Effect of Chemical versus Mechanical Oocyte Activation on Morphokinetic Embryonic Development in Oligoasthenoteratozoospermia Patients

doi:10.21203/rs.3.rs-3467532/v1

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Research Article

Effect of Chemical versus Mechanical Oocyte Activation on Morphokinetic Embryonic Development in Oligoasthenoteratozoospermia Patients

https://doi.org/10.21203/rs.3.rs-3467532/v1

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Objective

Assisted oocyte activation (AOA) is applied in male factor infertility for improving the successful fertilization rate. However, the safety and efficacy of AOA technique remain unknown. The aim of this study is the comparison of two different mechanical and chemical AOA methods on pre-implantation morphokinetic development of embryos derived from oligoasthenoteratozoospermia patients.

Methods

This study was applied in 65 cycles achieved between 2020 and 2022; 137 embryos were derived from ICSI-chemical AOA cycles and 124 embryos from ICSI-mechanical AOA cycles. Morphokinetic characteristics of all embryos were monitored via a time-lapse system.

Results

Our results showed similar morphokinetic development in both early and late-stage embryonic development in two different groups of this study.

Conclusions

Our results suggest that both mechanical and chemical methods AOA have the same effect on morphokinetic pattern of pre-implantation embryo development in oligoasthenoteratozoospermia.

Artificial oocyte activation

Intra cytoplasmic sperm injection

Ionomycin

Morphokinetic

Time-lapse system

Nowadays male factors are involved in 30–50% of all cases of infertility [1, 2]. However, nearly 30% of all cases of infertility have been identified as unexplained cases [3]. For overcoming these problems of infertility, intracytoplasmic sperm injection (ICSI) is the technique optimal for reaching fertilization and pregnancies in the cases of severe oligoasthenoteratozoospermia and unexplained infertility. In this method, a single sperm is injected directly into the oocyte, which is characterizes as a standard method [4, 5]. Currently, ICSI is a suitable technique for the treatment of various infertility cases [6]. The successful rate of fertilization by the ICSI method can be different from 70–80%. However, failed fertilization rate occurred in some cases with recurrent fertilization failure. Therefore, the ICSI method could not support fertilization failure even, in infertile couples with normal semen characteristics [7]. Thus, the 1–5% fertilization failure is not unexpected in ICSI cycles [8]. Although, the most common factor for ICSI failure is characterized in unexplained infertility [9]. However, a series of events that occur during fertilization guarantee a success rate of fertilization. Among these factors, oocyte activation (OA) after sperm injection is the essential factor for the successful rate. Both sperm and oocyte contribute to oocyte activation failure. Any defect in both of them could prevent mature oocytes undergoing activation and inhibit complete fertilization which is associated with ICSI failure up to 40% [10, 11]. Among factors that threaten the fertilization process, artificial oocyte activation (AOA) after the ICSI process could suggest in oligoasthenoteratozoospermia and globozoospermia patients [12, 13].

Following sperm entry into the ooplasm, intracellular calcium oscillations occurred from endoplasmic reticulum stores. It has been accepted that increases in the calcium level have an essential role in downstream signals leading to oocyte activation and the beginning of cleavage in fertilized oocytes. Therefore, an artificial oocyte activation technique could mimic this process in the ooplasm. In spermatozoa, the specific phospholipase C-zeta (PLCz) has a key role in intracellular calcium oscillations [14, 15]. Therefore, failed fertilization is not unexpected in male factor abnormalities such as globozoospermia and oligoasthenoteratozoospermia which are associated with a deficiency in the PLCz cascade [16, 17]. Overcoming failed fertilization could be employed by either mechanical [18, 19], electrical [20] or chemical stimuli [21], in order to induce artificial Ca2 + increases in the ooplasm. Although, the fertilization rate could be improved following ICSI-AOA in comparison with ICSI only, however, some studies indicated that the efficacy and safety of these methods remain unknown. For this purpose, we used the time-lapse technique. In the time-lapse technology pri-nucleus (PN) and embryo development could be surveyed in multiple observations [22, 23]. Since PN valuation is the most important factor for confirmation of fertilization, the time-lapse approach could be more suitable for comparing two mechanical and chemical oocyte activation methods. Thus, the aim of this study was to compare the effect of chemical AO with mechanical AO on fertilization, cleavage, and pregnancy rates in oligoasthenoteratozoospermia patients by monitoring the time-lapse approach.

Patients, ovarian stimulation and oocyte retrieval

The study was done on 65 couples in the period between 2020 and 2022, the male partners with oligoasthenoteratozoospermia undergoing ICSI cycles were included in this study. Written informed consent was obtained, in which patients agreed to share the outcomes of their own cycles for research purposes, and the study was approved by the Hormozgan University of Medical Sciences (IR.HUMS.REC.1398.002). The patients with surgically retrieved spermatozoa from testis or epididymis were excluded. The average female age was between 25–38 years and the cases with any female factors infertility like polycystic ovary syndrome, endometriosis, poor ovarian responders, and known genetic abnormally were excluded. In this study, ovarian stimulation was conducted using standard protocols, either long or short stimulation with an agonist or antagonist. Once at least three follicles reached ≥ 19 mm in diameter, ovulation was triggered with the administration of a single dosage of 250 µg recombinant human chorionic gonadotropin (hCG, Ovidrel, Sereno). After 35–36 h, oocyte retrieval was achieved under transvaginal ultrasound guidance.

ICSI, culture, embryo transfer, and pregnancy

Cumulus–oocyte–complexes (COC) were isolated from ovarian retrieved fluid and washed with HEPES-buffered medium (gamete medium; Cook, Brisbane, Australia). Then the COC was denuded with hyaluronidase enzyme to remove granulosa cells. After oocytes were incubated in a cleavage medium for 1–2 h, ICSI was performed. For ICSI procedures, spermatozoa were prepared by gradient swim-up method and placed into polyvinylpyrrolidone (PVP) solution (Invitrogen) in a standard plastic dish. Moreover, the mature oocytes were placed in droplets of the gamete medium and were injected using a microinjection needle in the same dish. Injected oocytes were placed at the cleavage medium and incubated in the time-lapse incubator. After 16–18 h, the presence of pronuclei was checked and the fertilization rate was assessed. On day 3, the cleaved embryos were placed and incubated in a new dish with blastocyst medium droplets covered with mineral oil until day 5 after microinjection.

Embryo transfer was performed on day 5. In this study, the oocytes were divided into two groups: group A (ICSI with chemical AOA, n = 278 and group B (ICSI with mechanical AOA, n = 256).

Chemical artificial oocyte activation:

Chemical oocyte activation was done as described previously [10]. Immediately following the ICSI procedure, mature oocytes were inserted in a 50-µl droplet of activation medium containing 10 µmol/l ionomycin (MP Biomedical, USA) in culture medium (cleavage medium; Invitrogen). The droplet was covered by mineral oil (Invitrogen) and incubated for 15 min at 37ᵒ C. then, the treated oocytes were washed with a culture medium and finally inserted in the time-lapse incubator with standard situations in 6% CO2, 5% O2, and 89% N2. After 18 h the PN formation was checked. 3 days after sperm injection the cleavage rate was assessed and for further evaluation oocytes were cultured in the blastocyst medium (Invitrogen) and incubated under mentioned states.

Mechanical artificial oocyte activation:

In order to mechanical oocyte activation, the oocyte was stimulated by vigorous cytoplasmic aspiration via a microinjection needle during the ICSI procedure [24]. This manually cytoplasmic stimulation leads to an increase in oocyte Ca 2 + level and improves the fertilization process [19]. After ICSI, the injected oocytes were cultured in the cleavage medium (Invitrogen) and inserted in the time-lapse incubator. All assessments were the same as the group A oocytes until day 5 after injection.

Embryo evaluation and selection by the time-lapse system

The Time-lapse system was organized by capturing an individual embryo every 20 minutes at 7 different focal planes. All the events related to embryo development are annotated together with the corresponding hour post-ICSI insemination (hpi). All morphokinetic data were recorded as mean ± SD hpi. In the time-lapse system. The morphokinetic timing nomenclature was based on the guidelines of Ciray et al. [25]. 3 predictive parameters are considered as a selection of high-quality embryos. These parameters include 1) cc2: time between division to 2 cells and division to 3 cells (≤ 11.9 h), 2) s3: time between division to 3 cells and subsequent division to 4 cells (≤ 0.76 h), and 3) t5 = time of division to 5 cells: 48.8–56.6 hpi. On the other hand, three parameters that were considered negative factors for implantation include, direct cleavage from the one- to the three-blastomere embryo, uneven blastomere size at the two-cell stage, and multinucleation at the four-cell stage. By considering these predictive factors the high-quality embryos were selected for transfer on day 5 after ICSI insemination. Moreover, other quantitative morphokinetic parameters wer analyzed; including appearance of the 2 pronuclei (t2PN), divisions to 2-cell through 8-cell stages (t2 to t8) and full blastocyst stage (tB). After 14 days from embryo transfer, the positive βhcg test was recorded as a positive chemical pregnancy.

Statistical analysis

All the characteristics including morphokinetic parameters of embryos, cleavage rate, high embryo quality rate, blastocyst formation rate, and chemical pregnancy rate were compared between groups A and B by using Pearson’s chi-square test (two-tailed) by GraphPad prism software. A p-value of < 0.05 was considered statistically significant.

The effect of ionomycin in comparison with mechanical activation on fertilization rate in oligoasthenoteratozoospermia patients:

In this study from 65 cycles, 588 oocytes were retrieved. Total oocytes were divided into two groups A and B. In group A, 278 oocytes were inseminated by the ICSI method and then incubated in ionomycin and monitored by time-lapse system. In group B, 256 oocytes after mechanical activation during insemination by ICSI methods, were monitored by the time-lapse system for assessing embryonic development. In group A, oocytes 137/278 (49.28%) showed 2PN, indicating activation and fertilization. In group B among 256 activated oocytes, 124 (48.43%) showed 2PN. The statistical analysis revealed that no significant differences were seen between the two groups regarding fertilization rate P > 0.05 (Table 1).

Table 1

ICSI outcomes and blastocyst development in sibling oocytes of 65 infertile couples following chemical and mechanical artificial oocyte activation.
No. of oocytes	2PN	3PN	Good quality embryos d3	Compact embryos	Blastocyst formation
278	137 (49.28%)	5 (1.79%)	76 (27.33%)	68 (24.46%)	59 (21.22%)
256	124 (48.43%)	4 (1.56%)	64 (25%)	55 (21.48%)	48 (18.75%)
P-value	0.34	0.21	0.11	0.08	0.07

Pn: pronuclei

The effect of ionomycin in comparison with mechanical activation on morphokinetic parameters at an early stage of embryo development in oligoasthenoteratozoospermia patients:

The morphokinetic parameters and irregular cleavage of embryonic development were compared between group A and group B embryos in the time-lapse monitoring group (Table 2 &3). Our results showed that the time between division to 2 cells and division to 3 cells (cc2) in both groups was statistically similar (13.2 ± 1.5 hpi vs. 12.9 ± 1.1 hpi, respectively, p > 0.05). Moreover, the time between division to 3 cells and subsequent division to 4 cells (s3) was not statistically different between the two groups (0.77 ± 0.69 hpi vs. 0.78 ± 0.71 hpi, respectively, p > 0.05). Also, there were no significant differences in the time from insemination to 5 cells (t5) between chemical and mechanical oocyte activation (61.1 ± 3.8 hpi vs. 63.5 ± 2.7 hpi, respectively, p > 0.05).

Table 2

Irregular cleavages in chemical and mechanical artificial oocyte activation in sibling oocytes
	Chemical AOA	Mechanical AOA
No. of fertilized oocytes	137	126
Irregular chaotic division	24 (17.51%)	20 (15.87%)
Trichotomous mitosis	15 (10.95%)	12 (9.52%)
1 to 3 cells	9 (6.56%)	8 (6.34%)
2 to 4 cells, 3 to 5 cells	4 (2.91%)	5 (3.96%)
Cell fusion	7 (5.11%)	6 (4.76%)
Planar embryos	3 (2.19%)	3 (1.92%)
Regularly cleaved embryos	75	73

Irregular cleavages were assessed according to Ciray et al. (2014) and Desai et al. (2018). P > 0.05

The effect of ionomycin in comparison with mechanical activation on morphokinetic parameters at a late stage of embryo development in oligoasthenoteratozoospermia patients:

The morphokinetic parameters of the later stages of embryonic development were also compared between chemical and mechanical oocyte activation groups in the time-lapse monitoring group (Table 3). Our results showed that the time from insemination to initiation of blastulation (tIB) was a little late in the mechanical oocyte activation group in comparison with chemical OA (104.4 ± 4.5 vs. 107.1 ± 6.2 hpi, respectively, p > 0.05). Moreover, the time from insemination to the formation of a full blastocyst (tFB) in the mechanical oocyte activation group was slightly slower than that of the chemical OA group (108.3 ± 6.0 vs. 110.8 ± 7.2 hpi, respectively, p > 0.05). Also, the time from insemination to the formation of an expanded blastocyst (tEB) was not different between both groups (112.9 ± 8.1 vs. 114.2 ± 7.6 hpi, respectively, p > 0.05). Thus, our results showed that none of the factors in morphokinetic parameters were statistical significance in the late stage of embryonic development between the embryos from chemical and mechanical oocyte activation.

Table 3

Annotated morphokinetic parameters of all embryos that made it to the blastocyst stage in chemical and mechanical artificial oocyte activation in sibling oocytes
parameters	Chemical AOA		Mechanical AOA		P-value
	Mean	Range	Mean	Range
tPN	5.2	1.2–9.3	6.1	1.9–10.4	0.45
t2	29.3	22.2–35.5	29.9	23.1–35.7	0.15
cc2	12.9	10.56–15.03	13.2	10.98–15.4	0.33
S3	0.77	0.56–0.98	0.79	0.58 − 0.100	0.41
t5	61.1	55.23–68.1	63.5	53.21–69.32	0.28
t8	73.1	51.4–87.1	74.2	50.3–88.5	0.34
tIB	104.4	95.6–112	107.1	96.3-119.5	0.76
tFB	108.3	97.5-115.3	110.8	99.2-116.7	0.66
tEb	112.9	99.8–130	114.2	100.1-131.8	0.12

Pn: pronuclei.

The effect of ionomycin in comparison with mechanical activation on chemical pregnancy rate in oligoasthenoteratozoospermia patients:

During embryo transfer, we have no significant difference in the percentage of each morphological grade of the transferred blastocysts between the two groups (p > 0.05). Therefore, our result showed non-significant differences in a chemical pregnancy rate in both groups (Table 4)

Table 4

Chemical pregnancy outcomes in 65 study patients divided into transfers from chemical and mechanical AOA embryos.
	Chemical AOA	Mechanical AOA
Embryos transferred	36	29
Positive hCG	16 (44.44%)	13 (44.82)

P > 0.05

Male factor infertility could impair the fertilization rate and could be one of the most causes of assisted reproductive technique [26]. Low fertilization rate compromise ART outcomes in couple with severe male factor like oligoasthenoteratozoospermia [27]. For overcoming this problem artificial oocyte activation was highlighted. Investigators suggested using calcium ionophore A23187 or ionomycin for artificial oocyte activation (AOA) as an effective treatment in male factor infertility [28]. Also, artificial oocyte activation has effective results on fertilization rate, embryonic development competency, and pregnancy rate. However, the safety and efficacy of these methods remain unknown [29].

The present study aims to compare the effects of two mechanical and chemical AOA after ICSI on embryonic development and pregnancy rate in oligoasthenoteratozoospermia by time-lapse monitoring. For this purpose, in group A, immediately after microinjection mature oocytes were cultured with the ionomycin under the oil for 15 min. In group B, vigorous cytoplasmic aspiration as mechanical AOA was performed by microinjection needle during ICSI.

Injected oocytes were incubated in a time-lapse system. The embryonic development in the early and late stages was monitored by the time-lapse system. The final goal of oocyte activation and selection of the most competent embryo by the time-lapse system was to increase the chance of successful pregnancy in severe male factor patients. Regarding equal conditions for embryo transfer, our results showed that there were not any differences in both chemical pregnancy rate in both groups.

Fertilization frailer is extremely high in infertile couples, especially in those with severe male factors such as oligoasthenoteratozoospermia [30]. Some studies demonstrated an improvement in pregnancy outcomes for artificial oocyte activation [21]. The present studies showed that although the 2PN formation was higher in group A in comparison with Group B, however, it is not statistically different. In this regard, Martínez et al. in their cohort study showed that ionomycin could shorter the time to 2PN in comparison with conventional ICSI. They suggested that ionomycin could improve the increases of Ca2 + in ooplasmic after ICSI without induction of any defect on oocyte morphokinetic and related embryos [31].

Oscillation of Ca2 + lead to inactivation of maturation promoting factors and consequently, progress meiotic resumption and extrusion of the second polar body. Therefore they revealed that the t2PN was shorter than the control group [31]. These findings were proved by a previous study that calcium oscillation has a key role in oocyte response to the fertilization process via an effect on CaMKII modulation [32]. In parallel, the use of mechanical oocyte activation in sibling oocytes in group B revealed that ooplasm aspiration during sperm injection is associated with the reasonable time for 2PN formation in similar oocytes in the group A. This data could be confirmed with the previous study showed that aspiration of ooplasmic, near the mitochondria region, has effective results in fertilization rate [19]. Their studies indicated that mechanical oocyte activation could apply instead of conventional ICSI in the cases of repeated fertilization failure. However, we could not find a study that analyzes the morphokinetic of the oocytes and embryos following mechanical oocyte activation. Meanwhile, current progress in the time-lapse system has provided novel morphokinetic markers for choosing competent embryos for transfer. For this goal, we have combined these two approaches in the IVF clinic. Here we showed that the early stages of development of embryos were similar in both groups according to the normal pattern. It has been suggested that the shorter time for s3 is a key factor for the prediction of blastocyst formation and embryo implantation [33]. The assessment of cc2, s3, and t5 as predictive factors for the blastocyst formation although are shorter in the group A oocytes however were not significant statistically. Moreover, we showed a synchronization cleavage in the s3 stages until the 8-cell embryos in both groups. The results of shebl et al. study with shorter s3 and cleavage of blastomeres in an orchestrated manner in fertilized oocytes which are treated by ionophore are consistent with our results [29]. However, there is controversy about applying chemical oocyte activation in fertilization failure cases. One hypothesis for this controversy could come from different kinds of chemical activation components. For instance, in the study by Nikiforaki et al. the two different AOA methods showed that the usage of ionomycin instead of A23187 was more effective for overcoming fertilization failure [28]. Another hypothesis could be due to the use of different groups of patients in different study designs. Therefore in a shebl et al. study, we arrange sibling oocytes in the two groups for comparing chemical and mechanical OA methods [29]. In this study, as opposed to the Martinez et al. study that included couples with a complete fertilization failure [31], we used the couples with the male factor infertility with oligoasthenoteratozoospermia features without female factors infertility. Because it seems that individualization of artificial oocyte activation could be accompanied by specific results for overcoming fertilization failure. In our line shebl et al. indicated the Ca2 + oscillation is faster in the cases with male factor infertility like PLCζ gene mutations than other factors that caused fertilization failure [6, 29].

Our results also showed that there were no significant differences in implantation rate between the chemical oocyte activation (Group A) and mechanical oocyte activation (Group B). Moreover, the implantation rate was similar in both groups. It was shown that by mechanical oocyte activation, mitochondria were accumulated

Collectively, our data reveal the distinct benefits of combining oocyte activation and time-lapse monitoring to select competent blastocysts for transfer to patients with oligoasthenoteratozoospermia. In this regard, the results from Meseguer et al. indicated that monitoring and selecting most component embryos with a time-lapse system has an effective role in the development of clinical pregnancy and implantation rates in comparison with conventional systems [34]. During the fertilization process, the mitochondria from the peri-cortical zone migrate to the center of the ooplasm. These mitochondria have an essential role in Ca2 + oscillation [19]. It seems that PLCz could accelerate the accumulation of mitochondria near the sperm injection site. Therefore, in the patients with the PLCz mutation, assisted mechanical accumulation of mitochondria near the sperm injection location could help the fertilization process and overcome fertilization failure.

Ethics approval and consent to participate

The present research was approved by the ethics committee of the Fertility and Infertility Research and Clinical Center of Hormozgan University of Medical Sciences (# IR.HUMS.REC.1398.002). We confirm that all experiments were performed in accordance with relevant guidelines and regulations. Moreover, a signed informed consent form was obtained from all participants.

Consent for publication

Not applicable. Our manuscript does not contain data from any individual person.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Competing interests

The authors declare that they have no competing interests in this section.

Funding

This manuscript was funded by the Fertility and Infertility Research and Clinical Center of Hormozgan University of Medical Sciences.

Authors' contributions:

F.E and M.A wrote the main manuscript.

F.E analyzed the data.

F.E prepared tables.

F.E and M.A reviewed the final revision of the manuscript.

All authors reviewed the manuscript.

All Authors read and approved the manuscript.

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Effect of Chemical versus Mechanical Oocyte Activation on Morphokinetic Embryonic Development in Oligoasthenoteratozoospermia Patients

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Abstract

Objective

Methods

Results

Conclusions

Introduction

Material and Methods

Patients, ovarian stimulation and oocyte retrieval

ICSI, culture, embryo transfer, and pregnancy

Chemical artificial oocyte activation:

Mechanical artificial oocyte activation:

Embryo evaluation and selection by the time-lapse system

Statistical analysis

Results

The effect of ionomycin in comparison with mechanical activation on fertilization rate in oligoasthenoteratozoospermia patients:

Discussion

Declarations

Ethics approval and consent to participate

Competing interests

Funding

References

Additional Declarations

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