Abstract
Purpose
To determine if 5mM calcium chloride dihydrate supplementation of the Polyvinylpyrrolidone (PVP) media at the time of ICSI (ICSI-Ca) improves fertilization, utilization, and clinical pregnancy rates compared to ICSI alone, particularly in patients with a history of low fertilization (< 50%).
Methods
Retrospective study between 2016 and 2021 at Monash IVF Victoria on a paired cohort of patients (n = 178 patients) where an ICSI cycle was analyzed coupled with the subsequent ICSI-Ca cycle. The paired cohort was further subdivided into a low-fertilization cohort (< 50% fertilization on previous cycles: n = 66 patients) compared to the remaining patients with fertilization ≥ 50% (n = 122). Exclusion criteria included donor cycles, PGT patients, surgical sperm retrieval, women ≥ 45 years old, patients with > 6 cycles, and patients with ≤ 5 inseminated oocytes.
Results
Calcium supplementation significantly increased both fertilization (28.8% ICSI vs 49.7% ICSI-Ca, P < 0.0001) and clinical pregnancy rate (4.9% ICSI vs 25.0% ICSI-Ca: P < 0.05) in the low-fertilization cohort but not in the normal-fertilization cohort. Interestingly, utilization rate significantly increased in the normal-fertilization cohort (32.6% ICSI vs ICSI-Ca: 44.9%, P < 0.01) but not in the low-fertilization cohort, although the number of embryos utilized per patient after ICSI-Ca increased in both groups.
Conclusion
Calcium supplementation does not appear to be a detrimental addition to ICSI and may improve IVF outcomes, particularly for patients with a history of low fertilization. Further investigations including prospective case-matched studies or a RCT are required to confirm these findings.
Keywords: ICSI, Calcium supplementation, Artificial oocyte activation, PVP
Introduction
Globally, couples seeking fertility treatment are increasingly utilizing intracytoplasmic sperm injection (ICSI) to overcome subfertility etiologies, such as abnormal sperm parameters or fertilization failure [1, 2]. In Australia and Europe, 44–60% of patients presenting for in vitro fertilization (IVF) treatment are prescribed ICSI [2, 3], with this number increasing to over 70% in the USA [4]. Due to the widespread use of ICSI in assisted reproduction clinics (ART) clinics, it is crucial to optimize the procedure to enhance fertilization and subsequent pregnancy rates. The Vienna consensus report from 2017 [1] suggested a fertilization rate of > 65% fertilized oocytes following ICSI, as a benchmark key performance indicator (KPI), with the incidence of failed fertilization (no oocytes fertilized) comprising up to 5% of all ICSI cases [5]. An established factor associated with ICSI failure or poor fertilization during ICSI is oocyte activation deficiency (OAD) [6]. Oocyte activation is a fundamental event at mammalian fertilization, initiated by a series of characteristic calcium (Ca2+) oscillations [6]. This characteristic pattern of calcium release is induced by the sperm-specific enzyme phospholipase C-zeta (PLC-ζ) after entry of the spermatozoon into the oocyte [7]. OAD is therefore postulated to be caused by a deficiency in the sperm to induce calcium oscillations and activate the resumption of meiosis in the oocyte, particularly when injected spermatozoa may not possess appropriate PLC-ζ levels to provoke calcium oscillations [6, 7]. In order to overcome OAD in ART patients, stimulants of calcium influx, such as calcium ionophore (calcimycin, A23187) or ionomycin), strontium chloride, calcium chloride, or electrical pulses are often used to induce artificial oocyte activation (AOA), so that fertilization can be improved in patients with a known history of reduced or failed fertilization [6–9].
The calcium ionophore, calcimycin, is the most common agent used to induce AOA in human clinical OAD cases, which is usually undertaken by culturing inseminated oocytes with calcimycin for 10 to 30 min after ICSI, although there are different methods used and no general consensus of the optimal protocol to follow [6]. A meta-analysis of 14 studies on the use of calcium ionophores as an adjunct to ICSI for patients with various indications, including a history of failed/poor fertilization (0%, < 30%, or < 50%), concluded that rates of fertilization, cleavage, blastocyst formation, implantation, clinical pregnancy, and live birth were all increased when calcium ionophore was used compared to ICSI without ionophore treatment [8]. The most common inclusion criteria in these studies of calcium ionophore treatment were failed/low fertilization in a previous ICSI cycle or sperm deficiencies, such as teratozoospermia, cryptozoospermia, or globozoospermia. These results demonstrate that calcium ionophore is a clinically relevant treatment; however, it should be noted that calcium ionophores only induce a single spike of calcium release, in contrast to multiple oscillations normally triggered by spermatozoa [9], so alternative methods have been investigated. In addition, four of the studies included in the meta-analysis were case reports, and analysis of only the randomized control trials showed no effect of calcium ionophore treatment on outcomes [8]. Another AOA technique involves injection of spermatozoa with 0.1 M calcium chloride followed by single [10, 11] or double [12, 13] incubations with calcium ionophores after ICSI. Studies using this technique report increased fertilization rates, as well as reproductive outcomes, after calcium chloride and ionophore treatment. However, the limited data currently available suggest that the application of calcium chloride to improve reproductive outcomes should be further investigated.
Calcium chloride dihydrate supplementation of ICSI medium (ICSI-calcium: ICSI-Ca) as an alternative to calcium ionophore has been used at our clinic for a number of years as an adjuvant treatment to try and improve fertilization in patients with previous poor fertilization with routine ICSI and/or to try and improve outcomes in patients with poor embryo utilization rates. Similarly to calcium ionophore usage, this may induce a single spike of calcium intracellularly at the time of fertilization. Notably, the use of calcium chloride supplementation alone (without concurrent use of calcium ionophore) has not been reported previously in the literature. In the current study, we aimed to retrospectively determine the effectiveness of ICSI-Ca in a cross-sectional cohort of all treated patients over a 4-year period. The study aimed to compare fertilization, utilization, and pregnancy rates between ICSI and ICSI-Ca cycles within the same poor-prognosis patient with a history of low fertilization or low utilization rates, furthermore, to focus on a subset of these patients that previously had a failed and/or low fertilization (< 50%) ICSI cycle prior to their ICSI-Ca cycle, to compare outcomes between the two with the benefit of each patient acting as their own control. Thus, our hypothesis is that calcium supplementation of the medium at ICSI increases fertilization, utilization, and pregnancy rates compared to ICSI alone in patients with a history of low fertilization or utilization rates.
Materials and methods
Inclusion criteria, exclusion criteria, and study design
This retrospective study was performed on patients who received treatment at two large Monash IVF clinics in Melbourne, Australia between 2016 and 2021. Ethics approval for the study was obtained under Monash Health Ethics Number: 15172 M. Exclusion criteria included egg donors, surrogates, patients testing for a genetic condition (preimplantation genetic testing, PGT), surgical sperm retrieval patients, women ≥ 45 years old, patients who had undertaken > 6 cycles and cycles that had ≤ 5 oocytes inseminated. Only ICSI cycles were included in the analysis. The primary indications for ICSI use were male infertility factor, failure of fertilization with standard IVF, and/or idiopathic infertility. The primary indications for ICSI-Ca use were reduced fertilization in a previous ICSI cycle (< 50%) and/or poor utilization in the previous cycle.
Of the treatment cycles undertaken during this period, the “ICSI only cohort” includes 4422 cycles from 3260 patients who had only conventional ICSI cycles without calcium supplementation to use as a comparator for the subgroup analysis to benchmark the poor prognosis cohort where ICSI-Ca was prescribed. The “paired cohort” consists of 356 cycles from 178 patients that had one standard ICSI cycle immediately preceding one ICSI-Ca cycle, also excluding any other cycles from these patients to include only the single pair of cycles. Any patient who had an ICSI-Ca cycle without having a previous standard ICSI cycle was thus excluded from analysis. Subsequently, this cohort was divided into two further subsets based on the fertilization success of their ICSI cycle; the low-fertilization cohort (66 patients), where the ICSI cycles from these patients had < 50% fertilization in their ICSI cycle and the normal-fertilization cohort (112 patients) who had ≥ 50% fertilization in their standard ICSI cycle (as represented in Fig. 1).
Fig. 1.
Retrospective study design and cohorts of patients included in the study
Ovarian stimulation and oocyte retrieval
Ovarian stimulation was conducted as previously described [14]. Briefly, patients underwent either a gonadotrophin-releasing agonist (GnRH) or a GnRH antagonist cycle with recombinant FSH. Oocyte retrieval (oocyte pick up (OPU)) was scheduled 35 or 36 h after human chorionic gonadotrophin (hCG) trigger. Cumulus oocyte complexes (COCs) were incubated in 1 mL G-IVF PLUS (Vitrolife, Goteburg, Sweden) after oocyte collection. At 39 h post trigger, COCs were denuded in 0.5 mL of 75 IU/mL hyaluronidase (Hyalase, Sanofi-aventis, NSW, Australia) in G-IVF PLUS (Vitrolife), and stripped oocytes were incubated in 0.5 mL G-1 PLUS (Vitrolife) prior to insemination. All oocytes and embryos were cultured at 37 °C in 6% CO2, 5% O2, 89% N2 in benchtop incubators (MINC, Cook Medical, Brisbane, Australia or PLANER, Origio, Malov, Denmark).
ICSI and ICSI-calcium supplementation
ICSI and ICSI-Ca were both performed 40 h post trigger. Sperm were prepared for ICSI either by swim-up or gradient centrifugation (SpermGrad, Vitrolife), dependent on sample initial motility. In brief, swim-up was performed by layering 1 mL of semen under 1 mL G-IVF PLUS in a 14-mL conical tube (Vitrolife) and incubated at 37 °C, 6% CO2 for 15 min before taking the uppermost layer of purified sperm. Gradient preparation required layering 1.5 mL of 90% and 45% SpermGrad (Vitrolife) underneath 1 mL semen and centrifuging for 15 min at 16 to 115 g dependent on sperm concentration and motility. The pellet was washed twice with 5 mL G-IVF PLUS followed by centrifugation for 10 min at 7 to 115 g. The final pellet was resuspended in G-IVF PLUS to a concentration of 1 to 2 million/mL. Prepared sperm were placed in a 10 µL polyvinylpyrrolidone (PVP) drop in an ICSI dish (Vitrolife) under oil [OVOIL (Vitrolife)]. One or two oocytes were placed in each 10 µL G-1 PLUS drop covered in OVOIL of the same ICSI dish. Standard ICSI was conducted as previously described [15]. In brief, sperm were immobilized in a 10 µL PVP drop and aspirated before injection into MII oocytes. For ICSI-Ca, sperm were placed into a 10 µL PVP drop and immobilized before moving to a secondary PVP-Ca drop (5 µL PVP mixed with 5 µL 10 mM CaCl2 (Sigma-Aldrich, St Louis, USA) in G-1 PLUS). Sperm were washed through the PVP-Ca drop, aspirated with PVP-Ca and injected into MII oocytes. Inseminated eggs were immediately moved to 25 µL G-1 PLUS drops under OVOIL in a microwell dish (Vitrolife), one egg per drop, for ongoing culture.
Embryo culture, transfer and freezing
Embryos were cultured in sequential media (G-1 PLUS/G-2 PLUS, Vitrolife) in 25 µL drops under oil in a microwell dish (Vitrolife) after ICSI. Oocytes were assessed for fertilization 16 to 18 h post insemination. Embryo assessments were performed between day 2 and day 6. Blastocysts were developmentally classified [16] and quality graded by assessing the trophectoderm and ICM, as described previously [17], with the overall grade of each embryo (A, B, C, or D) corresponding to the lowest grade given, i.e.: trophectoderm or inner cell mass. Based on clinical prognosis, up to two embryos were transferred on day 3 (cleavage transfer) or 5 (blastocyst transfer), with a preference for day 5 single embryo transfer (ET). The minimum requirement for transfer on day 3 was 4-cells or more, and on day 5, the embryo must have begun the compaction process. Supernumerary embryos of suitable quality (C grade or above) were routinely frozen on days 5 and 6.
Clinical outcomes and definitions
The primary outcome measure was fertilization rate (FR) per MII oocyte injected, with secondary outcomes, including abnormal fertilization rate (either one or greater than two pronuclei observed at the fertilization check), oocyte degeneration rate post injection, embryo utilization rate (UR), clinical pregnancy (CPR) per embryo transfer, as indicated by the presence of a gestational sac on ultrasound at 6 weeks post transfer, implantation rate (total number of embryos transferred/total number of gestational sacs), and live birth rate (LBR) defined as deliveries per embryo transfer, with singletons or multiple births considered a single pregnancy [18]. UR, regardless of day of transfer, was defined as a percentage of the total number of embryos either transferred or cryopreserved per fertilized oocyte.
Statistical analyses
Normality of the datasets was tested with the Shapiro-Wilks test. Fisher’s exact test was used for categorical data. All continuous data were non-parametric, and Mann–Whitney U and Wilcoxon signed-rank test for paired samples were used for statistical analyses. All statistical analyses were undertaken using R (R Foundation for Statistical Computing, Vienna, Austria). P values < 0.05 were considered to be statistically significant, and all data are presented as mean ± SD unless otherwise stated.
Results
Demographics and outcomes in conventional ICSI cycles
To demonstrate the outcomes of the poor prognosis cohort who undertook calcium supplementation during the study period, an initial comparison of demographics and outcomes was conducted between the ICSI cycle from patients in the paired cohort (patients with an ICSI cycle and their subsequent ICSI-Ca cycle) and compared to the ICSI cycles of patients who had not had any calcium supplementation (“ICSI only”).
The “ICSI only” cohort consisted of 3260 patients who undertook 4422 ICSI cycles and no ICSI-Ca cycles during the study period. The “ICSI cycles from paired cohort” group consisted of 178 ICSI cycles, one from each of the 178 patients in the paired cohort. There were fewer oocytes collected and fewer MII oocytes injected in the ICSI cycles from the paired cohort compared to the ICSI only cohort (P < 0.05, Table 1). In addition, the paired cohort patients compared to patients within the ICSI only cohort were also more likely to have lower fertilization (51.3% ± 25.2 vs 65.7% ± 20.3, Table 1; P < 0.0001), higher degeneration rate (11.5% ± 12.5 vs 9.9% ± 12.4, Table 1; P < 0.05), lower utilization (37.7% ± 30.6 vs 42.3% ± 26.7, Table 1; P < 0.05), as well as reduced clinical pregnancy rates (6.7% vs 34.5%, Table 1; P < 0.0001), and live birth rate (3.3% vs 32.8%, Table 1; P < 0.0001). There was no significant difference between the two groups for female age, percentage of fresh ETs, or the mean number of embryos transferred.
Table 1.
Comparison of cycles from patients in the ICSI only cohort and conventional ICSI cycles from the paired cohort
| Parameter | ICSI only cohort | ICSI cycles from paired cohort | P value |
|---|---|---|---|
| Patients (n) | 3260 | 178 | |
| Number of cycles | 4422 | 178 | |
| Mean age female | 36.7 ± 4.4 | 37.0 ± 3.6 | NS |
| Mean (no.) oocytes collected | 13.7 ± 7.1 | 12.1 ± 5.5 | < 0.05 |
| Mean (no.) MII oocytes injected | 10.7 ± 5.6 | 9.3 ± 4.1 | < 0.05 |
| Fertilization rate (%) | 65.7 ± 20.3 | 51.3 ± 25.2 | < 0.0001 |
| Degeneration rate (%) | 9.9 ± 12.4 | 11.5 ± 12.5 | < 0.05 |
| Fresh ETs (% all cycles) | 2092 (47.3%) | 90 (50.6%) | NS |
| Mean (no.) embryos transferred | 1.2 ± 0.4 | 1.2 ± 0.4 | NS |
| Utilization rate (%) | 42.3 ± 26.7 | 37.7 ± 30.6 | < 0.05 |
| Implantation rate (%) | 29.4% (723/2461) | 5.6% (6/108) | < 0.0001 |
| Clinical pregnancy rate (%) | 34.5% (723/2090) | 6.7% (6/90) | < 0.0001 |
| Live birth rate (%) | 32.8% (685/2090) | 3.3% (3/90) | < 0.0001 |
Values are mean ± SD for continuous data or % (n/total) for categorical data. P < 0.05 was considered to be statistically significant. MII metaphase II, NS not significant, ET embryo transfer
Paired cohort analysis
The paired cohort consisted of 178 patients, each with one ICSI cycle followed by one subsequent ICSI-Ca cycle. There was no significant difference in female age, number of oocytes collected or injected degeneration, and abnormal fertilization rates or the percentage or day of ET (Table 2). However, the fertilization rate was significantly increased after ICSI-Ca treatment (ICSI: 51.3% ± 25.2 vs ICSI-Ca: 55.8% ± 22.6, Table 2 and Fig. 2; P < 0.05). Notably, seven patients had complete fertilization failure in their ICSI cycle, and one of these patients subsequently had fertilization failure as well in their ICSI-Ca cycle. In contrast, only one patient had fertilization in their ICSI cycle and total fertilization failure in their ICSI-Ca cycle, with no notable differences observed between the treatment in both cycles so the reason is unclear. No significant difference was observed between the frequency of fertilization failure between the two groups.
Table 2.
Comparison of paired outcomes for all paired cycles and patients with both ICSI and the subsequent ICSI-Ca cycles, based on their initial ICSI cycle being either normal (≥ 50%) or low (< 50%) fertilization
| Parameter | All | Low-fertilization cohort | Normal-fertilization cohort | ||||||
|---|---|---|---|---|---|---|---|---|---|
| ICSI | ICSI-Ca | P value | ICSI | ICSI-Ca | P value | ICSI | ICSI-Ca | P value | |
| Patients (n) | 178 | 66 | 112 | ||||||
| Number of cycles | 178 | 178 | 66 | 66 | 112 | 112 | |||
| Mean age female | 37.0 ± 3.6 | 37.6 ± 3.5 | NS | 37.0 ± 3.7 | 37.5 ± 3.7 | NS | 37.0 ± 3.5 | 37.7 ± 3.4 | NS |
| Mean (no.) oocytes collected | 12.1 ± 5.5 | 12.3 ± 5.4 | NS | 12.3 ± 5.3 | 12.4 ± 5.0 | NS | 12.0 ± 5.6 | 12.2 ± 5.6 | NS |
| Mean (no.) MII oocytes injected | 9.3 ± 4.1 | 9.4 ± 4.1 | NS | 9.1 ± 4.3 | 9.1 ± 3.8 | NS | 9.4 ± 4.0 | 9.6 ± 4.0 | NS |
| Fertilization rate (%) | 51.3 ± 25.2 | 55.8 ± 22.6 | < 0.05 | 28.8 ± 13.2 | 49.7 ± 23.9 | < 0.0001 | 64.5 ± 20.7 | 59.4 ± 21.1 | <0.05 |
| Degeneration rate (%) | 11.5 ± 12.5 | 11.0 ± 12.8 | NS | 15.0 ± 13.4 | 9.6 ± 11.2 | < 0.05 | 9.5 ± 11.5 | 11.8 ± 13.6 | NS |
| Abnormal fertilization rate (%) | 4.1 ± 7.8 | 4.7 ± 8.39 | NS | 4.3 ± 8.6 | 2.6 ± 5.7 | NS | 4.1 ± 7.3 | 5.9 ± 9.4 | NS |
| Fresh ETs (% all cycles) | 50.6% (90/178) | 52.2% (93/178) | NS | 62.1% (41/66) | 60.6% (40/66) | NS | 43.8% (49/112) | 47.3% (53/112) | NS |
| Cleavage stage ET | 43.3% (39/90) | 52.7% (49/93) | NS | 61.0% (25/41) | 42.5% (17/40) | NS | 28.6% (14/49) | 60.4% (32/53) | < 0.05 |
| Blastocyst stage ET | 56.7% (51/90) | 47.3% (44/93) | NS | 39.0% (16/41) | 57.5% (23/40) | NS | 71.4% (35/49) | 39.6% (21/53) | < 0.05 |
| Mean (no.) embryos transferred | 1.2 ± 0.4 | 1.4 ± 0.5 | < 0.0001 | 1.2 ± 0.4 | 1.3 ± 0.4 | NS | 1.2 ± 0.4 | 1.6 ± 0.5 | < 0.0001 |
| Mean (no.) embryos utilized | 2.7 ± 1.1 | 3.3 ± 1.3 | < 0.05 | 2.5 ± 0.7 | 3.3 ± 1.4 | NS (0.07) | 2.8 ± 1.3 | 3.2 ± 1.3 | NS (0.07) |
| Utilization rate (%) | 37.7 ± 30.6 | 45.0 ± 30.0 | < 0.05 | 46.5 ± 36.2 | 45.2 ± 32.9 | NS | 32.6 ± 25.7 | 44.9 ± 28.3 | < 0.0001 |
Values are mean ± SD for continuous data or % (n/total) for categorical data. P < 0.05 was considered to be statistically significant. MII, metaphase II, NS not significant, ET embryo transfer
Fig. 2.
Comparison of fertilization rate among study groups. Entire paired cohort (ICSI n = 178, ICSI-Ca n = 178), low-fert cohort (ICSI n = 66, ICSI-Ca n = 66), and normal-fert cohort (ICSI n = 112, ICSI-Ca n = 112). Fertilization rate was expressed as a percentage of MII oocytes injected at ICSI. Error bars represent SEM. *P < 0.05, **P < 0.001
There were significantly more embryos utilized and transferred per ET following ICSI-Ca treatment (ICSI: 2.7 ± 1.1 vs ICSI-Ca: 3.3 ± 1.3 embryos utilized; ICSI: 1.2 ± 0.4 vs ICSI-Ca: 1.4 ± 0.5 embryos per ET; P < 0.0001, Table 2). Subsequently, the utilization rate was increased after ICSI-Ca treatment (ICSI: 37.7% ± 30.6 vs ICSI-Ca: 45.0% ± 30.0, Table 2; P < 0.05). Calcium supplementation also significantly increased implantation rate (ICSI: 5.6% vs ICSI-Ca: 15.8%; P < 0.05, Fig. 3), clinical pregnancy rate (ICSI: 6.7% vs ICSI-Ca: 22.6%; P < 0.05, Fig. 3), and live birth rate (ICSI: 3.3% vs ICSI-Ca: 16.1%; P < 0.05, Fig. 3) compared to ICSI alone.
Fig. 3.
Implantation, clinical pregnancy, and live birth rates (% of ETs) for patients among study groups. Paired cohort (ICSI n = 90, ICSI-Ca n = 93), low-fert cohort (ICSI n = 41, ICSI-Ca n = 40), and normal-fert cohort (ICSI n = 49, ICSI-Ca n = 53). n is the number of ETs. *P < 0.05
Low-fertilization paired cohort analysis
In the paired cohort, 66 patients had ICSI cycles with < 50% fertilization in their initial cycle and utilized ICSI-Ca on their subsequent cycle. There was no significant difference in the mean number of MII oocytes injected (ICSI: 9.1 ± 4.3 vs ICSI-Ca: 9.1 ± 3.89; P > 0.1, Table 2) or percentage of fresh ETs following calcium supplementation (ICSI: 62.1% vs ICSI-Ca: 60.6%; P > 0.1, Table 2). There was a significant decrease in degeneration rate following calcium supplementation but no change in the abnormal fertilization rate (ICSI: 15.0% ± 13.4 vs ICSI-Ca: 9.6% ± 11.2 degeneration rate; P < 0.05, ICSI: 4.3% ± 8.6 vs ICSI-Ca: 2.6% ± 5.7 abnormal fertilization rate; P > 0.1, Table 2). There was however, a significant increase in fertilization rate (ICSI: 28.8% ± 13.2 vs ICSI-Ca: 49.7% ± 23.9; P < 0.0001, Table 2 and Fig. 2), implantation rate, and clinical pregnancy rate (ICSI: 4.2% vs ICSI-Ca: 20%; P < 0.05, Fig. 2, and ICSI: 4.9% vs ICSI-Ca: 25.0%, P < 0.05 respectively; Fig. 3). There was also a small but non-significant increase in live birth rate following calcium supplementation with the use of ICSI-Ca (ICSI: 2.4% vs ICSI-Ca: 15%; P = 0.057, Fig. 3). Calcium supplementation also did not affect utilization rates in this cohort (ICSI: 46.5% ± 36.2 vs ICSI-Ca: 45.2% ± 32.9; P > 0.1, Table 2).
Normal-fertilization paired cohort analysis
Within the study period, 112 patients received calcium supplementation who had ≥ 50% fertilization in their previous ICSI cycle; however, they had poor utilization, with the utilization rate in their ICSI cycle even lower than that in the low-fertilization cohort (low-fert cohort: 46.5% ± 36.2 vs normal-fert cohort: 32.6% ± 25.7, P < 0.05). These normal-fertilization patients showed a small but significant decrease in fertilization rate after calcium supplementation relative to their initial ICSI cycle (ICSI: 64.5% ± 20.7 vs ICSI-Ca: 59.4% ± 21.1, P < 0.05; Fig. 2 and Table 2); however, they had a significantly improved embryo utilization rate (ICSI: 32.6% ± 25.7 vs ICSI-Ca: 44.9% ± 28.3, P < 0.05; Table 2). There was no significant difference in either degeneration rate or abnormal fertilization rate (Table 2). In addition, although the fertilization rate was slightly decreased following calcium supplementation, the utilization rate was increased, which led to no significant change in the actual number of embryos utilized per cycle (ICSI: 2.8 ± 1.3 vs ICSI-Ca: 3.2 ± 1.3; Table 2). Clinical pregnancy rate and implantation rate were not significantly different between the two groups (Fig. 3); however, there was a small but non-significant increase in the live birth rate after calcium supplementation (ICSI: 4.1% vs ICSI-Ca: 17.0%, P = 0.054; Fig. 3).
Discussion
This is the first study to describe the efficacy of calcium supplementation alone to the ICSI media (PVP) on outcomes in poor prognosis patients. This protocol involved directly injecting medium with increased calcium chloride dihydrate concentration concurrently with the spermatozoa into the oocyte at the time of ICSI. Notably, using a powerful paired cycle within patient analysis that compared ICSI-Ca to a standard ICSI cycle, calcium supplementation at ICSI improved outcomes such as fertilization, implantation, and pregnancy rate for patients who had a history of low fertilization (< 50%). For patients with normal fertilization, calcium supplementation resulted in a reduction in fertilization rate, but an increase in utilization rate, although no differences were evident in clinical pregnancy and live birth rates. These findings support the use of calcium supplementation for patients with a history of low fertilization. Previously, the ability of calcium chloride injection alone to induce oocyte activation has only been reported in porcine oocytes [19] and in human ART has only been used in conjunction with calcium ionophore exposure [10–13]. Therefore this novel approach to AOA, using only calcium chloride supplementation shows promise in a clinical setting.
The current study demonstrated that calcium supplementation in patients with a history of low fertilization resulted in a significant increase to fertilization rates. In comparison, studies using calcium ionophores in a patient cohort with low fertilization (< 30%, < 33%, or < 50%) have shown similar increases in fertilization ranging from 47.7 to 67% [12, 20–22]. Consequently, the current study showed an increase in fertilization tends to lead to a small increase in the number of embryos utilized, from 2.5 to 3.3 per cycle but most striking is the positive effect on implantation and clinical pregnancy rate. Notably, there was a fivefold (20%) increase in mean clinical pregnancy rate to 25% in the ICSI-Ca cycle compared to the ICSI only cycle of the low-fert cohort. Murugesu et al. [8] previously reported a pregnancy rate increase from 15.6 to 22.6% following AOA with calcium ionophore in a meta-analysis, although their patient population was significantly larger considering data from 14 studies. In the current study, it appears that this increase in fertilization in the calcium supplementation cycles led to an increase, though not quite statistically significant, in live birth rate. Nevertheless, increasing the power of analysis by combining both low and normal fertilization cohorts does result in a statistically significant increase in births for the entire paired cohort after calcium supplementation compared to ICSI alone. The live birth rate per ET in the paired cohort increased from 3.3 to 16.1% following calcium supplementation, in comparison to 9.1 to 26.8% after calcium ionophore treatment in the Murugesu meta-analysis [8]. It is also important to note that the rate of abnormal fertilization was not affected by ICSI-Ca treatment. Degeneration rate was also unchanged in the paired and normal fertilization cohorts, although notably it was decreased after ICSI-Ca treatment in the low fertilization cohort; the reason for this is unclear and requires further investigation.
In the present study, calcium supplementation did have a small but marked negative impact on fertilization rates in the normal fertilization cohort. Fortunately, the increase in utilization rate offsets the lower fertilization rate in these patients, with the utilization rate increasing from 32.6 to 44.9%, which is consistent with the utilization rate in our general patient population who have standard ICSI only. However, it is important to highlight that this may be attributed to an increased use of cleavage embryos, thus, increased number of embryos at transfer as observed in our results. Alternatively, the increase in utilization rate and the trend toward higher clinical pregnancy and live birth rates in the normal fertilization cohort is consistent with the suggestion that AOA may also influence embryo development, and the benefits lie beyond fertilization alone [23, 24]. However, the mechanism by which calcium supplementation at ICSI influences embryo development is not well understood. It is hypothesized that a lack of intracellular calcium stores results in cleavage anomalies and embryo arrest; therefore, a boost of calcium at the time of insemination may replenish stores and remove blocks in further cell cleavage [23]; however, further research into this area is warranted.
During ICSI with calcium supplementation, the spermatozoa are washed through an increased calcium concentration solution prior to injection into the oocyte. This technique may affect the action of the spermatozoa, activation of the oocyte or both, as calcium is not only important for oocyte activation but is also a critical electrolyte required for sperm capacitation and subsequent induction of the acrosome reaction [25]. Induction of both sperm capacitation [26] and the acrosome reaction in mammals prior to ICSI is known to improve both fertilization and embryo development [27–29]. Capacitation is an important physiological process sperm must undergo to be able to fertilize an oocyte. In vivo, mammalian sperm capacitation occurs in the female reproductive tract [25]; however, in vitro this is simulated in capacitation medium that requires serum albumin, Ca2+, and NaHCO3− [30]. The increase in intracellular calcium during capacitation of spermatozoa is mediated by both intracellular release from the acrosome [31] and opening of calcium channels in the membrane to facilitate influx of external Ca2+ ions [32, 33], with external Ca2+ ions contributing to the majority of the rise in calcium levels during capacitation [34]. In addition, in the current study at the time of ICSI with calcium supplementation, a small amount of calcium is also directly injected into the oocyte with the spermatozoa. Thus, it is likely that calcium is affecting the oocyte during calcium supplementation, as the contact with spermatozoon is transient, it cannot be ruled out that the effects seen are influencing the oocyte, as well as the spermatozoon.
Due to the retrospective nature of the study, not all constraints and variables can be controlled for or tested. Using a paired analysis with one cycle in each treatment group per patient helps eliminate many potential confounders. Moreover, low responders (≤ 5 oocytes) and outliers with a high number of cycles (greater than six) were also excluded from the study. However, one potential limitation was that during ICSI-Ca, the medium used at the final stage of injection has a 50% reduction in PVP concentration, due to the dilution with calcium-enriched medium, so this may also be contributing to the improvements identified. A recent study has shown an increase in fertilization and embryo development with lowering PVP concentrations (10%, 7%, and 5%) but they did not report on pregnancy rates [35]. In the calcium supplementation protocol described in this study, calcium chloride dihydrate is dissolved in medium that has a calcium ion concentration of 1.1 mM [36], which is similar to multiple different culture media brands available on the market (1.1 to 2.2 mM Ca2+), effectively increasing the calcium concentration tenfold to ~ 10 mM, which is then used to supplement the PVP with a 50:50 dilution of PVP to calcium medium. A combination of decreased PVP concentration and increased calcium concentration may assist in inducing the acrosome reaction prior to ICSI, leading to more efficient pronuclear formation, thus improving outcomes; however, further research needs to be undertaken to confirm this hypothesis.
The findings of the current study show that calcium supplementation at ICSI may improve outcomes for poor prognosis patients; however, there are limitations that need to be acknowledged largely due to the studies’ retrospective nature and lower patient numbers in the subgroup analysis. A prospective case-matched or randomized control trial (RCT) to further investigate effects is warranted, particularly to control for standardization of treatment, for example, the dilution of PVP, the amount of calcium injected, and the timing of sperm incubation in calcium prior to injection. Determining the best indications for use would also be useful by looking at the etiology of patient cohorts in which calcium ionophore has been shown to be beneficial, such as globozoospermia, teratozoospermia, and poor embryo development [8]. A comparison between calcium ionophore and calcium supplementation in a sibling oocyte study would also determine the effectiveness of this treatment in comparison to the current commercially available treatment for AOA, calcium ionophore. If increasing the concentration of calcium at ICSI benefits patients, particularly those with a history of low fertilization by increasing both fertilization and pregnancy rates, this may be a relatively simple way of improving outcomes by altering the media composition used at the time of microinjection.
Conclusion
In conclusion, using a paired analysis of ICSI and ICSI-Ca cycles in patients with a history of low fertilization (< 50%), the current study found that fertilization and clinical pregnancy rates were significantly increased by calcium supplementation compared to ICSI alone. However, the same benefits were not observed in patients presenting normal fertilization. Importantly, due to the nature of our study, prospective case-matched and/or RCT studies are required to further validate the clinical application of our calcium supplementation procedures in patients presenting a history of low fertilization.
Author contribution
SP, FH, MG, and DZ designed the study. SP conducted all statistical analyses, interpreted results, and drafted the article. All authors (SP, FH, BV, MG, and DZ) interpreted results, reviewed the manuscript and provided substantial advice through the data analysis work, and contributed intellectually to the writing or revising of the manuscript and approval of the final version.
Funding
This study is funded by Monash IVF Group, Victoria.
Declarations
Ethics approval
Ethics approval for the study was obtained under Monash Health Ethics Number: 15172 M.
Conflict of interest
The authors declare no competing interests.
Footnotes
Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Mark P Green and Deirdre Zander-Fox contributed equally to this manuscript
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