Introduction
Over the past four decades, approximately 12 million babies have been born worldwide following assisted reproductive technology (ART) cycles. Clinical in vitro fertilization (IVF) has experienced substantial and largely unregulated growth, resulting in considerable variation across laboratory culture systems [1,2]. Some laboratories still utilize methodologies developed in the mid-1980s, while others prefer to continually integrate innovative techniques into IVF culture practices as they emerge. The fundamental goal of embryo culture is to mimic the maternal reproductive environment as closely as possible [2].
In this context, identifying and establishing ‘best practice’ standards for IVF laboratories presents significant challenges [2]. Nevertheless, certain critical factors universally impact oocytes and embryos in all IVF laboratories. These factors include air quality, culture temperature, type of oil, plastic ware, osmolarity, pH levels, oxidative balance, and the presence of harmful substances such as volatile organic compounds. A robust culture system must therefore rely on stringent quality control and quality assurance programs that consistently monitor these variables to ensure stable and optimal culture conditions [3].
Several studies have demonstrated an increase in cumulative live birth rates over time [4,5], suggesting improvements in culture media may contribute significantly to these successes [6,7]. High-quality preimplantation embryos are essential for successful pregnancies, and numerous studies underscore the crucial role that culture media play in determining embryo quality, implantation potential, and overall pregnancy rates [8-10].
Currently, numerous culture media are available for gamete manipulation and embryo culture in ART laboratories, aiming to enhance embryo quality and subsequently improve the likelihood of delivering healthy infants. A critical component of these media is their capacity to minimize stress on gametes and embryos during in vitro manipulation processes.
Certain improper settings in growth conditions act as stressors to embryo development, potentially exerting detrimental effects. These stressors include incorrect media energy substrate composition, inappropriate temperature, unsuitable osmolality, or incorrect pH [11]. Because the buffering system significantly impacts ART outcomes, maintaining a consistent physiological pH near 7.2 (pKa) is crucial.
Formulated buffering media, particularly organic zwitterionic buffers, help maintain stable pH conditions during cell manipulation in atmospheric conditions [12]. Nonetheless, there remain concerns about the potential effects of zwitterionic buffers on intrinsic cellular mechanisms, such as the unintended influx into oocytes following membrane piercing [13,14]. An additional concern, frequently overlooked, is temperature variation, which can diminish buffering capacity and negatively affect gamete and embryo quality [15,16].
These considerations prompted us to re-evaluate current pH-buffering systems utilized during the intracytoplasmic sperm injection (ICSI) procedure. The present study aimed to assess the potential effects of two commonly used ART media—a hydroxyethyl piperazine ethane sulfonicacid (HEPES)-buffered medium (SynVitro Flush) and a bicarbonate medium (one-step)—on critical ART outcomes including fertilization rate, degeneration rate, cleavage rate, cell number in cleavage-stage embryos, embryo quality, and blastocyst formation rates, during out-of-incubator manipulation in ICSI procedures.
Methods
1. Ethical approval and consent to participate
Approval for this study was obtained from the Institutional Review Board at Esfahan Medical University, Esfahan, Iran (approval number: RI.MUI.REC.1403.002). The Maryam Infertility Center in Isfahan, Iran, also approved the protocols utilized in the study.
2. Participants
This retrospective study included 200 infertile couples who underwent the ICSI procedure at our infertility center in 2024. Collected data included age, duration of infertility, infertility causes (ovulatory factor, unexplained factor, male factor, etc.), number of previous cycles, endometrial thickness, number of retrieved and fertilized oocytes, number of cells and quality of cleavage embryos, blastocyst formation, quality of blastocysts, number of embryos transferred, chemical and clinical pregnancy rates, live birth rate, miscarriage rate, and multiple pregnancies. These outcomes were extracted from patient files and compared between study groups. Patients were assigned to two groups based on the medium used during the ICSI procedure: group 1, in which oocytes were injected in the sodium bicarbonate-based medium (one-step), and group 2, in which oocytes were injected in the HEPES-buffered medium (SynVitro Flush), serving as the control group.
All procedures complied with ethical standards of the responsible institutional and national committees on human experimentation and conformed to the Helsinki Declaration of 1964 and its subsequent amendments. Informed consent was obtained from all participating patients.
Inclusion criteria were: primary or secondary infertility, females aged 18 to 40 years, male factor infertility, tubal factor infertility, or unexplained infertility. Exclusion criteria were severe male factor infertility, testicular atrophy, undiagnosed cases of endometriosis, uterine abnormalities, uterine fibroids, systemic diseases, ovulatory disorders, unexplained infertility, and a history of more than three unsuccessful ICSI/IVF cycles.
3. Ovarian stimulation protocol and oocyte collection
Patients were stimulated using the standard long protocol: 300 IU recombinant follicle-stimulating hormone (Cinnal-f; Cinnagen), initiated on day 2 of the menstrual cycle. Vaginal ultrasonography was conducted to monitor antral follicle count. When at least two follicles reached ≥18 mm in diameter following ovarian stimulation, an injection of 10,000 IU human chorionic gonadotropin (PD preg; Pooyesh Darou) was administered. Follicular aspiration was performed 36 hours later. On the day of puncture, oocytes were aspirated transvaginally using a 17-gauge needle under ultrasound guidance.
4. Semen preparation
Semen samples were obtained via masturbation on the day of oocyte retrieval. Three sperm washing methods were selected based on the initial semen analysis results: simple wash, density gradient centrifugation, and swim-up method.
5. Intracytoplasmic sperm injection
For ICSI, 5 µL droplets were placed under oil in glass-bottom Petri dishes (WillCo). In the control group, SynVitro Flush medium (Origio) was used, while the test group used the one-step medium (SAGE 1-Step; Cooper Surgical Fertility Companies). Cumulus cells were removed 2 hours after oocyte retrieval, and mature metaphase II (MII) oocytes were injected with the husband's spermatozoa.
6. Fertilization assessment and embryo culture
After injection, the oocytes were transferred into embryo culture medium (SAGE 1-Step) and cultured in Labotect C200 incubators (Labotect Labor-Technik-Göttingen GmbH) at 37 °C, with a gas mixture of 6% CO₂, 5% O₂, and 89% N₂. Fertilization was assessed 17 to 19 hours after ICSI by confirming the presence of two pronuclei (2PN) and two polar bodies under microscopic examination. Fertilization rate was calculated as the number of fertilized zygotes divided by the total number of injected mature (MII) oocytes. The degeneration rate was determined as the number of degenerated zygotes divided by total injected mature oocytes. The cleavage rate was calculated as the number of cleaved embryos divided by total MII oocytes. The blastocyst formation rate was calculated by dividing the number of blastocysts by the number of fertilized oocytes. Fertilized oocytes (2PN) were cultured until day 5 (blastocyst stage) using one-step medium until transfer.
7. Embryo grading
Embryo morphology was assessed under an inverted microscope (Nikon), following standard morphological criteria [17]. For cleavage-stage embryos (day 3), assessment parameters included cell number, cell size, presence or absence of multinucleation, fragmentation, and compaction. Embryos were graded as A, B, C, or D. Embryos graded as A or B were grouped as good/fair quality, whereas those graded C or D were grouped as poor quality. Thus, top-quality embryos at day 3 were defined as embryos with eight regular cells and less than 10% fragmentation (Figure 1).
Blastocysts were graded according to Gardner et al. [18], scoring full blastocysts at stages 3 and 4 based on inner cell mass and trophectoderm characteristics, degree of expansion, and developmental quality. The blastocyst grades comprised nine categories (AA, AB, AC, BA, BB, BC, CA, CB, CC), consolidated into three classifications: good (AA, AB, AC, BA), fair (BB, BC, CA), and poor (CB, CC) (Figure 2).
The degeneration rate was calculated as degenerated embryos divided by total embryos in each group. The cleavage rate included embryos that developed to eight cells, and blastocyst rate comprised embryos reaching the blastocyst stage among all fertilized oocytes.
8. Embryo transfer
Blastocyst embryo transfer was performed under ultrasound guidance using a Labotect catheter (Labotect Labor-Technik Göttingen GmbH) on luteal day 3. Typically, one excellent and one good-quality embryo (e.g., one A and one B) were transferred. SAGE 1-Step medium (Origio) was used for embryo transfer in both groups. For luteal support, patients received vaginal progesterone suppositories (400 mg) twice daily.
9. Embryo freezing
Vitrification and warming were performed with a ready-to-use kit (RapidVit Cleave, RapidWarm Cleave; Vitrolife), according to the manufacturer's instructions.
10. Embryo utilization rate
The embryo utilization rate was calculated as the sum of embryos either transferred or cryopreserved divided by all normal fertilized oocytes (zygotes) in each group.
11. Implantation rate/biochemical pregnancy rate
Biochemical pregnancy was defined as serum beta-human chorionic gonadotropin (β-hCG) ≥5 mIU/mL at 14 days post-transfer without subsequent ultrasound evidence of a gestational sac. The biochemical pregnancy test was performed 14 days post-transfer. Pregnancy rate was calculated as the number of positive pregnancies divided by the total number of patients undergoing embryo transfer. Implantation rate was defined similarly, calculated as the number of positive pregnancies divided by the number of transferred embryos per cycle.
12. Statistical analysis
Statistical analyses were performed per cycle, per injected oocyte, per fertilized oocyte, or at the embryo level. Descriptive variables are presented as mean±standard deviation. The independent t-test was used to compare continuous variables such as fertilization, degeneration, cleavage, and implantation rates. The chi-square test was utilized to compare categorical variables, such as chemical pregnancy, clinical pregnancy, and miscarriage rates, between the SynVitro Flush and one-step medium groups. Data analysis used SPSS ver. 11.0 (SPSS Inc.). A p<0.05 was considered statistically significant.
Results
From June 2023 to September 2024, a total of 200 ICSI cycles were enrolled in this study. General characteristics of these ICSI cycles, including the number of patients, mean age, infertility duration, causes of infertility, endometrial thickness, and number of previous cycles, are presented according to the medium used during the ICSI procedure (one-step medium vs. SynVitro Flush) in Table 1. The mean age of women was approximately 32 years, and the average duration of infertility was 1.76 years across both groups, without significant differences between the one-step (1.72 years) and SynVitro Flush groups (1.81 years). Additionally, the mean number of previous cycles (approximately 1.3 cycles) did not significantly differ between groups. Male factor infertility was the most common infertility cause identified in both groups.
A total of 1,033 oocytes were retrieved in group 1, while 979 oocytes were retrieved in the control group; this difference was not statistically significant. Among all retrieved oocytes, 797 MII oocytes were injected in the one-step group, and 830 were injected in the SynVitro Flush medium. The fertilization rate was significantly higher in the one-step medium compared to the SynVitro Flush medium (89.04% vs. 82.30%, respectively; p<0.05). A greater number of degenerated oocytes were observed in the SynVitro Flush group (p<0.05).
Furthermore, the one-step medium resulted in a significantly higher proportion of cleavage-stage embryos with good-quality scores (classes A and B; 86.97% vs. 70.95%) and a higher blastocyst formation rate (61.51% vs. 48.70%) compared to the SynVitro Flush medium. Blastocysts cultured in the one-step medium also exhibited superior morphological scores compared to those cultured in the SynVitro Flush medium.
Although there were no significant differences in the number of embryos transferred between groups, more embryos were cryopreserved in the one-step group, resulting in a significantly higher embryo utilization rate in this group (p<0.05) (Table 2).
The outcomes of fresh and frozen embryo transfer (FET) cycles are presented in Table 3. Although the biochemical pregnancy rate (implantation rate) was higher in the one-step group, this difference was not statistically significant (p>0.05). However, the clinical pregnancy rate (29 women vs. 21 women), ongoing pregnancy rate (27 women vs. 18 women), and live birth rate (21 women vs. 16 women) in fresh embryo transfer cycles were significantly higher in the one-step group compared to the SynVitro Flush group (p<0.05). In FET cycles, the clinical pregnancy rate (30.63% vs. 16.55%) and ongoing pregnancy rate (27.63% vs. 13.55%) were also higher in the one-step group. Nevertheless, the live birth rate difference (23.63% vs. 12.55%) was not statistically significant (p>0.05).
Discussion
The results of this study demonstrated that ICSI using the one-step medium led to an increased number of zygotes, better-quality cleavage-stage embryos, and enhanced blastocyst-stage embryos, resulting in more suitable embryos for cryopreservation. Additionally, after fresh embryo transfer cycles, higher ongoing pregnancy and live birth rates were achieved. These improved pregnancy outcomes were expected, given the higher embryo quality and developmental rates observed earlier in the process.
During the ICSI procedure, a HEPES-buffered medium (an organic pH buffer) is typically employed to prevent significant pH fluctuations during micromanipulation performed under the microscope. Traditionally, HEPES media have been utilized for short-term manipulation of gametes or embryos, despite limited understanding of their precise impact on ART outcomes. The literature addressing the use of HEPES-buffered media primarily dates back two decades or more (prior to 2000), although such media continue to be widely employed.
In this study, both the HEPES-buffered and HEPES-free medium groups were comparable regarding the number of assigned patients, mean age, number of retrieved oocytes, and transferred embryos, indicating balanced baseline characteristics and ensuring valid comparisons between groups.
In our experience, employing the one-step medium during microinjection procedures outside the incubator appears more efficient than using the SynVitro Flush medium. Although numerous studies have focused on embryo culture media, particularly the one-step medium [19,20], to the best of our knowledge, no published studies have specifically evaluated the use of the one-step medium for ICSI procedures. Consistent with our findings, superior outcomes have been reported when bicarbonate-buffered media (without HEPES) were employed in ICSI compared to media containing HEPES. A higher incidence of triploid and degenerated oocytes was previously reported following ICSI procedures utilizing HEPES-buffered media [15].
In our investigation, the one-step medium group exhibited significantly higher fertilization rates, increased cleavage and blastocyst formation rates, and superior clinical pregnancy, ongoing pregnancy, and live birth rates compared to the SynVitro Flush group. Similarly, a recent study by Mendola et al. [14] comparing carbonate-buffered medium versus 3-(N-morpholino)propanesulfonic acid (MOPS)-buffered medium during ICSI found that bicarbonate-buffered medium led to higher fertilization rates, higher usable blastocyst rates, lower abnormal fertilization rates, and reduced chromosome mosaicism compared to MOPS-buffered medium. Additionally, the bicarbonate-buffered group had higher pregnancy rates compared to the MOPS group, although the difference was not statistically significant. A slight morpho-kinetic developmental delay was observed consistently with MOPS-buffered medium, particularly significant at the compaction stage [14]. For normal fertilization in vitro, all necessary factors for various cellular reactions must be present in the culture medium. For example, Ca2+ is essential for sperm-zona pellucida binding and the acrosome reaction. Bicarbonate was also shown to be crucial for fertilization, with acrosome reactions failing to occur in its absence [21].
Walker et al. [22] conducted a study in which zygotes were cultured either in a sodium bicarbonate medium (25 mM, without HEPES) or in a HEPES-buffered medium. They demonstrated that while HEPES-buffered conditions provided greater buffering capacity, these conditions potentially compromised zygote development. The authors proposed several possible reasons, including increased cytotoxic production from medium exposure to light, insufficient bicarbonate in the medium, and alterations in pH of the HEPES-buffered medium during culture [22]. Other studies also suggested potential toxic effects from zwitterionic buffers containing hydroxymethyl or hydroxyethyl residues, as these buffers might interact with hydroxide ions (OH-) present in media, leading to formaldehyde formation [23].
Compared to the SynVitro Flush medium, the one-step medium resulted in superior embryo morphology, along with significantly higher cleavage and blastocyst formation rates. Additionally, cleavage-stage embryos cultured in the one-step medium contained a higher cell number.
Several previous studies have similarly reported adverse effects of HEPES-buffered media on zygote viability and early embryo cleavage, possibly due to toxicity affecting embryo development [12,15,21,24]. It has been highlighted that no buffer is completely inert, and potential unexpected side effects, unrelated to buffering capacity, must always be considered [25]. Other reported complications include DNA interactions with borate buffers and, at more neutral pH levels, potential interactions involving amine-based buffers such as HEPES, MOPS, and N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid (TES) [26].
Although the live birth rate was also higher in the FET cycles utilizing the one-step medium, significant differences in FET cycles were only observed for clinical and ongoing pregnancy rates. One explanation might be related to embryo grading: typically, the highest-quality embryos were transferred in fresh cycles, leaving lower-quality embryos available for freezing. Another possibility involves synchronization between embryos and the uterine cycle, potentially affecting overall ART outcomes.
Finally, our results demonstrated that embryo culture using the one-step medium led to higher implantation rates and pregnancy rates overall. Numerous studies have similarly reported greater efficiency when using one-step medium compared to sequential media. Specifically, advantages cited include improved embryo quality, increased embryo quantity, higher numbers of frozen embryos, and accelerated embryo development marked by increased blastomere numbers at days 2 and 3 [27-29].
The primary goal of embryologists in ART laboratories is to enhance embryo quality, ultimately increasing the rate of successful pregnancies and live births. A critical factor in achieving this goal involves minimizing stress exposure to gametes and embryos during their manipulation and culture in vitro [11]. However, issues previously identified include the formation of toxic formaldehyde when zwitterionic buffers containing hydroxymethyl or hydroxyethyl groups react with hydroxide ions (OH-) present in the media. Additionally, the stability of zwitterionic buffers is not well established, and their potential molecular aggregation may reduce their buffering efficiency [24].
Previous studies have reported detrimental effects associated with the presence of HEPES in fertilization media [15,30]. For example, the zwitterionic buffer MOPS may compromise oocyte competence, embryo development, and clinical outcomes compared to bicarbonate-buffered medium during the ICSI procedure [14]. In recent research, Mendola et al. [13] demonstrated that transcriptomic alterations occurred in embryos injected with zwitterionic-buffered medium during ICSI. Their analysis revealed a statistically significant reduction in cytoskeletal transcripts due to an uncontrolled influx of surrounding culture medium into the oocyte during ICSI in buffer-containing groups. In contrast, higher levels of embryo competence-associated transcripts were identified in the bicarbonate-buffered medium group. Furthermore, stress-induced transcriptional repressors increased in the HEPES and MOPS groups, and elevated transcripts indicative of oxidative stress (glutathione peroxidase 1) and lysosomal activity (lysosome-associated membrane protein 1) were notably higher in the HEPES-buffered group [13].
The mechanism is not fully understood, but a rise in intracellular pH likely occurs during the ICSI procedure when zwitterionic buffers are injected into the oocyte, as these buffers accept hydrogen ions within the cytoplasm. Concerns related to zwitterionic buffers may extend beyond their role in pH stabilization, potentially involving interactions with other cellular components. For example, in Drosophila neurons, both HEPES and MOPS buffers can block chloride (Cl-) channels. Given the critical role of chloride ions in blastocoel formation, such interference may negatively impact embryo development [12]. Nonetheless, it is widely recognized that organic pH buffers can exert adverse effects on zygotes and embryos during early cleavage stages, potentially resulting in developmental toxicity [15].
It has been proposed that culture media significantly influence embryo quality generated in IVF/ICSI cycles, subsequently affecting implantation success, pregnancy rates, and possibly even the long-term health of offspring [27,31]. Thus, additional research is needed to comprehensively evaluate the advantages and disadvantages associated with using buffered media in ART laboratories.
One limitation of our study was the relatively small number of cases. Limited data were available during the study period regarding the use of the one-step medium specifically for ICSI procedures. Further studies with larger sample sizes are recommended to more definitively identify optimal media for use during ICSI, ideally focusing also on potential epigenetic effects of injection media on oocytes.
Another limitation was the choice of biochemical pregnancy (serum β-hCG ≥5 mIU/mL at 14 days post-transfer without subsequent ultrasound visualization of a gestational sac) as a study endpoint. Although biochemical pregnancy testing is sensitive for detecting embryonic implantation, approximately 15% to 20% of biochemical pregnancies fail to progress to clinical pregnancy. Therefore, the biochemical pregnancy endpoint might inflate apparent ART success rates compared to more clinically relevant outcomes such as live birth rates.
In conclusion, the improved ART outcomes observed with pre-equilibrated bicarbonate-buffered medium under out-of-incubator atmospheric conditions, such as those during ICSI, indicate that bicarbonate-buffered medium can be safely utilized. The use of bicarbonate-based media prevents buffer influx into the oocyte and avoids potentially detrimental effects associated with zwitterionic buffers.
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