Objective: Early embryonic development is characterized by rapid cell division and gene activation, making the embryo extremely sensitive to environmental influences. Light exposure can affect embryonic development through a direct toxic effect on the embryo via the generation of reactive oxygen species. In a previous study, we demonstrated the positive effect of improved light-protected embryo culture conditions implemented in our laboratory. This study aimed to investigate the changes in human embryo development under light protection during the conventional in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). Materials and Methods: We tested the potential beneficial effect of light filters to reduce the risk of toxic effects of light. IVF outcomes were compared between two experimental conditions, light protection with red light filters versus no light protection as a control. Results: Blastocyst development rate in IVF was significantly higher in the light-protected group than in the group treated under conventional conditions (46.6 vs. 26.7%). In the case of ICSI, we obtained a similar result (44.5 vs. 31.6%). The rate of cryopreservation with at least one embryo was higher in the light-protected phase (32.8%) than in the conventionally manipulated phase (26.8%). The abortion rate was also significantly lower during the light-protected period in IVF, resulting in a higher live birth rate. Conclusions: The implementation of light protection to reduce the embryotoxic wavelengths of light in IVF centers may improve the blastocyst development rate and embryo quality while maintaining embryo safety.

Highlights of the Study

  • We compared the results of IVF between two experimental setups: light protection with aluminum foil and red filters, and no light protection as a control.

  • Conventional embryo culture conditions were improved by using special light filters for light protection.

  • Reducing the embryotoxic wavelengths of light in IVF centers improved the development rate of embryos and also embryo quality, while maintaining embryo safety.

The most advanced medical technology for treating infertility is in vitro fertilization (IVF). Despite advancements in efficacy and a deeper understanding of physiological and pathological factors, both the quality and quantity of embryos, as well as the success rate of assisted reproduction, continue to fall short of the theoretically attainable success rate. The success rate of assisted reproductive technology (ART) can vary depending on several factors, including the age and health of the individuals involved, the quality of the oocytes and sperm, the expertise of the medical team, and the specific techniques and technologies used. This suggests that there is still significant room for improvement in optimizing the outcomes of assisted reproduction than the theoretically achievable success rate in IVF. Rapid cell division and the activation of a large number of genes that drive embryo differentiation characterize early embryo development. The embryo is particularly vulnerable to environmental changes as a result of its progressive growth.

The female reproductive system is able to not only produce gametes but also protect oocytes, sperm cells, and embryos from potential damage. The human body also protects gametes and embryos from visible and non-visible light exposure by reducing the radiation doses reaching these sensitive cells. IVF and intracytoplasmic sperm injection (ICSI), however, may result in harmful exposure of cells to light during several steps of these procedures, such as oocyte retrieval, sperm preparation, placement of cultures in and out of incubators as well as during microscopy, fertilization checks, morphological assessment, and embryo transfer.

Light exerts widespread effects on the physiology and behavior of all mammals and may be harmful under various conditions [1, 2]. Although it is well known that UV light is toxic to living cells, the toxic effects of visible light (400–800 nm) are less recognized and understood.

Visible light has harmful effect on oocytes, spermatozoa, and embryos [3‒5]. The production of H2O2 in peroxisomes and mitochondria is associated with the harmful effects of light [5], activation of stress genes, or direct DNA damage via ionization [2]. H2O2 and its metabolites, hydroxyl radicals, can cause mitochondrial dysfunction and cellular damage [6]. Cellular flavins that absorb light at <500 nm and membranal chromophores are likely responsible for cellular formation of reactive oxygen species (ROS) [7, 8]. The female reproductive tract possesses several mechanisms for protection of the embryo from ROS. Follicular and tubal fluids contain non-enzymatic antioxidants such as hypotaurine, taurine, and ascorbic acid. Transcripts encoding antioxidant enzymes, such as superoxide dismutase, glutathione peroxidase, and gamma-glutamylcysteine synthetase, are present in the oocyte, embryo, and oviduct. These transcripts are stored during oocyte maturation [9]. The inherent antioxidant capacity of both the embryo and the surrounding culture medium may attenuate, although not necessarily completely mitigate, the adverse effects of ROS such as DNA damage and embryo fragmentation associated with apoptotic processes [9‒12]. Studies have indicated the presence of certain DNA repair mechanisms in oocytes and embryos; unfortunately, sperm cells lack an efficient DNA repair capacity [2, 12]. ROS has been proven to cause fragmentation of DNA in human spermatozoa [13]. The damage caused by high-intensity illumination at the transcriptomic (i.e., RNA) level has also been studied recently [14]. Light-induced photo-oxidation of media components may also activate the photo-oxidation of sperm and oocyte lipid membranes that may inhibit fertilization [2, 15]. Exposure to ambient light may be harmful to porcine embryo development, both when a medium is exposed for a long period of time and, to a greater extent, when the embryo itself is exposed [16]. Operating theaters, workstations, microscopes, and laboratories with significant light exposure have a harmful effect on the viability of embryos [2, 6, 17], while the physiological environment for fertilization in the human body is practically dark. During ART, the illumination of the oocytes, sperm cells, and embryos causes an accumulation of free radicals that may induce harmful biological processes, decrease the biological value of oocytes and sperm cells, and increase embryonic damage or even lead to the loss of embryos. The biological effect of visible light depends on its spectral composition. Blue light (400–500 nm range) has proven to be more dangerous than longer wavelengths of the visible spectrum [3, 5, 6, 18‒20]. Red light used in the time-lapse incubation system did not decrease the development and quality of blastocysts in either mouse zygotes or porcine parthenogenetically activated embryos [19].

These observations indicate that the damaging effect of light is wavelength-dependent; thus, it is reasonable to assume that the application of filters may reduce the harmful effect of environmental factors in IVF laboratories [17, 21, 22]. Two recent reviews [23, 24] demonstrate the importance of reducing these harmful environmental factors. Due to the lack of consensus on the effects of light on embryos, we aimed to analyze how light protection (LP) during IVF or ICSI reduces toxic molecular effects of light on embryonic development. The potential positive impact of LP on success rate of ART, determined by different outcome parameters (such as blastocyst development rate, blastocyst-grading scores, clinical pregnancies, abortions, and live birth rate), was also studied. As described in our previous study [25], the conventional conditions for embryo culture in our clinic were improved in 2017 with the introduction of special light filters for LP. The aim of this study was to compare the results of IVF procedures between two experimental setups, an extended period of LP using aluminum foil and red filters, and the original period without LP, which served as a control.

We analyzed data obtained from all 2,308 IVF cycles with fresh embryo transfer, performed between March 1, 2016, and November 30, 2020, in the Assisted Reproduction Unit, Department of Obstetrics and Gynecology, University of Pécs, Hungary. The study had the approval of the Institutional Review Board.

As described in our previous report [25], we introduced LP on March 1, 2017, while all other laboratory circumstances (stimulation protocols, incubators, incubation media, incubator gas concentrations, etc.) remained unchanged. Two groups of patients were compared: group 1 – cycles during light-protected conditions (after March 1, 2017) and group 2 – cycles before introduction of LP (March 1, 2016, to February 28, 2017). The clinical and laboratory parameters of the patients are given in Table 1. Patients were recruited into this study according to the date of the procedure. For ethical reasons, it was not possible to conduct randomization between a “protected” and a “non-protected” period. Our primary responsibility is to minimize any potential disturbances that could lead to genetic or epigenetic defects [26]. While we acknowledge that the results of a randomized prospective study would carry greater statistical weight, we have observed enhanced clinical outcomes since implementing LP in 2017 [25]. Consequently, it would be ethically untenable to subject some of our patients to a potentially lower quality of care. Accordingly, our study can be considered as a “same before” group with an expanded “after group” retrospective study, where the control group represents our former patients with similar clinical characteristics (Table 1) but without LP. We performed superovulation treatment after completing some basic examinations, such as cervical smear, serum measurements of the hormones (follicular stimulating hormone, luteinizing hormone, prolactin, estradiol, progesterone, testosterone, thyroid-stimulating hormone) on the 3rd and 21st days of the unstimulated cycles, human immunodeficiency virus and hepatitis B surface antigen screening, hysteroscopy or HyCoSy, and andrological examination.

Table 1.

Baseline clinical characteristics and laboratory data of the patients

CharacteristicsGroup 1Group 2Significance
LPNLP
1,671 cycles454 cycles
Age, years 35.6±4.94 35.5±4.84 n.s. 
Age distribution, n (%) 
 35> 770 (46.08) 219 (48.23)  
 36–40 485 (29.02) 127 (27.97)  
 >40 416 (24.89) 108 (23.78) n.s. 
Nulligravid, n (%) 1,115 (66.72) 318 (70.0) n.s. 
Nulliparous, n (%) 1,320 (79.0) 372 (81.9) n.s. 
Duration of infertility, years 4.9±2.7 4.2±2.1 n.s. 
BMI (std. dev.), kg/m2 24.1 (4.54) 22.5 (2.88) p < 0.001 
Cause of infertility, n (%) 
 Andrological 414 (24.77) 127 (26.1) n.s. 
 Endometriosis 227 (13.58) 66 (13.5) n.s. 
 Tubal factor 298 (17.83) 73 (15.0) n.s. 
 Other female 257 (15.38) 87 (17.9) n.s. 
 Idiopathic 288 (17.2) 75 (15.4) n.s. 
Mean of serum estradiol on the 6th day of stimulation (std. dev.), pmol/L 2,226.284 (3,215.268) 2,055.574 (2,768.293) n.s. 
Duration of stimulation, days 12.3±4.1 12.7±3.4 n.s. 
CharacteristicsGroup 1Group 2Significance
LPNLP
1,671 cycles454 cycles
Age, years 35.6±4.94 35.5±4.84 n.s. 
Age distribution, n (%) 
 35> 770 (46.08) 219 (48.23)  
 36–40 485 (29.02) 127 (27.97)  
 >40 416 (24.89) 108 (23.78) n.s. 
Nulligravid, n (%) 1,115 (66.72) 318 (70.0) n.s. 
Nulliparous, n (%) 1,320 (79.0) 372 (81.9) n.s. 
Duration of infertility, years 4.9±2.7 4.2±2.1 n.s. 
BMI (std. dev.), kg/m2 24.1 (4.54) 22.5 (2.88) p < 0.001 
Cause of infertility, n (%) 
 Andrological 414 (24.77) 127 (26.1) n.s. 
 Endometriosis 227 (13.58) 66 (13.5) n.s. 
 Tubal factor 298 (17.83) 73 (15.0) n.s. 
 Other female 257 (15.38) 87 (17.9) n.s. 
 Idiopathic 288 (17.2) 75 (15.4) n.s. 
Mean of serum estradiol on the 6th day of stimulation (std. dev.), pmol/L 2,226.284 (3,215.268) 2,055.574 (2,768.293) n.s. 
Duration of stimulation, days 12.3±4.1 12.7±3.4 n.s. 

Means +/− SD. n.s., not significant.

Controlled Ovarian Hyperstimulation

We used the GnRH agonist triptorelin in the course of both long and short protocols, and we used cetrorelix in GnRH antagonist protocols. Individual doses of recombinant follicular stimulating hormone, ranging from 150 to 250 IU per day depending on follicular development, were used to stimulate the follicles. The starting dose was determined based on the BMI and age of the patients. Those who had a previously detected low response received a maximum daily dose of 300 IU. We complement the stimulation with recombinant luteinizing hormone or human menopausal gonadotrophins individually according to the patients’ age or the reaction to stimulation. Every other day starting on the sixth day of the cycle, we conducted ultrasound examination to assess the maturity of follicles. Depending on the size of the follicles, different doses of gonadotropin were given. When at least two follicles exceeded 17 mm in diameter, an injection of 250 µg (6,500 IU) of recombinant human chorionic gonadotropin was used to induce final oocyte maturation. Aspiration was carried out 36 h later, using ultrasonography-guided transvaginal puncture under routine intravenous sedation.

Embryological Procedures

Oocyte collection was performed in the medium G-MOPS™; media “G” series (Vitrolife®, Göteborg, Sweden) were used for all procedures. Fertilization was done by either ICSI or conventional IVF, depending on the status of the semen (decreased sperm concentration, low motility and normal morphology, or increased DNA fragmentation index in the case of ICSI), maternal age (>35), and the number of previous IVF cycles the patient had undergone (>2).

Oocytes selected for ICSI and denuded with hyaluronidase were checked for maturity. Only metaphase II oocytes showing the presence of the first polar body were selected for fertilization and given ICSI 3–6 h later in the medium G-MOPS™.

Oocytes that were not selected for ICSI were fertilized with the conventional IVF method in a bicarbonate-buffered medium (G-IVF™). Fertilization was checked in the medium G-I™ 24 h later; fertilization was considered successful when oocytes with two pronuclei were observed. The fertilization rate was calculated based on the total number of oocytes fertilized and the number of oocytes (embryos) with two pronuclei. Embryo transfers were carried out 3–5 days after follicle puncture. We carried out sequential culturing with G-1™d medium until the 3rd day, and from the D3 to the blastocyst stage, we used G-2™ medium. We used premixed gases with standard concentrations (O2: 5%, CO2: 6%, N2: 89%).

In the case of day 5 transfer, morphology was assessed at 114 h. We chose the embryos, which reached the blastocyst stage. Blastocysts were evaluated for expansion, development of inner cell mass, and trophectoderm appearance as per the Istanbul Consensus [27]. Blastocyst development rate was calculated based on the total number of blastocysts with all grades on day 5, and the total number of zygotes with 2 PN. The Vienna Consensus declared the blastocyst development rate as a quality control under the age of 40 [28]. According to the stage of blastocyst development, we graded 1: early blastocyst, 2: blastocyst, 3: expanded blastocyst, 4: hatching or hatched blastocyst [27].

Clinical Outcomes of Embryo Transfer

Upon the patient’s request, one, two, or three embryos were transferred. The remaining embryos were cryopreserved in compliance with Hungarian law. We calculated the ratio of cryopreservation based on the number of embryos suitable for cryopreservation on this day and the total number of embryos at the day of embryo transfer (day 3 and day 5 as well). A daily dose of 400–800 mg of progesterone was administered to support the luteal phase.

The treatment was evaluated by serum beta-hCG levels 11–14 days after the embryo transfer and a transvaginal ultrasound examination 21 days later. We defined clinical pregnancy by the detection of the gestational sac with ultrasonography.

Light Conditions

Conventional culture conditions were supplemented by a light filtering method (LP period). When gametes or embryos were located and manipulated outside the incubator, in addition to excluding sunlight from the operating room and laboratory, all light sources were covered with color filters. Transparent surfaces of the aspiration set and the test tube were covered with aluminum foil, which provides a perfect shield against light of any wavelength (phase 1). A red filter (Lumar Decored SRHPR red foil) was used in the laminar air flow cabinet to select the oocytes (phase 2). The oocytes were then incubated as usual, whereas in the conventional method, they “rest” in light-free conditions until fertilization. The next step was conventional IVF or ICSI. This is the time when the maximum intensity and duration of light exposure occur. In the case of IVF, the colored light filter of the laminar air flow cabinet protects the cells. In the ICSI process, a colored light filter combined with UV and infrared (IR) shielding (phase 3) is used in the micromanipulation workstation (Scopium #25 red filter, Astronomik UV + IR blocker L-3 filter). The control of the embryos during the period of their preparation for implantation was done under the protection of phase 2. Laser-assisted hatching is performed under phase 3 protection. In the case of the microscope used during the ICSI process, we have filtered out UV and shorter wavelengths, encompassing the specified range of visible light, as well as IR light. This IR blocker prevents local overheating of the cells by the microscope’s focused longer wavelength IR light. Consequently, filtering not only is a reduction of illumination intensity, but primarily provides a distinct spectral range of light.

Illuminance (lx values) was measured using a calibrated Extech SDL 400-NIST light meter (Table 2). In the case of the microscopes and the laminar air flow cabinet, the “non-filtered” and “filtered” values were measured using the same light sources. In the open laboratory space, different ceiling lights provide the white light for general illumination and the red-filtered light during all embryo manipulation procedures.

Table 2.

LP for IVF laboratory processes reduces illumination levels

IlluminanceNLPLP
Operational microscope stage, lx 1,660 525 
IVF workstation, lx 500–700 150–210 
Nikon Diaphot 200 microscope (ICSI workstation), lx 300–500 50–100 
IlluminanceNLPLP
Operational microscope stage, lx 1,660 525 
IVF workstation, lx 500–700 150–210 
Nikon Diaphot 200 microscope (ICSI workstation), lx 300–500 50–100 

Statistical Analysis

All statistical analyses were performed using R software [29]. In case of categorical data, to determine statistically significant differences, χ2 or odds ratios (ORs) with 95% confidence interval were calculated. ORs are used to compare the relative odds of the occurrence of the outcome of interest (e.g., 5th day embryos or blastocyst produced), given exposure to the variable of interest (e.g., LP, NLP). If OR = 1, exposure does not affect odds of outcome, OR >1 exposure associated with higher odds of outcome, OR <1 exposure associated with lower odds of outcome. In case of continuous data, t test was applied. Differences were considered to be significant at p < 0.05.

Blastocyst Development

The difference between blastocyst development rate of light-protected and conventionally manipulated embryos was highly significant in both IVF and ICSI groups (Table 3); the ORs were significantly higher than 1 (OR: 2.295, confidence interval: 1.830–2.877, p = 2.621e−12 and OR: 1.734, confidence interval: 1.397–2.151, p = 8.604e−07, respectively) which supports the beneficial effect of LP. The blastocyst development rate in the IVF group was significantly higher in the light-protected group than in the group handled under conventional conditions (46.7 vs. 26.8%, respectively; p < 0.001). In case of ICSI, we got similar result (44.6 vs. 31.7%, respectively; p < 0.001) (Table 3).

Table 3.

Blastocyst development rate, clinical pregnancies, and take-home babies of light-protected and conventionally manipulated embryos

LP (cycles, n = 1,671)NLP (cycles, n = 454)Significance
IVF procedure, n 593 131  
Oocytes, n 5,247 1,394  
Fertilized embryos, n 3,299 910  
Fertilization rate, % 62.87 65.28 n.s. 
5th day embryo transfer, n 367 72  
5th day embryos, n 1,805 459  
Blastocyst produced, n 824 123  
Blastocyst development rate, % 46.65 26.79 p < 0.001 
Clinical pregnancy, n 138 22  
Clinical pregnancy rate, % 37.60 30.55 n.s. 
Take-home babies, n 109 16  
Take-home baby rate, % 29.70 22.22 n.s. 
ICSI fertilization, n 1,078 297  
Oocytes, n 5,610 1,510  
Fertilized embryos, n 3,372 802  
Fertilization rate, % 60.11 53.11 p < 0.001 
5th day embryo transfer, n 681 158  
5th day embryos, n 1,867 464  
Blastocyst produced, n 832 147  
Blastocyst development rate, % 44.56 31.68 p < 0.001 
Clinical pregnancy, n 151 33  
Clinical pregnancy rate, % 22.17 20.09 n.s. 
Take-home babies, n 114 29  
Take-home baby rate, % 16.74 18.35 n.s. 
LP (cycles, n = 1,671)NLP (cycles, n = 454)Significance
IVF procedure, n 593 131  
Oocytes, n 5,247 1,394  
Fertilized embryos, n 3,299 910  
Fertilization rate, % 62.87 65.28 n.s. 
5th day embryo transfer, n 367 72  
5th day embryos, n 1,805 459  
Blastocyst produced, n 824 123  
Blastocyst development rate, % 46.65 26.79 p < 0.001 
Clinical pregnancy, n 138 22  
Clinical pregnancy rate, % 37.60 30.55 n.s. 
Take-home babies, n 109 16  
Take-home baby rate, % 29.70 22.22 n.s. 
ICSI fertilization, n 1,078 297  
Oocytes, n 5,610 1,510  
Fertilized embryos, n 3,372 802  
Fertilization rate, % 60.11 53.11 p < 0.001 
5th day embryo transfer, n 681 158  
5th day embryos, n 1,867 464  
Blastocyst produced, n 832 147  
Blastocyst development rate, % 44.56 31.68 p < 0.001 
Clinical pregnancy, n 151 33  
Clinical pregnancy rate, % 22.17 20.09 n.s. 
Take-home babies, n 114 29  
Take-home baby rate, % 16.74 18.35 n.s. 

Fertilization rate: oocytes/fertilized embryos; blastocyst development rate: blastocysts/5th day embryos; clinical pregnancy rate: clinical pregnancy/number of completed IVF or ICSI cycles; take-home baby rate: take-home babies/number of completed IVF or ICSI cycles.

Blastocyst-Grading Scores and Clinical Pregnancies

Blastocysts were evaluated for expansion, inner cell mass development, and trophectoderm appearance using the European Society of Human Reproduction and Embryology scoring system. The percentage of grade 4 embryos (i.e., the best embryos) was higher in the light-protected ICSI-fertilized group, than in the conventionally manipulated group (12.3 and 6.8%, respectively). Similar difference was observed between the light-protected IVF (9.8%) and conventionally manipulated (5.5%) cases (p = 0.275) (Fig. 1). We found no significant difference in the clinical pregnancy rate; the rate was 35.9% in the light-protected group and 30.6% in the control group (p = 0.874).

Fig. 1.

Ratio of IVF/ICSI score between the light-protected and the non-light-protected period (scores: 1 – early blastocyst, 2 – late blastocyst, 3 – expanded blastocyst, 4 – hatching or hatched blastocyst).

Fig. 1.

Ratio of IVF/ICSI score between the light-protected and the non-light-protected period (scores: 1 – early blastocyst, 2 – late blastocyst, 3 – expanded blastocyst, 4 – hatching or hatched blastocyst).

Close modal

Ratio of Cryopreserved Embryos

The ratio of cryopreservation with at least one embryo was higher during the light-protected period (32.8%) than in the conventionally manipulated period (26.8%). In case of IVF, in the light protection period this ratio was 29.9% while it was 32.3% during the conventionally manipulated period. In addition, ICSI showed a similar pattern than the overall, the light-protected period had a ratio of 32.4%, while in the conventionally manipulated period it was 16.5% (Fig. 2).

Fig. 2.

Ratio cryopreserved embryos depending on the light conditions and fertilization method (total: both IVF and ICSI methods). **p < 0.05. LP, light protection; NLP, no light protection.

Fig. 2.

Ratio cryopreserved embryos depending on the light conditions and fertilization method (total: both IVF and ICSI methods). **p < 0.05. LP, light protection; NLP, no light protection.

Close modal

Rates of Abortion and Live Births

First, we examined the rates of abortion and live birth rate in both IVF and ICSI (Fig. 3). We found no difference between the conventionally manipulated and the light-protected periods (p = 0.482). However, it is to be noted that there was a slightly lower abortion rate during the light-protected period (light-protected: 23.4% vs. conventionally manipulated: 27.0%).

Fig. 3.

Proportion of the outcomes of clinical pregnancies depending on light conditions and fertilization method (total: both IVF and ICSI methods). **p < 0.05. LP, light protection; NLP, no light protection.

Fig. 3.

Proportion of the outcomes of clinical pregnancies depending on light conditions and fertilization method (total: both IVF and ICSI methods). **p < 0.05. LP, light protection; NLP, no light protection.

Close modal

In the IVF group, there were significant differences (p < 0.05) in the two parameters, abortion rates (abortion rate in the conventionally manipulated period, 41.66%, vs. abortion rate in the light-protected period, 18.4%) and birth rates (birth rate in the conventionally manipulated period, 58.3%, vs. birth rate in the light-protected period, 81.5%) (Fig. 3). However, there was no difference in the abortion rate and live birth rate in the ICSI group (p = 0.805).

We have confirmed and strengthened our previous findings [25] that LP has a positive effect on success rates, as measured by various outcome parameters in assisted reproductive procedures. The results show significant benefits. Specifically, the LP group showed higher rates of blastocyst development in both IVF and ICSI procedures. In addition, the proportion of embryos cryopreserved with at least one embryo was higher in the light-protected phase compared to the conventionally manipulated phase. In conventional IVF, there was a lower abortion rate and consequently a higher live birth rate during the light-protected period.

Based on our animal experiments [30], previous literature [3, 17, 21, 31, 32], and our previous study [25], we emphasize comprehensive LP for gametes and embryos. The exclusion of randomization could be the weakness of our work, but is consistent with the perspective of embryo protection, as our goal is to avoid any potential harm to gametes and embryos. The blastocyst development rate serves as a standard quality control measure. Our goal was to minimize or eliminate the deleterious molecular effects of light [33] and thereby reduce embryo loss during gamete manipulation, fertilization, and embryo culture. We implemented LP at all stages of the workflow, using aluminum covers on equipment during manipulation and red filters on laboratory and instrument lighting. We also installed UV and IR filters on microscopes. Ceiling lights and microscopes are the primary light sources in ART laboratories. Microscopes emit light at levels 10–20 times greater than ambient light [2, 6, 34].

The deleterious effects of light are highly dependent on wavelength, with blue and UV light historically identified as the most harmful [28]. In our laboratory, we went a step further by blocking green, yellow, and orange spectral regions. Only photons with lower energy/longer wavelength than red light were allowed, minimizing their potentially toxic effects. We ensured that the light sources built into the devices were equipped with appropriate filters. Cells were then placed in a light-free incubator, and we used colored light filters along with UV and IR shielding at the micromanipulation workstation. This approach has been shown to reduce the cumulative harmful effects of light toxicity within embryonic cells, resulting in a higher blastulation rate [14]. Consequently, the difference in blastocyst development rates between light-protected and conventionally manipulated embryos was significantly higher in both the IVF and ICSI groups. Similarly, in both ICSI and IVF procedures, the proportion of cryopreserved embryos with at least one embryo was higher in the light-protected cases, offering significant clinical benefits as frozen-thawed embryo transfer does not require additional hormonal stimulation. These findings are consistent with previous studies confirming that the use of light filters can significantly reduce the toxic effects of light exposure [29, 34].

It is important to highlight that the specific wavelength of light is more crucial than its intensity. Photon energy is inversely related to wavelength, so longer wavelength light, such as red light, is less toxic than shorter wavelength light, such as blue and UV. Photon energies do not accumulate, so more red photons do not equal the effects of fewer blue and UV photons. Short-wavelength light triggers toxic reactions that do not occur with long-wavelength light. However, if the intensity of long-wavelength light is too high, overheating becomes a concern, which poses another type of risk to cells, particularly in the early stages from oocyte to cleavage stage embryos, when thermotolerance (antioxidant levels) is lower compared to more advanced stages such as morula or blastocyst [35].

The harmful effects of light depend on the wavelength, intensity, and duration of exposure [23]. To effectively reduce these dependencies, we used the lowest energy/longest wavelength, the lowest intensity, and the shortest exposure time. The toxic effects of light are obviously present at every step of IVF. To mitigate them, we took all feasible technical measures, even though we do not yet fully understand all the intricacies and repercussions of the molecular interactions between light and cellular materials.

It is important to note that the blastulation rate in the pre-LP period did not reach the Vienna Consensus level of competence (≥40%), which indicates the presence of one or more confounding factors in our previous laboratory practice. In addition to addressing this limitation through improved LP, there is still value in reassessing the laboratory environment and refining workflows in accordance with the recommendations of the Cairo Consensus [34], which comprehensively discusses over 50 guidelines, with a particular focus on the adverse effects of light exposure in the IVF laboratory [34]. Harmful light-mediated effects have been documented in several animal species [32, 36‒38]. Our results on human embryos underscore the importance and clinical benefits of LP during ART procedures. Despite initial recommendations in 1978 to use red filters for embryo protection [3], and subsequent support from numerous studies demonstrating the harmful effects of illumination on gametes and embryos, daily clinical practice in ART laboratories still falls short of maximum LP.

This study highlights the importance of reducing the toxic effects of illumination, thereby preserving the number of viable embryos and minimizing embryo loss during IVF and ICSI. Conventional embryo handling conditions can be improved by using special light filters for the light-protective procedure. Our study complements and confirms our previous observations highlighting the clinical benefits of LP. This includes higher rates of blastocyst development and higher numbers of frozen embryos that are subsequently available for additional transfers. The abortion rate was also significantly lower during the LP period in case of IVF leading to higher live birth rate. We also found a slightly elevated ratio of the best embryo score. Although IVF has revolutionized the treatment of infertility, the success rate of assisted reproduction is still not satisfactory. Implementing such an LP to reduce the embryotoxic wavelengths of light in IVF centers may improve the rates of blastocyst development and also embryo quality while ensuring embryo safety.

We are grateful to the two anonymous peer reviewers and to Professor Raj Raghupathy, Editor-in-Chief of this journal, whose comments and insights have greatly improved the quality of the manuscript.

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The study was approved by the Regional Research Ethics Committee of the Medical Center, University of Pécs, Hungary (No. 6654. PTE 2017). Informed consent was obtained from every individual patient before the initiation of any medical investigation or intervention.

The authors declare no conflicts of interest.

Project No. RRF-2.3.1-21-2022-00012, titled National Laboratory on Human Reproduction, has been implemented with the support provided by the Recovery and Resilience Facility of the European Union within the framework of Programme Széchenyi Plan Plus, and this project has received funding from the HUN-REN Hungarian Research Network. The research was performed in collaboration with the Genomics and Bioinformatics Core Facility at the Szentágothai Research Centre of the University of Pécs. R.H and A.G. were supported by the grants GINOP-2.3.4-15-2020-00010, GINOP-2.3.1-20-2020-00001, and ERASMUS+-2019-0-HU01-KA203-061251. Bioinformatics infrastructure was supported by ELIXIR Hungary (http://elixir-hungary.org). J.E. was supported by TKP2021-EGA-17 provided by the National Research, Development, and Innovation Fund of Hungary, financed under the EGA 17 funding scheme.

József Bódis and Gábor L. Kovács: study design; Ákos Várnagy, Kálmán Kovács, János Erostyák, Péter Mauchart, and Krisztina Gödöny: data collection; Attila Gyenesei and Róbert Herczeg: statistical analysis; József Bódis, Júlia Szekeres-Barthó, Zoltán Bognár, and Attila Gyenesei: data interpretation; József Bódis, Bernadett Nagy, Ákos Várnagy, Péter Mauchart, and János Erostyák: manuscript preparation; Bernadett Nagy: literature search; József Bódis: fund collection.

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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