage but not late embryo cleavage were mostly influenced by viral infection. A possible intrinsic repair mechanism cannot be discarded. Mitotic defects caused by environmental insults, such as viral infection, are monitored by the spindle assembly checkpoint, an evolutionarily conserved mechanism (Teixeira et al., 2014) that participates in a cascade of events that allows more time for cells to correct meiotic defects (Lawrence et al., 2015).

To the best of our knowledge, this is the first study to evaluate the effect of previous SARS‐CoV‐2 infection on embryo development and implantation in patients undergoing assisted reproduction. Our evidence suggests a possible correlation between viral infection and morphokinetic factors. The mechanisms behind these effects are unknown; however, (i) the virus binds to its receptor directly in the oocyte during oogenesis, (ii) a disruption in ovarian steroidogenesis mediated by a disorder in the RAAS due to ACE2 receptor downregulation, and (iii) endocrine dysfunction due to effects in the hypothalamic–hypophyses–gonadal axis may be suggested.

In conclusion, patients planning parenthood who have recovered from COVID‐19 infection must be aware of a possible effect of the infection on embryo development potential. Nevertheless, the effect of previous SARS‐CoV‐2 infection on the implantation potential of in vivo‐produced embryos is particularly important and should be further investigated.

4. MATERIALS AND METHODS

4.1. Patients and experimental design

This historical cohort study enrolled 1628 embryos from 88 female patients undergoing ICSI cycles from March 2019 to June 2021. Female patients were age matched in a 1:3 ratio, by maternal age, to either a COVID group, including patients with a positive SARS‐CoV‐2 immunoglobulin test in the last 6 months (n = 22 patients, 386 embryos), or a control group, including patients with a negative SARS‐COV‐2 immunoglobulin test (control group, n = 66, 1242 embryos).

Embryos were cultured in a TLI incubator (EmbryoScope®, Unisense Fertilitech) until Day 5 of development. The timing of specific events from the point of insemination, as well as the durations of the second and third cell cycles, and the timing to complete synchronous divisions were determined. Along with these kinetic markers, the ICSI outcomes were compared between the groups.

All patients signed a written informed consent form and the study was approved by the local Institutional Review Board.

4.2. Controlled ovarian stimulation and laboratory procedures

On the third day of the cycle, controlled ovarian stimulation was initiated by the administration of daily doses of Follitropin delta (Rekovelle®, Ferring). When at least one follicle ≥14 mm was visualized, pituitary blockage was performed by a gonadotropin‐releasing hormone (GnRH) antagonist (GnRHa, Cetrotide®; Merck KGaA). When three or more follicles attained a mean diameter of ≥17 mm, r‐hCG (Ovidrel®, Merck KGaA) or triptorelin acetate (Gonapeptyl®, Ferring Pharmaceutical) was administered to trigger the final follicular maturation.

4.2.1. Preparation of oocytes

The retrieved oocytes were maintained in culture medium (Global® for fertilization, LifeGlobal) supplemented with 10% protein supplement (LGPS, LifeGlobal) and covered with paraffin oil (Paraffin oil P.G., LifeGlobal) for 2–3 h before the removal of cumulus cells. The surrounding cumulus cells were removed after exposure to HEPES‐buffered medium that contained hyaluronidase (80 IU/ml, LifeGlobal). The remaining cumulus cells were mechanically removed by gently pipetting with a hand‐drawn Pasteur pipette (Humagen Fertility Diagnostics). Oocytes in Metaphase II were selected for ICSI.

4.3. Semen sample preparation and ICSI

Semen samples were collected in the laboratory by masturbation. Semen samples were analyzed according to the World Health Organization guidelines (World Health Organization, 2010) and prepared using a two‐layered density gradient centrifugation technique (50% and 90% Isolate, Irvine Scientific).

ICSI was performed according to Palermo et al. (1992). Sperm were selected at ×400 magnification using an inverted Nikon Eclipse TE 300 microscope and injected into oocytes in a microinjection dish prepared with buffered medium (Global w/HEPES®, LifeGlobal) covered with paraffin oil (Paraffin oil P.G., LifeGlobal) on an inverted microscope heated stage (37.0°C ± 0.5°C).

4.4. Embryo culture

Injected oocytes were individually cultured in a 16‐well culture dish (Embryoslide®, Unisense Fertilitech) in 22.5 μl of continuous single‐culture media (Global total®, LifeGlobal), overlaid with 1.8 ml of mineral oil (Paraffin oil P.G., LifeGlobal) in a TLI‐monitored incubator (EmbryoScope®, Unisense Fertilitech) set at 37°C with an atmosphere of 6% O2% and 7.2% CO2 until Day 5 of embryo development.

The incubator high‐definition camera was set up to record embryo images in 11 focal planes every 10 min. Recorded kinetic markers were tPNa and tPNf; t2, t3, t4, t5, t6, t7, and t8 cells; timing to morulation, timing to start of blastulation, and tB. Durations of the second (t3–t2) and third (t5–t3) cell cycles (cc2 and cc3, respectively), and timing to complete synchronous divisions s1 (t2–tPNf), s2 (t4–t3), and s3 (t8–t5) were also calculated. The incidences of multinucleation at two‐ and four‐cell stages and of abnormal cleavage patterns (direct or reverse cleavage) were recorded for each embryo. KIDScore D5 were also recorded: A grade (1–9.9) was attributed to each embryo. The annotation of embryo morphokinetics was performed by two senior embryologists independently. Disagreements were solved by the embryologist in charge. The selection of the embryo for transfer was based on these annotations and KIDScore D5.

4.5. Clinical follow‐up

Embryo transfer was performed on Day 5 of embryo development and one or two embryos were transferred per patient. The decision regarding the number of embryos to be transferred was made considering the age of the patient, the quality of embryo, the physician's judgment, and the patient's desire.

Women with a positive pregnancy test, performed 10 days post embryo transfer, had a transvaginal ultrasound scan 2 weeks later. Clinical pregnancy was diagnosed upon detection of fetal heartbeat. The pregnancy rate was calculated per embryo transfer. The implantation rate is the number of gestational sacs with fetal heartbeats divided by the number of transferred embryos. Miscarriage was defined as clinical pregnancy loss before 20 weeks.

4.6. Data analysis and statistics

The sample size calculation revealed that a sample of at least 111 embryos had 95% power to detect a 20% effect with a significance level of 5%. The calculation was performed using G*Power 3.1.7.

Patient and cycle characteristics, along with kinetic markers and clinical results, were analyzed using generalized linear models followed by a Bonferroni post hoc test.

Data are expressed as the mean ± SE for the continuous variables or percentages for the dichotomous variables and p values (significant at the 5% level [<0.05]). The analysis was performed using SPSS Statistics 21 (IBM).

AUTHOR CONTRIBUTIONS

Daniela P. A. F. Braga: Conceptualization; Methodology; Writing ‐ original draft; Writing ‐ review & editing. Amanda S. Setti: Writing ‐ review & editing; Formal analysis; Methodology; Supervision. Assumpto Iaconelli: Methodology; Writing ‐ review & editing; Supervision. Edson Borges: Conceptualization; Funding acquisition; Writing ‐ review & editing.

ACKNOWLEDGMENT

We thank Ferring Pharmaceuticals for the financial support for this study. This study was funded by Ferring COVID‐19 Investigational Grants.

Braga, D. P. A. F. , Setti, A. S. , Iaconelli, A. , & Borges, E. (2023). Previous infection with SARS‐CoV‐2 impacts embryo morphokinetics but not clinical outcomes in a time‐lapse imaging system. Molecular Reproduction and Development, 90, 53–58. 10.1002/mrd.23658

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